METHOD OF USING ABSCISIC ACID TO TREAT AND PREVENT DISEASES AND DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application relies on the disclosure of, and claims the benefit of the filing dates of U.S. provisional patent application number 60/771,033, filed 8 February 2006, U.S. provisional patent application number 60/821,720, filed 8 August 2006, and U.S. utility patent application number 11/672,223, filed 7 February 2007, the entire disclosures of all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

[002] The present invention relates to the field of medical treatments for diseases and disorders. More specifically, the present invention relates to biologically active compounds that treat and prevent insulin resistance, impaired glucose tolerance, prediabetes, type 2 diabetes, and obesity-related inflammation. Description of Related Art

[003] ' In spite of efforts by public health officials to encourage physical activity and reduce energy intake, the obesity rate in the U.S. and worldwide has continued to climb and it has reached epidemic proportions. According to estimates by the Center for Disease Control and Prevention in the year 2000, 30% of Americans are obese and 65% are overweight (1). One of the manifestations associated with this obesity epidemic is the growing number of people diagnosed with Non-Insulin Dependent Diabetes Mellitus (NIDDM). NIDDM is a widespread and debilitating disease characterized by insulin resistance and inflammation that can lead to coronary heart disease, hypertension, blindness, neuropathy, nephropathy, and limb amputations (2). It was recently estimated that 20.8 million Americans had NIDDM and 40.1% of middle-aged adults had prediabetes, a condition characterized by either impaired glucose tolerance or high blood fasting glucose concentrations (2). Future predictions indicate that 1 of 3 children born in 2000 will one day become diabetic (3). The

impending consequence is that millions of people, if not already, will soon become dependent on oral antidiabetic medications to maintain their quality of life. [004] One of the most effective of the currently available medications is the thiazolidinedione (TZD) class of insulin-sensitizing drugs. Subsequent to their use as oral antidiabetic agents it was discovered that TZDs function by binding to peroxisome proliferator-activated receptor gamma (PPAR γ) (4), a nuclear receptor expressed highly in immune cells, intestine, and adipose tissue (5). The nuclear receptor superfamily, which includes the vitamin D receptor (VDR) ₅ retinoid X receptor (RXR), PPAR α, and PPAR δ, consists of 48 ligand-induced transcription factors that respond to steroid and thyroid hormones, vitamins, lipid metabolites, and xenobiotics (6, 7). After binding of a synthetic or natural agonist, PPAR γ forms a heterodimer with RXR and undergoes a conformational change that allows it to recruit coactivators (8). These coactivators, which include members of the steroid receptor coactivator (SRC) family, assist the PPAR γ RXR complex in binding to specific PPAR response elements (PPREs) in the promoter regions by increasing histone acetylation, thereby altering chromatin structure and making it more accessible (8). A primary outcome of the PPAR-controlled transcriptional regulation of genes is a reduction in the hyperlipidemia, hyperglycermia, and hyperinsulinemia associated with insulin resistance (9), though the extent to which each tissue contributes to this response is still unclear.

[005] While PPAR γ is expressed in a number of different organs that contribute to glucose homeostasis, including skeletal muscle, pancreas, and liver, white adipose tissue (WAT) is believed to represent the primary site of TZD action (10, 11). Adipose tissue is an extremely bioactive organ that produces a number of hormone- like polypeptides called adipokines, which regulate a wide-range of metabolic, immune and inflammatory processes throughout the body (12). Problems arise, however, when adipocytes become hypertrophic and dysfunctional during the onset of obesity. Obesity promotes the secretion of pro-inflammatory adipokines, such as leptin, plasminogen activator inhibitor 1 (PAI-I), tumor necrosis factor alpha (TNF-cc) and interleukin 6 (IL-6), and a suppression in the secretion of adiponectin, an anti-

US2007/003366

inflammatory and glucose-sensitizing polypeptide (13, 14). The pro-inflammatory adipokines can disrupt insulin signaling by promoting serine phosphorylation of insulin receptor substate 1 (IRS-I) (15). Obesity is also associated with the infiltration of bone-marrow derived macrophages, which become the key producers of pro-inflammatory mediators in WAT (16). PPAR γ activation by synthetic agonists such as rosiglitazone reduces macrophage infiltration in WAT, a sign of WAT inflammation, and increases the number of smaller, more insulin-sensitive adipocytes in the subcutaneous region (17, 18). Adipokine production is also modulated to favor the production of adiponectin and inhibit the secretion of pro-inflammatory compounds (19).

[006] The side effects of TZDs such as weight gain, hepato toxicity and congestive heart failure have limited their use by millions of diabetic patients (9, 20). For instance, troglitazone (Rezulin ^® ) was launched in 1997 and withdrawn from the market in March of 2000 due to reports of serious liver injury when compared to other TZDs (21), while other Food and Drug Administration (FDA)-approved TZDs for NEDDM treatment, including rosiglitazone (Avandia ^® ) and pioglitazone (Actos ^® ), continue to be widely prescribed, concerns regarding their safety persist. In this regard, the FDA recommended that the presence of liver enzymes in blood of diabetic patients taking Avandia ^® be periodically monitored. Furthermore, due to the risk factors and side effects connected with TZDs and other oral antidiabetic agents, there are no preventative medications currently available for the millions of people with prediabetes. While the role of ABA as a phytohormone has been studied extensively, there have been no studies that explore the effect of ABA as a dietary supplement or in treatment of diabetes or inflammation.

[007] U.S. Patent No. 3,958,025 to Livingston teaches a method of treating a vitamin deficiency of abscisic acid in man, animal, or avian species. The patent does not address the fact that abscisic acid is not considered by those of skill in the art as an essential vitamin, nor its requirement in the diet. In addition, the method disclosed in U.S. Patent No. 3,958,025 does not teach the use of abscisic acid to treat or prevent diabetes and inflammation, including obesity-related inflammation.

2007/003366

SUMMARY OF THE INVENTION

[008] In response, at least in part, to the above-described needs, the present invention provides a method of treating and preventing insulin resistance, prediabetes, type 2 diabetes, impaired glucose tolerance, and/or obesity-related inflammation in an animal, including mammals and humans, in need thereof. The method comprises administering a therapeutically effective amount of one or more of the following substances: abscisic acid, esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, and structurally related compounds thereof. In embodiments, the method comprises repeating the administering at least once. [009] In addition, the present invention provides a composition for treating and/or preventing type 2 diabetes, insulin resistance, impaired glucose tolerance, prediabetes, and/or obesity-related inflammation, where the composition comprises an effective amount of one or more of the following substances: abscisic acid, esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, and structurally related compounds thereof. In general, the composition is a therapeutic or prophylactic composition that comprises a solvent or carrier for the active compound.

_/ _ B—R_-I__E-_F—_ D__E__S__C_R_I__P_T_-I_O_-N_— O__F__ T__H__E—_ D—R_ __A__W___I_N__G_S

[010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the written description, serve to explain certain principles and details of embodiments of the invention.

[011] Figure 1 shows a general structure for ABA (Fig. IA) and a structurally related compound (Fig. IB).

[012] Figure 2 shows transactivation of peroxisome proliferator-activated receptor γ (PPAR γ) in 3T3-L1 pre-adipocytes cultured with abscisic acid (ABA) (0, 1, 2.5, 5, 10, 50 μM) or rosiglitazone (Ros) (1 μM). Relative luciferase activity (RLA) was calculated as the ratio of chemiluminescence 10 seconds before and after the reaction was stopped. Data are presented as average RLA ± standard error. Data points with an asterisk are significantly different (P<0.05).

[013] Figure 3 shows the effect of dietary abscisic acid (ABA) on response to an intraperitoneal glucose tolerance test (IPGTT) given after 36 days of dietary supplementation. (A) Leptin receptor-deficient (db/db) mice were fed 0, 100, 200, 400, or 800 mg of a racemic mixture of AB A/kg diet. (B) Db/db mice were fed either 0 or 100 mg racemic AB A/kg. Data are presented as least square means ± standard error. Data points with an asterisk are significantly different (P<0.05). [014] Figure 4 shows the effect of dietary abscisic acid (ABA)-supplementation on mRNA expression of peroxisome proliferator-activated receptor γ (PPAR γ) and PPAR γ-responsive genes adiponectin, aP2, and CD36 in white adipose tissue. Expression of the house-keeping gene (β-actin) in both diets was approximately equal. Data are presented as least square means ± standard error expressed as picograms (pg) of complementary DNA (cDNA) per microgram (μg) total RNA. An asterisk signifies that groups are significantly different (P<0.05).

[015] Figure 5 shows the effect of dietary abscisic acid (ABA)-supplementation on white adipose tissue (WAT) histology (A) Area of adipocytes (micrograms ² ) was assessed using SigmaPro ^® 5.0 software. Representative photomicrographs of WAT from mice fed diabetogenic, high- fat diets, supplemented with either 0 (B) or 100 mg AB A/kg diet (C) at IOOX magnification. Data are presented as least square means ± standard error. An asterisk signifies that values are significantly different (P<0.05). [016] Figure 6 shows the effect of dietary abscisic acid (ABA)-supplementation on macrophage infiltration and tumor necrosis factor-cc (TNF-α) expression in abdominal white adipose tissue (WAT) of db/db mice. Number of macrophages was obtained by averaging the results of 5 different fields at 400X magnification. Data are presented as least square means ± standard error. TNF-α is expressed as picograms (pg) of complementary DNA (cDNA) per microgram total RNA. An asterisk signifies that values are significantly different (P<0.05).

[017] Figure 7 shows a bar graph of liver histology scores for treatment with ABA (Panel A), and staining of liver cells (Panels B and C).

[018] Figure 8, Panels A-C, show intramuscular adipose tissue (also called infiltrating adipose tissue) and the presence of macrophages; Figure 8, Panel D, shows a bar graph of size of adipocytes in intramuscular adipocyte tissue. [019] Figure 9 shows the effect of ABA on body weight, feed intake, fasting blood glucose, and plasma lipids. Panel A shows body weights; Panel B shows food intake; Panel C shows fasting blood glucose (FBG, milligrams/deciliter), and Panel D shows plasma triglycerides (TGs, mg/ml). Data are presented as least square means ± standard error of 10 mice. The error bars at some time points (Figures 9 A and 9B) are smaller than the symbols and cannot be seen in the figure. Data points with an asterisk are significantly different (PO.05).

[020] Figure 10 depicts a phenotypic analysis of macrophage subsets within the stromal-vascular fraction (SVF) of white adipose tissue (WAT). Panels (A) and (B) are representative dot plots from a control db/db mouse. The F4/80hi and F4/801o subsets differed significantly in (C) total amount of macrophages and (D) mean fluorescence intensities (MFI). Panels (E) and (F) are the MFI's of CCR2 and CCR5- expressing macrophages in each population, respectively. Panel (G) represents the percentage of macrophages in each population expressing either CCR2 or CCR5. Data are presented as least square means ± standard error of 10 observations. Data points with an asterisk or number sign indicate a significant difference (P<0.05) between the separate macrophage populations.

[021] Figure 11 shows various characteristics of macrophages in control-fed and ABA-fed mice. The figure illustrates that abscisic acid (ABA) decreases the numbers of F4/80hi macrophages in white adipose tissue. Panels (A), (B), and (C) are representative dot plots for total stromal-vascular fraction (SVF) macrophage infiltration and the percentages of CCR2+ macrophages in the F4/80hi and F4/801o subsets, respectively, for control-fed db/db mice. Panels (D), (E), and (F) are the respective dot plots for a representative ABA-fed db/db mouse. Panels (G), (H), and (T) indicate the contribution of SVF macrophages, F4/801o macrophages, and F4/80hi macrophages to the total amount of gated cells. The mean fluorescence intensities (MFIs) of each macrophage sub-population for (J) CCR2 and (K) CCR5. (L) is the

difference in MFI of total SVF macrophages between control and ABA-fed mice. Data are presented as least square means ± standard error of 10 observations. Data points with different superscripts are significantly different (P<0.05). [022] Figure 12 shows the effect of dietary ABA supplementation on white adipose tissue and liver gene expression, MCP-I protein levels, liver triglyceride concentrations, and hepatic steatosis scores. This figure illustrates that abscisic acid (ABA) induces PPAR gamma and suppresses the expression of MCP-I in white adipose tissue. Db/db mice were fed high-fat diets with (black bars) or without (empty bars) ABA (100 mg/kg) for 36 days. Gene expression in RNA isolated from stromal-vascular cells (SVCs) and adipocytes was analyzed by real-time qRT-PCR for (A) monocyte chemoattractant protein 1 (MCP-I), (B) peroxisome proliferator- activated receptor gamma (PPAR gamma), and (C) CD36. (D) Liver MCP-I expression, (E) plasma MCP-I (picograms/milliliter), and (F) liver triglycerides (TG, mg/gram tissue) were also assessed. Gene expression values are expressed as a relative ratio to the housekeeping gene beta-actin. Data are presented as least square means ± standard error of 10 observations. Data points with an asterisk are significantly different (P<0.05). (G) Liver histology scores. (H) and (I) are representative photomicrographs of liver histology specimens stained with hemotoxylin and eosin (H&E).

[023] Figure 13 shows that the beneficial effects of ABA on insulin resistance are abrogated in immune cell-specific PPAR γ null mice. PPAR gamma flfl; MMTV- Cre+ and PPAR gamma flfl; MMTV-Cre- littermates (wild-type phenotype) were fed high-fat diets with or without ABA (100 mg/kg). (A) Final body weights and (B) response to an intraperitoneal glucose challenge (2 g glucose/kg body weight) in fasted mice (6h) were assessed after 28 weeks of dietary supplementation. Data are presented as least square means ± standard error of 8 mice. The error bars at some time points (Figure 13B and C) are smaller than the symbols and cannot be seen in the figure. Data points with different superscripts are significantly different (P<0.05). [024] Figure 14 shows that the beneficial effects of ABA on the infiltration of monocytes and chemokine production in white adipose tissue are abrogated in

immune cell-specific PPAR γ null mice. Panels (A), (B), and (C) represent the effects of ABA on total macrophage infiltration and the percentage of F4/801o and F4/80hi ATM in PPAR gamma flfl; MMTV-Cre+ and PPAR gamma flfl; MMTV-Cre- mice. Panels (D) and (E) are representative dot plots for stromal-vascular fraction (SVF) infiltration of ATMs in PPAR gamma flfl; MMTV-Cre+ and PPAR gamma flfl; MMTV-Cre- mice fed the control high-fat diet. Panels (F) and (G) are the respective dot plots for AB A-fed mice. Real-time qRT-PCR was used to assess the expression of monocyte chemoattractant protein 1 (MCP-I) (Panel H). Data are presented as least square means ± standard error of 8 mice. The error bars at some time points (Figure 14B) are smaller than the symbols and cannot be seen in the figure. Data points with different superscripts are significantly different (P<0.05). [025] Figure 15 shows that ABA inhibits MCP-I promoter activity by acting through PPAR γ. 3T3-L1 pre-adipocytes were transfected with a MCP-I promoter luciferase reporter construct with (black bars) or without (empty bars) co-transfection with a pCMX.PPAR gamma expression plasmid. After 20 hours, cells were treated with the PPAR gamma antagonist GW9662 (GW, 30 μM), ABA (12.5 μM), rosiglitazone (Ros, 1 μM), ABA and GW, or Ros and GW. The results were normalized for Renilla activity. Data are presented as least square means ± standard error of 4 observations. Data points with an asterisk are significantly different (P<0.05).

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE

INVENTION

[026] The present invention provides new uses for abscisic acid and structurally related compounds. The term abscisic acid (abbreviated herein as ABA) herein refers to a plant hormone containing a trimethylcyclohexene ring with one or more hydroxy groups (for instance a 6-hydroxy group), a 3-oxo group and an unsaturated side chain in the sixth position of the trimethylcyclohexen ring containing cis-7, trans-9 double bonds, its non-toxic salts, active esters, active isomers, active metabolites, and mixtures thereof. Non-toxic salts include, for example, alkyl esters having from 1 to 6

03366

carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and mixtures thereof. Active isomers of abscisic acid include geometrical isomers and its non-toxic salts, e.g., sodium, potassium, calcium, and magnesium salts, and its active esters, e.g. , alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and mixtures thereof. Active optical isomers of abscisic acid include the (+)-enantiomer and the (-)-enantiomer and its non-toxic salts, e.g., sodium, potassium, calcium, and magnesium salts, and its active esters, e.g., alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and mixtures thereof. Active metabolites of abscisic acid include oxygenated abscisic acid analogs, including but not limited to, 8'-hydroxyABA, (+)-7'- hydroxyABA, 2'3'-dihydroABA, 8'-hydroxy-2',3'-dihydroABA and its non-toxic salts, e.g., sodium, potassium, calcium, and magnesium salts, and its active esters, e.g., alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and mixtures thereof. Structurally related compounds, include but are not limited to, compounds containing conjugated double bonds (e.g., conjugated dienes, trienes and tetraenes) in the unsaturated side chain and compounds containing a trimethylcyclohexene ring with or without hydorxy moieties. For ease of reference, all such compounds are referred to herein generally at times as abscisic acid or ABA. [027] Abscisic acid may be a substantially pure single chemical compound or a mixture of one or more abscisic acid compounds as defined above. For example, the abscisic acid may be in the form of an extract obtainable or obtained from plant extracts, either directly or following one or more steps of purification or it can be chemically synthesized.

[028] The abscisic acid used in the described methods may be in a free acid form or bound chemically through ester linkages. In its natural form, abscisic acid is heat stable. Abscisic acid may be used in its natural state or in a dried and powdered form. Further, the free acid form of abscisic acid may be converted into a non-toxic salt, such as sodium, potassium or calcium salts, by reacting chemically equivalent amounts of the free acid form with an alkali hydroxide at a basic pH. Figure 1 depicts ABA and an exemplary compound falling within the definition of abscisic acid and

structurally related compounds. Other structurally related compounds are known in the art, such as those disclosed by Hill et al. (45), which is hereby incorporated herein by reference.

[029] In general, the invention provides for use of abscisic acid and structurally related compounds, such as a compound selected from the group consisting abscisic acid, esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, structurally related compounds thereof, or combinations thereof, in the treatment and prevention of diseases and disorders associated with PPARγ expression and lymphocyte activity. For example, in embodiments, the invention relates to prevention and/or treatment of hyperglycemia, impaired glucose tolerance, insulin resistance, prediabetes, and type 2 diabetes, while in other embodiments, the invention relates to prevention and/or treatment of inflammation, including but not limited to obesity-related inflammation. The invention is based, at least in part, on the discovery that abscisic acid can affect the expression of PPAR γ, and that the effects are relevant to all diseases and disorders that involve expression or activity of PPAR γ. As used throughout this document, the term ABA and all of its forms are meant to include the following compounds: abscisic acid, esters thereof, pharmaceutically suitable salts thereof, metabolites thereof, structurally related compounds thereof, analogs thereof, or combinations thereof, as disclosed herein. While not being limited to any particular mode of action, it is possible that abscisic acid and its derivatives and structurally related compounds affect PPARγ expression and/or activity. However, the invention also contemplates other modes of action, such as by affecting expression or activity of any number of other cellular molecules, including, but not limited to, nuclear receptors that may be activated by ABA, including liver X receptor (LXR), retinoid X receptor (RXR), pregnane X receptor (PXR), vitamin D receptor (VDR), as well as nuclear receptor-independent mechanisms.

[0301 In addition, in general, the invention relates to inhibition of infiltration of macrophages into skeletal muscle and white adipose tissue and related inflammation. The inhibition can be found in vitro and in vivo. The effect results from exposing cells to ABA. In embodiments, the invention provides for treating subjects with

ABA ₃ for example as a dietary supplement, to reduce skeletal muscle macrophage infiltration, white adipose tissue macrophage infiltration, inflammation, or all three. It also provides for treating a subject to achieve these goals, and additionally to treat a subject suffering from diabetes, to treat a subject at risk for developing diabetes, or to prevent a subject from developing diabetes.

[031] In a first aspect, the invention relates to a method of affecting the expression of PPAR γ in a cell. In general, the method comprises contacting a cell with ABA in an amount or concentration sufficient to affect expression or activity of PPAR γ in the cell. For example, it can be exposing a cell for a sufficient amount of time for ABA to enter the cell and have an effect on PPAR γ expression or activity. The method can be practiced either in vitro or in vivo. Where practiced in vitro, the method can be used to study the expression of PPAR γ, to test other compounds for the ability to supplement or antagonize the effects of ABA on PPAR γ expression, or for any other reason of importance to a researcher. When practiced in vivo, the method can be used as a method of treating a subject for one or more diseases or disorders associated with PPAR γ expression. It also may be a method of treating a subject that has a predisposition or likelihood of developing a disease or disorder associated with PPAR γ expression. According to the method of this aspect of the invention, preferably, expression of PPAR γ is increased. The step of contacting a cell can be any action that causes ABA to physically contact one or more target cells. Thus, it can be by way of adding ABA directly to an in vitro culture of cells to be contacted, and allowing the ABA sufficient time to diffuse through the media and contact at least one cell. Likewise, it can be through addition of a dry composition comprising ABA to cells in an aqueous environment. Alternatively, it can be by way of administering ABA to a subject via any acceptable administration route, and allowing the body of the subject to distribute the ABA to the target cell through natural processes. Thus, the in vivo methods can be methods of localized or systemic delivery of ABA to a cell in animals, including all mammals and humans in particular. According to this aspect, ABA and its related compounds can be used to treat a

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subject therapeutically or prophylactically, and to prepare a composition for use in treating.

[032] In another aspect, the invention provides a method of treating a subject suffering from or at risk of suffering from a disease or disorder involving PPAR γ expression. In general, the method comprises administering ABA or a composition comprising ABA to a subject in need thereof, in an amount sufficient to affect the amount or activity of PPAR γ in at least one cell of the subject. In embodiments, the ABA affects the expression of the PPAR γ gene, resulting in a change in PPAR γ mRNA levels in a cell. Ih embodiments, the ABA affects the amount of PPAR γ protein in a cell, preferably through increase in expression of the PPAR γ gene. In embodiments, the ABA affects the activity of the PPAR γ protein in a cell, preferably by increasing the amount of ABA in the cell. In preferred embodiments, PPAR γ mRNA expression, PPAR γ-responsive gene expression, such as CD36, AP2 (fatty acid binding protein 4) and adiponectin, protein levels, and/or protein activity is increased in a cell of the treated subject. In general, the method comprises administering a sufficient amount for a sufficient time to see a change in PPAR γ expression or activity. Often, the amount administered and the amount of time is adequate to see a change in one or more clinical symptoms of a disease or disorder, or to stop progression of a disease or disorder from reaching a stage where one or more clinical symptoms are seen. According to this aspect, ABA and its related compounds can be used to treat a subject therapeutically or prophylactically, and to prepare a composition for use in treating.

[033] In another aspect, the invention provides a method of treating a subject suffering from or at risk of suffering from a disease or disorder involving dysregulation of skeletal muscle macrophages and/or inflammation of skeletal muscles. In general, the method comprises administering ABA or a composition comprising ABA to a subject in need thereof, in an amount sufficient to reduce or eliminate skeletal muscle inflammation due to production of pro-inflammatory cytokines. According to this aspect, ABA and its related compounds can be used to

treat a subject therapeutically or prophylactically, and to prepare a composition for use in treating.

[034] In the methods of treating, administering ABA can be through any known and acceptable route. Such routes include, but are not necessarily limited to, oral, via a mucosal membrane (e.g., nasally, via inhalation, rectally, intrauterally or intravaginally, sublingualis), intravenously (e.g., intravenous bolus injection, intravenous infusion), intraperitoneally, and subcutaneously. Administering can likewise be by direct injection to a site (e.g., organ, tissue) containing a target cell (i.e., a cell to be treated). Furthermore, administering can follow any number of regimens. It thus can comprise a single dose or dosing of ABA, or multiple doses or dosings over a period of time. Accordingly, treatment can comprise repeating the administering step one or more times until a desired result is achieved. In embodiments, treating can continue for extended periods of time, such as weeks, months, or years. Those of skill in the art are fully capable of easily developing suitable dosing regimens for individuals based on known parameters in the art. The methods thus contemplate controlling, but not necessarily eliminating, the disease or disorder.

[035] The amount to be administered will vary depending on the subject, stage of disease or disorder, age of the subject, general health of the subject, and various other parameters known and routinely taken into consideration by those of skill in the medical arts. As a general matter, a sufficient amount of ABA will be administered in order to make a detectable change in the amount or activity of PPAR γ protein or mRNA in at least one cell of the subject to whom the ABA is administered. Suitable amounts are disclosed herein, and additional suitable amounts can be identified by those of skill in the art without undue or excessive experimentation, based on the amounts disclosed herein.

[036] The ABA will be administered in a form that is acceptable, tolerable, and effective for the subject. Numerous pharmaceutical forms and formulations for biologically active agents are known in the art, and any and all of these are contemplated by the present invention. Thus, for example, the ABA can be

formulated in an oral solution, a caplet, a capsule, an injectable, an infusible, a suppository, a losenge, a tablet, a cream or salve, an inhalant, and the like. [037] In one aspect, the invention provides a method of treating or preventing a subject suffering from diabetes, or otherwise healthy individuals at risk for developing diabetes. According to the invention, the term "a subject suffering from diabetes" is used to mean a subject {e.g., animal, human) having a disease or disorder showing one or more clinical signs that are typical of diabetes. The term "a subject at risk for developing diabetes" is used to mean a subject in which one or more clinical signs of diabetes are not clearly shown, but who shows one or more sub-clinical signs that are typical of diabetes, or who has a family history that indicates a significant risk of developing diabetes, or who suffers from obesity or is overweight. In general, the method of treating or preventing according to this aspect of the invention comprises administering to the subject an amount of ABA that is effective in treating or preventing one or more symptoms or clinical manifestations of diabetes, or in preventing development of such symptom(s) or manifestation(s). [038] Thus, according to the methods of the invention, the invention can provide methods of treatment of diabetes. The methods of treatment can be prophylactic methods. In embodiments, the method is a method of treating type 2 diabetes (T2D). hi embodiments, the method is a method of preventing diabetes, such as type 2 diabetes. In embodiments, the method is a method of halting the progression of diabetes, such as type 2 diabetes. In yet other embodiments, the method is a method of improving the health status of a subject suffering from diabetes, such as type 2 diabetes. Accordingly, in embodiments, the invention provides a method of protecting the health, organs, and/or tissues of a subject suffering from diabetes or at risk for developing diabetes.

[039] Tn one exemplary embodiment of the invention, the method of treating diabetes comprises treating diabetes without causing significant weight gain in the subject being treated. That is, it has been found that the method of treating according to the present invention, which provides the treatment effect, at least in part, by affecting the expression and/or activation of PPAR γ in some cells, provides the

beneficial effect without causing a significant gain in weight, for example by fluid retention, in the subject being treated, as compared to other similar subjects not receiving the treatment. While not wishing to be bound by any particular theory as to why this effect is seen, it is likely that treatment with ABA, while causing an increase in PPAR γ expression in some cells, does not cause over-expression or over- activation, as is commonly seen with some other {e.g., synthetic) PPAR γ agonists currently known for treatment of diseases associated with PPAR γ. [040] In view of the above-mentioned molecular basis for at least part of the effect seen, the present invention provides a method of treating diabetes by increasing the expression of PPAR γ in at least one cell of the subject being treated. As with other methods of the invention, the method comprises administering ABA to a subject suffering fjrom diabetes, where the ABA is administered in an amount sufficient to increase the expression, activity, or amount of PPAR γ in at least one cell of the subject, hi embodiments, the diabetes is type 2 diabetes. In the method, the cell(s) in which PPAR γ expression, level, or activity is increased can be any cell, from any tissue or organ, in the subject treated. In preferred embodiments, the cell(s) are white adipocyte tissue (WAT) cells, pancreatic cells, or both. In certain treatment methods, the methods do not cause an equivalent increase in PPAR γ expression, level, or activity in liver cells, as compared to the increase seen in WAT and/or pancreatic cells. In embodiments, no detectable increase in PPAR γ mRNA or protein is seen in a liver cell of a subject being treated. One exemplary embodiment of this aspect of the invention is a method of treating diabetes in which expression of PPAR γ is increased in certain cells of the subject, but not other cells, and in which the level of expression is not so high as to cause serious (or, in embodiments, any) noticeable or detectable deleterious effects on the short-term or long-term health of the subject. For instance, uncontrolled over-activation of PPAR γ in the liver could lead to liver injury. In treating diabetes according to the methods of the present invention, one effect that may be seen is an increase in interscapular brown adipose tissue (BAT) mass, which is a positive effect in the context of treatment of diabetes.

[041 ] In yet another aspect of the invention, a method of lowering glucose levels is provided. The method comprises administering ABA to a subject suffering from diabetes or at risk of suffering from diabetes, or otherwise having acute or long-term high glucose levels in blood or tissues. The ABA is administered in an amount sufficient to lower the glucose levels in the patient, and especially to lower levels of free glucose in the blood of the subject. Lowering can occur at any time under any physiological condition, but is preferentially seen with regard to the subject's fasting glucose level. In a related method of the invention, a method of increasing the glucose tolerance of a subject is provided. The method comprises the same steps as other methods of the invention, and is similarly based, at least in part, on the underlying mechanisms of action of ABA, and the surprisingly selective nature of the effects of ABA on certain cells, but not others. In addition, like the other methods, it is based, at least in part, on the low toxicity of ABA and, as a corollary, the high activity of ABA in affecting PPAR γ expression.

[042] Another aspect of the invention provides for effects on cells. These effects can be seen in vitro and in vivo. Certain effects have been discussed above, such as the effects on WAT, pancreatic cells, and BAT. In summary, the effects are to increase the levels of expression of PPAR γ in WAT and pancreatic cells, and to increase the mass of BAT. Additional effects provided by the methods of the invention include reducing the size of certain adipocytes and therefore preventing adipocyte hypertrophy and dysregulation (i.e., insulin resistance). Effects on liver cells and the liver in general are also seen as a result of practicing the methods of the invention. For example, a reduction in lipid accumulation in hepatocytes can be seen as an effect of the treatment (either in vivo administering or in vitro contacting) of the methods of the invention. As an outcome of this effect, the methods also provide a means for reducing the size of the liver of a subject, such as one suffering from diabetes (e.g., type 2 diabetes) or at risk of suffering from diabetes. [043] In addition to the methods provided above, the invention also provides methods that affect the immune system and the physiological effects of immune system functions. In general, the methods of this aspect of the invention are, like

those above, based at least in part on the effect of ABA on PPAR γ expression, level, and/or activity. The methods of this aspect of the invention can be considered as any of the following, or any combination of the following: methods of decreasing T cell proliferation, in particular, in diabetics or those at risk for developing diabetes; methods of suppressing excessive immune responses, including autoimmune responses, (in both diabetics and non-diabetics); and methods of protecting pancreatic beta cells from lipotoxicity and damage associated with immunoinflammatory responses. The methods of this aspect of the invention generally comprise administering to a subject in need ABA in an amount sufficient to produce the desired effect, whether- it be preventing overactivation of the immune system, decrease in T cell proliferation, or beta cell protection. The amount to be administered can be any suitable amount, as can be determined by application of known parameters in view of the amounts disclosed herein. Administration may be through any suitable route and by any suitable regimen, in accordance with the discussion above. Of course, where applicable (e.g., methods of inhibiting T cell proliferation), the methods according to this aspect of the invention maybe in vitro or in vivo methods. In the method of decreasing T cell proliferation, the method can be, among other things, a method of reducing the number of CD4+ T cells, a method of preventing or treating autoimmune disorders including type 1 diabetes, inflammatory bowel disease, multiple sclerosis or arthritis. Likewise, in the method of down-regulating overactivation of the immune system, the method can be, among other things, a method of reducing the number of CD4+ T cells, a method of reducing the number of CD8+ T cells, or both. It can also be, among other things, a method of reducing interferon γ (EFN γ) expression or levels, or production of IFN γ, in and around T cells or by T cells, antigen-presenting cells (macrophages or dendritic cells), or in the subject's body in general. [044] For example, exposing cells, such as skeletal muscle cells or adipose tissue cells, to ABA can result in inhibition or a reduction in skeletal muscle macrophage infiltration, which can lead to a reduction or inhibition of inflammation resulting from the effects of these cells. This can be thought of either as an effect on the immune system, per se, or an effect on immune system function. Regardless of the way in

which one considers the effect, the method provides a useful, convenient, and safe mode of treating a subject in need of such treatment. For example, it can benefit those suffering from inflammation in muscles or bone/muscle junctions, such as at tendons and ligaments. It can further benefit those suffering from diabetes, or those having a predisposition to diabetes, by reducing the number of macrophages in tissues and environments, where they can contribute to development or continuance of diabetes or a diabetic or pre-diabetic state.

[045] As such, the methods can provide methods of reducing inflammation, including obesity-related inflammation. The methods can reduce inflammation systemically (i.e., throughout the subject's body) or locally (e.g., at the site of administration or the site of inflammatory cells, including but not limited to T cells and macrophages). In treating or preventing obesity-related inflammation according to the methods of the present invention, one effect that may be seen is the decrease in the number of macrophages infiltrating the white adipose tissue and skeletal muscle tissue and a down-regulation of tumor necrosis factor- alpha expression in adipose tissue and skeletal muscle tissue. The methods can thus also be considered methods of affecting or altering the immune response of a subject to whom the ABA is administered.

[046] In view of the above methods, it should be evident that the present invention provides ABA for use in contacting cells, such as in treating cells of a subject. The above discussion focuses on the use of ABA alone or as part of a composition for use in what could generally be considered a pharmaceutical or medical setting. However, it is to be understood that the ABA or compositions comprising ABA can be used in treatment of subject by way of nutritional supplements, such as through dietary supplements. In such supplements, the ABA can be present in doses that are suitable for daily (or more often) administration. Typically, for dietary supplements, the ABA is presented in a form suitable for oral ingestion, such as by way of pill, capsule, tablet, caplet, powder, liquid, or the like. As with the forms for use in medical settings, typical additives can be included, such as colorants, flavorants, binders, gums, and the like.

[047] For example, the ABA may be present as a functional food ingredient, either as a stand-alone ingredient (such as would be seen with sugar, salt, pepper, etc.) or as an ingredient included in the food during processing or packaging. In such situations, the ABA and compositions comprising it would include ABA at an appropriate amount for oral ingestion. It is envisioned that this amount would be considerable less, on a gram of product ingested basis, than the amount used for pharmaceutical use in treating diabetes, prediabetes, impaired glucose tolerance, and insulin resistance or prophylactically treating those at risk for developing prediabetes, diabetes, impaired glucose tolerance, and insulin resistance. [048] In yet another form of the composition, a nutritional supplement is provided for treatment of diabetes or for treatment of those at risk for developing diabetes. In this form, the ABA is provided at a higher concentration than the food supplement form, but still at a lower dose than the "pharmaceutical" dose. This form can be considered a supplement for periodic administration of ABA to a subject. For example, it can be likened to a vitamin pill for periodic supplementation of various vitamins and minerals for a person, but rather than providing vitamins and minerals, it would provide ABA to enable the various beneficial effects discussed in this document, as well as others. The dietary supplement may comprise, in addition to ABA, any number of other substances, which are well known as suitable for ingestion by animals and humans. For example, it may contain fillers, binders, gums, colorants, and the like.

[049] As should be evident, the ABA may be provided in a pharmaceutically acceptable form. Thus, ABA can be provided in a form that is suitable for administration to a subject in need of it. It also may be present as a component of a composition, and in particular, a pharmaceutical composition. The ABA may be provided as a purified or semi-purified substance, or as a part of a simple or complex composition. Where present as part of a composition, the composition as a whole should be biologically tolerable at the amount to be exposed to a living cell. Thus, the composition may comprise toxic or otherwise deleterious substances when in its as- produced state, but be rendered non-toxic at a later date by further treatment or simply

by dilution. The pharmaceutical composition may comprise any number of substances in addition to ABA, such as, but not limited to, water, salts, sugars, buffers, biologically active compounds, drugs, etc.

[050] The ABA and compositions of the invention can be provided in any suitable form and container. Thus, in another aspect, the invention provides for a container containing ABA or a composition comprising ABA. In this aspect, the ABA or composition will be provided in the container in an amount that is sufficient for at least one use in a method according to the invention. Thus, it can be provided in an amount and in a form that is sufficient for one or more in vitro treatments of a cell for research purposes. It can also be provided in an amount and form that is sufficient for one or more in vivo treatments of a diabetic or person susceptible to developing diabetes. One of skill in the art can immediately contemplate the various numerous other amounts, forms, and uses for the various in vitro and in vivo applications, and thus all such amounts, forms, and uses need not be detailed here. [051] The container of the invention can be any container, fabricated in any shape and from any suitable material. It thus can be made from plastic, glass, paper or a paper product, metal, or some other polymeric material. It can be in any shape and size, such as in the shape of a tube, vial, ampoule, packet, pouch, wrapper, can, bottle, and box. Those of skill in the medical, dietary supplement, and food arts will immediately recognize the various other shapes, materials, and sizes that are suitable, and therefore these need not be detailed herein.

[052] In yet an additional aspect, the ABA, compositions, and/or containers, or combinations of these, can be provided in the form of a kit. For example, two or more containers containing a pharmaceutical formulation according to the invention may be provided together in a single package, referred to herein as a kit. Likewise, ABA and some or all reagents and supplies necessary for performing an in vitro assay according to the invention can be provided in a single package or kit. Numerous configurations of supplies and reagents may be included in the kit, in accordance with similar kits for pharmaceutical, dietary supplementation, and/or research that are known in the art.

[053] Definitions - Unless otherwise stated, the following definitions are used throughout the present application:

[054] Analysis of Variance (ANOVA): Arithmetic process for partitioning the overall variation in data sets into specific components based on sources of variation.

It has been used to determine whether numerical differences between treatment groups are statistically significant.

[055] Adipogenesis: the process by which new adipocytes or fat storage cells are generated.

[056] Allele: one of a number of viable DNA codings of the same gene.

[057] Conjugated diene: a molecule containing two double bonds separated by a single bond.

[058] Db/db mice: Term used to define a type of mouse which lacks both alleles of a long isoform of leptin receptor. This deficiency results in a high predisposition to developing type 2 diabetes. Reference is made to Experiments 1 and 3-6 (infra.) for further discussions on Db/db mice.

[059] Enantiomer: optical isomer; chemical classification of molecules based on their ability to rotate the plain of polarization clockwise (+) or anti-clockwise (-).

[060] Glycemia: concentration of glucose in blood.

[061] Hyperglycemia: increased concentrations of glucose in blood beyond the normal ranges.

[062] Hyperinsulinemia: increased concentrations of insulin in blood beyond the normal ranges.

[063] Insulinemia: concentration of insulin in blood.

[064] Insulin resistance: inability of tissues to respond to insulin and take up glucose from the blood.

[065] Substantially pure: having a purity of at least 90% by weight, preferably at least 95 % by weight such as at least 98%, 99% or about 100% by weight.

[066] Type 2 diabetes or Non-insulin dependent Diabetes Mellitus: term referring to a common type of diabetes caused by an unresponsiveness of cells to the actions of insulin. If the cells do not respond to insulin, they are unable to take up

glucose from blood, which results in glucotoxicity. In addition, the cells are deprived from the energy derived from glucose oxidation.

[067] Abscisic Acid (ABA): The term used herein refers to a plant hormone containing a trimethylcyclohexene ring with one or more hydroxy groups (for instance a 6-hydroxy group), a 3-oxo group and an unsaturated side chain in the sixth position of the trimethylcyclohexen ring containing cis-7, trans-9 double bonds its non-toxic salts, active esters, active isomers, active metabolites, active structurally related compounds and mixtures thereof. Non-toxic salts include, for example, alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and triglycerides, and mixtures thereof. Active isomers of abscisic acid include geometrical isomers and its non-toxic salts, e.g., sodium, potassium, calcium and magnesium salts, and its active esters, e.g. , alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and mixtures thereof. Active optical isomers of abscisic acid include the (+)-enantiomer and the (-)-enantiomer and its non-toxic salts, e.g., sodium, potassium, calcium and magnesium salts, and its active esters, e.g., alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and mixtures thereof. Active metabolites of abscisic acid include oxygenated abscisic acid analogs, including but not limited to, 8'- hydroxyABA, (+)-7'-hydroxyABA, 9"-hydroxyABA, 2'3'-dihydroABA, 8'-hydroxy- 2',3'-dihydroABA, neophaseic acid (neoPA) and its non-toxic salts, e.g., sodium, potassium, calcium and magnesium salts, and its active esters, e.g., alkyl esters having from 1 to 6 carbon atoms in the alkyl group, as well as mono-, di- and tri- glycerides, and active structurally related compounds thereof, including but not limited to compounds with conjugated double bonds (e.g., conjugated dienes, conjugated trienes conjugated tetraenes) in the side chain and compounds containing a trimethylcyclohexene ring, with or without hydroxy groups; active analogs thereof such as those described in The Merck Index, Eighth Edition, page 1711; active conjugated abscisic acids thereof such as β-D-glucopyranosyl abscisate (ABAGE) and abscisic acid-l'-β-D-glucopyranoside (ABAGS), and mixtures thereof.

[068] Abscisic acid may be a substantially pure single chemical compound or a mixture of one or more abscisic acid compounds as defined above. For example, the abscisic acid may be in the form of an extract obtainable or obtained from plant extracts, either directly or following one or more steps of purification. [069] The abscisic acid used in the described methods may be in a free acid form or bound chemically through ester linkages. In its natural form, abscisic acid is heat stable. Abscisic acid may be used in its natural state or in a dried and powdered form. Further, the free acid form of abscisic acid may be converted into a non-toxic salt, such as sodium, potassium or calcium salts, by reacting chemically equivalent amounts of the free acid form with an alkali hydroxide at a basic pH. [070] The abscisic acid may be a substantially pure single chemical compound or a mixture of one or more abscisic acid compounds as defined above. The term substantially pure means having a purity of at least 90% by weight, including all specific integers above 90%. Preferably it has a purity of at least 95 % by weight, such as at least 98%, 99%, or 100% or about 100% by weight. For example, the abscisic acid may be in the form of an extract obtainable or obtained from plants, either directly or following one or more steps of purification. [071] Administration - In the course of the method of the present invention, a therapeutically effective amount of abscisic acid compound can be administered to an animal, including mammals and humans, in many ways. While in the preferred embodiment, the abscisic acid compound is administered orally or parenterally, other forms of administration such as through medical compounds or aerosols are also contemplated.

[072] For oral administration, the effective amount of abscisic acid may be administered in, for example, a solid, semi-solid, liquid, or gas state. Specific examples include tablet, capsule, powder, granule, solution, suspension, syrup, and elixir agents. However, the abscisic acid compound is not limited to these forms. [073] To formulate the abscisic acid of the present invention into tablets, capsules, powders, granules, solutions, or suspensions, the abscisic acid compound is preferably mixed with a binder, a disintegrating agent and/or a lubricant. If necessary,

the resultant composition may be mixed with a diluent, a buffer, an infiltrating agent, a preservative and/or a flavor, using known methods. Examples of the binder include crystalline cellulose, cellulose derivatives, cornstarch, and gelatin. Examples of the disintegrating agent include cornstarch, potato starch, and sodium carboxymethylcellulose. Examples of the lubricant include talc and magnesium stearate. Further, additives, which have been conventionally used, such as lactose and mannitol, may also be used.

[074] For parenteral administration, the abscisic acid compound of the present invention may be administered rectally or by injection. For rectal administration, a suppository may be used. The suppository may be prepared by mixing the abscisic acid of the present invention with a pharmaceutically suitable excipient that melts at body temperature but remains solid at room temperature. Examples include but are not limited to cacao butter, carbon wax, and polyethylene glycol. The resulting composition may be molded into any desired form using methods known to the field. [075J For administration by injection, the abscisic acid compound of the present invention may be injected hypodermically, intracutaneously, intravenously, or intramuscularly. Medicinal drugs for such injection may be prepared by dissolving, suspending or emulsifying the abscisic acid of the invention into an aqueous or nonaqueous solvent such as vegetable oil, glyceride of synthetic resin acid, ester of higher fatty acid, or propylene glycol by a known method. If desired, additives such as a solubilizing agent, an osmoregulating agent, an emulsifier, a stabilizer, or a preservative, which has been conventionally used may also be added. While not required, it is preferred that the composition be sterile or sterilized. [076] For formulating the abscisic acid of the present invention into suspensions, syrups or elixirs, a pharmaceutically suitable solvent may be used. Included among these is the non-limiting example of water.

[077] The abscisic acid compound of the present invention may also be used together with an additional compound having other pharmaceutically suitable activity to prepare a medicinal drug. A drug, either containing ABA as a stand-alone

compound or as part of a composition, may be used in the treatment of subjects in need thereof.

[078] The abscisic acid of the present invention may also be administered in the form of an aerosol or inhalant prepared by charging the abscisic acid in the form of a liquid or fine powder, together with a gaseous or liquid spraying agent and, if necessary, a known auxiliary agent such as an inflating agent, into a non-pressurized container such as an aerosol container or a nebulizer. A pressurized gas of, for example, dichlorofluoromethane, propane or nitrogen may be used as the spraying agent.

[079] Abscisic acid may be administered to an animal, including mammals and humans, in need thereof as a pharmaceutical or veterinary composition, such as tablets, capsules, solutions, or emulsions. In a preferred embodiment of the invention, the free acid form of punicic acid is administered. However, administration of other forms of abscisic acid, including but not limited to esters thereof, pharmaceutically- suitable salts thereof, metabolites thereof, structurally related compounds thereof, analogs thereof, and combinations thereof, in a single dose or a multiple dose, are also contemplated by the present invention.

[080] Abscisic acid may also be administered to an animal in need thereof as a nutritional additive, either as a food or nutraceutical supplement.

[081] The terms "preventing" or "treating", "treating" or "ameliorating" and similar terms used herein, include prophylaxis and full or partial treatment. The terms may also include reducing symptoms, ameliorating symptoms, reducing the severity of symptoms, reducing the incidence of the disease, or any other change in the condition of the patient, which improves the therapeutic outcome.

[082] The abscisic acid is preferably used and/or administered in the form of a composition. Suitable compositions are, preferably, a pharmaceutical composition, a foodstuff or a food supplement. These compositions provide a convenient form in which to deliver the abscisic acid. Compositions of the invention may comprise an antioxidant in an amount effective to increase the stability of the abscisic acid with respect to oxidation.

[083] The amount of abscisic acid that is administered in the method of the invention or that is for administration in the use of the invention is any suitable amount. It is preferably from about 0.000 Ig to about 20g (more preferably 0.0 Ig to Ig, such as 0.05g to 0.5g) of abscisic acid or derivative thereof per day. Suitable compositions can be formulated accordingly. Those of skill in the art of dosing of biologically active agents will be able to develop particular dosing regimens for various subjects based on known and well understood parameters. [084] A preferred composition according to the invention is a foodstuff. Food products (which term includes animal feed) preferably contain a fat phase, wherein the fat phase contains abscisic acid. The foodstuffs are optionally used as a blend with a complementary fat. For example, the fat may be selected from: cocoa butter, cocoa butter equivalents, palm oil or fractions thereof, palmkernel oil or fractions thereof, interesterified mixtures of those fats or fractions thereof. It may also contain liquid oils, such as those selected from: sunflower oil, high oleic sunflower oil, soybean oil, rapeseed oil, cottonseed oil, fish oil, safflower oil, high oleic safflower oil, corn oil, and MCT-oils. Examples of suitable foodstuffs include those selected from the group consisting of margarines, fat continuous or water continuous or bicontinuous spreads, fat reduced spreads, confectionery products such as chocolate or chocolate coatings or chocolate fillings or bakery fillings, ice creams, ice cream coatings, ice cream inclusions, dressings, mayonnaises, cheeses, cream alternatives, dry soups, drinks, cereal bars, sauces, snack bars, dairy products, clinical nutrition products, and infant formulations.

[085] Other non-limiting examples of compositions are pharmaceutical compositions, such as in the form of tablets, pills, capsules, caplets, multiparticulates (including granules, beads, pellets and micro-encapsulated particles); powders, elixirs, syrups, suspensions, and solutions. Pharmaceutical compositions will typically comprise a pharmaceutically acceptable diluent or carrier. Pharmaceutical compositions are preferably adapted for administration parenterally {e.g., orally). Orally administrable compositions may be in solid or liquid form and may take the form of tablets, powders, suspensions, and syrups, among other things. Optionally,

the compositions comprise one or more flavoring and/or coloring agents. In general, therapeutic and nutritional compositions may comprise any substance that does not significantly interfere with the action of the ABA on the subject. [086] Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the art of pharmacy. The compositions of the invention may contain 0.01-99% by weight of abscisic acid. The compositions of the invention are generally prepared in unit dosage form. Preferably the unit dosage of abscisic acid is from lmg to lOOOmg (more preferably from 50mg to 500mg). The excipients used in the preparation of these compositions are the excipients known in the art. [087] Further examples of product forms for the composition are food supplements, such as in the form of a soft gel or a hard capsule comprising an encapsulating material selected from the group consisting of gelatin, starch, modified starch, starch derivatives such as glucose, sucrose, lactose, and fructose. The encapsulating material may optionally contain cross-linking or polymerizing agents, stabilizers, antioxidants, light absorbing agents for protecting light-sensitive fills, preservatives, and the like. Preferably, the unit dosage of abscisic acid in the food supplements is from lmg to lOOOmg (more preferably from 50mg to 500mg). [088] Dose - The method of the present invention can comprise administering a therapeutically effective amount of abscisic acid compound to an animal in need thereof. The effective amount of abscisic acid depends on the form of abscisic acid compound administered, the duration of the administration, the route of administration (e.g., oral or parenteral), the age of the animal and the condition of the animal, including mammals and humans.

[089] For instance, an amount of abscisic acid effective to treat or prevent type 2 diabetes, prediabetes, impaired glucose tolerance, or insulin resistance in an animal can range from 0.1-10,000 mg/kg/day. A preferred effective amount of abscisic acid is 1. to 5,000 mg/kg/day, with a more preferred dose being 2 to 100 mg/kg/day. The upper limit of the effective amount to be administered is not critical, as abscisic acid is relatively non-toxic as long as the recipient's diet contains the necessary essential nutrients. The effective amount of abscisic acid is most effective in treating or

preventing type 2 diabetes, pre-diabetes, impaired glucose tolerance, and insulin resistance of an animal when administered to an animal for periods ranging from about 7 to 100 days, with a preferred period of 15 to 50 days, and a most preferred period of 30 to 42 days.

[090] An amount of abscisic acid is most effective in preventing over-activation of the immune system can range from 0.1 to 500 mg/kg/day, with a preferred dose of 1 to 150 mg/kg/day.

[091] When the effective amount of the abscisic acid compound of the present invention is administered in a nutritional, medical or veterinary composition, the preferred dose ranges from about 0.01 to 2.0% wt/wt to the food or nutraceutical product.

[092] Preparation abscisic acid - Abscisic acid has previously been extracted from leaves of Lupin (Lupinus cosentinii), Apricot (Prunus armeniacά), Avocado (Persea Americana), Sunflower (Helianthus annuus), Grapevine (Vitis viniferά), Tomato (Lycopersicon esculentum), Spinach [Spinada oleracea), Orange (Citrus sinensis) and Mango (Mangifera indicά) (46). ABA and its metabolites have also been isolated from Brassica napus and Brassica rapa seed (47) and could also be isolated from fruits and any other plant materials. The abscisic acid compound has been extracted from plant leaves through many procedures, including: 1) methanol extraction; 2) cold water extraction or 3) boiling water extraction (Loveys, 1988). For the methanol extraction samples of leaf material were homogenized in aqueous methanol, the homogenate was centrifuged and the pellet re-extracted with methanol. Water was added to the combined supematants before evaporation. The resulting extract was adjusted to apH of 2.5 and the abscisic acid compound extracted with three washes of ethyl acetate. The ethyl acetate extracts can be further purified by chromatography. The cold water and boiling water methods consist on homogenization of plant materials in cold or boiling water, respectively prior to the ethyl acetate extraction.

EXAMPLES

[093] The invention will be further explained by the following Examples, which are intended to be purely exemplary of the invention, and should not be considered as limiting the invention in any way. In the Examples and throughout this specification, all percentages, part and ratios are by weight unless indicated otherwise. [094] Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor γ (PPAR γ) currently used in the treatment of Non- Insulin Dependent Diabetes Mellitus (NIDDM). Despite their efficacy, TZDs are associated with a number of side effects, such as weight gain, hepatotoxicity, and congestive heart failure that have limited their use by millions of diabetic patients. We have investigated the ability of abscisic acid (ABA), a naturally occurring phytochemical, to activate PPAR γ. We found that ABA induced transactivation of PPAR γ in 3T3-L1 pre-adipocytes in vitro. We next examined the effect of dietary ABA-supplementation (100 mg/kg) on glucose tolerance, obesity-related inflammation, and mRNA expression of PPAR γ and PPAR γ-responsive genes in white adipose tissue (WAT) of db/db mice fed high fat diets. Dietary ABA- supplementation for 36 days ameliorated fasting blood glucose and glucose tolerance during a glucose tolerance test, and increased mRNA expression of PPAR γ and its responsive genes (i.e., adiponectin, aP2, and CD36) in WAT. We also found that adipocyte hypertrophy, tumor necrosis factor-α (TNF-α) expression, and macrophage infiltration in WAT were significantly attenuated in ABA-fed mice. These findings indicate that ABA can be used as a nutritional and pharmaceutical intervention against diabetes, and in particular NEDDM, and against inflammation, including obesity- related inflammation.

[095] An objective of the studies presented below was to determine whether the naturally occurring phytochemical, ABA, was a novel PPAR γ ligand that could be used as a safer alternative to TZDs. The results from the transfection assay demonstrated for the first time that ABA activated PPAR γ in 3T3-L1 pre-adipocytes in vitro. Furthermore, we found that ABA was 10 times less potent than the synthetic agonist rosiglitazone. Other natural substances, including fatty acids and eicosanoids

(27), components of oxidized low-density lipoproteins (29), have also been found to activate PPAR γ in vitro. However, PPAR γ activation in vitro does not always result in transcriptional regulation of PPAR-responsive genes or beneficial health effects in vivo (7, 27).

[096] ABA is a widely studied naturally occurring phytohormone that plays a key role in many different processes in plants, including stress response, leaf abscission, growth and development, and germination (39-42). Interestingly, ABA is also a key component of sugar sensitivity, as plants deficient of ABA are glucose insensitive (42, 43). While ABA is ubiquitously found in plants, and therefore consumed by humans as a part of the daily diets, its natural content ranges from 5-15 mg/kg in leaves (44). Thus, the doses that can be attained by vegetable intake would be significantly lower than those required for efficacy against non-insulin dependent diabetic and pre- diabetic states. This is the first study to explore the effect of this bioactive molecule in the prevention of NIDDM. Our finding that ABA is a novel PPAR γ ligand opens the possibility that this compound could be used as a safe and effective alternative to the synthetic agonists for the 20.8 million Americans with NIDDM and the millions more who have prediabetes. There are no preventative medications currently available to the latter group. The potential for ABA as an everyday nutritional supplement for the treatment or prevention of NIDDM is promising, and as more is learned about other uses of PPAR γ agonists for treating chronic diseases such as atherosclerosis and inflammatory bowel disease, this potential will only continue to grow.

[097] Example 1: Research Design and Methods

[098] A. Transfection of 3T3-Ll preadipocytes

[099] Transformation plasmid DNA was performed using One Shot TOPlO

(Invitrogen, Carlsbad, CA). Briefly, 3 μl of the ligation reaction were added to a thawed 50 μl vial of One Shot ^® cells for each reaction. The vials were then set on ice for 30 minutes, followed by 30 seconds incubation in a 42°C water bath. Afterwards,

250 μl of pre-warmed S.O.C. medium was added to each vial. Vials were then

secured on their sides in a microcentrifuge rack and were placed in a Gyromax 727 orbital shaking incubator (Amerex Instruments, Lafayette, CA) and incubated at 37°C at 225 rpm. After 1 hour incubation, 100 μl of each transformation vial was spread on separate, labeled LB agar plates (Sigma- Aldrich, Milwaukee, WI), inverted, and incubated at 37°C overnight. Colonies were selected and isolated using Plasmid Maxi Kit (Qiagen, Valencia, CA) according to manufacturer's instructions. After isolation, the plasmids were transfected into 3T3-L1 pre-adipocytes (American Type Culture Collection, Manassas, VA) using the Targefect-BAC transfection kit. (Targeting Systems, Santee, CA). Briefly, 3T3-L1 cells were cultured in complete DMEM high glucose media (Invitrogen) at 37°C for 2 days until confluence. After digestion with 2 ml trypsin (Mediatech, Herndon, VA), the cells were resuspended in 6 ml high glucose DMEM (Mediatech) and then aliquoted into a 24 well plate. Following an overnight incubation at 37°C in a humidified atmosphere containing 5% CO ₂ , 50 μl of the transfection complex was added to new media for each well. Each 50 μl transfection complex contained 0.8 μg plasmid DNA and 1.5 μl Targefect (Targeting Systems). After another overnight incubation at 37°C, racemic ABA (1, 2.5, 5, 10, and 50 μM) and rosiglitazone (1 μM) were added to their respective wells in duplicate and incubated at 37°C for 24 hours.- On the day of the reading, the cells were washed with 500 μl PBS and lysed with 100 μl lysis reagent (Promega, Madison, WI). After 15 minutes, lysed cells were collected and luciferase activity for each concentration was calculated using the Dual-Luciferase reporter assay system (Promega) and TD- 20/20 Single-Tube Luminometer (Turner Biosystems, Sunnyvale, CA) according to manufacturers' instructions. Relative luciferase activity (RLA) was calculated as a ratio of the chemiluminescence 10 seconds after the Luciferase Assay Reagent II (Promega) was added over the chemiluminescence 10 seconds after the Stop&Glo Reagent (Promega).

[100] B. Mice and Dietary Treatments

[101] Four week-old, male BKS.Cg -+Lepr ^db /+Lepr ^db /OlaHsd (db/db) mice were purchased from Harlan Laboratories (Indianapolis, IN) and maintained at the animal facilities at Virginia Polytechnic Institute and State University in a room maintained at

75° F, with a 12:12 h light-dark cycle starting from 6:00 AM. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Virginia Polytechnic Institute and State University and met or exceeded requirements of the Public Health Service/National Institutes of Health and the Animal Welfare Act.

[102] Experiment 1 : mice (n=25) were fed high-fat (>40% energy from fat), isocaloric, isonitrogenous diets containing 0, 100, 200, 400, and 800 mg of a racemic (±) ABA mixture/kg diet for 36 days (Table 1) (Sigma). These doses were chosen based on the standard concentrations of TZDs utilized in the treatment of NIDDM. The mice were weighed and their food intake was recorded daily. On day 36 of the study fasted mice (12h) were bled through the caudal vein and fasting blood glucose concentrations were determined. To investigate the ability of ABA to normalize glucose concentrations, an intraperitoneal glucose tolerance test (IPGTT) was performed on day 36 on the fasted mice. Briefly, mice were bled at 0, 10, 25, and 45 minutes following the administration of glucose (2g/kg) for measurement of plasma glucose levels. Following the IPGTT, the mice were killed by CO ₂ narcosis and WAT was excised and stored in RNA later (Ambion, Austin, TX), a commercial solution that inhibits RNases. Weights were recorded for WAT, interscapular brown adipose tissue (BAT), and liver.

[103] Experiment 2: The ABA dose utilized (100 mg AB A/kg) was based on the results obtained from the IPGTT of the ABA titration study {i.e., Experiment 1). Mice in experiment 2 were fed high-fat diets containing 0 or 100 mg AB A/kg for 36 days. Their body weights and food intake was recorded daily, and blood was collected on day 36 of the study during an IPGTT. This IPGTT included one additional 100 minutes time point. Following the IPGTT the mice were killed and WAT was collected and stored in RNA later (Ambion) for RNA isolation and gene expression analyses and in 10% buffered neutral formalin for histological evaluation. Weights were recorded for abdominal WAT, BAT, and liver.

[104] C. Histopathology

[105] Hepatic and adipose sections were fixed in 10% buffered neutral formalin, embedded in paraffin, cut at thicknesses of 7 microns and later stained with hematoxylin and eosin (H&E) for histological examination of adipocyte hypertrophy and inflammatory lesions. Hepatic sections were scored based on the amount of lipodystrophy observed according to the Nonalcoholic steatohepatitis (NASH) scoring method, with a score of 0 signifying <5% of the cells contain lipid droplets and a score of 4 signifying that >75% of the cells contained lipids and a magnification of 400X (23). The final score was the average of five randomly chosen fields. The area of adipocytes from WAT was analyzed using Sigma Pro ^® 5.0 software at a magnification of 10OX, and macrophage infiltration was determined by averaging the number of macrophages observed in five different fields at 400X. [106] D. Quantitative Real-Time Reverse Transcriptase PCR

[107] Total RNA was isolated from WAT using the RNA isolation Lipid Tissue

Minikit (Qiagen) according to the manufacturer's instructions. Total RNA (1 μg) was used to generate complementary DNA (cDNA) template using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The total reaction volume was 20 μl with the reaction incubated as follows in the Tetrad Thermocycler: 5 minutes at 25°C, 30 minutes at 52°C, 5 minutes at 85°C, hold at 4°C. PCR was performed on the cDNA using Taq DNA polymerase obtained from Invitrogen and using previously described conditions (24, 25). Each gene amplicon was purified with the MiniElute PCR Purification Kit (Qiagen) and quantitated on an agarose gel. These purified amplicons were used to optimize real-time PCR conditions and to generate standard curves in the real-time PCR assay. Primer concentrations and annealing temperatures were optimized for the iCycler iQ system (Bio-Rad) for each set of primers using the system's gradient protocol (Table 1). PCR efficiencies were maintained at 100% for each primer set during optimization and also during the real-time PCR of sample DNA.

[108] Complementary DNA (cDNA) concentrations for β-actin, PPAR γ, PPAR a, PPAR δ, adiponectin, CD36, aP2, and TNF-α were examined by real-time

quantitative PCR using and iCycler IQ System and the iQ SYBR green supermix (Bio-Rad). SYBR green I is a general double-stranded DNA intercalating dye and, therefore, may detect nonspecific products and primer/dimers in addition to the amplicon of interest. In order to determine the number of products produced during the real-time PCR, a melting curve analysis was performed on each product. Realtime PCR was used to measure the starting amount of nucleic acid of each unknown sample of cDNA on the same 96-well plate. Results axe presented as starting quantity of cDNA (picograms) per microgram of mRNA.

[109] Table 1. Oligonucleotide Sequences for Quantitative Real-Time PCR.

Primer Sequence Length Ace. No. SEQ ID NO.

PPAR αF 5'TGGGGATGAAGAGGGCTGAGS' 143 NM_011144 1

PPAR aR 5'GGGGACTGCCGTTGTCTGTS' 2

PPAR δF 5 ' ACAGTGACCTGGCGCTCTTC3 ' 96 U10375 3

PPAR δR 5'TGGTGTCCTGGATGGCTTCTS' 4

PPAR γF 5 'CAGGCTTGCTGAACGTGAAG3 ' 117 NM Ol 1146 5

PPAR γR 5'GGAGCACCTTGGCGAACAS' 6

CD36F 5'CCGGGCCACGTAGAAAACA3' 156 NM_007643 7

CD36R 5'CCTCCAAACACAGCCAGGAC3' 8 β-actinF 5'CCCAGGCATTGCTGACAGG3' 141 X03672 9 β-actinR 5'TGGAAGGTGGACAGTGAGGCS' 10

AdiponectinF 5αCAAGGCCGTTCTCTTCACC3' 123 NM_009605 11

AdiponectinR 5'CCCCATCCCCATACACCTG3 ' 12 aP2F 5'TCTCTTATCAAAGGCTCTACTTCCS' 78 NM_001442 13 aP2R 5'CAAAATTCCATCCAGGCCTCT3' 14

TNF-αF 5αCTGCCAGAAGAGGCACTCC3' 137 M 13049 15

TNF-αR 5'CGATCACCCCGAAGTTCA3' 16

F, forward; R, reverse. PCR primer pairs were designed for an optimal annealing temperature of 57°C and product length shorter than 157 base pairs.

[110] E. Statistics

[111] Data were analyzed as a completely randomized design. To determine the statistical significance of the model, analysis of variance (ANOVA) was performed using the general linear model procedure of Statistical Analysis Software (SAS) as previously described (24). A P < 0.05 was considered to be significant. When the model was significant, ANOVA was followed by Sheffe's multiple comparison

method. Non-parametric data were analyzed by using the Mann- Whitney U test followed by a Dunn's multiple comparison's test.

[112] Example 2: ABA activates PPAR γ in 3T3-L1 pre-adipocytes in vitro

[113] In order to establish whether ABA was a novel ligand of PPAR γ in vitro, we cultured increasing concentrations (1, 2.5, 5, 10, and 50 μM) of racemic ABA with 3T3-L1 pre-adipocytes that were transfected with a PPAR γ-Luciferase plasmid. In concurrence with our hypothesis, ABA significantly increased the relative luciferase activity of the transfected cells beginning at the 10 μM ABA concentration (Figure 2). No additional increase in PPAR γ activity was observed with the 50 μM ABA concentration. Moreover, the PPAR γ activation obtained from 10 μM ABA was similar to that observed with 1 μM rosiglitazone, indicating that ABA is 10-fold less potent than the synthetic PPAR γ ligand in vitro.

[114] Example 3: Dietary ABA lowers fasting blood glucose and improves glucose tolerance

[115] To confirm our in vitro findings suggesting that ABA is a novel PPAR γ ligand, we performed two separate in vivo experiments using prediabetic db/db mice. Experiment 1 was a dose-titration study, where we fed 4-week old db/db mice (n=25) diabetogenic, high fat diets containing either 0, 100, 200, 400, or 800 mg racemic ABA mixture/kg diet. Experiment 2 was designed as a replicate of experiment 1 in which db/db mice (n=14) were fed the most effective dose of ABA or a control diet. On day 36, mice fed the ABA-supplemented diets had significantly lower fasting blood glucose (FBG) concentrations, regardless of the amount of dietary ABA (i.e., 100 to 800 mg), than mice fed an isocaloric and isonitrogenous control diet without ABA (Table T). These in vivo results are in agreement with the transfection findings showing no difference in PPAR γ transactivation between 10 and 50 μM ABA.

[116] Table 2: Composition of Experimental Diets'

Dietary Treatments

Ingredient Control lOO mg ABA 200 mg ABA 400 mg ABA 800 mg ABA

Casein 232 232 232 232 232

L-cystein 3 3 3 3 3

DL-Methionine 3.5 3.5 3.5 3.5 3.5

Corn Starch 137 137 137 137 137

Maltodextrin 150 150 150 150 150

Sucrose 162.595 162.595 162.595 162.595 162.595

Cellulose 50 50 50 50 50

Cholesterol 1.9 1.9 1.9 1.9 1.9

Mineral Mix (AIN-93) ² 40.60 40.60 40.60 40.60 40.60

Calcium phosphate dibasic 4.64 4.64 4.64 4.64 4.64

Vitamin Mix (AIN-93) ³ 16.24 16.24 16.24 16.24 16.24

Choline Bitartrate ⁴ ■ , 5 5 5 5 5

Tert-butylhydroquinone ' ⁵ ' 0.02 0.02 0.02 0.02 0.02

Vitamin K, phylloquinone 0.005 0.005 0.005 0.005 0.005

Soybean Oil 30 30 30 30 30

Lard 163.5 163.5 163.5 163.5 163.5

Abscisic Acid (ABA) - 0.100 0.200 0.400 0.800

* Provides approximately 19.6% fat, 0.2% total cholesterol and 4.4 kilocalories/g (kcal/g) it obtains 40% kcal from fat. Kcal density is approximately 16% higher than typical AIN-93G-based diets.

Supplied per kg of mineral mix: 357 g calcium carbonate, 196 potassium phosphate monobasic, 70.78 potassium citrate, 74 g sodium chloride, 46.6 g potassium sulfate, 24.3 g magnesium oxide, 6.06 g ferric citrate, 1.65 g zinc carbonate, 0.63 g magnesium carbonate, 0.31 g cupric carbonate, 0.01 g potassium Iodate, 0.01025 sodium selenate, 0.00795 ammonium paramolybdate, 1.45 g sodium- metasilicate, 0.275 chromium potassium sulfate, 0.0174 lithium chloride, 0.0815 boric acid, 0.0635 sodium fluoride, 0.0318 nickel carbonate, hydroxide, tetrahydrate, 0.0066 ammonium vanadate, and 220.716 sucrose.

Supplied per kg of vitamin mix: 3 g nicotinic acid, 1.6 g calcium pentotenate, 0.7 g pyridoxine HCL, 0.6 g thiamin HCL, 0.6 g riboflavin, 0.2 g folic acid, 0.02 g D-biotin, 2.5 g vitamin B ₁₂ (0.1% mannitol), 15 g DL-α tocopherol acetate (500 IU/g), 0.8 g vitamin A palmitate (500,000 ILVg), 0.2 vitamin D ₃ (cholecalciferol, 500,000 IU/g), 0.0075 vitamin K (phylloquinnone), 974.705 g sucrose. § The choline bitartrate concentrations have been increased from 2.5 g/kg in regular AIN-93G diets to 5 g/kg due to increased kcal density of high fat diets.

' ' Antioxidant

[117] In order to test the ability of ABA to activate PPAR γ in vivo, we fed prediabetic db/db mice 0, 100, 200, 400, and 800 mg of aracemic ABA mixture/kg as part of a high-fat diet for 36 days. Mice consuming the ABA-supplemented diets had

significantly lower fasting blood glucose levels and responded better to an IPGTT than mice fed the control diet. Moreover, all ABA-supplemented diets appeared to have a similar effect in improving glucose homeostasis, regardless of the amount of ABA from 100 to 800 mg. This finding agrees with in vitro findings which showed that 10 and 50 μM ABA induced similar PPAR γ transactivation. Commensurate with the enhancement in glucose tolerance, we found that the expression of PPAR γ and PPAR γ responsive genes adiponectin, CD36, and aP2 were significantly increased in WAT from db/db mice fed the ABA-supplemented diet. The increase in adiponectin production is significant in itself as it has been shown to promote insulin- stimulated glucose disposal in muscle (30), decrease hepatic glucose production (31), and increase the oxidation of intramuscular fatty acids (32). Consequently, it is possible that the some of the improvements in glucose homeostasis brought on by dietary ABA-supplementation are mediated by this important adipokine. [118] In addition to having lower FBG, mice fed ABA also had an improved response to the IPGTT on day 36 (Figure 3). The 100 mg AB A/kg dose elicited the most favorable response in glucose homeostasis during the IPGTT (Figure 3A). Based on these findings, however, in addition to the FBG readings showing no differences among the ABA diets from 100 to 800 mg/kg, we chose the 100 mg/kg dose of ABA to continue on with further experiments. The EPGTT at the end of Experiment 2 verified the positive glucose normalizing effects of ABA as glucose levels were significantly lower at every time point up to 100 minutes (Figure 3B).

[119] Example 4: Dietary ABA-supplementation increased WAT PPAR γ and

PPAR γ-responsive gene expression and reduced WAT inflammation [120] To determine whether the glucose normalizing effects of ABA were associated with activation of PPAR γ in WAT, we used quantitative real-time PCR to measure the expression of PPAR γ and PPAR γ-responsive genes in WAT of ABA- and control-fed mice. We found that dietary ABA-supplementation resulted in a significant increase in WAT expression of PPAR γ (PO.01) and the PPAR γ responsive genes adiponectin (PO.05), aP2 (P<0.02), and CD36 (P<0.01) (Figure 4).

There were no significant differences in mRNA expression of either PPAR α or PPAR δ (data not shown). Histological analysis confirmed that WAT from ABA-fed mice had significantly more small adipocytes and fewer hypertrophic adipocytes than WAT from control-fed mice (Figure 5), which correlated with a significantly lower number of macrophages infiltrating WAT in ABA-fed mice in comparison to mice fed the control diet (P<0.05) (Figure 6A). In line with the lower numbers of infiltrating macrophages, TNF-α mRNA expression was also down-regulated in WAT from ABA-fed mice (P<0.05) (Figure 6B).

[121] Another main effect of PPAR γ activation in WAT is the reduction in hypertrophic adipocytes, which are less insulin responsive and secrete more pro- inflammatory adipokines than normal-sized adipocytes (16, 18, 33). By promoting adipogenesis and adipocyte differentiation, TZDs create numerous smaller adipocytes which enhance the absorption of triglycerides, glucose, and free fatty acids from the bloodstream (18). These changes are also associated with a significant reduction in the expression of macrophage surface markers CD68, CDl 1, and F4/80 in WAT (17). The macrophages in stromal vascular fraction are main producers of WAT-derived TNF-α (16), which impairs insulin signaling by increasing serine phosphorylation of IRS-I (15). Histological analysis of WAT revealed that mice receiving the ABA- supplemented diet had significantly more small adipocytes than control mice. This attenuation in adipocyte hypertrophy was associated with significant reductions in both macrophage infiltration and TNF-α expression.

[122] Example 5: Dietary ABA-supplementation had no effect on body weight, feed intake or WAT weight

[123] A common side effect of synthetic PPAR γ agonists is increased body weight brought on by fluid retention (26). As expected, the db/db mice from both the control and ABA-fed groups gained a substantial amount of weight throughout the 36 day trial (Table 3).

[124] Table 3: Effect of abscisic acid (ABA) on fasting glucose levels on day 36 of dietary supplementation.*,**,***

Experiment 1 Experiment 2 n,> _t <W _A RA _/W ι Fasting glucose (rag/dL) Fasting glucose met ⁽ mgAtf A/κgj (mg/dL)

0 mg (control) 326. T 353. l ^a

100 mg 219.6 ^b 190.7 ^b

200 mg 210.2 ^b -

400 mg 185.6 ^b -

800 mg 202.0 ^b _

Pooled SEM 21.60 22.89 ANOVA P value 0.0113 0.0005

* Least squares means values in a column with different superscripts are significantly different (P < 0.05).

** P-value of main effects of diet (D) during 36-day period. Data were analyzed as a completely randomized design.

*** Pooled standard error of the mean

[125] However, as opposed to findings with TZDs, there were no significant differences in body weights between mice consuming ABA and control diet, including no differences in abdominal WAT. Interscapular BAT, however, was significantly increased in mice fed ABA. The mice in the ABA-supplemented diet finished the trial weighing approximately 2.2 grams less than the control mice (41.34 g vs 43.54 g), though none of the numerical differences in weigh data from any of the time-points were statistically significant. Total feed intake also did not differ significantly between treatment groups (data not shown).

[126] Table 4: Effect of dietary abscisic acid ( ABA)-Su pplementation on body organ weight. * ** ***

Initial body Final body WAT% Body Liver% Body BAT% Body weight (a) weight (g) Weight Weight Weight control 19.63 43.54 5.74 5.74 0.91

ABA 19.83 ^■ 41.34 6.06 5 16**** 1.37

Pooled SEM ⁶ 0.864 1.282 0.258 0.125 0.106

ANOVA P value 0.865 0.248 0.476 0.027 0.007

* Organs were excised and weighed on day 36 of experiment.

** Least squares means values in a column with an asterisk are significantly different

(P < 0.05).

*** jP-value of main effects of diet during the 36 day period. Data were analyzed as a completely randomized design

**** Pooled standard error of the mean

[127] While ABA and synthetic PPAR γ agonists (e.g., rosiglitazone) elicit similar effects on adipose tissue morphology, treatment with ABA did not induce any weight gain, a major adverse side effect associated with TZDs. Initially thought to be related to the increase in adipogenesis, it is now believed that that the main contributor to TZD-associated weight gain is increased fluid retention via the PPAR γ-dependent induction of the Na ⁺ transporter ENaC (26). Conditional knockout of PPAR γ in the renal collecting ducts prevents TZD-induced weight gain (26). Approximately 10-15% of patients using TZDs are forced to discontinue treatment due to edema, and the increase in extracellular volume from excess fluid retention also poses a major problem for individuals with preexisting congestive heart failure (34, 35). The lack of weight gain in our study suggests that fluid retention either does not occur or is very minimal in ABA-fed mice, and we suspect this may be due to the lower affinity of ABA for PPAR γ in comparison with TZDs. This hypothesis is supported by the lower potency of ABA when compared to rosiglitazone in 3T3-L1 cells. The significant increase in BAT may also have contributed to the slight, but

insignificant, reduction in weight of ABA-fed mice when compared to controls. The finding that ABA increases BAT is consistent with previous publications which found that PPAR γ activation by synthetic agonists such as TZDs increases BAT differentiation (36).

[128] Example 6: Dietary AB A-supplementation ameliorated hepatic steatosis

[129] To determine whether ABA had any hepatotoxic effects, we first determined whether there were any significant differences in liver sizes between the ABA and control-fed mice on day 36. We found that the livers from ABA-fed mice were significantly smaller than those from the controls (P<0.03). H&E-stained hepatic sections were then examined histologically at 400X magnification to assess lipodystrophy by using the NASH scoring method. We determined that hepatic steatosis was significantly mitigated in db/db mice that consumed ABA (P<0.02) (see Figures 7A and 7B).

[130] Due to the hepatotoxic effects of TZDs, we determined whether dietary AB A-supplementation had any effect on liver morphology. We demonstrated that the livers of ABA-supplemented mice were significantly smaller than those from the controls. On closer examination, we found that hepatocytes from ABA-fed mice had significantly reduced lipid accumulation. This finding is contrary to results one would expect to observe with synthetic PPAR γ agonists, since rosiglitazone treatment has been shown to exacerbate hepatic steatosis by inducing de novo lipogenesis (37) and troglitazone was withdrawn from the market by the FDA due to reports of severe liver injury or death. We believe that the different effects of TZDs and ABA on hepatic steatosis are due to ABA's weaker affinity for PPAR γcombined with adiponectin secretion from WAT. It has been demonstrated that treatment of obese mice with adiponectin ameliorates hepatic steatosis by reducing fatty acid synthesis and uptake while increasing fatty acid oxidation (38). Because production of adiponectin mRNA was increased in WAT following ABA treatment, this adipokine may have played a role in the improving hepatic health.

[131] Example 7: Dietary ABA-supplementation decreased CD4+ and CD8+ T cell proliferation ex vivo. [132] Isolation of lymphocytes and cell culture conditions

[133] Single cell suspensions were prepared from the spleen by gently crushing the spleens excised during the necropsy procedure between the frosted ends of microscope glass slides previously autoclaved. Tissue debris were removed by settling, erythrocytes were eliminated by hypotonic lysis and cells finally resuspended in complete RPMI (cRPML 10 % fetal bovine serum (Hyclone, Logan, UT), 25 mM HEPES buffer (Sigma, St. louis, MO), 100 units/ml penicillin (Sigma), 0.1 m/ml streptomycin (Sigma), ImM sodium pyruvate (Sigma), 1 mM nonessential aminoacids (Sigma), and 2 mM essential amino acids (Mediatech), and enumerated with a Coulter Zl Single Particle Counter (Beckman Coulter, Miami, Florida). [134] Lymphocyte Blastogenesis Test

[135] Wells of 96- well, round bottom microtiter plates were seeded with 2X10 ⁵ splenocytes in a total volume of 200 ml per well. Cells were cultured in cRPMI media with or without 2.5 μg/ml of Concanavalin A (Sigma) for 4 days. On day 4, 0.5 mCi of methyl- [ ³ Thymidine] was added to each well, and cells we incubate for 20 additional hours. Well contents were harvested onto glass fiber filters using cell harvester, and incorporated radioactivity measured by liquid scintillation counting. Samples were run in triplicate for each animal and ex-vivo treatment. Stimulation indices were calculated by dividing counts per minute of mitogen-stimulated wells by counts per minute of unstimulated wells.

[136] Carboxy-fluorescein diacetate succinidimyl ester

[137] A total of 30 x 10 ⁶ splenocytes were labeled with the green fluorescent dye carboxy-fluorescein diacetate succinidimyl ester (CFSE) (Molecular Probes) following manufacturer's instructions. Briefly, cells were aliquoted into 15 ml polystyrene conical tubes and centrifuged at 400 x g for 5 min. After eliminating the supernatant, cells were resuspended in 1 ml of RPMI 1640 with 2 mM CFSE. Cells were incubated with the dye for 7 min at room temperature and the reaction was

stopped by the addition of 9 ml of RPMI. To eliminate excess CFSE, cells were washed 2 times in RPMI 1640 and finally resuspended in complete RPMI. [138] Splenocytes at 10 ⁶ cells/ml were seeded in 96-well, round-bottom microtiter plates and incubated incomplete RPMI with or without Concanavalin A (Sigma) at 2.5 μg/ml. After 5 days in culture, cells were harvested and labeled for the analysis cell surface marker expression by flow cytometry. Cell surface staining was conducted by incubating cells in 100 ml of combined biotinylated rat anti mouse CD4 (clone) and phycoerithrin labeled rat anti-mouse CD8α (clone) diluted in FACS buffer (PBS containing 5% of fetal bovine serum (Hyclone) and 0.01% of sodium azide (Sigma)) for 20 min at 4°C. Following 2 washes in Fluorescence activated cell sorting (FACS) buffer, cells were incubated with streptavidin-PeCy5 (Pharmingen, San Diego, CA) for the biotinylated anti-mouse CD4 antibody. Cells were analyzed by 3-color flow cytometry (CFSE, phycoerythrin and PeCy5) in a FACScalibur instrument (Becton Dickinson). Ten thousand events were acquired, and data were analyzed using the Cell Quest Pro™ software (Becton Dickinson). Proliferation was assessed by the decrease in fluorescent intensity of CFSE staining. A Relative Proliferation Index was calculated by dividing the percentage of CD4 ⁺ or CD8α cells that proliferated in Concanavalin A-stimulated cultures by the percentage of CD4 ⁺ or CD8α cells that proliferated in cultures maintained with media alone. [139] ABA modulates the proliferation of CD4+ and CD8+ T-cells

[140] Evidence has shown that PPAR γ agonists inhibit T-cell proliferation by blocking the production of interleukin-2 (IL-2) (Yang et al. J. Biol. Chem. 2000). To determine if ABA had any effect on T-lymphocyte proliferation, we performed two different proliferation assays, flow cytometry and LBT. Our results from flow cytometry show that after the first generations, both CD4+ and CD8+ concanavalin A- induced proliferation significantly decreases in the ABA supplemented diet. This finding is supported by the numerical differences in the LBT data, which measures total lymphocyte proliferation for only the last generations (Table 5). [141] These results are consistent with recent findings showing that proliferation induced by anti-CD3, another polyclonal stimulator of T cell proliferation, was

decreased in lymphocytes derived from the spleen of ABA- fed mice (stimulation index 30.12 in controls versus 8.55 in ABA-fed mice). However, proliferation of mesenteric lymph node-derived lymphocytes was augmented 10-fold following ABA feeding and prostaglandin E2 (PGE2) production was decreased by ABA. While polyclonal stimulation of T cells (mitogen- or anti-CD3 -induced stimulation) is generally detrimental and it can be associated with the onset of autoimmune and allergic responses, discreet antigen-specific responses to pathogens are beneficial and contribute to resistance against infectious diseases. We next measured the ability of ABA to modulate antigen-specific responses to influenza virus antigens following immunization with an inactivated influenza virus vaccine. We found that ABA maintained or enhanced the ability of T cells to proliferate in response to influenza virus antigens (stimulation index 4.86 in controls versus 8.61 in ABA-fed mice). This enhanced proliferative ability was associated with increased production of interleukin- 2 (IL-2) in ABA-fed mice (5 vs 31.67 picograms IL-2/ml of cell supernatant). [142] Table 5 Lymphocyte Proliferation Assays. ¹ ' ² ' ³

Flow Cytometry LBT

Total

CD4+ T cells CD8+ T cells lymphocytes

Diet

RPIl RPI2 RPIl RPI2 SI (mgABA/kg)

0 12.43 47.55* 3.086 212.01* 21.36 100 17.72 30.56 5.261* 63.26 9.22

Pooled SEM ⁴ 4.26 2.87 0.53 13.19 7.08

ANOVA P value 0.38 0.0019 0.015 0.0002 0.25

' Flow cytometry and LBT were performed to assess lymphocyte proliferation, RPl (relative proliferation index) J is the proliferation oft

2 generations ofCD4 and first 4 generations of CDS cells. RPI2 is a measure of the last 3 and 4 generations ofCD4 and CDS T-cells, respectively. The stimulation index (SI) is an assessment of total T [ymphocy te proliferation during the LBT assay.

² Least squares means values in a column with an asterisk are significantly different (P < 0.05).

³ P-value of main effects of diet (D) during 36 day period. Data were analyzed as a comp lately randomized design.

* Pooled standard error of the mean.

[143] Example 8: Effect of ABA on Skeletal Muscle Inflammation

[144] Recent evidence has indicated that obesity-induced insulin resistance (IR) is associated with white adipose tissue (WAT) macrophage infiltration and inflammation. We fed either control or abscisic acid (ABA)-supplemented high-fat diets to db/db and lean C57BL6/J mice for 36 days and measured fasting blood glucose levels. Flow cytometry was utilized to assess the macrophage infiltration, including those expressing chemokine receptors 2 and 5 (CCR2 and CCR5), in abdominal WAT and SM. Tumor necrosis factor a (TNF-α) and monocyte chemoattractant protein-1 (MCP-I) expression in both tissues was quantified by real time qRT-PCR. Dietary ABA-supplementation significantly decreased CCR2 ⁺ and CCR5 ⁺ macrophage infiltration into WAT. In SM, ABA attenuated hypertrophy of intermuscular adipose tissue (HVLAT). MCP-I expression in stromal vascular cells (SVCs) and SM was significantly downregulated in ABA-fed mice. This example shows that obesity-induced IR is significantly associated with SM inflammation and that PPAR γ ligands can improve insulin sensitivity by inhibiting SM inflammation. [145] For this Example, the following materials and methods were used:

[146] Mice and dietary treatments. Six to eight week-old BKS.Cg -+LePr ¹ " ³

/+Lepr ^db /OlaHsd (db/db) mice and their lean littermates were housed at the animal facilities at Virginia Polytechnic Institute and State University in a room maintained at 75° F, with a 12:12 h light-dark cycle starting from 6:00 AM. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Virginia Polytechnic Institute and State University and met or exceeded requirements of the Public Health Service/National Institutes of Health and the Animal Welfare Act.

[147] Mice (n=17) were fed high-fat diets containing 0 or 100 mg of racemic ABA (Sigma Aldrich, St. Louis, MS)/kg diet for 36 days. On day 36, fasted mice (12h) were sacrificed by CO ₂ narcosis and blood was withdrawn directly through the heart for assessment of fasting glucose levels with an Accu-Chek ^® Glucometer (Roche, Indianapolis, IN). Abdominal WAT and skeletal muscle from the left hindlimb (gastrocnemius, plantaris, and soleus) were then excised to generate single

cell suspensions for flow cytometry. SM from the right hindlimb were also placed in 10% buffered neutral formalin for histological evaluation and stored in RNA later (Ambion, Austin, TX), a commercial solution that inhibits RNases. [148] WAT Digestion. Abdominal WAT was excised, weighed, minced into small <10 mg pieces and placed into digestion media consisting of DMEM (Mediatech, Herndon, VA) supplemented with 2.5% HEPES (Mediatech) and 10 mg/ml fatty-acid poor bovine serum albumin (FAB-poor BSA, Sigma), Liberase Blendzyme 3 (0.03 mg/ml, Roche) and DNase I (50 U/ml, Qiagen, Valencia, CA). Samples were incubated in a rotating 37°C water bath for 90 minutes and then filtered through a 250 μm nylon mesh (Sefar America Inc., Depew, New York) to remove undigested particles and centrifuged at 4°C at 2235 rpm for 10 minutes. The floating portion (adipocyte-rich portion), was then removed and digested for an addition 60 minutes at 37°C. The pellet, consisting of stromal vascular cells (SVCs), was washed with DMEM and centrifuged at 4°C at 2235 rpm for 10 minutes. The supernatant was discarded and erythrocytes were lysed by incubating the SVCs in 2 ml erythrocyte lysis buffer for 2 minutes before stopping the reaction with 9 ml IX PBS. Cells were then centrifuged again at 4°C at 2235 rpm for 10 minutes, suspended in 1 ml of IX PBS, and enumerated with a Zl Single Particle Counter (Beckman Coulter, Fullerton, CA). The SVCs were resuspended in FACS buffer at a concentration of 2 x 10 ⁶ cells/ml. The cells not used in the resuspensions were centrifuged for 5 minutes at 13,000 rpm, resuspended in RLT lysis buffer (Qiagen) containing 1% β- mercaptoethanol, and immediately frozen at -80 ⁰ C for RNA isolation and gene expression analyses.

[149] Quantitative real-time reverse transcriptase PCR. Total RNA was isolated from SVCs and SM using the RNA isolation Minikit (Qiagen) according to the manufacturer's instructions. Total RNA (1 μg) was used to generate complementary DNA (cDNA) template using the iScript cDNA Synthesis Kit (Bio- Rad, Hercules, CA). The total reaction volume was 20 μl with the reaction incubated as follows in an MJ MiniCycler: 5 minutes at 25°C, 30 minutes at 52°C, 5 minutes at 85 ⁰ C, hold at 4°C. PCR was performed on the cDNA using Taq DNA polymerase

obtained from Invitrogen and using standard conditions. Each gene amplicon was purified with the MiniElute PCR Purification Kit (Qiagen) and quantitated on an agarose gel by using a DNA mass ladder (Promega). These purified amplicons were used to optimize real-time PCR conditions and to generate standard curves in the realtime PCR assay. Primer concentrations and annealing temperatures were optimized for the iCycler iQ system (Bio-Rad) for each set of primers using the system's gradient protocol. PCR efficiencies were maintained between 92 and 105% and correlation coefficients above 0.98 for each primer set (Table 6) during optimization and also during the real-time PCR of sample DNA. [150]

Table 6. Oligonucleotide sequences for quantitative real-time PCR. ¹ ' ²

Primer Sequence Length Ace. No. SEQ ID NO. β-actinF 5'CCCAGGCATTGCTGACAGGS' 141 X03672 17 β-actinR S'TGGAAGGTGGACAGTGAGGCS ' 18

TNF-αF 5αCTGCCAGAAGAGGCACTCC3' 137 NMJ)13693 19

TNF-αR 5'CGATCACCCCGAAGTTCA3' 20

MCP-IF 5'CTTGCCTAATCCACAGACTG'3 146 AJ238892 21

MCP-IR 5'GCCTGAACAGCACCACTA'3 22

¹ F, forward; R, reverse. PCR primer pairs were designed for 78 and 157 base pairs. Annealing temperatures are 57°C for β-actin and TNF-α, 54°C for MCP-I, and 51.9 ⁰ C for MIP- lα.

² When plotting threshold cycle versus log starting quantity (pg), standard curves had slopes between -3.1 and -3.7; PCR efficiencies between 92 and 105 and R ² above 0.98.

[151] Complementary DNA (cDNA) concentrations for genes of interest were examined by real-time quantitative PCR using an iCycler IQ System and the iQ SYBR green supermix (Bio-Rad). A standard curve was generated for each gene using 10- fbld dilutions of purified amplicons starting at 5 pg of cDNA and used later to calculate the starting amount of target cDNA in the unknowns. SYBR green I is a general double-stranded DNA intercalating dye and, therefore, might detect nonspecific products and primer/dimers in addition to the amplicon of interest. In order to determine the number of products synthesized during the real-time PCR, a melting curve analysis was performed on each product. Real-time PCR was used to measure the starting amount of nucleic acid of each unknown sample of cDNA on the

same 96- well plate. Results are presented as starting quantity of target cDNA (picograms) per microgram of total RNA.

[152] Histopathology. SM specimens were fixed in 10% phosphate buffered neutral formalin, embedded in paraffin, cut at thicknesses of 5 microns and later stained with hematoxylin and eosin (H&E) for histological examination of IMAT. The average area of adipocytes in IMAT was calculated using Image Pro Plus ^® software at a magnification of 10OX.

[153] Statistical Analyses. Data were analyzed as a completely randomized design. To determine the statistical significance of the model, analysis of variance (ANOVA) was performed using the general linear model procedure of Statistical Analysis Software (SAS). A probability value (P) < 0.05 was considered to be significant. When the model was significant, ANOVA was followed by Sheffe's multiple comparison method. Non-parametric data were analyzed by using the Mann- Whitney C/ test followed by a Dunn's multiple comparison's test. Correlations between macrophage infiltration and fasting blood glucose levels were assessed with Pearson's Correlation Coefficient. [154] Results

[155] Adipocyte Hypertrophy in Intermuscular Adipose Tissue

[156] Data obtained and presented as part of this invention support the conclusion that SM inflammation, brought on by adipocyte hypertrophy and macrophage infiltration in IMAT, contributes significantly to the systemic insulin resistance induced by obesity. Following histological examination of SM specimens from control-fed obese and lean mice, one of our initial observations was that adipocytes in IMAT were extremely large. We also observed that IMAT in the db/db mice, regardless of diet, was significantly more hypertrophic than the lean mice (Figure 8, Panels A-D). More specifically, Figure 8 illustrates the effect of obesity on adipocyte hypertrophy in intermuscular adipose tissue (IMAT). Db/db and lean C57BL6/J mice were fed high-fat control diets or one supplemented with abscisic acid (ABA, 100mg/kg diet) for 36 days. Illustrated in the Figure are representative photomicrographs of IMAT in (Panel A) control-fed lean, (Panel B) ABA-

supplemented db/db, and (Panel C) control-fed db/db mice taken at IOOX magnification. Average adipocyte areas (Panel D) were calculated using Image-Pro

Plus ^® software. Data are presented as least square means ± standard error. Data points with different subscripts are significantly different (PO.05).

[157] We have previously demonstrated that dietary ABA-supplementation significantly attenuates adipocyte hypertrophy in abdominal WAT of db/db mice. In similar regard, IMAT hypertrophy was also significantly attenuated as a result of

ABA-supplementation.

[158] Skeletal Muscle Inflammation

[159] Db/db mice were fed either a control high-fat diet or one supplemented with 100 mg/kg all racemic abscisic acid (ABA). Real-time qRT-PCR was utilized to assess mRNA expression of tumor necrosis-α (TNF-α), monocyte chemoattractant protein- 1 (MCP-I) in SM.

[160] We next measured TNF-α expression in SM and observed that TNF-α was approximately 2-fold higher in control- fed db/db mice when compared to ABA-fed db/db mice (-P=0.03) and control-fed lean mice (P=0.03).

[161 ] Example 9: Further Characterization of the Role of ABA in PPAR γ

Activity

[162] Obesity is associated to a low-grade chronic inflammation characterized by macrophage infiltration into white adipose tissue (WAT). However, the phenotype of adipose tissue macrophage (ATM) subsets, and the mechanisms by which insulin- sensitizing compounds modulate subset-specific macrophage infiltration remain unknown. In the following set of experiments, we used a loss-of-function approach to investigate whether the peroxisome proliferator-activated receptor (PPAR) γ agonist, abscisic acid (ABA), ameliorates insulin resistance through a mechanism dependent on immune cell PPAR γ. We characterized two phenotypically distinct ATM subsets in db/db mice based on the amount of F4/80 and chemokine receptors (CCR) expressed on their surfaces. The F4/80 ^hiεh macrophages were more abundant in WAT and expressed greater surface concentrations of chemokine receptor 2 (CCR2) and

CCR5 when compared to F4/80 ^|OW macrophages. ABA significantly decreased CCR2 ⁺ F4/80 ^hi macrophage infiltration into WAT and suppressed monocyte chemoattractant protein- 1 (MCP-I) expression in WAT and plasma. Furthermore, it decreased MCP-I promoter activity in 3T3-L1 cells. The deficiency of PPAR γ in immune cells, including macrophages, impaired the ability of ABA to suppress infiltration of F4/80 ^hi macrophages into WAT ₅ repress WAT MCP-I mRNA expression and improve glucose tolerance. We provide in vivo molecular evidence that ABA improves insulin sensitivity and obesity-related inflammation by inhibiting MCP-I expression and F4/80 ^hi ATM infiltration through a PPAR γ-dependent mechanism.

[163] To further characterize the findings discussed above, the study discussed below aimed to dissect the expression of PPAR γ-responsive and inflammatory genes by adipocytes versus stromal vascular cells (SVC)s in WAT. We also phenotypically characterized the subsets of adipose tissue macrophages (ATM) targeted by ABA and determine whether ABA ameliorates insulin resistance and obesity-related inflammation through a mechanism dependent upon immune cell PPAR γ. Our data demonstrate that SVCs, and not the adipocytes, are the main producers of PPAR γ- responsive genes and chemokines in WAT. In addition, we provide in vivo molecular evidence suggesting that ABA ameliorates glucose tolerance and obesity-related inflammation by suppressing MCP-I expression and F4/80 ^hi macrophage infiltration into WAT through a mechanism dependent on immune cell PPAR γ. [164] Materials and Methods [165] Mice and dietary treatments

[166] BKS.Cg -+Lepr ^db /+LePr ⁰ VOIaHSd (db/db), PPAR γ flfl; MMTV-Cre ⁺ and PPAR γ flfl; MMTV-Cre" mice in a C57BL/6J background were housed at the animal facilities at Virginia Polytechnic Institute and State University in a room maintained at 22°C, with a 12:12 h light-dark cycle starting from 6:00 AM. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Virginia Polytechnic Institute and State University and met or exceeded requirements

of the Public Health Service/National Institutes of Health and the Animal Welfare Act.

[167] Db/db mice (n=l 0) were fed high- fat diets with a previously described ingredient composition (48) containing 0 or 100 mg of racemic ABA (Sigma Aldrich, St. Louis, MS)/kg diet for 36 days. On day 36, fasted mice (12h) were sacrificed by CO ₂ narcosis and blood was withdrawn directly from the heart for assessment of fasting glucose levels with an Accu-Chek ^® Glucometer (Roche, Indianapolis, IN). Plasma was also collected for analysis of MCP-I protein levels and plasma triglycerides (TGs). Abdominal WAT was then excised to generate single cell suspensions for flow cytometry. Liver specimens were placed in 10% buffered neutral formalin for histological evaluation. Liver samples were also stored in RNA later (Ambion, Austin, TX), a commercial solution that inhibits RNases, for RNA isolation and quantitative gene expression analyses. PPAR γ floxed mice expressing the Cre transgene (e.g., PPAR-γ fl/fl; MMTV-Cre) undergo premature termination of translation following the loss of the exon 2 of PPAR γ due to the enzymatic activity of the recombinase on genomic DNA (49, 50). PPAR γ flfl; MMTV-Cre ⁺ and PPAR γ flfl; MMTV-Cre ^" littermate mice were fed high-fat diets with or without ABA (100 mg/kg). After 28 weeks, the mice were fasted for 6 hours and bled through the caudal vein for assessment of fasting blood glucose concentrations by using an Accu-Chek ^® Glucometer (Roche, Indianapolis, EN). The mice were then subjected to an intraperitoneal glucose tolerance test (2 g/kg body weight), with determination of glucose levels at 15, 30, and 90 minutes post-challenge. At the 90 minute timepoint, mice were sacrificed by CO ₂ narcosis. Abdominal WAT was then excised to generate single-cell suspensions for flow cytometry or placed in 10% buffered neutral formalin for histological evaluation and stored in RNA later (Ambion, Austin, TX) for RNA isolation and quantitative gene expression analyses. [168] WAT Digestion

[169] Abdominal WAT was excised, weighed, minced into small <10 mg pieces and placed into digestion media consisting of DMEM (Mediatech, Herndon, VA) supplemented with 2.5% HEPES (Mediatech) and 10 mg/ml fatty-acid free bovine

serum albumin (FAB-poor BSA, Sigma), Liberase Blendzyme 3 (0.03 mg/ml, Roche) and DNase I (50 U/ml, Qiagen, Valencia, CA). Samples were incubated in a rotating 37°C water bath for 90 minutes and then filtered through a 250 μm nylon mesh (Sefar America Inc., Depew, New York) to remove undigested particles and centrifuged at 4°C at 1,000 x g for 10 minutes. The pellet, consisting of stromal vascular cells (SVCs), containing endothelial cells, pre-adipocytes, macrophages and T cells, was washed with DMEM and centrifuged at 4°C at 1,000 x g for 10 minutes. The supernatant was discarded and erythrocytes were lysed by incubating the SVCs in 2 ml erythrocyte lysis buffer for 2 minutes before stopping the reaction with 9 ml IX phosphate-buffered saline (PBS). Cells were then centrifuged again at 4°C at 1,000 x g for 10 minutes, suspended in 1 ml of IX PBS, and enumerated with a Zl Single Particle Counter (Beckman Coulter, Fullerton, CA). The SVCs were resuspended in FACS buffer (IX PBS, 1% normal goat serum, 0.2% sodium azide) at a concentration of 2 x 10 ⁶ cells/ml. The SVCs not used in the resuspensions were centrifuged for 5 minutes at 10,000 x g, resuspended in RLT lysis buffer (Qiagen) containing 1% β- mercaptoethanol, and immediately frozen at -80 ⁰ C for RNA isolation and gene expression analyses. [170] Flow Cytometry

[171] SVCs (2 x 10 ⁵ cells) were seeded into 96-well plates and centrifuged at

4°C at 1,800 x g for 4 minutes. The cells were then incubated in the dark at 4°C for 20 minutes in FcB lock (20 μg/ml; BD Pharmingen), and then for an additional 20 minutes with fluorochrome-conjugated primary antibodies anti-F4/80-PE-Cy5 (5 μg/ml), anti-CD 1 Ib-FITC (2 μg/ml) (eBioscience), anti-CCR5-PE or anti-CCR2-PE (R&D systems, Minneapolis, MN). The specific antibody combinations used were F4/80, CDl Ib, CCR5 and F4/80, CDlIb, CCR2. After incubation with primary antibodies, cells were centrifuged at 4 ⁰ C at 1,800 x g for 4 minutes and washed with 200 μl of FACS buffer. After washing, cells were suspended in .200 μl PBS and 3- color data acquisition was performed on a FACS Calibur flow cytometer. Data analyses were performed by using the CellQuest software (BD).

[172] Quantitative real-time reverse transcriptase PCR [173] Total RNA was isolated from SVCs and liver using the RNA isolation Minikit (Qiagen) according to the manufacturer's instructions. RNA from the adipocyte fraction and WAT was isolated using the Lipid Minikit (Qiagen). Total RNA (1 μg) was used to generate complementary DNA (cDNA) template using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The total reaction volume was 20 μl with the reaction incubated as follows in an MJ MiniCycler: 5 minutes at 25 ⁰ C, 30 minutes at 52°C, 5 minutes at 85°C, hold at 4°C. PCR was performed on the cDNA using Taq DNA polymerase obtained from Invitrogen and using previously described conditions (50). Each gene amplicon was purified with the MiniElute PCR Purification Kit (Qiagen) and quantitated on an agarose gel by using a DNA mass ladder (Promega). These purified amplicons were used to optimize real-time PCR conditions and to generate standard curves in the real-time PCR assay. Primer concentrations and annealing temperatures were optimized for the iCycler iQ system (Bio-Rad) for each set of primers using the system's gradient protocol. PCR efficiencies were maintained between 92 and 105% and correlation coefficients above 0.98 for each primer set (Tables 1 and 6) during optimization and also during the realtime PCR of sample DNA.

[174] Complementary DNA (cDNA) concentrations for genes of interest were examined by real-time quantitative PCR using an iCycler IQ System and the iQ SYBR green supermix (Bio-Rad). A standard curve was generated for each gene using 10- fold dilutions of purified amplicons starting at 5 pg of cDNA and used later to calculate the starting amount of target cDNA in the unknown samples. SYBR green I is a general double-stranded DNA intercalating dye and may therefore detect nonspecific products and primer/dimers in addition to the amplicon of interest. In order to determine the number of products synthesized during the real-time PCR ₃ a melting curve analysis was performed on each product. Real-time PCR was used to measure the starting amount of nucleic acid of each unknown sample of cDNA on the same 96-well plate. Results are presented as starting quantity of target cDNA (picograms) per microgram of total RNA.

[175] Hepatic Steatosis, plasma MCP-I and liver triglyceride content [176] Liver specimens were excised and immediately fixed in 10% phosphate buffered neutral formalin, embedded in paraffin, cut at thicknesses of 5 microns and stained with hematoxylin and eosin (H&E) for histological examination. For hepatic triglyceride assessment, livers fragments (50—100 mg) were extracted for 16 hours at 4°C with 4 ml CHCl ₃ /methanol (2:1, vol/vol). Two milliliters of 0.6% NaCl were then added to the extract, and the mixture was centrifuged at 2,000 x g for 20 minutes. Lipids were isolated by nitrogen evaporation, dissolved in 100 μl isopropanol, and quantified with a triglyceride assay kit (Stanbio). The Triglyceride Assay Kit (Sigma) and Ready-set-go MCP-I ELISA (ebioscience) were used to quantify plasma TGs and MCP-I, respectively, according to manufacturer's instructions. [177] Transfection of 3T3-L1 preadipocvtes

[178] A pCMX.PPAR γ expression plasmid (kindly provided by Dr. R.M.

Evans, The SaIk Institute, San Diego, CA) and pMCP-l-514(enh) luc construct (51) were purified using Qiagen's Maxi kit (Valencia, CA). 3T3-L1 cells (American Type Culture Collection, Manassas, VA) were grown in 24-well plates in DMEM high glucose medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) until 60-70% confluence. Before transfection, the medium was changed to 1% FBS. The cells cultured in 24-well plates were co-transfected with 0.8 μg plasmid DNA and 1 ng of pRL reporter control plasmid per well using F-I transfection reagents (Targeting Systems, Santee, CA) according to the manufacturer's protocol. Transfection efficiencies were determined by cotransfecting the cells with a pcDNA™ 3.1/His//αcZ control vector at 24 h. The transfected cells were then treated with either a racemic ABA mixture (12.5 μM; Sigma) or rosiglitazone (1 μM; Cayman Chemicals, Ann Arbor, MI) for 24 hours. Designated wells were also treated with the PPAR γ antagonist GW9662 (30 μM; Cayman Chemicals). Transfected cells were harvested in reporter lysis reagent. Luciferase activity, normalized to pRL activity in the cell extracts was determined by using the dual luciferase reporter assay system (Promega, Madison, WI) in a TD-20/20 Single-Tube Luminometer (Turner Biosystems, Sunnyvale, CA). Relative luciferase activity (RLA) was calculated as a

ratio of the chemiluminescence 10 seconds after the Luciferase Assay Reagent II

(Promega) was added over the chemiluminescence 10 seconds after the Stop&Glo

Reagent (Promega).

[179] Statistical Analyses

[180] For db/db experiment, data were analyzed as a completely randomized design. To determine the statistical significance of the model, analysis of variance

(ANOVA) was performed using the general linear model procedure of Statistical

Analysis Software (SAS). In the experiment using PPAR γ flfl; MMTV-Cre ⁺ and

PPAR γ flfl; MMTV-Cre ^" mice, data were analyzed as a 2 x 2 factorial arrangement within a completely randomized design. The statistical model utilized was Y _ijk = μ +

Genetic Background _j + Diet,- + (Genetic Background x DietX _j + Error _ijk . To determine the statistical significance of the model, analysis of variance (ANOVA) was performed using the general linear model procedure of SAS, and probability value (P)

< 0.05 was considered to be significant. When the model was significant, ANOVA was followed by Fisher's Protected Least Significant Difference multiple comparison method. Non-parametric data were analyzed by using the Mann- Whitney U test followed by a Dunn's multiple comparison's test.

[181] Results

[182] ABA improves fasting glucose and lowers plasma triglycerides in db/db mice without affecting body weight

[183] Figure 9 shows that ABA ameliorates fasting glucose concentrations without inducing body weight gain. The effect of ABA on body weight, feed intake, fasting blood glucose and plasma lipids is shown in the figure. Db/db mice were fed control or ABA-supplemented (lOOmg/kg diet) high-fat diets for 36 days. Panel (A): Body weights; and Panel (B): food intake were assessed weekly. After dietary treatment, plasma was withdrawn from fasted mice (12h) to obtain fasting blood glucose (Figure 9C; FBG, milligrams/deciliter) and plasma triglycerides (Figure 9D; TGs, mg/ml). Data are presented as least square means ± standard error of 10 mice. The error bars at some time points (Figures 9A and 9B) are smaller than the symbols

and cannot be seen in the figure. Data points with an asterisk are significantly different (PO.05).

[184] ABA-supplementation to db/db mice for 36 days significantly lowers fasting glucose levels and improves their response to an intraperitoneal glucose tolerance test (48). Similar to these previous findings, ABA did not alter food intake or body weights of the db/db mice throughout the course of the experiment (Figure 9 A and B). In this study, the db/db mice fed the ABA-supplemented diet had significantly lower fasting blood glucose (FBG) and plasma TGs concentrations when compared to mice fed the control high-fat diet (Figure 9C and D).

[185] The stromal-vascular fraction CSVF) contains two phenotvpicallv distinct macrophage sub-populations

[186] Figure 10 shows phenotypic analyses of macrophage subsets within the stromal-vascular fraction (SVF) of white adipose tissue (WAT). Panels (A) and (B) are representative dot plots from a control db/db mouse. The F4/80 ^hi and F4/80 ¹⁰ subsets differed significantly in (C) total amount of macrophages and (D) mean fluorescence intensities (MFI). (E) and (F) are the MFIs of CCR2 and CCR5- expressing macrophages in each population, respectively. (G) represents the percentage of macrophages in each population expressing either CCR2 or CCR5. Data are presented as least square means ± standard error of 10 observations. Data points with an asterisk or number sign indicate a significant difference (P<0.05) between the separate macrophage populations.

[187] More specifically, to characterize the phenotypical differences in ATM in the SVF, we examined surface expression of F4/80 and CDl Ib and chemokine receptors (CCR2 and CCR5). SVCs were isolated from adipocytes by gradient centrifugation and subsequently gated on the immune cell population based on forward versus side scatter (Figure 10A). We identified two pheno typically distinct sub-populations of F4/8O ⁺ CD1 Ib ⁺ macrophages in the SVF of WAT which differed in their mean fluorescence intensity (MFI) for F4/80, yielding a macrophage subset expressing high surface concentrations of the F4/80 molecule (F4/80 ^hi ) and a subset expressing low surface concentrations of the F4/80 molecule (F4/8O ¹⁰ ) (Figure 1OB,

1 OC). The F4/S0 ^hi population was more abundant in WAT than the F4/80 ¹⁰ subset, encompassing approximately 75% of total macrophages versus 25% in control-fed db/db mice, respectively (Figure 10B). The F4/80 ¹ " macrophage subset also expressed higher surface concentrations of CCR2 and CCR5 when compared to the F4/80 ¹⁰ subset (Figure 10G).

[188] ABA inhibits F4/80 ^hi macrophage infiltration into WAT

[189] After characterizing these two macrophage sub-populations, we next determined whether ABA targeted one of these populations or both. We found that ABA significantly suppressed F4/80 ^bi macrophage infiltration into WAT (P=0.05) but had no effect on the F4/80 ^low ATM. The results are shown in Figure 11. The figure shows that ABA decreases the numbers of F4/80 ¹ " macrophages in white adipose tissue. Panels (A), (B), and (C) are representative dot plots for total stromal-vascular fraction (SVF) macrophage infiltration and the percentages of CCR2+ macrophages in the F4/80 ^hi and F4/8O ¹⁰ subsets, respectively, for control-fed db/db mice. Panels (D), (E), and (F) are the respective dot plots for a representative ABA-fed db/db mouse. Panels (G), (H), and (I) indicate the contribution of SVF macrophages, F4/80 ^!c macrophages, and F4/80 ^hl macrophages to the total amount of gated cells. The mean fluorescence intensities (MFIs) of each macrophage sub-population for (J) CCR2 and (K) CCR5. Panel (L) is the difference in MFI of total SVF macrophages between control and ABA-fed mice. Data are presented as least square means ± standard error of 10 observations. Data points with different superscripts are significantly different (PO.05).

[190] In addition, the amount of F4/80 expressed in the surface of F4/80 ^hl macrophages administered ABA, as measured by MFI, was significantly lower than that in mice fed the control diet (Figure 1 IL). The differences observed in the F4/80 ^hl population in mice fed ABA were due primarily to a reduction in a subset of F4/80 ^hi CDl Ib ⁺ CCR2 ⁺ ATM (P=0.006).

[191] ABA decreases MCP-I mRNA expression in WAT. MCP-I protein concentrations in plasma and attenuates liver triglyceride accumulation and hepatic steatosis

[192] A significant reduction in the infiltration of the F4/8O ^hi CD 11 b ⁺ CCR2 ⁺ macrophage subset into WAT could be due to attenuated CCR2 surface expression in monocytes, repression of CCR2 ligand expression in WAT, or both. CCR2 and its principal ligand, MCP-I, have been recently linked to the development of obesity- induced insulin resistance (52-54). To determine whether ABA inhibited MCP-I expression in WAT, we assessed levels of MCP-I mRNA content in the stromal vascular fraction (i.e., macrophages, T cells, fibroblasts, preadipocytes and endothelial cells) and in the adipocyte fraction. Both WAT fractions produce MCP-I in obese mice and humans (16).

[193] Figure 12 shows that ABA induces PPAR γ and suppresses the expression of MCP-I in white adipose tissue. Specifically, the effect of dietary ABA supplementation on white adipose tissue and liver gene expression, MCP-I protein levels, liver triglyceride concentrations and hepatic steatosis scores is shown in the figure. Db/db mice were fed high-fat diets with (black bars) or without (empty bars) ABA (100 mg/kg) for 36 days. Gene expression in RNA isolated from stromal- vascular cells (SVCs) and adipocytes was analyzed by real-time qRT-PCR for (A) monocyte chemoattractant protein 1 (MCP-I), (B) peroxisome proliferator-activated receptor γ (PPAR γ), and (C) CD36. (D) Liver MCP-I expression, (E) plasma MCP- 1 (picograms/milliliter), and (F) liver triglycerides (TG, mg/gram tissue) were also assessed. Gene expression values are expressed as a relative ratio to the housekeeping gene β-actin. Data are presented as least square means ± standard error of 10 observations. Data points with an asterisk are significantly different (PO.05). (G) Liver histology scores. (H) and (I) are representative photomicrographs of liver histology specimens stained with hemotoxylin and eosin (HScE). [194] We found that ABA decreased the concentrations of MCP-I mRNA by almost 15-fold in SVCs. There was also a significant reduction in MCP-I mRNA in the adipocyte fraction following ABA-supplementation (P=O.05), though this

reduction was not as drastic as the difference we observed in SVCs (see Figure 12A). PPAR γ mRNA expression was higher in both SVCs and adipocytes (Figure 12B), but expression of the well-known PPAR γ -responsive gene and fatty acid transporter, CD36, was only significantly elevated in SVCs (Figure 12C). We did not see significant differences in the mRNA expression of MIP-I a or the innate immune receptor toll-like receptor 4 (TLR-4) in either SVCs or adipocytes. [195] We next determined whether the suppressed MCP-I expression observed in WAT of ABA-fed mice resulted in decreased plasma MCP-I concentrations. MCP- 1 protein concentrations were significantly decreased in plasma, but not in liver, from ABA-fed mice (Figures 12D and 12E), suggesting that WAT-derived MCP-I may elicit systemic actions. In a recent study, MCP-1-overexpressing mice under control of the aP2-promoter (i.e., adipose tissue specific) had a significantly elevated risk of developing hepatic steatosis (53). Upon histological examination of liver specimens, we found that the hepatic steatosis was less severe in ABA-fed mice (Figure 12G). In line with this histological finding, the amount of liver triglycerides was significantly lower in mice fed the ABA-supplemented diet as compared to mice fed the control diet (Figure 12F). [196] Targeted deficiency of PPAR γ expression in immune cells impairs the ability of ABA to improve glucose tolerance

[197] The greatest differences in MCP- 1 and the PPAR γ-responsive gene CD36 induced by ABA occurred in SVCs, suggesting that immune cell PPAR γ (and not adipocyte PPAR γ) is the molecular target for ABA in WAT. To determine whether the ability of ABA to normalize plasma glucose concentrations following a GTT was mediated through immune cell PPAR γ, we next fed high-fat diets supplemented with or without ABA to PPAR γ flfl; MMTV-Cre ⁺ mice, which do not express PPAR γ in hematopoetic cells, and PPAR γ flfl; MMTV-Cre ^' littermates (wild-type phenotype). After 28 weeks of high-fat feeding, the mice were overweight (Figure 13A). Similar to our findings with db/db mice, ABA did not increase body weights in either MMTV-Cre ⁺ and MMTV-Cre ^" and did not effect food intake. The deficiency of PPAR γ in immune cells abrogated ABA-induced normalization of fasting blood

glucose concentrations 90 minutes following an intraperitoneal GTT (Figure 13B and 13C).

[198] More specifically, Figure 13 shows the beneficial effects of ABA on insulin resistance are abrogated in immune cell-specific PPAR γ null mice. PPAR γ flfl; MMTV-Cre ⁺ and PPAR γ flfl; MMTV-Cre ^" littermates (wild-type phenotype) were fed high-fat diets with or without ABA (100 mg/kg). (A) Final body weights and (B) response to an intraperitoneal glucose challenge (2 g glucose/kg body weight) in fasted mice (6h) were assessed after 28 weeks of dietary supplementation. Data are presented as least square means ± standard error of 8 mice. The error bars at some time points (Figure 13B and C) are smaller than the symbols and cannot be seen in the figure. Data points with different superscripts are significantly different (P<0.05). [199] Deficient PPAR γ expression in immune cells impairs the ability of ABA to suppress MCP-I expression and infiltration of the F4/80 ¹ " macrophage subset into WAT

[200] To determine whether the observed differences in glucose tolerance corresponded to phenotypic changes in SVF macrophage subsets, we investigated the effect of ABA on both F4/80 ^hi and F4/8O ¹⁰ ATM. Dietary ABA-supplementation significantly decreased both total SVF macrophage infiltration and the amount of F4/80 ^hl macrophages in mice expressing PPAR γ in immune cells (i.e., PPAR γ fl/fl; MMTV-Cre-). However, the deficiency of PPAR γ in immune cells (i.e., PPAR γ fl/fl; MMTV-Cre+) abrogated the ability of ABA to suppress infiltration of F4/80 ^hi into WAT. Of note, surface CCR2 was expressed by a greater percentage of ATM from tissue-specific PPAR γ null mice.

[201] In Figure 14, results are shown that confirm that the beneficial effects of ABA on the infiltration of monocytes and chemokine production in white adipose tissue are abrogated in immune cell-specific PPAR γ null mice. The effect of dietary abscisic acid (ABA)-supplementation and tissue-specific deletion of peroxisome proliferator-activated receptor γ (PPAR γ) in immune cells on adipose tissue macrophage (ATM) phenotype is shown. (A), (B), and (C) represent the effects of ABA on total macrophage infiltration and the percentage of F4/80 ¹⁰ and F4/80 ^hi ATM

in PPAR γ flfl; MMTV-Cre ⁺ and PPAR γ flfl; MMTV-Cre ^" mice. (D) and (E) are representative dot plots for stromal-vascular fraction (SVF) infiltration of ATMs in PPAR γ flfl; MMTV-Cre ⁺ and PP AR γ flfl; MMTV-Cre ^" mice fed the control high-fat diet. (F) and (G) are the respective dot plots for ABA-fed mice. Real-time qRT-PCR was used to assess the expression of monocyte chemoattractant protein 1 (MCP-I) (H). Data are presented as least square means ± standard error of 8 mice. The error bars at some time points (Figure 14B) are smaller than the symbols and cannot be seen in the figure. Data points with different superscripts are significantly different CPO.05).

[202] We examined whether immune cell PPAR γ was required for the suppressive actions of ABA on WAT MCP-I mRNA expression. ABA decreased both WAT and plasma MCP-I levels in mice expressing PPAR γ in immune cells (Figure 14H). However, in line with our findings on F4/80 ¹ " macrophage infiltration into WAT, the ability of ABA to suppress the expression of MCP-I in WAT was significantly impaired in tissue-specific PPAR γ null mice, suggesting that ABA acts through a mechanism requiring the expression of PPAR γ in immune cells. The SVF of WAT contains macrophages, T cells, endothelial cells, fibroblasts and pre- adipocytes, but the only cell subsets in this fraction lacking the PPAR γ gene in the tissue-specific PPAR γ null mouse would be the cells of hematopoietic origin i.e., bone marrow-derived macrophages and T cells. Thus, the differences in glucose tolerance observed might be attributed to one or both immune cell subsets. [203] ABA inhibits MCP-I promoter activity through PPAR γ

[204] To further examine the effect of ABA on MCP-I transactivation, 3T3-L1 pre-adipocytes were transfected with a MCP-I promoter luciferase reporter construct and also co-transfected with a pCMX PPAR γ expression plasmid to increase endogenous PPAR γ activity. Cells were then treated with ABA (12.5 μM), rosiglitazone (1 μM), the PPAR γ antagonist GW9662 (30 μM), or a combination of agonist and inhibitor. We previously found that ABA (12.5 μM) and rosiglitazone (1 μM) were equally effective in inducing PPAR γ transactivation (48). In this study, co-transfection with the PPAR γ expression vector significantly enhanced the ability

of both ABA and rosiglitazone to inhibit MCP-I promoter activity (Figure 15). The effects on MCP-I transactivation by both compounds were significantly inhibited by co-treatment with GW9662.

[205] Figure 15 thus shows that ABA inhibits MCP-I promoter activity by acting through PPAR γ. 3T3-L1 pre-adipocytes were transfected with a MCP-I promoter luciferase reporter construct with (black bars) or without (empty bars) co-transfection with a pCMX.PPAR γ expression plasmid. After 20 hours, cells were treated with the PPAR γ antagonist GW9662 (GW, 30 μM), ABA (12.5 μM), rosiglitazone (Ros, 1 μM), ABA and GW, or Ros and GW. The results were normalized for Renilla activity. Data are presented as least square means dfc standard error of 4 observations. Data points with an asterisk are significantly different (P<0.05). [206] Discussion of Example 9 and Figs 9-15

[207] The obesity and T2D epidemics follow a similar demographic pattern (1,

55), but little is known about the factors linking obesity to insulin resistance and T2D at the cellular and molecular level. The "FFA efflux theory" proposes that adipocyte dysfunction and extra-adipose fat storage are the central components in the pathogenesis of insulin resistance, highlighting the importance of adipocytes as cellular targets (56). hi contrast, the "endocrine theory" suggests that proinflammatory mediators play an essential immunopathogenic role, favoring immune cells as central cellular targets for therapies against T2D (57). ζPAR γ is expressed in both adipocytes and immune cells and has been identified as an important therapeutic target both in insulin resistance and T2D. In this document, we show that PPAR γ and its responsive gene CD36 are expressed at higher concentrations in SVCs than in adipocytes and that treatment with a novel insulin-sensitizing PPAR γ agonist induces PPAR γ-responsive gene expression primarily in SVCs. These results are also suggestive that TZDs may elicit their insulin-sensitizing actions through PPAR γ expressed in immune cells infiltrating the WAT . Hence, PPAR γ activation in immune cells may be considered a central regulator of insulin resistance and obesity- related inflammation. In line with this conclusion, obesity-induced insulin resistance is associated with the infiltration of bone-marrow-derived monocytes into WAT and

differentiation into ATM (16, 17). The studies presented herien phenotypically characterized the subsets of ATM targeted by ABA and investigated whether this compound ameliorates insulin resistance and obesity-related inflammation through a mechanism dependent upon immune cell PPAR γ.

[208] We have previously found that ABA improves glucose tolerance and reduces total ATM infiltration and TNF-α expression in WAT of db/db mice (58). We report for the first time the presence of two phenotypically distinct subsets of macrophages in the SVF of WAT which differ in the amount of surface F4/80 (e.g., F4/8O ¹⁰ and F4/80 ^hi ). The newly identified F4/80 ^hi subset is more abundant (e.g., 75% of the macrophages in the SVF) and expresses higher surface concentrations of CCR2 and CCR5 in comparison to the F4/8O ¹⁰ population.

[209] The expression of F4/80 is tightly regulated according to the physiological status of cells. Because the precursor of tissue macrophages, the bone-marrow derived blood monocyte, expresses lower surface F4/80 than its mature counterparts (58), the F4/80 ¹ " ATM subset is probably a mature cell type that contributes to obesity- related inflammation, whereas F4/80 ^|OW ATM may be monocytes, which have recently transmigrated into the WAT. Alternatively, a difference in the ontogeny of F4/80 ^hl and F4/80 ¹ " macrophages could account for these two distinct ATM subsets. The SVF, in addition to containing bone marrow-derived cells of myeloid origin, includes pre-adipocytes that can differentiate into adipocytes or F4/80-expressing macrophages of mesenchymal origin (59). Resident macrophages from different organs differ considerably in the amount of F4/80 expressed on their surfaces (60). Moreover, the limited expression of chemokine receptors on the majority of F4/80 ^l0 ATMs may be indicative of a predominantly resident population (61).

[210] After phenotypically characterizing these two ATM subsets, we next examined the ability of ABA to differentially modulate their numbers in WAT. We found that ABA decreased the amount of F4/80 ^hi CCR2 ⁺ ATM but had a negligible effect on the F4/8O ¹⁰ population. Although the effect of synthetic PPAR γ ligands on the infiltration of F4/80 ^hi CCR2 ⁺ ATM has not been studied, the reduction of this subset of ATM by ABA is in line with the well-characterized ability of PPAR γ to

suppress CCR2 expression. At the molecular level, the CCR2 gene contains two promoters which axe both repressed by PPAR (62) and TZDs downregulate the MCP- 1 -induced chemotactic response in THP-I monocytes (63). Moreover, in obese mice matched for adiposity, Ccr2 deficiency decreased macrophage content and WAT inflammation while ameliorating hepatic steatosis (52). Our data supports the contention that ABA activates PPAR γ in monocytes, which, in turn, suppresses surface CCR2 expression and infiltration of F4/80 ^hi ATM.

[211] We next determined whether ABA downregulated the expression of MCP- 1, the main ligand for CCR2, in WAT. Similar to CCR2, MCP-I is inhibited following TZD treatment in both SVCs and adipocytes (64). We found a 15-fold decrease in MCP-I mRNA in the SVF of WAT and a 4-fold decrease in plasma MCP- 1. The decrease observed in the SVCs was far greater than the approximately 2-fold decrease Xu et al. obtained following similar short-term intervention with rosiglitazone in ob/ob mice (17). In addition to recruiting macrophages and directly impairing insulin signaling by blocking IRS-I and Akt phosphorylation (53). In line with the suppressed expression of MCP-I mRNA in WAT and decreased concentrations of MCP-I protein in plasma of ABA-fed mice, we observed a decreased severity in high-fat diet-induced hepatic steatosis which could not be attributed to differences in MCP-I mRNA expression in the liver. In addition to the decreased systemic MCP-I concentrations, the ABA-induced reduction in plasma TGs may contribute to the improvement of liver steatosis. To further characterize the role of PPAR γ in mediating ABA-induced repression of MCP-I, we co-transfected 3T3- Ll pre-adipocytes with a MCP-I reporter construct and a PPAR γ expression vector. The presence of the expression vector significantly enhanced the ability of both ABA and rosiglitazone to inhibit MCP-I promoter activity. The effects of both compounds were also inhibited by the selective PPAR γ antagonist GW9662, suggesting that the ABA-induced repression of MCP-I expression in WAT is mediated through the inhibitory actions of activated PPAR γ on the MCP-I promoter. [212] Because macrophages are the main producers of MCP-I in WAT, we next examined whether the deletion of PPAR γ in immune cells would impair the ability of

ABA to ameliorate insulin resistance and obesity-related inflammation. For this purpose, we used a mouse model in which the PPAR γ gene has been disrupted in immune cells. While several studies have been performed on muscle specific (65), until now, little was known about the importance of immune cell PPAR γ in the regulation of insulin resistance and T2D. The PPAR γ fl/fl; MMTV-Cre ⁺ mice lack PPAR γ in immune and epithelial cells but express it at normal concentrations in adipocytes. Thus, they represent an excellent model to dissect the immune cell- dependent mechanisms of PPAR γ action. We have previously used this loss-of- function approach to characterize in vivo the mechanism by which conjugated linoleic acid, another natural agonist of PPAR γ, prevents intestinal inflammation (50). Our present findings indicate that the loss of PPAR γ in immune cells impairs the ability of ABA to improve glucose tolerance, suppress F4/80 ^hl ATM infiltration, and down- regulate MCP-I expression in WAT, suggesting that the protective actions of ABA on insulin resistance are mediated, in part, through a mechanism dependent upon expression of PPAR γ in immune cells.

[213] In summary, we have characterized for the first time two phenotypically distinct subsets of ATM (F4/80 ^hi and F4/8O ¹⁰ ) and demonstrated that a novel PPAR γ agonist decreases the infiltration of F4/80 ^hi ATM, MCP-I expression in WAT, MCP-I promoter activity, and ameliorates insulin resistance through a mechanism requiring immune cell PPAR γ. These data further support the theory that immune cell-derived pro-inflammatory mediators are components in the pathogenesis of insulin resistance and T2D.

[214] It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

W 2

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