CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claim priority to United States Patent Application U.S. 63/128,473, filed December 21 , 2020, the entire contents of which is hereby incorporated by reference.

FIELD

[0002] The present disclosure relates generally to metformin prevents hyperglycaemia-associated, oxidative stress-induced vascular endothelial dysfunction: essential role for the orphan nuclear receptor, Nr4a1 (Nur77). Prevention of hypergycaemic endothelial dysfunction by Metformin

BACKGROUND

[0003] Metformin, first used clinically in the late 1950s, remains a drug of first- choice for type-2 diabetics. In contrast with many newer therapeutic diabetes drugs, metformin was not designed for a specific cellular therapeutic target. Rather, its development came from observations that French lilac (Galega officinalis)-demed guanidines could treat “sweet urine” disease. Notwithstanding its clinical utility, the therapeutic mechanisms whereby biguanides work clinically are still unclear. It is now evident that metformin has a number of putative targets that contribute to its clinical effectiveness, quite apart from its facilitation of the actions of insulin (Nafisa et al., 2018).

SUMMARY

[0004] In one aspect there is provided a method of treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, comprising: administering a therapeutically effective amount of metformin or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, in a subject in need thereof, the subject comprising endothelial cells comprising Nr4A1 protein.

[0005] In one aspect there is provided a method of treating diabetes, comprising: selecting a subject with diabetes, said subject comprising endothelia cells comprising Nr4A1 protein, administering a therapeutically effective amount of metformin , or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, to said subject.

[0006] In one example, said diabetes is Type 1 diabetes, Type 2 diabetes, or gestational diabetes.

[0007] In one example, said treatment protects the endothelium from hyperglycaemia-induced dysfunction.

[0008] In one example, said subject is a human.

[0009] In one aspect there is provided a method of treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, comprising: administering a therapeutically effective amount of Cytosporone B , or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, in a subject in need thereof, the subject comprising endothelial cells comprising Nr4A1 protein.

[0010] In one aspect there is provided a method of treating diabetes, comprising: selecting a subject with diabetes, said subject comprising endothelia cells comprising Nr4A1 protein, administering a therapeutically effective amount of Cytosporone B, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, to said subject.

[0011] In one example, said diabetes is Type 1 diabetes, Type 2 diabetes, or gestational diabetes.

[0012] In one example, said treatment protects the endothelium from hyperglycaemia-induced dysfunction.

[0013] In one example, said subject is a human.

[0014] In one aspect there is provided a use of a therapeutically effective amount of metformin, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, in treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes in a subject in need thereof, the subject comprising endothelial cells comprising Nr4A1 protein.

[0015] In one aspect there is provided a use of metformin for treating diabetes, comprising: selecting a subject with diabetes, said subject comprising endothelia cells comprising Nr4A1 protein, use of a therapeutically effective amount of metformin , or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, on said patient. [0016] In one example, said diabetes is Type 1 diabetes, Type 2 diabetes, or gestational diabetes.

[0017] In one example, said use protects the endothelium from hyperglycaemia- induced dysfunction.

[0018] In one example, said subject is a human.

[0019] In one aspect there is provided a use of a therapeutically effective amount of Cytosporone B, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, for treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, the subject comprising endothelial cells comprising Nr4A1 protein.

[0020] In one aspect there is provided a use of a therapeutically effective amount of Cytosporone B, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, for treating a subject, comprising: selecting a subject with diabetes, said subject comprising endothelia cells comprising Nr4A1 protein, administering a therapeutically effective amount of Cytosporone B to said subject.

[0021] In one example, said diabetes is Type 1 diabetes, Type 2 diabetes, or gestational diabetes.

[0022] In one example, said treatment protects the endothelium from hyperglycaemia-induced dysfunction.

[0023] In one example, said subject is a human.

[0024] In one aspect there is provided a kit for treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, comprising: metforminand , or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, a container, and optionally instructions for the use thereof, the subject comprising endothelial cells comprising Nr4A1 protein.

[0025] In one example, said diabetes is Type 1 diabetes, Type 2 diabetes, or gestational diabetes.

[0026] In one example, said treatment protects the endothelium from hyperglycaemia-induced dysfunction.

[0027] In example, said subject is a human.

[0028] In one aspect there is provided a kit for treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, comprising: Cytosporone B, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, and a container, and optionally instructions for the use there of, the subject comprising endothelial cells comprising Nr4A1 protein.

[0029] In one example, said diabetes is Type 1 diabetes, Type 2 diabetes, or gestational diabetes.

[0030] In one example, said treatment protects the endothelium from hyperglycaemia-induced dysfunction.

[0031] In one example, said subject is a human.

BRIEF DESCRIPTION OF THE FIGURES

[0032] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0033] Figure 1 : metformin reduces hyperglycaemia-induced endothelial dysfunction for acetylcholine (muscarinic) and 2fl_l (PAR2)-stimulated vasorelaxation. A) Scheme explaining the incubation of metformin along with different glucose concentrations followed by wire-myography assay. (B-E) Concentration-effect curves for acetylcholine (ACh: B,C) and 2fl_l (D,E)-induced vasorelaxation in wild-type aortic rings incubated with either 10 or 25 mM glucose without or with 1 or 500 pM metformin for 48h. Data points represent the means ± SEM (error bars) for measurements done with up to six independently assayed tissue rings obtained from at least 8 independently harvested aorta tissue segments for each agonist concentration.

Error bars smaller than the symbols are not shown. Statistically significant differences are indicated by an asterisk *. For panels B and D: *P<0.05, comparing 25 mM glucose cultures either untreated (open squares) or 1 pM metformin-treated tissues (Solid squares) and *P<0.05, comparing metformin-treated tissues maintained in 25 mM glucose with metformin-treated tissues maintained in 10 mM glucose. For panel C, *P<0.05, for the reduction in ACh-mediated relaxation caused by 500 pM metformin, compared with untreated tissues. Panel D: ns = no significant difference between the hyperglycaemia-induced reduction in response to PAR2 activation (2fl_l) in the absence or presence of 500 pM metformin.

[0034] Figure 2: An Nr4a1 antagonist reverses the ability of metformin to attenuate hyperglycaemia-induced vascular endothelial dysfunction as assessed by a tissue bioassay. A) Scheme outlining the protocol to demonstrate the block by the Nr4a1 antagonist, TMPA, of the protective effect of metformin to preserve endothelial dysfunction after culture of wild-type (WT) aortic rings in 25 mM glucose. (B) Concentration-effect curves for ACh-induced relaxations in wild-type aortic rings cultured in 25 mM glucose and 50 pM TMPA without or with 1 pM metformin for 48 hours. (C) Concentration-effect curves for 2fl_l-induced relaxations in wild-type aortic rings cultured in 25 mM glucose without or with 1 pM metformin for 48 hours in the absence (open and solid squares) or presence of 50 pM TMPA (solid diamonds). Data points represent the means ± SEM (bars), for n=8. Error bars smaller than the symbols are not shown. ns=not significant.

[0035] Figure 3: Aorta tissues from Nr4a1-hull mice display reduced muscarinic/acetylcholine and PAR2 (2-fLI)-mediated vasorelaxation compared with wild-type tissues and express lower levels of eNOS. Freshly isolated aorta rings from either wild-type (WT: open squares) or Nr4a1-null mice (solid squares) were constricted with 2.5 pM phenylephrine and the relaxant effects of increasing concentrations of the acetylcholine (ACh) (A) or the PAR2-selective peptide agonist, 2fl_l (B) was measured as described in methods. C) Representative western blot analysis showing the total eNOS in aortic segments from Nr4a1-null and WT mice relative to the signal for beta- actin. D) Histograms showing the abundance of eNOS relative to the beta-actin signal as measured by densitometry for aorta tissues from wild-type (WT: right-hand histogram) and from Nr4a1-null mice (left-hand histogram) *P<0.05, comparing the response to acetylcholine (ACh) for WT vs Nr4a1-null tissues: mean±SEM (bars) for n = 6 and for total eNOS in WT vs Nr4a1-null tissues (n=5).

[0036] Figure 4: metformin does not reduce hyperglycaemia induced endothelial dysfunction in Nr4a1-null tissues. A) Scheme showing the time frame for organ culture of Nr4a1-null aorta rings in 10 or 25 mM glucose, with or without either 1 or 25 pM metformin prior to wire myograph bioassays (B-E). B,C: Concentration-effect curves for acetylcholine-induced vasorelaxation in Nr4a1-null aortic rings cultured in 10 or 25 mM glucose without or with 1 pM (Panel B) or 25 pM metformin (Panel C) for 48h. D, E: Concentration-effect curves for 2-fLI-induced vasorelaxation in Nr4a1-null aortic rings incubated in 10 or 25 mM glucose, without or with either 1 pM (Panel D) or 25 pM (Panel E) metformin for 48h. Data are presented as mean ± SEM (bars) for n=6 for each group. Error bars smaller than the symbols are not shown. ns= not significant.

[0037] Figure 5: metformin treatment in vivo improves acetylcholine and PAR2 (2fLI)-mediated vasorelaxation in aortic ring preparations from wild-type but not from Nr4a1-null streptozotocin (STZ)-induced diabetic mice. Aorta rings from either wild-type (Panels B & D) or Nr4a1-null (Panels E & G) streptozotocin-diabetic mice, treated or not with metformin in vivo, according to the schema in Panel A, were isolated after 2 weeks of metformin treatment and evaluated by wire myography for their vasorelaxant responses to increasing concentrations of either acetylcholine (ACh) (Panels B & C) or 2fl_l (Panels D & E), as described in methods. Data represent the mean relaxation responses ±SEM (bars) for 6 independently assayed aorta rings. *P<0.05, comparing tissues from untreated vs metformin-treated wild-type and Nr4a1-null mice. There was no significant (ns) difference for the relaxation responses for the tissues from the Nr4a1 -null-derived tissues, whether or not the mice were treated with metformin. [0038] Figure 6: metformin’s ability to prevent hyperglycaemia-induced ROS production in cultured aorta-derived endothelial cells is blocked by an NR4A1 antagonist. Wild-type mouse aorta-derived primary endothelial cell cultures were incubated as outlined in Methods, with either 25mM (Panels A to C; G to L) or 5 mM glucose (Panels D to F) in the absence (Panels A to F) or presence (Panels G to L) of 50 |iM metformin. The metformin-treated cultures also did (Panels J to L) or did not (Panels G to I) contain the Nr4a1 antagonist, TMPA. Increased cellular ROS observed after 1 hour was indicated by increased Cell-ROX green fluorescence. Nuclei are stained blue with DAPI.

[0039] Figure 7: metformin reduces intracellular ROS in wild-type but not Nr4a1-null aortic endothelial cells. Wild-Type (WT: Panels A to C) or Nr4a1-null (Panels D to F) aorta-derived endothelial cells were incubated for 24 hours with either 5mM or 25mM glucose (G), without (Panels A,B, D, E) or in combination with (Panels C and F) 5pM metformin. Increased ROS is indicated by increased Cell-ROX green fluorescence; nuclei are stained blue with DAPI. The histograms in Panel (G) show the corrected ‘green’ fluorescence intensity (arbitrary units) calculated by fluoresence yield analysis of at least 6 equivalent fields from 3 independent microscopic images, as outlined in methods. * P<0.05 for reduction of fluorescence for wild-type cells in 25 mM glucose with metformin, compared with metformin-untreated cells, ns: no significant difference for Nr4a1-null cells in 25 mM glucose with metformin compared with metformin-untreated Nr4a1-null cells.

[0040] Figure 8: metformin improves the oxygen consumption rate (OCR) in wild-type, but not in Nr4a1-null mouse aortic segments and endothelial cells exposed to hyperglycaemia. Aorta rings from either wild-type (Panel A) or Nr4a1-null mice (Panel B), with the endothelium side facing up in the Seahorse chamber were incubated with 25mM glucose for 24 hours without (solid blue circles) or with (solid red squares) metformin and the oxygen consumption rates (OCR) were measured as in Methods. The OCR shown on the Y-axis was normalized to the protein content of the tissues using wave software as in Methods. (C) Primary replicate monolayer cultures of endothelial cells derived from either wild-type (red and blue symbols) or Nr4a1-null mice (magenta and black symbols) were maintained under hyperglycaemic conditions (25 mM glucose) for 24 hours in the absence (blue and black solid circles) or presence (red and magenta squares) of 10pM metformin. The oxygen consumption rates (OCR) of six replicate monolayers were then measured as outlined in Methods. Values represent the means ± SEM (bars) for n=6 for each data point. *P<0.05, comparing wild type tissues maintained in 25mMG/24 hours + 10pM metformin vs wild type tissues maintained without metformin in 25mMG/24 hours in figure 8A; and comparing wild type endothelial monolayers maintained in 25mMG/24 hours + 10pM metformin vs wild type cell monolayers maintained without metformin or Nr4a1-null cell monolayers maintained in 25mMG/24 hours + 10pM metformin. No significant effect of metformin to increase the oxygen consumption rate was observed for tissues or cells derived from the Nr4a1-null mice (Compare red vs blue symbols in figure 8B and magenta vs black symvbols in Figure 8C).

[0041] Figure 9: metformin at a concentration that prevents endothelial oxidative stress (10 M) does not affect Complex I and Complex III oxygen consumption rates in mouse aortic endothelial cells. (A-B) Scheme showing the experimental procedures done to study the mitochondrial complex-mediated respiration in primary cultures of wild-type mouse aortic endothelial cells. (C-D) Cells were assessed for mitochondrial OCR upon maintaining them in 25mM glucose (G)/24 hours in the absence (solid blue circles) or presence of either 10 pM (solid red squares) or 500 pM (magenta triangles) metformin. (E to H) Quantification of the OCR for complexes I to IV shows that 10 pM metformin does not affect Complexes I and III, whereas at 500pM, metformin compromises both complexes I and III. *P<0.05, for Panels E & G, comparing cells maintained in 25mM G/24 hours without or with 10 pM metformin vs 25mM G/24h+ 500 pM metformin. *P<0.05 for Panel F for cells maintained in 25mM glucose(G)/24 hours versus cells maintained in 25mM G/24h with either 10 pM or 500 pM metformin. *P<0.05 for Panel H for cells maintained in 25mM G/24 hours without or with 500 pM metformin versus cells maintained in 25mM G/24 hours with 10 pM metformin.

Histograms snow the means ±SEM (bars) for n = 5 replicate monolayers. [0042] Figure 10: metformin treatment preserves mitochondrial integrity in aortic endothelial cells exposed to hyperglycaemia. (A-D) Electron microscopic images of wild-type primary cultures of aorta-derived endothelial cells incubated with 10 mM (Low glucose: Panel A) or 25 mM (High glucose: Panels B to D) glucose concentrations for 24 hours in the absence (Panels A and B) or presence of either 10 pM (Panel C) or 500 pM (Panel D) metformin. Circular and spindle-shaped organelles of different area size were observed (Panel F). Baseline ‘spindle’ mitochondrial morphology comprising elongated structures with visible cristae are denoted with yellow arrows. Circular morphology, indicative of increased oxidative activity and increased mitochondrial fission is denoted by green arrows. The ratio of rounded to spindle structures are associated with increased oxidative stress. The ratio was calculated as outlined in Materials and Methods (Histograms, Panel E) to quantify the degree of change of morphology that was caused by a switch from low (10 mM) to high glucose (25 mM) and reversed in the presence of 10 pM, but not 500pM metformin. Panel F shows the 2D- size measure of circular and spindle shaped mitochondria (A.U). *P<0.05, comparing high glucose alone vs high glucose plus 10 pM metformin. Data represent the mean ratios (green/yellow) +/- SEM (bars) from measurements done for equivalent image areas for at least 6-10 cells per condition.

[0043] Figure 11 : metformin rescues endothelial vasorelaxant action subsequent to hyperglycaemia- induced dysfunction at a concentration that can activate AMPKinase. As shown by the schema in Panel A, intact vascular organ cultures were maintained for 48 hours under hyperglycaemic conditions (25 mM glucose) and were then supplemented or not by the addition of either 1 or 10 pM metformin for a further 12 hours. Tissues were then recovered from the culture medium and evaluated for the vasorelaxant actions of acetylcholine (ACh: Panel B) and 2fLI (Panel C). D and E: Based on the protective action of 10 pM metformin, its impact on AMPKinase activationphosphorylation in cultured endothelial cells was assessed by western blot analysis (Panels D and E). Densitometry measurements of the activation/phosphorylation of AMPKinase, normalized to the beta-actin signal illustrated in the representative gel in Panel D, are shown by the histograms in Panel E. *P< 0.05 for vasorelaxant responses improved by either 1 or 10 10 pM metformin compared with metformin-untreated tissues. Values represent the means +/- SEM (bars) for measurements done with 6 independently-assayed tissues. Error bars smaller than the symbols are not shown. *P<0.05 for increases shown in Panel E in phospho-AMPK for endothelial cells maintained in 25mM g/24h +10 .M or 500 .M metformin, compared to cells maintained in 25mM G/24 oursh without metformin. Values for histograms in panel E represent the average densitometric ratio of phospho-AMPK/beta-actin +/- SEM (bars) for n = 3. Increased activation of AMPK by its agonist, AICAR, is shown in Panels D and E.

[0044] Figure 12: metformin docks with NR4A1 in silico and the interactions of metformin with NR4A1. (A) Representation of NR4A1 (ribbon model) interacting with metformin (stick model). (B) Active site residues of metformin (ball and stick model) along with bond lengths. (C) Surface representation of Nr4a1 pocket within which metformin binds.

[0045] Figure 13. NR4A1 -agonist, cytosporone B protects against hyperglycaemia in mouse microvascular endothelial cells at low (50 nM) but not high (500-1000nM) concentrations. (A-B) Concentration-effect curves for ACh (Panel B) and 2-fLI (Panel A)-induced vasorelaxation in wild-type aortic rings maintained for 48 hours in 25 mM glucose without (solid circles) or with 50 nM (solid diamonds), 500 nM (solid squares) or 10000 nM cytosporone B. Data are presented as means ± SEM (bars), n=6 for each group. * (Panel A) and ** (Panel B), P <0.05, comparing responses of untreated tissues vs tissues treated with 50 nM cytosporone B.

[0046] Figure 14. Molecular dynamics of metformin-NR4A1 complex. (A)

RMSD plot of Nr4a1 as a function of time. (B) Backbone RMSF plot. (C) Total energy of the system during simulation. D) Average potential energy of the protein. (E) Number of H-bonds between metformin and Nr4a1. (F) Distance of H-bonds present between the metformin and Nr4a1. G) SASA plot as a function of time.

[0047] Figure 15. Isolation of a biotinylated-metformin/Nr4a1 complex using Neutravidin affinity beads.

[0048] The complex between solubilized myc-tagged or mRFP-tagged Nr4a1 and biotinylated metformin was harvested using Neutravidin cross-linked magnetic beads as outlined in Methods. Proteins attached to biotinylated metformin (Bio-Met) on the neutravidin beads were dissociated from biotinyl-metformin by the addition of 100 pM free metformin, and the proteins eluted were analyzed by western blot detection of either myc- tagged (Panel A) or mRFP-tagged Nr4a1as outlined in Methods. No signal was observed when the Neutravidin bead harvesting procedure was done using either the metformin- free biotin C10 linker (Bio-CONH-Ci ₀ : N1 , Panels A and B) or the biotin-free metformin- C10 linker construct (metformin-NH-Ci ₀ : N2, Panels A and B). DETAILED DESCRIPTION

[0049] Abbreviations

[0050] ACh - Acetylcholine, an endothelial muscarinic receptor agonist

[0051] AICAR - 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPKinase

[0052] CsnB - Cytosporone B, 3,5-Dihydroxy-2-(1-oxooctyl)-benzeneacetic acid, ethyl ester

[0053] 2fLI— 2-furoyl-Leu-lle-Gly-Arg-Leu-amide, a potent and selective PAR2 agonist

[0054] L-NAME - Nw-Nitro-L-arginine methyl ester hydrochloride

[0055] MMEC -mouse microvascular endothelial cells

[0056] NO - Nitric Oxide

[0057] NR4A1/Nr4a1- Human nuclear receptor 4A1 (murine gene designated as Nr4a1 ; formerly designated Nur77). Both designations (human NR4A1 ; mouse Nr4a1) are used interchangeably in the text.

[0058] OCR - Oxygen consumption rate

[0059] ODQ - Guanylate cyclase inhibitor [1 H-[1 ,2,4]oxadiazolo-[4, 3- a]quinoxalin-1-one]

[0060] PAR2 - Proteinase-activated receptor 2

[0061] PE - Phenylephrine

[0062] mRFP - monomeric red fluorescent protein

[0063] ROS - Reactive oxygen species

[0064] STZ - Streptozotocin

[0065] THPN - an NR4A1 agonist,- 1-(3,4,5-trihydroxyphenyl)-nonan-1-one

[0066] TMPA - an NR4A1 antagonist, ethyl 2-[2,3,4-trimethoxy-6-(1- octanoyl)phenyl]acetate

[0067] In one aspect there is provided a method of treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, comprising: administering a therapeutically effective amount of metformin, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, in a subject in need thereof, the subject comprising endothelial cells comprising Nr4A1 protein. [0068] In one aspect, there is provided a method of treating diabetes, comprising: selecting a subject with diabetes, said subject comprising endothelia cells comprising Nr4A1 protein administering a therapeutically effective amount of metformin, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, to said subject.

[0069] In one aspect there is provided a method of treating a subject having diabetes, suspected of having diabetes, or at risk of developing diabetes, comprising: administering a therapeutically effective amount of Cytosporone B, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, in a subject in need thereof, the subject comprising endothelial cells comprising Nr4A1 protein.

[0070] In one aspect there is provided a method of treating diabetes, comprising: selecting a subject with diabetes, said subject comprising endothelia cells comprising Nr4A1 protein, administering a therapeutically effective amount of Cytosporone B, or a tautomer thereof, or a pharmaceutically acceptable salt, or a solvate thereof, or a functional derivative thereof, to said subject.

[0071] The term “functional derivative” as used herein refers to a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original compound. A functional derivative or equivalent may be a natural derivative or is prepared synthetically.

[0072] Also encompassed as prodrugs or "physiologically functional derivative". [0073] The term “physiologically functional derivative” as used herein refers to compounds which are not pharmaceutically active themselves but which are transformed into their pharmaceutically active form in vivo, i.e. in the subject to which the compound is administered.

[0074] The term “prodrug” as used herein, refers to a derivative of a substance that, following administration, is metabolized in vivo, e.g. by hydrolysis or by processing through an enzyme, into an active metabolite.

[0075] In some examples, there is described a composition comprising a compound as described herein, and a pharmaceutically acceptable carrier, diluent, or vehicle.

[0076] As used herein, “diabetes” or “diabetes mellitus” refer to a chronic metabolic disorder of multiple aetiology, characterized by chronic hyperglycaemia with disturbance of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effect of diabetes mellitus includes long-term damage, dysfunction and failure of various organs. Diabetes mellitus is usually divided into two major categories:

[0077] Type 1 diabetes (formerly insulin-dependent diabetes mellitus) usually develop in childhood or adolescence and are prone to ketosis and acidosis. Type 1 diabetes accounts for around 10% of all diabetes.

[0078] Type 2 diabetes (formerly non-insulin-dependent diabetes mellitus) includes the common major form of diabetes which results from defect(s) in insulin secretion, almost always with a major contribution from insulin resistance. Type 2 diabetes accounts for around 90 % of all diabetes.

[0079] Diabetes also encompasses “gestational diabetes”. Gestational diabetes refers to a condition in which a woman without diabetes develops high blood sugar levels during pregnancy.

[0080] The term “prediabetes” refers to a condition wherein the subject has a blood glucose level higher than normal, but not yet enough to be classified as diabetes. Prediabetes increases the risk of developing a type-2 diabetes.

[0081] “Diabetes” and “prediabetes” can be diagnosed by measuring glycemia.

[0082] As used herein, “glycemia” refers to the glucose level in plasma, “glycemia” is generally expressed as mg of glucose per deciliter of plasma, g of glucose per liter of plasma, or as mmol of glucose per liter of plasma.

[0083] Diabetes and prediabetes can be screened based on plasma glucose criteria, either by the fasting plasma glucose (FPG) test, the A1 C test, or the oral glucose tolerance test (OGTT). T

[0084] As used herein, “hyperglycemia” is a higher than normal fasting blood glucose concentration, usually 126 mg/dL or higher. In some studies hyperglycemic episodes were defined as blood glucose concentrations exceeding 280 mg/dL (15.6 mM). [0085] As used herein, “hypoglycemia” is a lower than normal blood glucose concentration, usually less than 63 mg/dL (3.5 mM). Clinically relevant hypoglycemia is defined as blood glucose concentration below 63 mg/dL or causing patient symptoms such as cognitive impairment, behavioral changes, pallor, diaphoresis hypotonia, flush and weakness that are recognized symptoms of hypoglycemia and that disappear with appropriate caloric intake. Severe hypoglycemia is defined as a hypoglycemic episode that required glucagon injections, glucose infusions, or help by another party. [0086] As used herein, “subjects at risk of developing type-2 diabetes” refers to a subject having prediabetes and/or having one or several of the risk factors such as a family history of diabetes, a history of heart disease or stroke, health conditions such as polycystic ovary syndrome, high blood pressure, abnormal cholesterol levels and/or obesity, age, ethnicity, and lifestyle habits such as physical inactivity and high glucose/sweetener content diet.

[0087] The term “treatment”, “treat”, or “treating” as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0088] The term "amelioration" or "ameliorates" as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.

[0089] The term "symptom" of a disease or disorder is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease.

[0090] A "treatment regimen" as used herein refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication.

[0091] A compound or composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.

[0092] In treating a subject, a therapeutically effective amount may be administered to the subject.

[0093] As used herein, the term “therapeutically effective amount” refers to an amount that is effective for preventing, ameliorating, or treating a disease or disorder (e.g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn's disease, e.g., cancer). [0094] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0095] The compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot I for example, subcutaneously or intramuscularly.

[0096] Compounds and/or compositions comprising compounds disclosed herein may be used in the methods described herein in combination with standard treatment regimes, as would be known to the skilled worker.

[0097] Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[0098] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[0099] EXAMPLES

[00100] Vascular pathology is increased in diabetes due to reactive-oxygen- species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1 to 50 pM) protects tissue-intact and cultured vascular endothelial cells from hyperglycaemia/ROS-induced dysfunction, typified by reduced agonist-stimulated endothelium-dependent vasorelaxation in response to muscarinic or Proteinase- activated-receptor-2 (PAR2) agonists. Metformin not only attenuated hyperglycaemia- induced ROS production in aorta-derived endothelial cell cultures, but also prevented hyperglycaemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues, in accord with our observing a direct metformin-Nr4a1 interaction. Our data indicate a critical role for Nr4a1 in metformin’s endothelial-protective effects, observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-l or complex-ill oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations, requiring the expression of Nr4a1 , protect the vasculature from hyperglycaemia-induced dysfunction in addition to metformin’s action to enhance insulin action in diabetics.

[00101] Of note for the data we will present, are metformin’s beneficial cardiovascular effects, due largely to its ability to protect the vascular endothelium from hyperglycaemia-induced dysfunction (Ding et al., 2019; Kinaan et al., 2015; Mather et al., 2001 ; Triggle and Ding, 2017; Zilov et al., 2019). This dysfunction is attributed to hyperglycaemia-generated reactive oxygen species (ROS) that compromises endothelial function (Brownlee, 2001 ; Shah and Brownlee, 2016). To date, the cellular effects of metformin have been commonly attributed to the inhibition of mitochondrial complex I (El- Mir et al., 2000); (Owen et al., 2000), resulting in the activation of AMP-Kinase (AMPK). AMPK in turn is believed to mediate many of the actions of metformin, including the reduction of cholesterol synthesis (Carling et al., 1989; Carling et al., 1987; Lee et al., 2010) and the enhancement of endothelial nitric oxide synthase (eNOS) to improve vascular vasorelaxant function (Cheng et al., 2014; Driver et al., 2018). However, since high metformin concentrations (>500 pM) are required to inhibit complex I (El-Mir et al., 2000) (Kinaan et al., 2015; Owen et al., 2000), whereas clinical plasma metformin levels range from 1-20 micromolar (Christensen et al., 2011); (Graham et al., 2011); (Scheen, 1996), it is unlikely that metformin-mediated inhibition of complex 1 explains its therapeutic action in diabetics. We therefore focused on metformin concentrations within the therapeutic range (1 to 50pM).

[00102] Such metformin concentrations, matching therapeutic blood levels, have been reported to protect cultured rat endothelial cells from hyperglycaemia-induced oxidative stress (Ouslimani et al., 2005). This action of metformin would be in accord with: 1 . The ability of metformin to improve diabetic vascular endothelial function in vivo (Mather et al., 2001); and 2. Our findings that endothelial function can be protected from hyperglycaemia-induced dysfunction by minimizing endothelial-damaging ROS-meditated oxidative stress (El-Daly et al., 2018). In terms of this likely ‘antioxidant’ mechanism for metformin’s action on the vasculature, we sought to identify another ‘partner’ that might play a role in its action.

[00103] Our attention was drawn to the ability of the ‘orphan nuclear receptor’, NR4A1/Nr4a1/Nur77, to modulate carbohydrate metabolism in a way that reflects metformin’s actions (Mohankumar et al., 2018; Zhang et al., 2018). Significantly, metformin can up-regulate the transcription of NR4A1 ; and metformin’s action in cultured murine thigh-muscle-derived C2C12 myoblasts requires NR4A1 expression (Mohankumar et al., 2018). Since metformin improves vascular endothelial function in type 2 diabetics in vivo (Mather et al., 2001), we hypothesized that, as for our previous findings (El-Daly et al., 2018), metformin would preserve diabetic endothelial function by minimizing hyperglycaemia-induced endothelial oxidative stress. Further, given the requirement of Nr4a1 for metformin’s action in mouse C2C12 cells (Mohankumar et al., 2018), we also hypothesized that metformin’s vascular action would be linked to the expression of NR4A1 .

[00104] To test our hypotheses, we evaluated metformin’s effects in multiple settings: 1 . Vascular organ cultures coupled with a bioassay to assess hyperglycaemia- induced vascular endothelial dysfunction in mouse wild-type and Nr4a1 -null-derived aorta rings (El-Daly et al., 2018; Pulakazhi Venu et al., 2018), 2. Primary aorta-derived wildtype and mouse Nr4a1-null endothelial cell cultures in which ROS production was elevated by hyperglycaemia, 3. Tissue and cell mitochondrial complex-l, complex-ll, complex-ill and complex-IV function (oxygen-consumption rates) for wild-type and Nr4a1- null-derived samples (aorta rings and endothelial cells) cultured at either high (25 mM) vs low (5-10 mM) glucose, and 4. An in-vivo streptozotocin(STZ) diabetes model, using metformin treatment of both wild-type and Nr4a1-null STZ-diabetic mice. Isolated aorta tissues from the metformin-treated and untreated mice were evaluated for hyperglycaemia-impaired endothelial vasodilator function.

[00105] Further, to assess a potential physical link between NR4A1 and metformin, we used an in-silico docking approach, to determine if metformin can potentially interact directly with NR4A1. That approach was paralleled with an avidin ‘pulldown’ approach, using biotinylated metformin to determine if Nr4a1 might interact biochemically with metformin in solution. Our data indicate that indeed, metformin at therapeutic concentrations (1-50 pM) can protect the endothelium from hyperglycaemia-induced ROS-associated dysfunction, but only for Nr4a1 -expresssing tissues. [00106] We found that metformin, at therapeutic concentrations (1-50pM), prevents hyperglycaemia-induced endothelial vasodilator dysfunction by attenuating reactive oxygen-species-induced damage, whereas high metformin (>100pM) impairs vascular function. However, this action of metformin requires the expression of the orphan nuclear receptor, NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin improves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.

[00107] Materials and Methods

[00108] Chemicals and other reagents. The PAR-activating peptide, 2-furoyl- LIGRLO-NH ₂ (2fLI) (purity > 95% validated by HPLC and mass spectral analysis), was synthesized in the University of Calgary, Health Sciences Centre peptide synthesis facility. Phenylephrine HCI, acetylcholine, L-arginine, L-NAME, indomethacin, sodium nitroprusside, and anhydrous glucose were purchased from Millipore Sigma, Burlington, MA (former Sigma-Aldrich); Both high glucose (4.5 g/L, 25 mM) as well as low glucose (1 g/L, 5.5 mM) Dulbecco's Modified Eagle's Medium (DMEM) used for endothelial cell and aorta ring organ cultures were purchased from Thermo Fisher Scientific (Waltham, MA). Metformin-hydrochloride was purchased from Cayman Chemicals, Ann Arbor Ml Cat. No 13118. The NR4A1 agonist, THPN [1-(3,4,5-trihydroxyphenyl)-nonan-1-one, Cat. No. 3063200] and antagonist, TMPA, [ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate Cat. number 492910], cytosporone B [3,5-Dihydroxy-2-(1-oxooctyl)benzeneacetic acid ethyl ester, Cat. No. 2997], the guanylate cyclase inhibitor, ODQ [ [1 H-[1 ,2,4]oxadiazolo- [4, 3-a]quinoxalin-1-one]Acetylcholine, Cat. No. 03636], streptozotocin [N- (Methylnitrosocarbamoyl)-a-D-glucosamine], (ACh) and phenylephrine (PE) were from MilliporeSigma (Oakville ON). Heparin for mouse anticoagulation was purchased from Leo Pharma (Thom hill, ON, Canada). Other basic chemicals were purchased from either MilliporeSigma (Oakville, ON) or WR (Radnor, PA).

[00109] Animals. We used Nr4a1+/+ (designated as Wild-Type (WT)) and Nr4a1- /- (Nr4a1-null) male mice on a C57/BI6 genetic background for our study. The mice were purchased from Jackson laboratory stock number 006187. Mice were used in keeping with the Canadian Council on Animal Care/2010/EU/63-approved procedures. For this study we used only mice of 2-3 months of age, bodyweight 20-25g. Nr4a1+/+ (hereafter termed wild-type/WT) and Nr4a1-Z- mice (also termed Nr4a1-null). Mice were bred in our facility and our colonies are refreshed yearly with mice purchased from the Jackson Laboratory (Bar Harbor, MA, USA). All experiments were performed with littermate controls. The number of animals per group was determined based on our previous publications in accord with the ARRIVE guidelines for reporting animal research (Kilkenny et al., 2010).

[00110] Ten-week-old male mice were used for experiments. Investigators were not blinded to the group allocation. Mice were housed at the Clara Christie Centre for Mouse Genomics at University of Calgary, in microisolator cages, with a standard 12 hour light/dark cycle, ambient temperature 23° C and were provided standard rodent diet (Envigo/Teklad LM-485) and water ad libitum.

[00111] Animal euthanasia with heparinization to obtain aorta tissue for organ culture procedures. Prior to euthanasia, animals were injected with heparin (0.1 mL of 100 U/mL, administered intraperitoneally) and then euthanized 10 minutes later by cervical dislocation, performed under isoflurane anaesthetic. Blood vessels were transcardially perfused with 1 mL of 100U/ml heparin. The descending aorta and abdominal aorta were dissected free of perivascular adipose and connective tissue and placed into ice-cold Krebs solution (115 mM NaCI, 25 mM NaHCO ₃ , 4.7 mM KCI, 1.2 mM NaH ₂ PO ₄ , 10.0 mM dextrose and 2.5 mM CaCI ₂ ), pH 7.4, aerated with 95% O ₂ and 5% CO ₂ .

[00112] Organ culture. Isolated aorta tissue was cut into rings of approximately 1 mm in length. The segments were then randomized in groups of three or more, and incubated in either normoglycaemic (5 or 10 mM) or hyperglycaemic (25mM) glucose (G)-containing media (DMEM High glucose media Cat. No SH30081.01) for 48 hours in the absence or presence of varying concentrations of metformin or cytosporone B as indicated. The euglycaemic 5 or 10 mM glucose-containing media was prepared by diluting the high glucose medium with DMEM containing OmM glucose (XF Assay Medium modified DMEM 0 mM glucose media from Agilent, Santa Clara CA, Cat. No 102365-100). Cultures were maintained in a humidified incubator at 37°C under an atmosphere of 5% CO ₂ in room air for 48h, with or without additions as indicated. After 48 hours, the tissues were recovered from the culture medium and mounted in a wiremyograph for the evaluation of endothelial function (below). In an alternate protocol, cultures that had been maintained in a hyperglycaemic medium (25 mM glucose) for 48 hours in the absence of metformin were then supplemented or not with either 1 or 10 pM metformin and were maintained for a further 12 hours prior to their isolation for the wire myograph vasorelaxation bioassay. [00113] Tissue Bioassays/Wire-myography procedures for evaluating endothelial function. After a period of organ culture (24 to 48 hours), the aorta ring tissues were recovered from the culture medium, transferred to tissue bioassay medium (Krebs solution, pH 7.4) and mounted in a Mulvany-Halpern myograph organ bath (610 multimyograph system coupled to Charts system software, AD instruments, Colorado Springs, CO, USA) for bioassay measurements. Alternatively, rings were used for measurements of mitochondrial function using the ‘Seahorse’ apparatus (see below). All assays to measure endothelium-dependent tissue vasorelaxation were performed at 37°C in Krebs buffer aerated with 5% CO ₂ in room air. A resting tension of 1 g (4.8 mN) was maintained for 1 h prior to and during all experiments. After a 60 min equilibration period, tissue viability was verified by monitoring a contraction in response to the addition of 80 mM KCI to the organ bath. The presence of vasoconstriction confirms the viability of the tissues. Next, the integrity of the endothelium was verified by contracting the tissue with phenylephrine (PE: 2.5 pM) followed by monitoring a relaxation caused by Acetylcholine (ACh, 3pM). A prompt ACh-mediated relaxation response was used to verify that the endothelium was functionally intact. Tissues were washed 3 times after reaching an equilibrium tension and allowed to re-equilibrate in bioassay buffer for 20 min prior to the next addition of agonists to the organ bath. After the responsiveness of the tissues to PE- induced contraction and endotheliumndependent relaxation had been validated, the following experimental protocols were pursued.

[00114] Vasorelaxant responses. Concentration-effect curves for endothelium dependent vasorelaxation induced by ACh and the PAR2D selective agonist, 2DfuroylD LIGRLODNH ₂ (2nfl_l) , were measured upon first contracting the tissues with phenylephrine (PE) (2.5 pM) to a plateau tension, followed by the addition of increasing concentrations of ACh or 2 fLI to the organ bath. Relaxant responses were also evaluated in the presence of inhibitors where tissues were pre-treated with putative endothelium-targeted inhibitors (e.g. L-NAME) for 20 minutes prior to contracting the tissues with PE and then adding an endotheliumndependent vasorelaxant agonist (ACh or 2 DfLI) to the organ bath. Relaxation responses were then calculated as % PE contraction, according to the equation: Relaxation, %PE = [(tension PE alone - tension with PE in the presence of vasodilator)/tension PE alone] x 100.

[00115] Endothelial cell isolation and generation of primary cultures. Mouse aortic endothelial cells were isolated from dissected aorta tissue as described previously (Wang et al., 2016). In brief, isolated aortic segments were placed on Matrigel (Corning Matrigel® Matrix GFR, LDEV-free) with the endothelium side facing the gel and were supplemented with DMEM-5mM glucose containing D-valine CDB-131 -US biological life sciences, MA, USA) media supplemented with human epidermal growth factor (5ng/ml), Vascular Endothelial Growth Factor (2ng/ml), Endothelial Cell Growth Supplement (Bovine hypothalamus extract: BT,203, Alfa Aesar, Cat no: CAAAJ64516-MF) (30pg/ml), hydrocortisone (1 pg/ml), heparin (0.75U/ml), glutamax, penicillin and streptomycin. Upon sprouting, cells were moved to gelatin-coated T-25 flasks by trypsinization and passaged when 80% confluent. During this step, the cells were washed twice with isotonic phosphate-buffered saline, pH7.4-1 mM EDTA for 15minutes and then dissociated for 3 to 5 minutes with 0.25% (w/vol) trypsin (approx. 0.1 mM enzyme) in isotonic phosphate- buffered saline, pH 7.4 containing 1 mM EDTA. The cells were transferred by scraping into new gelatin-coated T-25 flasks and allowed to attach for 20 minutes and then fed with the above endothelial cell growth medium. The medium was then changed to eliminate any contaminating cells and the cells were re-fed. The cells were allowed to grow to confluence, were lifted from the plate by trypsinization, plated and re-fed in gelatin-coated T-25 flasks and were used for assay from passage-3 and on. Next, cells expressing the endothelial cell phenotype were harvested by cell sorting, using expression of the CD102 marker for their identification. Sorted cells, reacting with FITC — labeled anti-CD102 (FITC Rat Anti-Mouse CD102 3C4 (mlC2/4) RUO 557444, BD Biosciences) were expanded and used for the study. Confirmation of the endothelial cell phenotype was verified by monitoring VE-Cadherin expression by immunohistochemistry as described previously (El-Daly et al., 2018).

[00116] Measurement of the oxygen consumption rate (OCR) in Aortic segments and cultured endothelial cells. Aortic segment mitochondrial oxygen consumption rate measurements were done as described previously (El-Daly et al., 2018). In brief, a single aorta tissue segment from a wild-type or Nr4a1-null mouse provided approximately 4 to 5 aorta tissue fragments per mouse. Aortic tissues were cut open and placed into the 24-well multiwell Seahorse islet plates (Agilent Santa Clara CA, Cat. No. 101122-100) with the endothelium side facing up, enclosed by the capture screen. This procedure enabled the tissues or endothelial cell monolayers to be held in place during the assay. The tissues or cell monolayers were first incubated in DMEM (Seahorse Bioscience North Billerica MA) containing either 25 mM or 10 mM glucose in the presence or absence of 10 or 500pM metformin for 24 hours in a humidified incubator under an atmosphere of 5% CO ₂ in air at 37 °C. The oxygen consumption rate (OCR) measurements were then performed using a Seahorse analyzer (Agilent XFe24 analyzer).

[00117] To normalize the tissue oxygen consumption rate data (below) to the protein content of the vascular sections, each sample was harvested immediately after the respirometry measurements were done and solubilized, to determine protein content. In brief, aorta tissue samples were put in protein lysis buffer containing protease inhibitors (PhosStop and complete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail: MilliporeSigma). Stainless steel beads were added and the samples were blended (Bullet Blender nextadvance.com, Troy, NY) for 15 min. Supernatant aliquots (10 pl) were added to 300 pl of precision red solution (Cytoskeleton. Inc., Denver, CO) and incubated for 5 to 10 min. The protein concentration was then calculated from the resulting O.D. measured at 600 nm according to the manufacturer's formula

(O.D. x 12.5 = mg/ml). The oxygen consumption rate data were normalized to the protein content of the tissue samples using the Seahorse wave software. In a similar way, endothelial cell monolayers obtained from both wild-type and Nr4a1-null mice were grown in a T25 flask to 80% confluency. At that point, both wild-type and Nr4a1-null cells were harvested by trypsinization, counted and seeded in endothelial growth medium-5 mM glucose as described above at 50,000 cells/well into XF-cell culture microplates (Agilent Technologies Mississauga ON, Cat. No. 102340-100). Allowing overnight for attachment, the cells were then switched to serum-free DMEM-25 mM glucose without or with supplementation with either 10 pM or 500 pM metformin and cultured for a further 24 h at 37 C. The cells were then taken from the incubator and studied for their oxygen consumption rates (OCR) using the Seahorse analyser. The data were analyzed using the mito-stress assay report generator supplied by Agilent technologies (Santa Clara CA). The oxygen consumption rate data (OCRs), obtained from 5 replicate monolayer cultures were normalized to protein levels analyzed after the assay as previously described (El- Daly et al., 2018).

[00118] Measurement of the oxygen consumption rate in permeabilized cells to study individual respiratory chain complex-mediated respiration. The activity of individual respiratory chain complexes was evaluated in permeabilized cells as described previously (Sumi et al., 2018). In brief, wild-type endothelial cell monolayers, prepared as described in the previous paragraphs were incubated for 24 h in 25mM Glucose-DMEM, treated or not with metformin (10 or 500 pM). Cells were then washed with Mitochondrial Assay Solution buffer (220 mM mannitol, 70 mM sucrose, 10 mM KH ₂ PO ₄ , 5 mM MgCI ₂ , 2 mM HEPES, 1 mM EGTA, 0.2% fatty acid- free bovine albumin, adjusted to pH 7.2 with KOH), and the medium was replaced with Mitochondrial assay solution (MAS) buffer supplemented with 10 mM pyruvate, 1 mM malate, 4 mM ADP, and 1 nM plasma membrane permeabilizer™. The cells were then loaded into the XFe24 Seahorse analyzer to measure respiration rates using cycles of 30 seconds mixing/30 seconds waiting/4 min measurement.

[00119] Protocol A: After the measurement of pyruvate-driven respiration, rotenone (final concentration 2 pM) was injected through port A to halt the complex I- mediated respiratory activity. Next, succinate (10 mM) was injected through port B to donate electrons at complex II, bypassing complex I inhibition.

[00120] The addition of antimycin A (2 pM) via port C inhibited complex III, and

N,N,N,N- tetramethyl-p-phenylenediamine (TMPD 0.1 mM), combined with ascorbate (10 mM), was subsequently injected through port D to measure complex IV activity. This procedure is shown in the schema illustrated in figure 9.

[00121] Protocol B: As an alternative approach, cells were initially supplemented with pyruvate to measure complex I activity. After injection of rotenone, duroquinol was injected to stimulate complex Ill-mediated respiration. This procedure is shown in the scheme illustrated in figure 9.

[00122] Measurement of reactive oxygen species (ROS) in cultured endothelial cells. Wild-type and Nr4a1 -null-derived endothelial cell monolayers generated as aorta-derived primary cell cultures as described in the above methods, were grown in DMEM-5 mM glucose and were switched to DMEM-25mM glucose in the presence or absence of metformin. Monolayers were incubated at 37° C for 1 hour and then stained for 30 minutes to detect reactive oxygen species using CellROX green (Thermo Fisher Scientific, Cat. No. C10444: reactive oxygen species = green colour). The cells were then fixed with 10% formalin for 15 minutes and washed with isotonic phosphate-buffered saline pH 7.4 (PBS), 3 times for 5minutes and permeabilized using

O.5% triton x-100 for 10 minutes. The fixed cells were washed to remove any triton x-100 and stained for 1 hour using hoechst 33342 to identify the nuclei. The samples were then washed with phosphate-buffered isotonic saline, pH 7.4, 3 times for 5minutes and visualized under florescence microscopy with excitation and emission at 485/520nm respectively. Images were taken using a 20x objective. Fluorescence intensity was quantified using imaged. Corrected cell fluorescence intensity (CTCF) flouresence was calculated according to the formula: cell fluorescence (CTCF) = Integrated Density - (Area of selected cell x Mean fluorescence of background readings). To compare the levels of ROS observed in the wild-type versus Nr4a1-null cells, morphometric analysis was done by integrating the mean fluorescence intensity observed in three independent equivalent image fields for each condition. Data were expressed as the mean fluorescence yield (arbitrary units) per monolayer field. In separate experiments done to evaluate the impact of hyperglycaemia on endothelial cells of a different tissue source, mouse microvascular endothelial cells (MMECs: ATTC, Manassas VA, Cat. No. CRL- 2460) were used. To assess the ability of metformin to mitigate ROS production, the MMECs were seeded into MatTek glass bottom dishes at 50,000 cells/plate. Cells were treated with mouse-derived physiological concentrations of glucose (11 mM) and high glucose (40mM) in Dulbecco's Modified Eagle Medium (DMEM, Gibco/Thermo Fisher Scientific) with and without 50pM metformin (Sigma) for 24 hours, followed by staining with dihydroethidium (DHE, Invitrogen/Thermo Fisher Scientific). Imaging was done using Carl Zeiss LSM 880 confocal microscope, and 5 to7 random images from equivalent microscopic field areas were obtained from each sample. Total fluorescence intensity was measured at excitation/emission wavelengths of 518/606nm, and was quantified using imaged as mean gray values. Values were normalized to normal glucose controls.

[00123] Streptozotocin (STZ) in vivo model of diabetes for wild-type and Nr4a1-null mice. Uncontrolled diabetes was induced as outlined in Figure 5A, with five consecutive daily subcutaneous doses of freshly prepared streptozotocin (STZ: 50 mg/kg), as described previously (Furman, 2015) following Basic protocol 1 for mice. Blood glucose levels were measured in tail clip blood samples one week after the first STZ injection using a One touch ultra -Test strips Code 25 glucometer. In brief, after STZ treatment of wild-type and Nr4a1-null animals, that resulted in sustained hyperglycaemia for 12 to 13 weeks (the same for both wild-type and Nr4a1-null mice), the mice were divided into two groups: Group 1 : STZ-only injected mice and Group 2: STZ- injected mice with a daily oral gavage administration of metformin (65mg/kg/day) for 2- weeks. This dosing is predicted to yield blood metformin concentrations of about 30 pM (Martin-Montalvo et al., 2013). Post 2-weeks of gavage, blood glucose levels were measured in all mice prior to euthanasia and were found to be equivalent for both wildtype and Nr4a1-null mice. Aortic segments were isolated from the two groups of wild-type and Nr4a1-null mice (STZ alone vs STZ animals also treated with metformin) and were used for vasorelaxant bioassays as outlined in Methods to evaluate vascular function as described above for muscarinic (ACh) and PAR2 (2fl_l)-mediated vasorelaxation. All experiments adhered to ARRIVE Guidelines (Kilkenny et al., 2010)

[00124] Transmission Electron Microscopy imaging of Aorta and endothelial cells. Wild-type mouse aortic endothelial cells were grown to confluency on a gelatin coated glass coverslip in growth medium as described above and switched to DMEM containing 25 mM glucose and incubated for a further 24 hours in the absence or presence of either 10 or 500 pM metformin. Post-incubation, cells were directly fixed with 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4) for a minimum of 2hours. Cells incubated with 25mM glucose without or with metformin were treated with same fixative. The specimens were washed in 0.1 M sodium cacodylate buffer at pH 7.4 before being post-fixed in 2% osmium tetroxide. The tissue was then dehydrated with graded acetone and then infiltrated with several changes of graded Epon: Acetone and then embedded in Epon resin. The sections were cut at 70 nm and stained with a 2% uranyl acetate and counterstained with a 4% lead citrate solution. Transmission Electron Microscope images were acquired on a Hitachi model H-7650 from 4000x -20000x magnification. The acquired images were then processed for mitochondria number, shape and circularity using Imaged. Morphometric analysis of the distinct mitochondrial morphologies (‘spindle’ vs ‘circular’) was manually counted using Imaged. In equivalent image areas, the proportion of circular to spindle-shaped mitochondria was taken as an index of increased metabolism due to hyperglycaemia exposure (Hackenbrock, 1966). [00125] Western blot detection of phospho-AMPKinase and phospho-eNOS. Wild-type endothelial cells were grown to 80% confluency endothelial cell growth medium (described above) in gelatin-coated 24 well multiple-well plates of 15.6mm diameter/well. Cells were then switched to either 5 mM or 25 mM glucose-DMEM for 24 hours.

Monolayers were subsequently treated or not for 1 hour at 37 °C with metformin (10pM or 500pM) or with the AMPKinase activator, AICAR (as a positive control: 500pM, added to DMEM-5 mM glucose wells: MilliporeSigma - Cat no A9978). After 1 h, the cells were then lysed and homogenized using ice-cold phosphoprotein lysis buffer containing NP40: 20 mM Tris-HCI, pH 7.5, 100 mM NaCI, buffer: MgCI ₂ , 1 mM EDTA, 1 mM EGTA 0.5% NP40, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na ₃ VO ₄ , 25 mM NaF and 1 mM dithiothreitol. The lysis buffer (1 mL) was also supplemented with 10 pl of Proteinase Inhibitor Cocktail set III (MilliporeSigma cat. no. 539134), containing 1 mg/ml eupeptin, 1 mg/ml aprottinin and 1 mM phenylmethylsulfonhl fluooride. Western blot analysis was done essentially as previously described (Mihara et al., 2013). Equivalent amounts of protein from each cell monolayer extract were heat-denatured at 92 °C for 6 min in denaturing Laemmli buffer and resolved on 4-20% gradient Novex Tris- Glycine gels (Thermo Fisher scientific) run at 120 V for 2 hours. Transfer of proteins onto PVDF membrane was done using a semi-dry method. The resolved proteins were transferred to PVDF membrane, blocked for 1 hour at room temperature in PBST buffer [Phosphate-buffered isotonic saline, pH 7.4, supplemented with 0.1% (v/v) Tween-20] containing 0.1% ECL Advance Blocking Agent (GE Healthcare, Waukesha, Wl). Western blot detection of phospho-eNOS and phospho-AMPK was performed using rabbit anti- phospho eNOS (Thr-495) and anti-phospho-AMPK antibodies (Cell Signalling Technology, Danvers MA, Cat. Nos. 9574 and 2535 respecitvely). Beta-actin was measured for loading control (Cell Signalling Technology, Danvers MA, Cat. no. 3700). After washing the membrane with PBST, the peroxidase activity was detected with the chemi-luminescence reagent ECL-Advance (GE Healthcare, Waukesha, Wl) using a Chemdoc imager (Biorad, Mississauga ON). Band intensities representing eNOS or p- AMPK were were quantified using Image J software, and normalized to the signal generated in the same lane on the same gel by re-reprobing for beta-actin (Cell Signaling Technology Cat. No. 3700. The measurements were done for a minimum of 3-replicates. [00126] Western blot detection of eNOS in aorta tissues. Western blot analysis, done essentially as described in the above section, was used to determine the abundance of vascular eNOS in wild-type and Nr4a1-null aortic tissues. The aortic tissues were excised and cleaned in Kreb’s buffer as described above. The tissues were immediately weighed and snap frozen in liquid nitrogen. Following this procedure, tissues were stored at -80 °C. The stored tissues were put into ice-cold NP40-containing lysis buffer (composition as above) and blended using stainless steel beads in a Bullet Blender (Nextadvance.com Troy NY) in NP40-proteinase-inhibitor-supplemented lysis buffer (above). Protein concentration was determined using precision red reagent (Cytoskeleton Inc Denver CO) as described previously (El-Daly et al., 2018). Equivalent amounts of protein from each tissue extract were analysed. Detection of total eNOS was performed using anti-rabbit eNOS antibody (Cell Signalling Technology, cat. no. 32027). The betaactin signal for each sample was measured as a gel-loading control (Cell Signalling Technology, Cat. no. 3700S). Band intensities representing eNOS were quantified using the Image J quantification (Rueden et al., 2017; http://rsbweb.nih.gov.ezproxy.lib.ucalgary.ca/ij/). eNOS levels were normalized for differences in protein loading by expressing the densitometry data in arbitrary units relative to the corresponding total protein and beta-actin band detected in the same sample. Data were obtained from a minimum of three replicate tissue samples.

[00127] Synthesis of Biotinylated metformin. The synthesis of biotin-tagged decyl amine (1), free of metformin, to serve as a ‘control’ was completed by the reaction of 1-decylamine (Sigma-Aldrich, CAS 2016-57-1) with biotin-pentafluorophenyl ester (prepared according to procedure reported by (Papatzimas et al., 2019), in dimethylformamide at room temerature for 1 hour. Compound 1 was purified by trituration with diethyl ether. S-Methyl-guanylisothiouronium iodide (2) was prepared according to the published procedure by (Wilkinson et al., 2011) from amidinothiourea (Combi-Blocks, CAS 2114-02-5) and methyl iodide (Sigma-Aldrich, CAS 74-88-4). Reaction of compound 2 with 1-decylamine in dimethylformamide at 60°C for 10 h produced crude metformindecylamine (3) that was purified using a Biotage Isolera Prime 30 chromatography instrument using a Biotage SNAP KP-NH column (MeOH/CHCh). Tetradecanedioic acid (Sigma-Aldrich CAS 821-38-5) was reacted with oxalyl chloride to produce the diacyl chloride intermediate, that was then reacted with aquoues NH ₃ to prepare the diamide. The diamide was then reduced with LiAIH ₄ to produce 1 ,14-diaminotetradecane. This diamine was reacted with Biotin-Pfp to prepare the monofunctionalized derivative. The intermediate was then reacted with compound 2 in dimethyl formamide at 60°C for 12 hours, and purified using a Biotage Isolera Prime 30 chromatography instrument using a Biotage NAP KP-NH column (MeOH/CHCh) to furnish the biotin-tagged metformin (4).

[00128] Using biotinyl metformin to assess metformin-Nr4a1 interactions [00129] Expression of C-terminally myc- and monomeric red-fluorescent protein (mRFP)-tagged mouse Nr4a1 in HEK cells. Mouse Nr4a1 with a C-terminal myc or mRFP tag was expressed in a human embryonic HEK293 cell background to serve as a source of Nr4a1 protein to test its interaction with biotinylated metformin. The coding cDNA sequence of mouse Nr4a1 was cloned into the pCDN3.1+ plasmid vector under the control of a CMV promoter. The coding sequence of Nr4a1 cDNA (double stranded DNA fragment of NM_010444.2, obtained from Integrated DNA Technologies, Inc., Coralville, Iowa) was cloned into the pCDNA 3.1+ EcoRI vector Xhol site with a linker. The C-terminal stop codon was replaced with a Xhol cleavage site and then fused to either a myc epitope peptide or a monomeric red fluorescent protein (mRFP) tag sequence. For Nr4a1 protein expression, HEK-background LX293 cells (Takara Bio USA, Mountain View, CA) were grown in 10% foetal calf serum-supplemented Dulbecco’s Modified Minimal essential medium (Thermo Fisher. Scientific) to 20% confluence in T75 flasks. When at 20% confluency, cells were transfected with pCDNA3.1-mNR4a1-mRFP or pCDNA3.1-mNr4a1-myc using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Cell growth medium was replaced the following day and the monolayer was cultured further overnight. Cells were washed and lifted with isotonic phosphate- buffered saline pH 7.4, containing 1 mM EDTA. Cells pellets were collected in 5 ml roundbottom polystyrene tubes by centrifugation at 200g for 3.5 minutes, resuspended in 500 |il of binding buffer (50 mM Hepes/KOH, pH 7.4, 78 mM KCI, 4 mM MgCI ₂ , 2 mM EGTA, 0.2 mM CaCI ₂ , 1 mM dithiothreitol and protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Sigma-Aldrich, Canada) that was prepared at 2x concentration for cell freezing. The cell suspension was snap frozen with liquid nitrogen, and stored at -80 °C until use.

[00130] Preparation of soluble Nr4a1 for use in ‘pulldown’ experiments. Cells, were thawed, cooled in ice-water and homogenized in 2x-concentrated binding buffer (above) to dissociate Nr4a1 from DNA using a polytron (Kinematica, PT10/35 with 5 mm saw tooth generator) at maximum speed, controller value at 9, for 30 seconds. The homogenate was clarified by centrifuge at 20000 g for 5 min. The supernatant was clarified further at 10000g for 10 min at 4 °C, and was then diluted with an equal volume of ice-cold deionized water to yield 1x-binding buffer.

[00131] Harvesting the biotinylated metformin-Nr4a1 complex. Duplicate samples of the lysate (0.25 mL each) in 1 .5 ml microfuge tubes were supplemented with the biotinylated metformin construct (4 ng in 2 gl DMSO) or with the same amounts of either the metformin-free biotinylated C10 linker or a biotin-free metformin-C10 linker construct. Binding was allowed to proceed on ice for 15 minutes. Neutravidin crosslinked magnetic beads (Sera-Mag SpeedBeads Neutravidin Magnetic Beads, GE Healthcare Life Sciences, Marlborough, MA) were used to harvest the biotinylated metformin and biotinylated linker from the cell extract. A 4 |il suspension, of beads per reaction was transferred to 100 |il of Hanks Buffered isotonic Saline Solution, pH7.4, containing 0.1 % (w/v) fatty acid-free Bovine serum albumin and washed 3 times with same buffer. The washed beads were resuspended in 20 |il of binding buffer for each reaction and were added directly to the cell lysates containing either biotinylated metformin or the control constructs (biotinylated metformin-free C10 linker; biotin-free metformin-C10 linker). Beads were sedimented magnetically using a magnetic particle concentrator (Dynal MPC, Oslo Norway) and Neutravidin-bead-bound protein was eluted from its metformin complex by incubation of the sedimented beads for 30 min. at 30 °C in 10 |il of binding buffer containing 100 pM metformin. The high metformin concentration was added to prevent a re-association of released proteins with the resin-bound biotinlated metformin. Eluted Nr4a1 was detected bywestern blot analysis of the bead- eluted solution as previously described (Mihara et al., 2013) with detection using an Nr4a1 antibody (mouse anti-nur77, cat. No. 554088, BD pharmigen/BD biosciences San Jose, CA).

[00132] In silico Molecular docking. The atomic coordinates of NR4A1 were downloaded from Protein Data Bank (PDB) (PDB ID: 2QW4). The ligand used for docking was metformin obtained from PubChem (CID-4091 ; https://pubchem.ncbi. nlm.nih.gov/compound/4091#section=3D-Conformer). Both the protein and the ligand were prepared using Autodock tools (Morris et al., 2009) and were converted to pdbqt files for docking. The ligand was docked into the binding pocket near N-terminal sequence of the ligand binding domain of Nr4A1that is situated towards the C-terminus of the intact protein (Lanig et al., 2015), by defining grid box dimensions with 1 A spacing and size of 52 X 52 X 58 pointing in X, Y, and Z directions using Autodock Vina (Trott and Olson, 2010). Default parameters were used during the docking simulations.

[00133] Molecular Dynamics Simulation

[00134] Molecular dynamic simulation was performed on the metformin-NR4A1 complex using Gromacs 2019.4 (Abraham et al., 2015) at 300 K temperature using Charmm36 force field (Huang et al., 2017). The ligand file from the complex of metformin- NR4A1was extracted using gmx grep module. The topology and force-field parameters of the ligand were generated using CGenFF server (Vanommeslaeghe et al., 2010). The complex was solvated with water molecules in a dodecahedron box with an edge margin of 2.0 A from each side. Simple point charge (spc216) water model was used to solvate the complex. Ions were added to replace the solvent molecules with monoatomic ions using gmx genion module. Energy minimization was performed using steepest-descent algorithm followed by equilibration and MD run. The resultant trajectories and calculated binding affinities (Kcal/mol) were thoroughly analyzed using different GROMACS modules including gmx energy, gmx trjconv, gmx rms, gmx hbond, gmx analyze, and gmx sasa. The three dimensional models were visualized and prepared using Pymol (Schrodinger, LLC.) and VMD (Humphrey et al., 1996) software.

[00135] Statistical analyses. Data are presented as means ± SEM. For multiple group comparisons, statistical significance was determined by one-way ANOVA using a Newman-Keuls post-hoc test to assess differences between groups. For two-group comparisons, an unpaired, two-tailed, Student's t test was employed. A p value < 0.05 was considered statistically significant.

[00136] Results

[00137] 1. metformin at low (1 pM: Fig. 1B, 1D) but not high concentrations

(500 pM: Fig. 1C, 1E) protects the endothelium from hyperglycaemia-induced endothelial dysfunction, maintaining acetylcholine (muscarinic) and PAR2 (2fl_l)- induced vasorelaxation.

[00138] When aorta rings were placed in organ culture for 48 hours under hyperglycaemic conditions (25 mM versus euglycaemic 10 mM glucose : schema shown in Fig. 1A), the vasorelaxant actions of both acetylcholine (Fig. 1 B) and the PAR2 agonist, 2-fLI (Fig. 1 D), were markedly compromised, compared with their actions on aorta rings cultured using euglycaemic glucose concentrations: compare open diamonds versus open squares, Figs 1 B and 1 D). However, the concurrent presence of 1 pM metformin in the 25 mM glucose-containing culture medium preserved both the muscarinic and PAR2- mediated vasorelaxant responses, shifting the concentration-response curves (solid squares Figs. 1 B & 1 D) towards those for tissues cultured at euglycaemic glucose concentrations (10 mM: open diamonds, Figs 1B & 1 D). The presence of 1 pM metformin in the organ cultures maintained at 10 mM glucose did not change the concentrationresponse curves (solid diamonds, Figs. 1 B & 1 D) compared to metformin-untreated tissues (open diamonds, Figs. 1 B & 1 D). Surprisingly, a high concentration of metformin (500 pM: Solid squares, Figs. 1C & 1 E) failed to maintain the vasorelaxant action of acetylcholine (Fig. 1C, solid squares) and 2fl_l (Fig. 1 E, solid squares) for tissues cultured under hypergycaemic conditions (25 mM glucose). All of the vasorelaxant responses in the tissues cultured under all conditions were abolished in the presence of 1 mM L- NAME, indicating the dependence on endothelial eNOS. In sum, our data showed that metformin in the concentration range from 1 to 100 pM was able to preserve endothelial function for tissues maintained at high glucose concentrations, whereas higher concentrations of metformin (> 250 - 500 pM) did not (Figs 1 B to 1 E and data not shown).

[00139] 2. An Nr4a1 antagonist reverses the ability of metformin to attenuate hyperglycaemia-induced vascular endothelial dysfunction as assessed by a tissue bioassay.

[00140] To assess the possible link between metformin action and the orphan nuclear receptor, Nr4a1 , an Nr4a1 antagonist, TMPA, was used (Zhan et al., 2012). As shown in Figss 2B and 2C (solid squares, B; solid diamonds, C), the presence of the Nr4a1 antagonist, TMPA (50 pM), in the hyperglycaemic culture medium prevented the ability of metformin to preserve the vasorelaxant response to muscarinic (2B) and PAR2 (2C) activation. The antagonist action thus pointed to a direct link between the actions of metformin and the function of Nr4a1 in the tissues.

[00141] 3. Metformin does not prevent hyperglycaemia-induced endothelial dysfunction in organ cultures of aorta tissue from Nr4a1-null mice.

[00142] To explore further, the link between Nr4a1 and the ability of metformin to preserve vascular endothelial function in the setting of hyperglycaemia, we used aorta ring preparations obtained from Nr4a1-null mice that were maintained in hyperglycaemic organ cultures with or without metformin (Fig. 3). Both acetylcholine and 2fl_l were able to cause vasorelaxation in the Nr4a1-null tissues, but the sensitivity of the Nr4a1-null tissues was slightly lower (solid squares versus open squares, Figs 3A & 3B) compared to the tissues from wild-type animals (open squares, Figs 3A & 3B). This lower sensitivity to the vasorelaxant agonists may relate to the slightly lower abundance of eNOS observed in the Nr4a1-null tissues, compared with those from wild-type animals (Figs 3C & 3D). Thus, the Nr4a1 -null-derived tissues were fully functional in terms of their endotheliumdependent vasorelaxant responses which were at a level comparable to the function of the wild-type tissues, although slightly less sensitive to endothelial agonists. Further, all vasorelaxant responses in the Nr4a1-null tissues were blocked by L-NAME combined with the guanylyl cyclase inhibitor, ODQ (data not shown).

[00143] The impact of hyperglycaemia on the endothelium-dependent vasorelaxant responses in the Nr4a1-null tissues maintained in organ cultures was next evaluated. As shown in Fig. 4, elevated glucose concentrations (25 mM versus 10 mM) were able to diminish very modestly the vasorelaxant responses to acetylcholine and 2fl_l (compare open diamonds vs open squares, Figs. 4B & 4D) in the Nr4a1-null tissues. However, in the presence of metformin (1 or 25 pM) the hyperglycaemic Nr4a1-null cultures failed to preserve the endothelial vasorelaxant function of either acetylcholine or 2fl_l (Figures 4B to 4E, solid squares) as it did for the tissues from the wild-type mice (Fig. 1 B & 1 D). These data supported the conclusion obtained using the Nr4a1 antagonist, TMPA, that Nr4a1 itself plays a key role for the ability of metformin to preserve endothelial vasorelaxant function in the setting of hyperglycaemia.

[00144] 4. Metformin administered in vivo preserves vascular endothelial vasorelaxant function in aorta tissues from streptozotocin-induced hyperglycaemic wild-type but not in tissues from hyperglycaemic Nr4a1-null mice. [00145] In view of the ability of metformin to preserve endothelial function in the hyperglycaemic murine vascular in vitro organ culture system for wild-type animals (Fig.

1) but not for Nr4a1 -null mice (Fig. 4), we sought to determine if vascular endothelial function could be preserved in the setting of streptozotocin-induced diabetic hyperglycaemia in vivo, as outlined by the scheme in Fig. 5A. The ambient blood glucose concentrations in the streptozotocin-treated wild-type and Nra1-null animals the same and were greater than 30 mM, compared with about 10 mM in the control STZ-untreated mice. In accord with data obtained previously using a mouse streptozotocin model (Furman, 2015), the endothelium-dependent vasorelaxant actions monitored for aorta rings isolated from these STZ-diabetic mice, measured in vitro for both acetylcholine and 2-fLI, were both markedly diminished, compared with the responses of tissues obtained from euglycaemic mice (Compare open diamonds in Figs 1 B and 1 D with open circles in Figs 5B and 5D). However, the vasorelaxant actions of both acetylcholine and 2-fLI were markedly preserved in aorta tissues obtained from the metformin-treated animals (Figs 5B and 5D: compare open versus solid circles). In contrast, the vasorelaxant actions of both acetylcholine and 2-fLI were blunted for aorta rings obtained from the Nr4a1-null animals whether or not the animals were treated with metformin (Figs 5C and 5E: compare open squares with solid squares). Thus, metformin administered in vivo was able to preserve vascular endothelial vasorelaxant function in the setting of streptozotocin-induced hyperglycaemia, but only for mice expressing Nr4a1.

[00146] 5. Metformin’s ability to prevent hyperglycaemia-induced ROS production in cultured aorta-derived endothelial cells is blocked by an NR4A1 antagonist.

[00147] In keeping with our published work (El-Daly et al., 2018), it is generally accepted that hyperglycaemia causes endothelial dysfunction by the generation of reactive oxygen species (ROS: Brownlee, 2001 ; Shah and Brownlee, 2016). Further, it has been reported that metformin can decrease the intracellular production of ROS in cultured bovine aorta-derived endothelial cells (Ouslimani et al., 2005). Therefore, using cultures of mouse-aorta-derived endothelial cells, as in our previous work (El-Daly et al., 2018), the production of reactive oxygen species was monitored in cells exposed to 25 mM versus 5 mM glucose (Fig. 6). In accord with the data obtained previously using bovine aorta-derived endothelial cells (Ouslimani et al., 2005), high glucose concentrations led to a marked increase in ROS (Fig. 6, GFP signal, top panels, B and C), that was not observed for cells cultured at 5 mM glucose (Fig. 6, second row of panels from top, E and F). The increase in ROS observed in the presence of 25 mM glucose was suppressed by cells cultured in the concurrent presence of 50 pM metformin (Fig. 6, third row of panels from top, H and I). However the Nr4a1 antagonist, TMPA, was able to reverse the ability of metformin to minimize the production of ROS in the presence of 25 mM glucose (Fig.6, fourth row of panels, K and L). For reasons we were not able to determine, the elevation of ROS, as indicated by the CellROX green reagent, was quite variable, frequently yielding lower signals than those shown for the representative experiment illustrated in Figure 6B/C. Notwithstanding, the presence of metformin uniformly reduced the hyperglycaemia-induced increase in ROS. Further, we assessed the ability of metformin to prevent the hyperglycaemia-induced increase in ROS in mouse microvascular endothelial cells (MMECs), as opposed to those derived from aorta. Metformin was also able to reduce the abudance of ROS in MMECs exposed to hyperglycaemia, as indicated by the reduction in the dihydroethidium (DHE) reactivity.

[00148] 6. Metformin does not attenuate hyperglycaemia-induced ROS in cultured endothelial cells from Nr4a1-null mice.

[00149] In contrast with the ability of metformin to attenuate the production of ROS in primary mouse aortic endothelial cells from wild-type mice exposed to 25 mM glucose (Fig. 6, compare panel 6B with panel 6H; and in Fig. 7, compare panel 7A with panels 7B and 7C), metformin was not able to eliminate ROS in the Nr4a1 -null-derived endothelial cells exposed to 25 mM glucose (Fig. 7, compare panel D with panels E and F). Even at a low glucose concentration (5 mM) the Nr4a1 -derived endothelial cells showed appreciable ROS activity compared to the wild-type cells (compare panels A and D, Fig. 7). Quantified cell fluorescence measurements showed that although 5pM metformin was able to reduce the increase in the CellROX signal in the wild-type endothelial cells, it was not able to diminish the fluorescence signal in the Nr4a1-null cells (Histograms, Fig. 7G). Taken together, the data indicated that the action of Nr4a1 is linked not only to the ability of metformin to preserve vascular endothelial vasorelaxant function in intact tissues exposed to hyperglycaemia (above), but also to the ability of metformin to attenuate the production of endothelial ROS caused by elevated concentrations of glucose in endothelial cell cultures.

[00150] 7. Metformin-preservation of hyperglycaemia-impaired mitochondrial oxygen consumption rate in aorta ring organ cultures and cultured aorta-derived endothelial cells is Nr4a1 -dependent. [00151] Although the precise mechanism whereby metformin enhances insulin action is uncertain, it has been suggested that metformin’s antidiabetic effects may relate to its ability to compromise mitochondrial complex I function (Discussed by Kinaan et al., 2015; Triggle and Ding, 2017). Of note, it has been pointed out that the impact on mitochondrial complex I function observed in vitro is found only at metformin concentrations more than an order of magnitude higher (e.g. 500pM) than those plasma levels (about 20pM) observed for individuals treated with metformin (Kinaan et al., 2015; Triggle and Ding, 2017). We therefore evaluated the impact of hyperglycaemic conditions (25 mM glucose) on the oxygen consumption rate for intact aorta ring tissues (Figure 8A) and cultured endothelial cells (Figure 8C) in the absence (solid blue circles, Figures 8A/8C) and presence (solid red squares, Figures 8A/8C) of metformin at a concentration in the range that we found to protect the endothelium from hyperglycaemia-induced dysfunction, and to prevent hyperglycaemia-induced endothelial ROS production (between 1 to 50 pM metformin: see Figures 1 and 6).

[00152] As shown in Figure 8A, in isolated aortic rings maintained in 25 mM glucose, metformin (10 pM: solid red squares) substantially increased the mitochondrial oxygen consumption rate from the much lower level caused by hyperglycaemia in the absence of metformin (Figure 8A: compare solid blue with solid red symbols). A comparable effect of metformin was observed in aorta ring-derived cultured endothelial cells exposed to 25 mM glucose (Figure 8C: compare solid blue versus solid red symbols). Thus, a concentration of metformin that preserves endothelial function both in intact tissues and in endothelial cultures improves rather than compromises mitochondrial function. Nonetheless, this concentration of metformin failed to improve the oxygen consumption rate either in hyperglycaemia-maintained aorta ring segments or for hyperglycaemia-exposed endothelial cell cultures obtained from the Nr4a1-null mice (Figure 8B, compare blue and red symbols; Figure 8C, compare blue and solid magenta squares). Thus, Nr4a1 was required to enable metformin to rescue the mitochondrial oxygen consumption rate that was diminished by hyperglycaemia.

[00153] We also found that in wild-type tissues maintained under hyperglycaemic conditions (25 mM glucose), that the addition of 10pM metformin improved the basal oxygen consumption rate, indicating metformin at this concentration didn’t affect mitochondrial Complex I to reduce the oxygen consumption rate. Also, the cellular respiration parameters indicating the ability to respond to stress, such as spare respiratory capacity, was improved. Moreover, the mitochondrial structural integrity as indicated by proton leak and the total ATP production remains unaffected . However, in Nr4a1-null tissues, glucose affected the mitochondrial respiration at the level of basal respiration. The Nr4a1-null tissues could not withstand the 25mM glucose incubation and metformin had a modest impact on the basal oxygen consumption rate. Nonetheless, we found no difference in ATP production or spare respiratory capacity. These data indicate that Nr4a1 is a critical factor for the function of metformin.

[00154] 8. Endothelial mitochondrial complex I and III function are compromised by high (500 pM) but not low (10 pM) concentrations of metformin; and complex II and IV functions impeded by hyperglycaemia are improved by low metformin concentrations.

[00155] To verify the differences in the impact of high (500 pM) versus low (10 pM) metformin concentrations on endothelial mitochondrial function, the cultured endothelial cell oxygen consumption rates were measured for cells exposed to 25 mM glucose in the absence or presence of either 10 pM or 500 pM metformin (Figures 9A to 9F) and cellular oxygen consumption rates were monitored (Figure 9). The protocols shown in the figure evaluated the effects of metformin on mitochondrial complex functions I to IV in samples exposed to 25 mM glucose. Whereas 10 pM metformin had no effect on Complexes I and

III (Figures 9C, solid red squares vs solid blue squares and middle histogram, Figure 9E), it appeared to enhance the hyperglycaemia-compromised oxygen consumption rate for Complexes II and IV (Figures 9F and 9H). This increase in complex II and IV enhanced mitochondrial spare respiratory capacity, in turn promotes cell survival. In contrast, 500 pM metformin, as expected, reduced the basal oxygen consumption rates for Complexes I, III and IV (Figures 9C, magenta triangles and 9 E, right-hand histogram). The data showed that, in agreement with the action of high concentrations of metformin on complex I function already reported in the literature, 500 pM metformin indeed reduced the Complex I oxygen consumption rate (right-hand histogram, Figure 9E). The Complex

IV activity reduced by 500 pM metformin points to a reduced conversion of oxygen to water, thereby making cells susceptible to hypoxia (Figure 9H). However, no effect of 10 pM metformin on Complex I was observed (Figure 9E, left-hand histogram); and the impacts of this low concentration and 500 pM metformin on the functions of complexes II to IV were variable, causing an inhibition, an enhancement (complexes II and IV: Figures 9F and 9H) or no effect on the oxygen consumption rates (Figures 9D to 9F). Previous studies have shown that longer inhibition of Complex IV can shut down mitochondrial function, resulting in cellular death. Thus, the impact of 10 pM metformin to reduce the effect of hyperglycaemia on Complex IV might counteract hyperglycaemia-induced apoptosis. The ability of 500 pM metformin to cause cell death has been reported elsewhere (Fontaine, 2018; Samuel et al., 2017; Szabo et al., 2014; Triggle et al., 2020). These data support the conclusion that the lower concentration of metformin functions differentially in response to hyperglycaemia and does not affect Complex I mitochondrial oxidative phosphorylation.

[00156] These data led us to assess the impact of low (10pM) and high (500pM) concentrations of metformin on the extracellular acidification rate (ECAR ) providing us a snapshot of glycolysis. We found that 500pM metformin treatment caused a basal increase in ECAR compared to 10pM metformin treatment, eventually contributing to a reduction in glycolysis, thereby pointing a differential role of 10pM metformin in the context of hyperglycaemia. These data indicate that cellular metabolism is dampened by 10pM metformin while 25mM glucose alone or in combination with 500pM metformin has an adverse impact on glycolysis.

[00157] 9. Mitochondrial morphology is changed by exposing cells and tissues to hyperglycaemia and this change is prevented by metformin.

[00158] It has been known for over 40 years that mitochondrial morphology changes from a spindle to circular shape, indicative of an increased mitochondrial metabolic state (Hackenbrock, 1966). We therefore monitored mitochondrial structure via electron microscopy, to evaluate the impact of hyperglycaemia on organelle shape in the setting of hyperglycaemia, which induces the production of ROS; and to assess the effect of metformin on hyperglycaemia-induced changes in mitochondrial morphology (Figure 10). We observed that the wild-type cells cultured in the presence of 5mM glucose for24h displayed large numbers of elongated ‘spindle-shaped’ mitochondrial structures with visible cristae (yellow arrows, Figure 10A), along with a proportion of mitochondria showing a ‘circular’ shape (green arrows, Figure 10A). However, compared with exposure to 10 mM glucose, cells incubated under hyperglycaemic conditions (25mM Glucose for 24 hours) displayed a much higher proportion of mitochondria with ‘circular’ shapes (green arrows), compared to elongated ‘spindle’ shapes (yellow arrows, Figure 10B). In contrast, in the concurrent presence of 10 pM metformin, the mitochondrial morphology of endothelial cells exposed to 25 mM glucose showed a more ‘normal’ elongated ‘spindle- shaped’ phenotype (Compare Figure 10C, and 10B), reflecting the relative abundance observed at a low glucose concentration (Panel A). Morphometric analysis was done to evaluate the impact of hyperglycaemia on mitochondrial shape, that reflects oxidative activity (Hackenbrock, 1966). The manual count of circular and spindle-shaped mitochondria in equivalent image fields of a set of 6 independent micrographs like the ones shown in Figures A to D, revealed a differential abundance of circular vs spindle- shaped mitochondria under the different conditions (Figure 10F). There was a marked increase in the relative abundance of circular vs spindle-shaped mitochondria when cells were switched from low (10mM) to high (25 mM) mM glucose (compare the two histograms on the left in Figure 10E). However, the presence of 10 pM metformin in the hyperglycaemic cultures prevented the increase in circular-shaped mitochondria caused by hyperglycaemia (compare first and third histograms from the left in Figure 10E). Surprisingly, treatment with 500 pM metformin led to a predominance of mitochondria with a circular shape (green arrows, Figures 10D to 10F), indicative of oxidative stress. The morphology changes can be correlated with the changes in the oxygen consumption rates shown in Figures 8A and 8C. Thus, the impact of metformin at low concentrations to improve the oxygen consumption rate of hyperglycaemia-exposed endothelial cells was reflected by its impact on mitochondrial morphology; and the morphology effect of a high metformin concentration was in agreement with the effect of 500 pM metformin to impair the oxygen consumption rate.

[00159] Taken together, these data show that at concentrations of metformin that reverse the ability of hyperglycaemia to compromise the mitochondrial oxygen consumption rate, metformin also preserves the morphology of mitochondria observed under euglycaemic conditions and prevents hyperglycaemia-induced vascular endothelial dysfunction.

[00160] 10. ‘Rescue’ of aorta rings exposed to hyperglycaemia for 48 hours; then treated with metformin for 12 hours.

[00161] Since endothelial vasorelaxant function was compromised when tissues were maintained under hyperglycaemic conditions, but protected when metformin was present in the culture medium (Figures 1 B and 1 D), it was of interest to determine if metformin could also reverse the impact of hyperglycaemia if added at 48 hours after the initiation of hyperglycaemia, instead of at the beginning of the culture period. As shown in Figures 11A-11C, the addition of metformin at the 48 hour time point was able, within another 12 hours, to re-establish the endothelial responses for the vasorelaxant function of both acetylcholine and 2fl_l (Figure 11 B and 11C). Thus, the action of metformin can be seen to be both ‘prophylactic’ and ‘therapeutic’ in terms of protecting the endothelium from hyperglycaemia-induced vasorelaxant dysfunction either before or after a tissue is exposed to hyperglycaemia.

[00162] 11. Metformin at a concentration that preserves vascular endothelial function increases AMPKinase phosphorylation.

[00163] Because the actions of metformin have been attributed to its activation of AMPKinase (Hawley et al., 2002), we sought to determine if the same concentration of metformin that could rescue endothelial function in intact vascular tissue could activate AMPKinase in isolated aortic endothelial cells incubated under hyperglycaemia conditions, followed by 10pM metformin treatment. Western blot analysis showed that this concentration of metformin caused an increase in phospho-AMPK that was up to 50% of the level stimulated by the AMPKinase agonist, AICAR (Figure 11 D, 11 E).-Our results support the likelihood that 10pM metformin is sufficient to activate phospho-AMPK in keeping with the effect of 500pM metformin treatment. However, that high metformin concentration activates AMPKinase at a level that, unlike 10pM metformin, not only compromises mitochondrial complex I function (Figure 9E) but also fails to prevent hyperglycaemia-induced endothelial dysfunction (Figures 1 D, 1 E). Thus, both low and high concentrations of metformin were able to activate AMPK, but only the low concentration of metformin was able to reverse hyperglycaemia-compromised endothelial vasodilator function. A direct link between the level of AMPK activation and the ability of metformin to preserve hypergycaemia-induced endothelial dysfunction could not therefore be established.

[00164] 12. Metformin is predicted to interact with NR4A1 by in silico modelling

[00165] The data described in the previous sections indicating a requirement for the expression of Nr4a1 to enable metformin to protect the endothelium from hyperglycaemia-induced dysfunction suggested that, as for other compounds with a structure related to metformin, Nr4a1 might be able to interact directly with metformin (Lanig et al., 2015). Molecular docking studies have identified a ligand binding region of Nr4a1 in its C-terminal domain. In keeping with the results outlined by (Lanig et al., 2015) we found that an in silico docking approach revealed a predicted accurate and preferred metformin docking site in NR4A1 that was the same as the one identified by Lanig et al. (2015) (Figure. 12A). The best-docked conformation was selected on the basis of binding affinity and interaction energy parameters. As per the findings of Lanig et al., 2015, metformin was docked in the binding site near N-terminus of the C-terminal domain of NR4A1 and showed close interactions with Leu228, Leu178, Val179, and Thr182.

Metformin would thus be predicted to bind closely in the NR4A1 binding cavity (Figure12C). The predicted interacting residues and atoms leading to the metformin- NR4A1 binding are shown in Figure 12B. The interacting residues in the binding pocket are shown in Figure 12C. The docking analysis clearly indicates that metformin could in principle interact closely within the previously proposed Nr4a1 ligand binding site. [00166] We compared the docking of metformin with another NR4A1 agonist, cytosporone B, also known to affect gluconeogenesis (Zhan et al., 2008). As found previously by (Zhan et al., 2012) and by us, it was possible to identify a putative docking site for cytosporone B with NR4A1 . Thus, the putative NR4A1 agonists, metformin and cytosporone B, both of which are implicated in regulating glucose metabolism, were able in principle to dock with NR4A1. The conformations of docked metformin, cytosporone B, and the NR4A1 agonist, THPN, are shown in Fig. 12. As mentioned, metformin showed close interactions with the defined active site residues, Asp137, Pro139, and Pro184 by forming H-bonds and salt-bridges (Figure. 12B and C). Cytosporone B showed interactions with more residues than metformin, including Asn1 , Asp137, Pro139, Ala140, Ala179, Glu183, Pro184, and Gln185, by forming conventional H-bonds, alkyl bonds, and pi-alkyl bonds. The surface representation of cytosporone B is shown in. Thus, cytosporone B might be expected to drive the NR4A1 ‘receptor’ in a way similar to, but distinct from the actions of metformin.

[00167] Like metformin, the NR4A1 -agonist, THPN (Wu and Chen, 2018), showed interactions with Asn1 , Asp137, Pro139, Ala140, Ala179, and Glu183 by forming conventional H-bonds, Van der wall’s interactions, alkyl bonds, and pi-alkyl bonds. The surface representation of the NR4A1 agonist, THPN, lying within the binding pocket. Further, TMPA, an NR4A1 -antagonist, showed conventional H-bonds and carbon hydrogen interactions with Asn1 , Glu175, and Gln185. TMPA fits in the predicted NR4A1 ligand binding pocket,. In sum, both metformin and the antagonist we used to modulate the effects of metformin on maintaining endothelial cell function in the setting of hyperglycaemia (TMPA) were able in principle to dock with NR4A1 ,

[00168] 13. Cytosporone B, like metformin, protects the endothelium from hyperglycaemia-induced dysfunction, but only at low concentrations.

[00169] Given that the in silica analyses indicated that cytosporone B can interact with NR4A1 , we predicted that it, like metformin, might protect the endothelium from hyperglycaemia-induced dysfunction. Indeed, it was found using the vascular ring organ culture test system, that low concentrations of cytosporone B in the nanomolar concentration range (50 nM) were able, like metformin, to preserve endothelial function for the vasorelaxant actions of both acetylcholine and 2fl_l (Figure 13a; 13b). However, exposure to higher concentrations of cytosporone B (500 to 1000 nM) appeared to be toxic, leading to a deterioration of both the endothelial cell vasorelaxant function and to the ability of phenylephrine to constrict the tissue. These effects of cytosporone B were possibly due to the ability of cytosporone B at these concentrations to promote cellular apoptosis over a 24 hour time frame (Zhan et al., 2008; Zhan et al., 2012). To conclude, the Nr4a1 -interacting ligand, cytosporone B, like metformin, was able to protect the endothelium from hyperglycaemia-induced dysfunction, but only at concentrations in the 10 to 50 nanomolar range.

[00170] 14. Molecular Dynamic (MD) calculations for metformin-Nr4a1 complex stability in a simulated in vivo environment.

[00171] Simulations were done to determine the structural stability in a simulated in vivo environment of NR4A1 after binding with metformin within a nanosecond time scale. This complex was selected based on the least binding affinity and was subjected to 30 ns MD simulations, and the results were analyzed. The Root Mean Square Deviation (RSMD), a crucial parameter to analyze the equilibration of molecular dynamic trajectories, was estimated for backbone atoms of the metformin and metformin-NR4A1 complex. Measurements of the backbone RMSD for the complex provided insights into the theoretical conformational stability of the metformin- NR4A1 complex in solution. Slight deviations can be seen during the time period of 10-20 ns. After that, a slight decrease in the RMSD value can be seen that remained stable afterwards (Figure 14A). The overall RMSD value fluctuated between 8-10 nm. The Root Mean Square Fluctuation (RMSF) calculates the standard deviation of atomic positions. The plot showed residual fluctuations in NR4A1at several regions during the simulation (Figure 14B). The calculated total energy and average potential energy was found to be -1.77e+006 KJ/mol and -2.175e+006 KJ/mol respectively (Figure 14C & 14D). As the molecular recognition between a receptor and a ligand lies in H-bonding, we calculated the number and distance of potential hydrogen bonds between metformin and NR4A1 in the putative in- silico complex. Multiple H-bonds can be seen in the plot during the time period of 10-30 ns (Figure 14E & 14F). This result implies that metformin can bind to NR4A1 with several H-bonds that remain stable during the 30ns simulation. The calculated average H-bond distance ranged between 0.2 to 0.35 nm. Solvent Accessible Surface Analysis (SASA) estimates the conformational changes in protein upon ligand binding. Fluctuations can be observed in SASA plot during the first 15ns simulation and no major change was observed afterwards showing the stable conformation of metformin-Nr4a1 complex (Figure. 14G). Based on these in-silico analyses, we conclude that the metformin-Nr4a1 complex can be stable and can be predicted to exist under physiological conditions in solution as well. The calculated binding affinities of the complexes: NR4A1 -metformin, NR4A1-cytosporone B, along with the NR4A1-THPN agonist, and NR4A1-TMPA antagonist complexes were found to be -4.3 Kcal/mol, -5.5 Kcal/mol, -5.3 Kcal/mol, and - 3.5 Kcal/mol respectively.

[00172] 15. Biotinylated metformin can interact directly with Nr4a1.

[00173] Given the above described in-silico data predicting a physical interaction between metformin and Nr4a1 , we tested the hypothesis that Nr4a1 could interact directly with metformin in a cell expression system. To this end, we synthesized biotinyl- metformin, along with a metformin-free biotin linker, in keeping with previously published work (Horiuchi et al., 2017). Murine Nr4a1 , incorporating either a myc or a monomric red- fluorescent-protein (mRFP) tag on its C-terminus was prepared and expressed in an HEK cell background as described in methods. Expression of the construct was verified by western blot analysis. Extracts of tagged Nr4a1 -expressing HEK cells were made and supplemented with either biotinyl-metformin or metformin-free biotinyl linker. The potential complex between biotinyl-metformin and tagged Nr4a1was adsorbed to neutravidin beads which were harvested and washed as outlined in Methods. The metformin-Nr4a1 complex adhering to the neutravidn beads was then dissociated in the presence of 100 pM free metformin, and the released proteins were solubilized in electrophoresis buffer for the conduct of western blot analysis to detect either the myc or mRFP-tagged Nr4a1 using and anti-Nr4a1/Nur77 antibdy. As shown in Figure 15, a western blot signal for Nr4a1 was detected for the analysis of the avidin-bead-pulldowns using biotinylated metformin (Bio-Met), but not for pulldowns using either the metformin-free C10 linker or the biotin-free metformin-C10 linker alone (Lanes N1 and N2, Figure 15A & 15B). For reasons we were not able to determine, the avidin bead pulldowns showed variable amounts of mRFP-Nr4a1 western blot reactivity, most likely depending on the efficiency of extraction of the tagged mRFP-Nr4a1 constructs from the HEK cell expression system. Nonetheless, no signal was ever detected from the pulldowns obtained using the metformin-free biotinylated linker control or the biotin-free metformin-linker control. We concluded that indeed metformin was able to interact with tagged myc/mRFP-Nr4a1 in the HEK cell extracts either on its own, or in a complex with other constituents.

[00174] DISCUSSION

[00175] 1. Main Findings. The main findings of our study were not only that 1 .

Metformin is able to protect the endothelium both in vitro and in vivo from hyperglycaemia-induced vasorelaxant dysfunction by minimizing oxidative stress (ROS production) but also 2. The ‘orphan nuclear receptor’, Nur77/NR4A1 is an essential partner in this action of metformin. The data thus point to an unexpected novel mechanism to explain the ability of metformin to maintain endothelial function in the setting of hyperglycaemia. This positive impact of metformin on vascular endothelial function was observed in vivo for diabetic patients some time ago (Mather et al., 2001), but the mechanism for this action was not determined. The ability of metformin to interact with NR4A1 was supported by our in-silico analysis identifying a potential docking of metformin with NR4A1 and the ability of tagged NR4A1 to accompany biotinylated metformin upon harvesting the biotin-metformin-NR4A1 complex from a cell extract using an avidin bead affinity isolation procedure. Of importance, the sequences of human NR4A1 and mouse Nr4a1 are highly homologous, especially in the predicted ligand binding pocket. Thus, our data for observing the interaction between biotinylated metformin with mouse Nr4a1 undoubtedly apply to human NR4A1 as well. It is possible, however, that in the avidin bead pulldown experiments, biotinylated metformin might have been bound to NR4A1 in a complex with other proteins. This possibility merits investigation in the future using a proteomic approach to analyse the metformin-NR4A1 complex that adhered to the avidin beads. Of note, our in-silico analysis showed that the NR4A1 antagonist, TMPA, is in principle also able to dock with NR4A1. Yet, TMPA blocks, rather than mimics the action of metformin. It is feasible that the ‘metformin- antagonist’ activity of TMPA, due to its occupying the NR4A1 metformin ligand binding site, may concurrently activate other signal pathways, as a ‘biased’ agonist/antagonist. It is possible that the differences in the actions of metformin and TMPA may be due to the higher predicted binding affinity for the metformin-NR4A1 complex (-4.3 Kcal/mol), compared with the TMPA-NR4A1 complex (-3.5 Kcal/mol). In this regard, cytosporone B, which acted like metformin in our vascular organ cultures had a higher predicted affinity for NR4A1 (-5.5 Kcal/mol) than either metformin or TMPA. This issue merits further investigation, since TMPA itself can trigger the release of NR4A1- bound constituents like LKB1 to enable their exit from the nucleus so as to affect cytosolic AMPK-regulated signalling (Zhan et al., 2012).

[00176] 2. Biphasic concentration-dependent actions of metformin and cytosporone B. Of note, the actions of metformin to protect the endothelium from hyperglycaemia-induced dysfunction were mimicked by another ligand, cytosporone B, that although structurally dissimilar from metformin, like metformin, has been predicted to interact directly with the NR4A1 ligand docking site and has been shown to be an agonist for NR4A1/Nur77 (Zhan et al., 2008). Similar to the effects of metformin, cytosporone B had a ‘biphasic’ action, protecting the endothelium at relatively low concentrations; but impairing vascular function (both endothelium and smooth muscle) at high concentrations. These deleterious actions of metformin and cytosporone B may be due to their interactions with effectors other than NR4A1 . However, it is also possible that both metformin and cytosporone B, which are structurally distinct, upon interacting with NR4A1 , can drive the receptor in a ‘biased’ manner to stimulate different signalling via distinct ligand-NR4A1 interactions that are concentration-dependent. This issue clearly merits further study. In principle, it may be possible to generate compounds that interact selectively with NR4A1 to stimulate only the ‘beneficial’ vascular protective signals and to avoid the deleterious effects observed at the relatively high concentrations of the two NR4A1 -interacting ligands. It is hoped that the in-silico analyses that we provide will aid the discovery of more selective NR4A1 -stimulating agonists that will avoid the cytotoxic actions observed for both cytosporone B and metformin.

[00177] 3. metformin action to prevent hyperglycaemia-induced endothelial dysfunction does not affect the mitochondrial complex I oxygen consumption rate. Of note, the concentrations of metformin that were able to preserve endothelial function under hypergycaemic conditions (e.g. 1 to 10 pM) were well below those found in other studies to impair mitochondrial complex I function (in the millimolar range: (El-Mir et al., 2000; Owen et al., 2000)as discussed by (Kinaan et al., 2015; Triggle and Ding, 2017). Thus, we found no impact on the mitochondrial oxygen consumption rate at concentrations of metformin that preserved endothelial function in the setting of hyperglycaemia (Figures 8 & 9). Of importance, the concentrations of metformin that we observed were able to improve hyperglycaemia-exposed endothelial cell function both in vitro and in the STZ-diabetes model in vivo are in the therapeutic plasma concentration range observed in metformin-treated type-2 diabetics. However, this concentration of metformin that did not affect mitochondrial complex I function, was able to cause an improvement in the function of Complexes II and IV. Thus metformin’s effect on Complexes II and IV in the setting of hyperglycaemia may be protective.

[00178] The low metformin concentration (10 pM) caused a modest increase in the phosphorylation of AMPKinase (Figure 11 D, 11 E). This result indicated that the activation of AMPKinase may possibly play a role in metformin’s endothelium-preserving function even though the low metformin concentration did not affect Complex I function. However, as already noted, the high concentration of metformin (500 pM) that on its own compromises endothelial cell function, also increases AMPK phosphorylation. Further the NR4A1/Nur77 antagonist, TMPA, that blocks the action of metformin to maintain endothelial function can liberate nuclear NR4A1 -bound LKB1 to migrate to the cytosol and activate AMPKinase (Zhan et al., 2012). Possibly, by interacting with NR4A1 , metformin, like TMPA, may also dissociate LKB1 from NR4A1 to result in LKB1 exit from the nucleus to activate AMPK. Thus, activation of AMPK may be unrelated to the endothelium-protective action of metformin and establishing the role of metformin- stimulated AMPK activation for preserving endothelial cell function is a challenge that we have elected to leave for future work.

[00179] 4. Implications for the use of metformin to treat diabetic vascular dysfunction. As alluded to above, from in-vivo measurements of endothelial vasodilator function done in human type 2 diabetic patients, it has been known for two decades now that metformin can improve blood vessel endothelial function (Mather et al., 2001). The results we report here suggest (e.g. Figure 11) that metformin may be not only ‘protective’ if administered prior to a hyperglycemic event but can also ‘rescue’ the endothelium from hyperglycaemia-induced dysfunction, even after a significant period of hyperglycaemia. Our data not only provide a mechanistic rationale for this action of metformin to protect the endothelium from hyperglycaemia-induced dysfunction by reducing oxidative stress, but also reveal an unexpected link between this action of metformin and the orphan nuclear receptor, NR4A1 . Thus, it will be of much interest to see if the ability of metformin to enhance insulin action is also tied to its interaction with NR4A1 and to assess if the action of metformin in other tissues like the liver is also dependent on NR4A1 . Since it is known that a subset of type 2 diabetics does not respond well to metformin treatment, it will be of value to explore the potential link between possible genetic polymorphisms in the NR4A1 gene and the success or failure of metformin to treat diabetics. Moreover, given the potential off-target effects that our data imply for the actions of metformin (e.g. high concentrations impair, rather than preserve endothelial function), the door would appear to be open, using the in-silico approach we describe here, to design more targeted NR4A1 agonists that might replace metformin as a therapeutic agent to protect the endothelium from hyperglycaemia-induced dysfunction.

[00180] Ethics

[00181] Animal experimentation: Institutional animal approval: all mouse work was done in accordance with University of Calgary institutional animal care and use committee approved protocol #AC15-0002

[00182] References:

[00183] Abraham MJ, Murtola T, Schulz R, Pall S, Smith JC, Hess B and Lindahl E (2015)GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1-2: 19-25.

[00184] Brownlee M (2001)Biochemistry and molecular cell biology of diabetic complications. Nature 414(6865): 813-820.

[00185] Carling D, Clarke PR, Zammit A and Hardie DG (1989)Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. Eur J Biochem 186(1-2): 129-136.

[00186] Carling D, Zammit VA and Hardie DG (1987)A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 223(2): 217-222.

[00187] Cheng YY, Leu HB, Chen TJ, Chen CL, Kuo CH, Lee SD and Kao CL (2014)Metformin-inclusive therapy reduces the risk of stroke in patients with diabetes: a 4-year follow-up study. J Stroke Cerebrovasc Dis 23(2): e99-105.

[00188] Christensen MM, Brasch-Andersen C, Green H, Nielsen F, Damkier P, Beck-Nielsen H and Brosen K (2011)The pharmacogenetics of metformin and its impact on plasma metformin steady-state levels and glycosylated hemoglobin A1c.

Pharmacogenet Genomics 21 (12): 837-850.

[00189] Ding H, Ye K and Triggle CR (2019)lmpact of currently used anti-diabetic drugs on myoendothelial communication. Curr Opin Pharmacol 45: 1-7.

[00190] Driver C, Bamitale KDS, Kazi A, Olla M, Nyane NA and Owira PMO (2018)Cardioprotective Effects of Metformin. J Cardiovasc Pharmacol 72(2): 121-127.

[00191] El-Daly M, Pulakazhi Venu VK, Saifeddine M, Mihara K, Kang S, Fedak PWM, Alston LA, Hirota SA, Ding H, Triggle CR and Hollenberg MD (2018)Hyperglycaemic impairment of PAR2-mediated vasodilation: Prevention by inhibition of aortic endothelial sodium-glucose-co-Transporter-2 and minimizing oxidative stress. Vascul Pharmacol 109: 56-71.

[00192] El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M and Leverve X (2000)Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem 275(1): 223-228.

[00193] Fontaine E (2018)PMC6304344 Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences. Front Endocrinol (Lausanne) 9: 753.

[00194] Furman BL (2015)Streptozotocin-lnduced Diabetic Models in Mice and Rats. Curr Protoc Pharmacol 70: 5 47 41-20.

[00195] Graham GG, Punt J, Arora M, Day RO, Doogue MP, Duong JK, Furlong TJ, Greenfield JR, Greenup LC, Kirkpatrick CM, Ray JE, Timmins P and Williams KM (2011 )Clinical pharmacokinetics of metformin. Clin Pharmacokinet 50(2): 81-98.

[00196] Hackenbrock CR (1966) Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol 30(2): 269-297.

[00197] Hawley SA, Gadalla AE, Olsen GS and Hardie DG (2002)The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes 51 (8): 2420-2425.

[00198] Horiuchi T, Sakata N, Narumi Y, Kimura T, Hayashi T, Nagano K, Liu K, Nishibori M, Tsukita S, Yamada T, Katagiri H, Shirakawa R and Horiuchi H (2017)PMC5437248 Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity. J Biol Chem 292(20): 8436-8446.

[00199] Humphrey W, Dalke A and Schulten K (1996)VMD: Visual molecular dynamics. Journal of Molecular Graphics 14(1): 33-38.

[00200] Kilkenny C, Browne WJ, Cuthill IC, Emerson M and Altman DG (2010)PMC2893951 Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8(6): e1000412.

[00201] Kinaan M, Ding H and Triggle CR (2015)PMC5588255 Metformin: An Old Drug for the Treatment of Diabetes but a New Drug for the Protection of the Endothelium. Med Prine Pract 24(5): 401-415.

[00202] Lanig H, Reisen F, Whitley D, Schneider G, Banting L and Clark T (2015)PMC4535767 In Silico Adoption of an Orphan Nuclear Receptor NR4A1. PLoS One 10(8): e0135246. [00203] Lee JP, Brauweiler A, Rudolph M, Hooper JE, Drabkin HA and Gemmill RM (2010)PMC3086825 The TRC8 ubiquitin ligase is sterol regulated and interacts with lipid and protein biosynthetic pathways. Mol Cancer Res 8(1): 93-106.

[00204] Martin-Montalvo A, Mercken EM, Mitchell SJ, Palacios HH, Mote PL, Scheibye-Knudsen M, Gomes AP, Ward TM, Minor RK, Blouin MJ, Schwab M, Pollak M, Zhang Y, Yu Y, Becker KG, Bohr VA, Ingram DK, Sinclair DA, Wolf NS, Spindler SR, Bernier M and de Cabo R (2013)PMC3736576 Metformin improves healthspan and lifespan in mice. Nat Commun 4: 2192.

[00205] Mather KJ, Verma S and Anderson TJ (2001)lmproved endothelial function with metformin in type 2 diabetes mellitus. J Am Coll Cardiol 37(5): 1344-1350.

[00206] Mihara K, Ramachandran R, Renaux B, Saifeddine M and Hollenberg MD (2013)PMC3829148 Neutrophil elastase and proteinase-3 trigger G protein-biased signaling through proteinase-activated receptor-1 (PAR1). J Biol Chem 288(46): 32979- 32990.

[00207] Mohankumar K, Lee J, Wu CS, Sun Y and Safe S (2018)PMC5888234 Bis-Indole-Derived NR4A1 Ligands and Metformin Exhibit NR4A1 -Dependent Glucose Metabolism and Uptake in C2C12 Cells. Endocrinology 159(5): 1950-1963.

[00208] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS and Olson AJ (2009)PMC2760638 AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30(16): 2785-2791.

[00209] Nafisa A, Gray SG, Cao Y, Wang T, Xu S, Wattoo FH, Barras M, Cohen N, Kamato D and Little PJ (2018)Endothelial function and dysfunction: Impact of metformin. Pharmacol Ther 192: 150-162.

[00210] Ouslimani N, Peynet J, Bonnefont-Rousselot D, Therond P, Legrand A and Beaudeux JL (2005)Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells. Metabolism 54(6): 829-834.

[00211] Owen MR, Doran E and Halestrap AP (2000)PMC1221104 Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 348 Pt 3: 607-614.

[00212] Papatzimas JW, Gorobets E, Maity R, Muniyat Ml, MacCallum JL, Neri P, Bahlis NJ and Derksen DJ (2019)From Inhibition to Degradation: Targeting the Antiapoptotic Protein Myeloid Cell Leukemia 1 (MCL1). J Med Chem 62(11): 5522-5540.

[00213] Pulakazhi Venu VK, Saifeddine M, Mihara K, El-Daly M, Belke D, Dean JLE, O'Brien ER, Hirota SA and Hollenberg MD (2018) Heat shock protein-27 and sex- selective regulation of muscarinic and proteinase-activated receptor 2-mediated vasodilatation: differential sensitivity to endothelial NOS inhibition. Br J Pharmacol 175(11): 2063-2076.

[00214] Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, and Eliceiri KW (2017) lmageJ2: Imaged for the next generation of scientific image data. BMC Bioinformatics 18 (1):529-555.

[00215] Samuel SM, Ghosh S, Majeed Y, Arunachalam G, Emara MM, Ding H and Triggle CR (2017)Metformin represses glucose starvation induced autophagic response in microvascular endothelial cells and promotes cell death. Biochem Pharmacol 132: I IS- 132.

[00216] Scheen AJ (1996)Clinical pharmacokinetics of metformin. Clin Pharmacokinet 30(5): 359-371.

[00217] Shah MS and Brownlee M (2016)PMC4888901 Molecular and Cellular Mechanisms of Cardiovascular Disorders in Diabetes. Circ Res 118(11): 1808-1829.

[00218] Szabo C, Ransy C, Modis K, Andriamihaja M, Murghes B, Coletta C, Olah G, Yanagi K and Bouillaud F (2014)PMC3976625 Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms. Br J Pharmacol 171 (8): 2099-2122.

[00219] Triggle CR and Ding H (2017)Metformin is not just an antihyperglycaemic drug but also has protective effects on the vascular endothelium. Acta Physiol (Oxf) 219(1): 138-151.

[00220] Triggle CR, Ding H, Marei I, Anderson TJ and Hollenberg MD (2020)Why the endothelium? The endothelium as a target to reduce diabetes-associated vascular disease.’. Can J Physiol Pharm.

[00221] Trott O and Olson AJ (2010)PMC3041641 AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31 (2): 455-461.

[00222] Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, Darian E, Guvench O, Lopes P, Vorobyov I and Mackerell AD, Jr. (2010)PMC2888302 CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J Comput Chem 31 (4): 671-690.

[00223] Wang JM, Chen AF and Zhang K (2016)PMC5226426 Isolation and Primary Culture of Mouse Aortic Endothelial Cells. J Vis Exp(118). [00224] Wilkinson RA, Pincus SH, Shepard JB, Walton SK, Bergin EP, Labib M and Teintze M (2011)PMC3019677 Novel compounds containing multiple guanide groups that bind the HIV coreceptor CXCR4. Antimicrob Agents Chemother 55(1): 255-263.

[00225] Wu L and Chen L (2018)PMC6236262 Characteristics of Nur77 and its ligands as potential anticancer compounds (Review). Mol Med Rep 18(6): 4793-4801. [00226] Zhan Y, Du X, Chen H, Liu J, Zhao B, Huang D, Li G, Xu Q, Zhang M, Weimer BC, Chen D, Cheng Z, Zhang L, Li Q, Li S, Zheng Z, Song S, Huang Y, Ye Z, Su W, Lin SC, Shen Y and Wu Q (2008)Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat Chem Biol 4(9): 548-556.

[00227] Zhan YY, Chen Y, Zhang Q, Zhuang JJ, Tian M, Chen HZ, Zhang LR, Zhang HK, He JP, Wang WJ, Wu R, Wang Y, Shi C, Yang K, Li AZ, Xin YZ, Li TY, Yang JY, Zheng ZH, Yu CD, Lin SC, Chang C, Huang PQ, Lin T and Wu Q (2012)The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK. Nat Chem Biol 8(11): 897-904.

[00228] Zhang L, Wang Q, Liu W, Liu F, Ji A and Li Y (2018)PMC6022324 The Orphan Nuclear Receptor 4A1 : A Potential New Therapeutic Target for Metabolic Diseases. J Diabetes Res 2018: 9363461.

[00229] Zilov AV, Abdelaziz SI, AlShammary A, Al Zahrani A, Amir A, Assaad Khalil SH, Brand K, Elkafrawy N, Hassoun AAK, Jahed A, Jarrah N, Mrabeti S and Paruk I (2019)Mechanisms of action of metformin with special reference to cardiovascular protection. Diabetes Metab Res Rev 35(7): e3173.

[00230] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00231] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00232] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.