COMBINATION OF GLP-1 AND GLP-2 FOR TREATING OR PREVENTING

METABOLIC DISEASES, DISORDERS AND SYNDROMES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/359,413, filed on July 7, 2016. The entirety of the aforementioned application is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. 3092-51000-059- 01S, awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.

BACKGROUND

[0003] Current methods of treating metabolic diseases, disorders and syndromes have numerous limitations, including limited pharmacological efficacy in restoring glycemic control and insulin sensitivity. Numerous embodiments of the present disclosure address the aforementioned limitations.

SUMMARY

[0004] In some embodiments, the present disclosure pertains to therapeutic compositions containing both glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2) or their derivatives for treating or preventing a metabolic disease in a subject (such as obesity, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), short- bowel syndrome (SBS), and intestinal deficiency of nutrient absorption after bariatric surgery); for treating or preventing a metabolic disorder in a subject (such as hyperphagia, hyperglycemia, postprandial hyperglycemia, hyperlipidemia, abnormal cholesterol levels, hypertension, increased blood pressure, excess body fat mass, and body adiposity); or for treating or preventing a metabolic syndrome (such as insulin resistance, insulin sensitivity, and glucose intolerance) in a subject. [0005] In some embodiments, the present disclosure pertains to methods of treating or preventing metabolic diseases, disorders or syndromes in a subject by administering the therapeutic compositions of the present disclosure to the subject. In some embodiments, the administration of the therapeutic compositions of the present disclosure to the subject can improve the subject's metabolic and physiological status. For instance, in some embodiments, the administration improves postabsorptive insulin sensitivity in the subject. In some embodiments, the administration preserves postprandial glycemic control in the subject.

FIGURES

[0006] FIGURE 1 illustrates a method of treating or preventing a metabolic disease, disorder or syndrome in a subject by administering a therapeutic composition that includes glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2).

[0007] FIGURE 2 shows images of pre-proglucagon (PPG) neurons and GLP-2 receptor (Glp2R)-containing neurons in the brainstem.

[0008] FIGURE 3 shows images of glucagon (Gcg)-cre expression by a Cre-dependent Rosa26-eGFP reporter.

[0009] FIGURE 4 shows images and schemes relating to the remote activation of hM3Dq- expressing PPG neurons mapped by c-Fos expression.

[0010] FIGURE 5 illustrates a protocol for quantifying glucose metabolism and insulin sensitivity.

[0011] FIGURE 6 shows images and schemes relating to the acute activation of hM3Dq- expressing PPG neurons induced acutely by clozapine-N-oxide (CNO). FIG. 6A shows PPG neurons indicated by mCherry (red) in the brainstem nucleus of the solitary tract (NTS) from the Gcg-Cre mouse locally infected with AAV hM3Dq-mCherry vectors with c-Fos expression (green). FIG. 6B shows hM3Dq-expressing PPG neurons excited by CNO (5 μΜ), as illustrated by representative traces of the whole-cell patch clamp. FIG. 6C shows PPG neurons labeled by GFP (green) from the Gcg-Cre: :Rosa26-eGFP mouse brainstem. FIG. 6D shows the location of the brainstem slice.

[0012] FIGURE 7 shows data indicating that the remote activation of PPG neurons in the NTS enhances glucose tolerance and insulin sensitivity in mice fed regular chow. FIG. 7A shows a 6 hour fasting glucose at 0 and 3.5 hour post injection of CNO (ip 1 mg/kg). Excitatory AAV- hM3Dq-mCherry vectors (100 nL) were injected into the Gcg-Cre mouse brainstem NTS. FIG. 7B shows blood glucose concentration of mice (at 3 weeks post viral injection) measured after ip glucose challenge. CNO (1 mg/kg) was ip injected 30 minutes prior to ip glucose tolerance test (GTT). FIG. 7C shows endogenous glucose production (EGP) during a basal period. FIG. 7D shows that the acute activation of PPG neurons in mice (at 3 weeks post viral injection) augments insulin sensitivity largely by further suppression of EGP and GNG. GIR, glucose infusion rate; Rd, rate of glucose disappearance; EGP, endogenous glucose production; GNG, gluconeogenesis. Mice were ip injected with CNO or saline prior to insulin clamp. n=6~8/ group. * or **P < 0.05 or 0.01.

[0013] FIGURE 8 shows data indicating that the remote activation of the DVC ChAT neurons enhances insulin sensitivity.

[0014] FIGURE 9 illustrates a proposed model for the activity of PPG neurons in glycemic control.

[0015] FIGURE 10 provides data demonstrating that glucose homeostasis and insulin sensitivity are impaired in GLP-1R/2R double knockout mice (DKO mice) that were fed a high fat diet (HFD). The insulin clamp (after a 12 hour fast) and postprandial kinetics (after a 6 hour fast) were performed prior to severe obesity (by 12-wk old). Fasting glucose in mice after overnight fast. n=8~10/ group. * or **P < 0.05 or 0.01 between vertical sleeve gastrectomy (VSG) vs sham.

[0016] FIGURE 11 provides data indicating that postprandial GLP-1 and PYY are elevated in high fat diet induced obese mice (DIO mice) after VSG. Blood samples were harvested 2 hours after re-feeding. Gut hormones were measured with ELISA. n=8~10/ group. **P < 0.01 within genotype between VSG vs sham; ^ab P < 0.05 within surgery between genotypes.

[0017] FIGURE 12 provides data indicating that VSG triggers postprandial negative neuroendocrine feedback on glycemic control via GLP-1R/2R. Postprandial kinetics was performed in mice fasted for 6 hours on day 28 after VSG, when body weight (BW) was not dramatically different. n=8~10/ group. * or **P < 0.05 or 0.01 within genotype between VSG vs sham.

[0018] FIGURE 13 demonstrates that VSG ameliorates insulin resistance in DIO mice via GLP-1R/2R. Basal glucose was measured in mice after a 12 hour fast. Insulin clamp was performed in 12 hour fasted mice on day 21 after VSG, when BW was not dramatically different. n=8~10/ group.†, * or **P < 0.10, 0.05 or 0.01 within genotype between VSG vs sham; ^ab P < 0.05 within surgery between genotype. [0019] FIGURE 14 provides data indicating that GLP-1R/2R are required for intestinal adaptation in DIO mice after VSG.

[0020] FIGURE 15 provides data indicating that DKO mice show a high incidence of hepatic steatosis and inflammation.

DETAILED DESCRIPTION

[0021] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word "a" or "an" means "at least one", and the use of "or" means "and/or", unless specifically stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.

[0022] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0023] Metabolic diseases, disorders and syndromes provide numerous public health concerns. For instance, obesity and diabetes are escalating global epidemics. Bariatric surgery is recommended as the most effective therapy for morbid obesity and diabetes. In particular, Roux- en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG), two widely used bariatric procedures, induce diabetes remission independent of weight loss. Furthermore, mechanisms for rapidly restoring glycemic control (GC) and insulin sensitivity (IS) are unknown.

[0024] Glucagon-like peptides 1 and 2 (GLP-1 and GLP-2, respectively) are co-transcribed and co-translated from the glucagon (Gcg) gene, post-translationally processed, and co-secreted from enteroendocrine L cells in the gut and pre-proglucagon (PPG) neurons in the nucleus of the solitary tract (NTS) of the brainstem in response to nutrients. GLP-1 and GLP-2 are key signals for the brain and pancreas to control energy balance and glucose homeostasis in rodents and humans. However, the pharmacological action of the brainstem GLP-1 -producing neurons is unknown on glycemic control and insulin sensitivity.

[0025] Moreover, GLP-1 and GLP-2 have been individually used to treat obesity, diabetes and gut diseases. Through distinct G protein-coupled receptors (GLP-1R and GLP-2R), GLP-1 augments pancreatic insulin secretion while GLP-2 enhances intestinal cell proliferation. However, the combined use of GLP-1 and GLP-2 for treating metabolic diseases, disorders and syndromes has not been envisioned or demonstrated.

[0026] In some embodiments, the present disclosure pertains to therapeutic compositions containing both glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2) or their derivatives for treating or preventing a metabolic disease in a subject (such as obesity, type 2 diabetes, NAFLD, NASH, short-bowel syndrome (SBS), and intestinal deficiency of nutrient absorption after bariatric surgery); for treating or preventing a metabolic disorder in a subject (such as hyperphagia, hyperglycemia, postprandial hyperglycemia, hyperlipidemia, abnormal cholesterol levels, hypertension, increased blood pressure, excess body fat mass, and body adiposity); or for treating or preventing a metabolic syndrome (such as insulin resistance and glucose intolerance) in a subject. In some embodiments, the present disclosure pertains to methods of treating or preventing metabolic diseases, disorders or syndromes in a subject by administering the therapeutic compositions of the present disclosure to the subject.

[0027] As set forth in more detail herein, the methods and therapeutic compositions of the present disclosure can have various embodiments. For instance, various methods may be utilized to administer the therapeutic compositions of the present disclosure to various subjects in order to treat or prevent various metabolic diseases, metabolic disorders or metabolic syndromes.

[0028] Therapeutic compositions

[0029] Therapeutic compositions generally refer to compositions that include GLP-1 and GLP-2. GLP-1 and GLP-2 may be in various forms in the therapeutic compositions of the present disclosure. For instance, in some embodiments, each of GLP-1 and GLP-2 can be in the form of synthesized peptides, recombinant peptides, secreted peptides, isolated peptides, lyophilized peptides, derivatized peptides, intact peptides, peptide fragments that include one or more receptor binding domains, and combinations of such forms.

[0030] In some embodiments, each of GLP-1 and GLP-2 can be in derivatized form. For instance, in some embodiments, each of GLP-1 and GLP-2 can include chemically modified amino acid sequences or amino acids conjugated with fatty acids.

[0031] In some embodiments, each of GLP-1 and GLP-2 can be in the form of synthesized peptides. In some embodiments, each of GLP-1 and GLP-2 can be in the form of recombinant peptides.

[0032] In some embodiments, each of GLP-1 and GLP-2 can be in the form of secreted peptides. In some embodiments, GLP-1 and GLP-2 may be secreted from nerve cells, endocrine cells, and combinations thereof. In some embodiments, GLP-1 and GLP-2 may be secreted from prepoglucagon (PPG) neurons. In some embodiments, GLP-1 and GLP-2 may be secreted from enteroendocrine L cells. In some embodiments, GLP-1 and GLP-2 may be secreted from their overexpressing cells.

[0033] In some embodiments, each of GLP-1 and GLP-2 can be in the form of peptide fragments that include one or more receptor binding domains. For instance, in some embodiments, GLP-1 can be in the form of a peptide fragment that includes a GLP-1R binding domain. In some embodiments, GLP-2 can be in the form of a peptide fragment that includes a GLP-2R binding domain.

[0034] In some embodiments, GLP-1 and GLP-2 can be in the form of separated peptides. In some embodiments, GLP-1 and GLP-2 can be linked to one another by a linking agent. In some embodiments, peptide fragments of GLP-1 and GLP-2 that include one or more receptor binding domains are linked to one another. In more specific embodiments, a GLP-1R- specific binding amino acid sequence and a GLP-2R- specific binding amino acid sequence are genetically or chemically linked to one another as a dual agonist to activate both GLP-1R and GLP-2R. [0035] The therapeutic compositions of the present disclosure may be in various forms. For instance, in some embodiments, the therapeutic compositions of the present disclosure may be in the form of a liquid, a solid, a gas, and combinations thereof. In some embodiments, the therapeutic compositions of the present disclosure may be in the form of a liquid, such as a syrup. In some embodiments, the therapeutic compositions of the present disclosure may be in the form of a solid, such as a pill.

[0036] The therapeutic compositions of the present disclosure may also include components in addition to GLP-1 and GLP-2. For instance, in some embodiments, the therapeutic compositions of the present disclosure can also include other active agents. In some embodiments, the other active agents can include, without limitation, peptides, hormones, chemicals, and combinations thereof.

[0037] In some embodiments, the therapeutic compositions of the present disclosure also include a carrier. In some embodiments, the carrier includes, without limitation, carbon-based nanomaterials, liposomes, polymers, micelles, microspheres, nanostructures, dendrimers, homing peptides, homing proteins (e.g., antibodies), and combinations thereof.

[0038] In more specific embodiments, the therapeutic compositions of the present disclosure include GLP-1 as a carrier that serves as a homing peptide for central delivery of agonists or activators to activate GLP-1 -producing neurons in the brainstem NTS. In some embodiments, the therapeutic compositions of the present disclosure include GLP-IR as a homing peptide for central delivery of agonists or activators to activate GLP-lR-expressing neurons in the brainstem DMV.

[0039] In some embodiments, the therapeutic compositions of the present disclosure also include formulation materials for modifying, maintaining, or preserving various parameters (e.g., pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution, rate of release, rate of adsorption, rate of penetration, and combinations thereof). In some embodiments, suitable formulation materials can include, without limitation, amino acids (e.g., glycine), antimicrobials, antioxidants (e.g., ascorbic acid), buffers (e.g., Tris-HCl), bulking agents (e.g., mannitol and glycine), chelating agents (e.g., EDTA), complexing agents (e.g., hydroxypropyl-beta-cyclodextrin), and combinations thereof. [0040] Administration of therapeutic compositions

[0041] Various methods may be utilized to administer the therapeutic compositions of the present disclosure to subjects. For instance, in some embodiments, the administration occurs by methods that include, without limitation, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, intrathecal injection, topical administration, central administration, peripheral administration, and combinations thereof. In some embodiments, the administration occurs by intravenous administration. In some embodiments, the administration occurs by central administration. In some embodiments, the administration occurs by peripheral administration.

[0042] Subjects

[0043] The methods and therapeutic compositions of the present disclosure may be utilized in various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject has been selected for or undergone bariatric surgery, such as roux-en- Y gastric bypass (RYGB) or vertical sleeve gastrectomy (VSG). In some embodiments, the subject is a non-human animal. In some embodiments, the non-human animal includes, without limitation, mice, rats, rodents, mammals, cats, dogs, monkeys, pigs, cattle and horses. In some embodiments, the subject suffers from a metabolic disorder, a metabolic syndrome, or a metabolic disease. In some embodiments, the subject suffers from a metabolic disease, such as nonalcoholic steatohepatitis (NASH), short-bowel syndrome (SBS), or combinations of such diseases.

[0044] Treatment or prevention of metabolic disorders, diseases or syndromes

[0045] The methods and therapeutic compositions of the present disclosure may be utilized to treat or prevent various metabolic disorders, diseases or syndromes. In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to treat various metabolic disorders, diseases or syndromes. In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to prevent various metabolic disorders, diseases or syndromes. In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to treat and prevent various metabolic disorders, diseases or syndromes. In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to treat and prevent nonalcoholic steatohepatitis (NASH) and short- bowel syndrome (SBS).

[0046] In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to treat or prevent metabolic syndromes. In some embodiments, the metabolic syndromes to be treated or prevented include, without limitation, insulin resistance, insulin sensitivity, glucose intolerance, and combinations thereof.

[0047] In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to treat or prevent metabolic diseases. In some embodiments, the metabolic diseases to be treated or prevented can include, without limitation, obesity, diabetes (e.g., type II diabetes), non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), short-bowel syndrome (SBS), intestinal deficiency of nutrient absorption after bariatric surgery, and combinations thereof.

[0048] In some embodiments, the methods and therapeutic compositions of the present disclosure may be utilized to treat or prevent metabolic disorders. In some embodiments, the metabolic disorders to be treated or prevented can include, without limitation, hyperphagia, hyperglycemia, postprandial hyperglycemia, hyperlipidemia, abnormal cholesterol levels, hypertension, increased blood pressure, excess body fat mass, body adiposity, intestinal deficiency of nutrient absorption, and combinations thereof.

[0049] Without being bound by theory, the methods and therapeutic compositions of the present disclosure may treat or prevent metabolic disorders, diseases and syndromes by various mechanisms. For instance, in some embodiments illustrated in FIG. 1, the administering of the therapeutic compositions of the present disclosure to a subject (step 10) can result in the dual activation of the GLP-1 and GLP-2 receptors in the cells of the subject (GLP-IR and GLP-2R, respectively) (step 12). Thereafter, the dual activation of GLP-IR and GLP-2R can result in the treatment (step 14) or prevention (step 16) of metabolic disorders, diseases or syndromes. [0050] The methods and therapeutic compositions of the present disclosure can be utilized to activate GLP-1R and GLP2-R in various cells by administering various combinations of exogenous GLP-1 and GLP-2 and their derivatives to a subject.

[0051] The administration of the therapeutic compositions of the present disclosure can treat or prevent metabolic disorders, diseases or syndromes by inducing various physiological effects in subjects. Such physiological effects can include, without limitation, augmentation of pancreatic insulin secretion, enhancement of intestinal cell proliferation, improvement of insulin sensitivity (e.g., restoration or enhancement of insulin sensitivity), improvement of glucose intolerance, preservation of glucose homeostasis (e.g., reducing endogenous glucose production and fasting glucose), deceleration in gastric emptying, decrease in body fat accumulation, decrease in hepatic steatosis and inflammation, decrease in food intake, decrease in blood pressure, improvement of intestinal blood flow and circulation, enhancement of intestinal adaption (absorption), and combinations thereof.

[0052] In some embodiments, the administration of the therapeutic compositions of the present disclosure can treat or prevent metabolic disorders, diseases or syndromes by improving insulin sensitivity in subjects. In some embodiments, the administration of the therapeutic compositions of the present disclosure can treat or prevent metabolic disorders, diseases or syndromes by preserving glucose homeostasis in subjects.

[0053] As such, the methods and therapeutic compositions of the present disclosure can have various applications. For instance, in some embodiments, the administration of the therapeutic compositions of the present disclosure can improve glucose intolerance and insulin resistance associated with obesity, type II diabetes, and various metabolic syndromes. In some embodiments, the administration of the therapeutic compositions of the present disclosure can prevent postprandial hyperglycemia associated with obesity, type II diabetes, and various metabolic syndromes. In some embodiments, the administration of the therapeutic compositions of the present disclosure can reduce endogenous glucose production (gluconeogenesis) and fasting glucose associated with obesity, type II diabetes, and various metabolic syndromes.

[0054] In some embodiments, the administration of the therapeutic compositions of the present disclosure can reverse non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) associated with obesity, type II diabetes, and various metabolic syndromes. In some embodiments, the administration of the therapeutic compositions of the present disclosure can reverse body adiposity (e.g., by decreasing body fat accumulation and food intake) and improve body composition in subjects associated with obesity, type II diabetes, and various metabolic syndromes. In some embodiments, the administration of the therapeutic compositions of the present disclosure can improve hypertension (e.g., by decreasing blood pressure) in subjects associated with obesity, type II diabetes, and various metabolic syndromes.

[0055] In some embodiments, the administration of the therapeutic compositions of the present disclosure can improve intestinal blood flow and intestinal adaptation after bariatric surgery. In some embodiments, the central and peripheral administration of the therapeutic compositions of the present disclosure can prevent and treat various metabolic disorders. In some embodiments, the administration of the therapeutic compositions of the present disclosure can improve glycemic control and restore insulin sensitivity in obesity and diabetes.

[0056] In some embodiments, the administration of the therapeutic compositions of the present disclosure can augment intestinal cell proliferation and absorption in total parental nutrition, short bowel syndrome, and intestinal function after bariatric surgery. In some embodiments, the central and peripheral administration of the therapeutic compositions of the present disclosure can prevent and treat various intestinal deficiency. In some embodiments, the central and peripheral administration of the therapeutic compositions of the present disclosure can be used to enhance intestinal adaptation and absorption after intestinal resection or bariatric surgery.

[0057] Additional Embodiments

[0058] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure herein is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0059] Example 1. Use of GLP-1 -producing neurons for the control of glucose homeostasis and insulin sensitivity [0060] In this Example, Applicants aimed to determine if acute activation of PPG neurons (i.e., GLP-1 -producing neurons) enhances peripheral glycemic control and insulin sensitivity. A pharmacogenetics mouse model for the remote control activation of prepoglucagon (PPG) neurons in the brainstem in vivo was established. First, Applicants generated a glucagon (Gcg) promoter-driven Cre transgenic mouse line in which Gcg-Cre expression was colocalized to GLP-1 -positive neurons in the brainstem. Second, Applicants created a remote control of activation of PPG neurons in an in vivo mouse model using the DREADD (designer receptors exclusively activated by designer drugs) approach.

[0061] Cre-dependent, excitatory AAV hM3Dq-mCherry vectors were injected into the brainstem NTS of 8-wk old Gcg-Cre mice. Next, validation of Gcg-Cre-dependent hM3Dq activation was established. In particular, hM3Dq expression in the brainstem was performed using a Gcg-Cre:Rosa26eGFP reporter mice, in which hM3Dq-mCherry was faithfully expressed in GFP-positive neurons (identified by Gcg-Cre mediated eGFP). Moreover, excitation of hM3Dq-mCherry expressing neurons was confirmed by the whole cell patch clamp. Membrane potential and firing rate of Gcg-Cre: hM3Dq-mCherry NTS neurons were recorded in brain slices.

[0062] In addition, central neural function of PPG neuronal activation was measured. Activation of hM3Dq-expressing neurons mediated neural circuitry was mapped by immunostaining c-Fos in the brain after ip administration of clozapine N-oxide (CNO, an exogenous and specific agonist for hM3Dq G protein-coupled receptor). Increased c-fos expression was mainly localized to central autonomic regions. Moreover, PPG neurons-mediated neurotransmitter profiling was quantified by LC-MS-based metabolomics.

[0063] As major neurotransmitters, GLP-1 and GLP-2 were released from activated PPG neurons. Upon CNO stimulus, the hM3Dq receptors rapidly activated infected PPG neurons in vivo.

[0064] In addition, the peripheral metabolic impact of PPG neuronal activation was studied. By utilizing a glucose tolerance test and a hyperinsulinemic euglycemic clamp, glucose homeostasis and insulin sensitivity were quantified in conscious Gcg-Cre mice injected with Cre-dependent hM3Dq vectors. [0065] Fasting glucose was decreased while glucose tolerance was enhanced in the Gcg-Cre mice after ip CNO. In addition, the glucose infusion rate (GIR) was increased and resulted from augmentation of insulin-mediated stimulation of glucose disposal (Rd) and insulin-mediated suppression of endogenous glucose production (EGP) upon ip CNO. Note that GIR indicates whole-body insulin sensitivity, while Rd represents peripheral insulin sensitivity and EGP hepatic insulin sensitivity, respectively.

[0066] The aforementioned results indicate that the acute activation of PPG neurons in the brainstem NTS improves glucose tolerance and augments insulin sensitivity, suggesting that PPG neurons play an important physiological role in glycemic control and insulin sensitivity.

[0067] Example 1.1. Methods

[0068] A pharmacogenetics mouse model for the remote activation of PPG neurons in vivo was established through the following steps. Glucagon (Gcg) promoter-driven Cre transgenic mouse line was generated and confirmed with GLP-1 immuno staining. Remote activation of the NTS PPG neurons in the mouse model was created using the DREADD approach. In particular, Cre- dependent, excitatory AAV hM3Dq-mCherry vectors were injected into the brainstem NTS of 8 wk-old, Gcg-Cre mice using stereotaxic coordinates.

[0069] Gcg Cre-dependent hM3Dq activation was validated by the following methods. The hM3Dq expression in the brainstem was validated using Gcg-Cre: :Rosa26-eGFP reporter mice. Acute activation of hM3Dq-expressing neurons was confirmed by the whole cell patch clamp. Membrane potential and firing rate of Gcg-Cre: :hM3Dq-mCherry NTS neurons were recorded in brains slices.

[0070] Metabolic significance of remote activation of PPG neurons in the brainstem was determined as follows. Remote activation of hM3Dq-expressing neurons-mediated neural circuitry was mapped by c-Fos immunostaining in the brain after ip injection of clozapine N- oxide (CNO). Upon CNO stimulus, the hM3Dq receptors rapidly activated infected PPG neurons in vivo. Glucose tolerance tests and hyperinsulinemic euglycemic clamps were employed to quantify glucose homeostasis and insulin sensitivity in conscious Gcg-Cre mice after Cre- dependent hM3Dq vectors were injected into their brainstem NTS. [0071] A dual stable isotopic tracer method for hyperinsulinemic euglycemic clamp was developed as follows. Mice were continuously infused with 6,6- H ₂ -D-glucose via a jugular catheter for 3 hours during a basal period and then for 3 hours during an insulin clamp (FIG. 5), whereas 6,6- H ₂ -D-glucose plus D-glucose were infused to maintain blood glucose level. Deuterium enrichments of glucose were measured after derivatization using GC-MS. Glucose kinetics was quantified at a steady-state in conscious mice.

[0072] Example 1.2. Experimental results

[0073] FIG. 2 shows the results of PPG neurons-mediated neural circuitry in the brainstem. As illustrated in FIGS. 2A-B, autonomic PPG neurons were labeled by Gcg promoter-driven eGFP (green) in the mouse brainstem NTS. As shown in FIG. 2C, Glp2r neurons were immunostained (green) where PPG neurons widely project. In addition, Phox2b neurons were labeled by Cre- dependent, Rosa26-td Tomato reporter (red).

[0074] The results show that Glp2r-positive neurons are co-localized to Phox2b neurons (yellow) in the DMV area, indicating an autonomic circuitry of NTS PPG neurons to DMV Phox2b neurons.

[0075] FIGURE 3 shows that the generated Gcg-Cre transgenic mouse line was validated by Cre-dependent Rosa26-eGFP reporter mouse. Gcg-Cre-mediated GFP expression was shown in distinct neurons in the brainstem NTS (FIG. 3A) and enteroendocrine cells in the gut (FIG. 3B). GFP-expressing cells were GLP-l/2-immunopositive.

[0076] FIGURE 4 shows data relating to the remote activation of Cre-dependent hM3Dq- expressing PPG neurons, as indicated by c-Fos expression. As indicated in FIG. 4A, a weak c- Fos expression was observed in the basal without CNO injection. FIG. 4B shows a symbolized map of the brainstem area. FIG. 4C shows that c-Fos expression was increased at 30 minutes after CNO injection. FIG. 4D shows an enlarged image of the squared region in FIG. 4C. The c-Fos-positive cells (green) were largely segregated from hM3Dq-expressing cells (red).

[0077] FIGURE 6 illustrates the activation of hM3Dq-expressing PPG neurons induced acutely by CNO. As shown in FIG. 6A, PPG neurons were indicated by mCherry (red) in the brainstem NTS from the Gcg-Cre mouse locally infected with AAV hM3Dq-mCherry vectors with c-Fos expression (green). As indicated in FIG. 6B, hM3Dqexpressing PPG neurons excited by CNO (5 μΜ) showed representative traces of the whole-cell patch clamp. As shown in FIG. 6C, PPG neurons were labeled by GFP (green) from the Gcg-Cre::Rosa26-eGFP mouse brainstem. FIG. 6D shows the location of the brainstem slice.

[0078] FIGURE 7 demonstrates that the remote activation of PPG neurons enhances glucose tolerance and insulin sensitivity in mice fed regular chow. FIG. 7A shows data relating to a 6 hour fasting glucose at 0 and 3.5 hours after injection of CNO (ip 1 mg/kg). AAVhM3Dq- mCherry vectors (100 nL) were injected into the Gcg-Cre mouse brainstem NTS. FIG. 7B shows blood glucose concentration of mice (at 3 weeks post viral injection). The blood glucose concentration was measured after an ip glucose challenge. CNO was ip injected 30 minutes prior to ipGTT. FIG. 7C shows data relating to endogenous glucose production (EGP) during a basal period in the mice (at 3 weeks post viral injection).

[0079] FIG. 7D shows insulin sensitivity of the conscious mice (at 3 weeks post viral injection) quantified by hyperinsulinemic euglycemic clamp coupled with dual stable isotopic tracers

( 2 ¾0 and 6,6- 2

H2-D-glucose, as shown in FIG. 5). Glucose kinetics was determined at the steady status. Remote activation of PPG neurons augments insulin sensitivity largely by further suppression of endogenous glucose production. Mice were ip injected with CNO or saline prior to an insulin clamp (n=6~8/ group. * or **P < 0.05 or 0.01).

[0080] FIGURE 8 shows that the remote activation of ChAT neurons in the DVC enhances hepatic insulin sensitivity in mice that were fed regular chow. FIG. 8A shows ChAT neurons labeled by ChAT-Cre-mediated tdTomato (red) in the brainstem DVC from a ChAT Cre::Rosa26-tdTomato reporter mouse. FIG. 8B shows GLP-2R colocalized to ChAT neurons in the DVC. FIG. 8C shows a protocol for quantifying insulin sensitivity in mice with remote activation of the DVC ChAT neurons. AAV-hM3Dq-mCherry vectors were injected into the ChAT Cre mouse brainstem DMV.

[0081] FIG. 8D shows insulin sensitivity of the conscious mice quantified by hyperinsulinemic eyglycemic clamp coupled with dual stable isotopic tracers (FIG. 5). Remote activation of ChAT neurons augments insulin sensitivity by further suppression of endogenous glucose production. Mice were ip injected with CNO (1 mg/kg) or saline prior to insulin clamp (n=6~8/ group. * or **P < 0.05 or 0.01). ChAT Cre mouse line is confirmed in Cre-mediated Rosa26-td-Tomato reporter and GLP-2R-immunoreactive neurons stained by immunohistochemistry, indicating GLP-2R-postive neurons are ChAT neurons in the brainstem.

[0082] In summary, the results indicate that NTS PPG neurons can be remotely activated with the DREADD approach, as confirmed by c-Fos expression after ip CNO. Moreover, the NTS PPG neurons infected with AAV hM3Dq particles can be acutely excited ex vivo upon CNO application. It is assumed that the hM3Dq activation would rapidly excite infected PPG neurons. In addition, remote activation of either PPG neurons (i.e., GLP-1 -producing neurons) or ChAT neurons (i.e., GLP-2R-expressing neurons) improves glucose tolerance and insulin sensitivity, suggesting that PPG-ChAT autonomic circuitry plays an important role in glycemic control and insulin sensitivity.

[0083] As illustrated in FIG. 9, Applicants propose a working model. In particular, activation of the NTS PPG neurons improves glycemic control and insulin sensitivity via fine-tuning autonomic outputs to peripheral tissues (liver), thereby suppressing hepatic glucose production (HGP), which involves Glplr/2r-positive, ChAT neurons in the brainstem DMV.

[0084] Example 2. GLP-1R/2R dual activation for improving postprandial glycemic control and insulin sensitivity in DIO mice after VSG

[0085] In this Example, Applicants provide data indicating that GLP-1R/2R dual activation is needed for improving postprandial glycemic control (PGC) and insulin sensitivity (IS); essential for improving non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH); and required for intestinal adaptation after bariatric surgery in diet-induced obese mice (DIO mice) after vertical sleeve gastrectomy (VSG). Mice with GLP1R/2R double knockout (DKO) displayed postprandial hyperglycemia and postabsorptive insulin sensitivity (FIG. 10). The DKO mice fed high-fat diet (HFD) showed severe glucose intolerance after gavage feeding (FIG. 10A), which was attributed to sustained high Ra and EGP (FIGS. lOB-C). Importantly, postprandial suppression of gluconeogenesis was blunted in the DKO mice (FG. 10D). The biphasic pattern of appearance rates of ingested glucose (Ra, meal) for initiating the neuroendocrine negative feedback was abolished (FIG. 10E). [0086] In the Ra, meal would represent the portal appearance rate of ingested glucose, a key player of the enteroinsular axis in postprandial glycemic control (PGC). For rapidly rising Ra, meal may be a physiological trigger (e.g., for glucose-sensing endocrine β cells in the pancreas and neurons in the brain) to initiate a negative-feedback neuroendocrine control of PGC. In fact, the DKO mice fed either HFD or regular chow had higher fasting glucose (FIG. 10F). Moreover, IS was further impaired in the DKO mice fed HFD as indicated by higher EGP and lower Rd during the insulin clamp (FIG. 10G). Body weight and fat mass increased in the DKO (FIG. 10H) due to reduced basal metabolic rate and increased feeding. This metabolic phenotype of the DKO mouse indicates that endogenous GLP1R/2R play an essential role in PGC and IS.

[0087] As shown in FIG. 11, postprandial GLP-1 and PYY were elevated after VSG while GIP (another incretin hormone) was not. As speculated, dietary ingredients are not digested fully in the proximal gut for enteroendocrine K cells to secrete GIP, while absorbed incompletely in the distal gut and thus used for enteroendocrine L cells to secret GLP.

[0088] The data in FIG. 12 demonstrates that the dual activation of endogenous GLP-1R/2R is necessary for VSG to restore postprandial glycemic control in DIO mice. Postprandial glucose intolerance (induced by HFD) was improved after VSG only in the wild type (WT) mice, not the DKO mice (FIG. 12A). The amount of glucose in the whole body (indicated inversely by the H2-glucose dilution) was significantly decreased at the baseline and postprandial state after VSG in the WT mice, but not the DKO mice) (data not shown).

[0089] The peak of Ra was higher and shifted left after VSG only in the WT mice (FIGS. 12C- D). In WT mice, VSG augmented postprandial glucose disappearance (Rd) occurred within an early phase after gavage feeding (FIG. 12B), whereas both EGP and gluconeogenesis (GNG) were reduced within late phases (FIGS. 12E-F).

[0090] Without being bound by theory, Rd might represent insulin-stimulated rapid translocation of glucose transporters responsible for uptake in adipose/muscle tissues, while GNG would result from insulin-inhibited transcription of gluconeogenic genes in the liver. However, VSG- improved postprandial glycemic kinetics was blunted in the DKO mice. [0091] The data in FIG. 13 demonstrates that the dual activation of endogenous GLP-1R/2R is necessary for VSG to improve postabsorptive insulin sensitivity in DIO mice. In the WT, VSG reduced basal glucose after a 12 hour fast (FIG. 13A) and enhanced whole-body insulin sensitivity (GIR) by augmenting insulin- stimulated Rd and insulin-inhibited GNG (FIGS. 13B- D). However, VSG-improved IS was fully blunted in the DKO DIO mice, suggesting that the dual activation of GLP-1R/2R is necessary for VSG to improve IS.

[0092] The data in FIG. 14 demonstrates that the dual activation of endogenous GLP-1R/2R are required for intestinal adaptation in DIO mice after VSG. In the WT, VSG showed intestinal adaptation by increased intestinal proliferative index (FIGS. 14 A-D). Note that proliferation is assessed by BrdU incorporation into the jejunum crypt. BrdU was immunostained in green. S = Sham; V = VSG. However, VSG-augmented intestinal proliferation was fully blunted in the DKO DIO mice (FIGS. 14 E-F), suggesting that the dual activation of GLP-1R/2R is necessary for VSG-induced intestinal adaptation and prevented intestinal deficiency from bariatric surgery.

[0093] The data in FIG. 15 demonstrates that the double knockout of endogenous GLP-1R/2R shows high incidence of hepatic steatosis and inflammation (i.e., nonalcoholic steatohepatitis (NASH)) in DIO mice. Histological inflammation and fibrosis is characterized only in the liver from the DIO mouse with DKO (FIG. 15 B) while hepatic steatosis is shown in both WT (FIG. 15 A) and DKO. The data indicate that endogenous GLP-1R/2R are important for pathological progression of NAFLD to NASH.

[0094] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.