AVOCATIN B FOR THE TREATMENT OF DISEASES AND CONDITIONS.

Abstract

A method for treating a disease or condition may be characterized by a metabolic disorder in a subject who needs it, which may include administering to the subject a therapeutically effective amount of avocatin B.

Description

AVOCATIN B FOR THE TREATMENT OF DISEASES AND CONDITIONS CROSS REFERENCE WITH RELATED PATENT APPLICATION This application claims priority and benefit from U.S. Provisional Application No. 62 / 899,402 filed on 09 / 12 / 2019 and entitled Avocatina B for the treatment of diseases and conditions, the entirety of which is incorporated herein by reference. FIELD OF INVENTION The present description generally refers to diet-induced obesity, and more particularly to the treatment of diet-induced obesity and associated pathologies with avocatin B. BACKGROUND OF THE INVENTION United States Patent Publication No. 2017 / 0304251 describes a method for treating leukemia comprising administering to a subject in need a therapeutically effective amount of a composition comprising a compound of Formula (I) and / or (II) having the structure: OR2 R RIO n OR2 R RIO η III where: I0 represents a single or double bond; R is OH when CR is C — R, and R is O when CR is C=R; n is 1, 3, 5 or 7; and R1 and R2 are independently hydrogen or acetyl and / or isomers, stereoisomers or solvates thereof and / or mixtures thereof. BRIEF DESCRIPTION OF THE INVENTION The teachings described in the present description may, in a broad sense, relate to a method for treating a disease or condition characterized by dysregulation of glucose-stimulated insulin secretion (GSIS) in a subject in need, the method comprising administering to the subject a therapeutically effective amount of avocatin B. The teachings described in the present description may, in another broad aspect, relate to a method for treating a disease or condition characterized by a metabolic disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in this description may be related to a method for treating a disease or condition characterized by insulin resistance in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description may be related to a method for treating a disease or condition characterized by reduced sensitivity to insulin in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in this description can be related to a method for rescuing glucose-stimulated insulin secretion (GSIS) in a subject who has obesity-associated lipotoxicity, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in this description can be related to a method for increasing glucose utilization in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description can be related to a method for modulating reactive oxygen species in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description can be related to a method for increasing glucose uptake in pancreatic tissue in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description can be related to a method for increasing glucose uptake in skeletal muscle tissue in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description can be related to a method for increasing glucose oxidation in pancreatic tissue in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description can be related to a method for increasing glucose oxidation in skeletal muscle tissue in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in this description can be related to a method for reversing insulin resistance in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. In another aspect, the teachings described in the present description can be related to a method for increasing insulin sensitivity in a subject who needs it, the method comprising administering to the subject a therapeutically effective amount of avocatin B. Other aspects of the teachings described in this description, which may be used in combination with any other aspect, including the broad aspect listed above, may include that the disease or condition is diet-induced obesity. The therapeutically effective amount of avocatin B can be administered in at least one daily dose. The at least one daily dose may comprise from approximately 25 mg to approximately 200 mg of avocatin B. The at least one daily dose may comprise around 50 mg of avocatin B. The teachings described in this description may, in another broad sense, relate to using a therapeutically effective amount of avocatin B to treat a disease or condition characterized by a metabolic disorder in a subject who needs it. The metabolic disorder may be characterized by dysregulation of glucose-stimulated insulin secretion (GSIS) in the subject. The metabolic disorder may be characterized by insulin resistance in the subject. The metabolic disorder may be characterized by reduced insulin sensitivity in the subject. The metabolic disorder may be characterized by lipotoxicity associated with obesity in the subject. In another aspect, the teachings described in this description may also include increasing glucose utilization in the subject. In another aspect, the teachings described in this description may also include the modulation of reactive oxygen species in the subject. In another aspect, the teachings described in this description may also include increasing glucose uptake in the pancreatic tissue of the subject. In another aspect, the teachings described in this description may also include increasing glucose uptake in skeletal muscle tissue in the subject. In another aspect, the teachings described in this description may also include increasing glucose oxidation in the pancreatic tissue of the subject. In another aspect, the teachings described in this description may also include the increase in glucose oxidation in the skeletal muscle tissue of the subject. In another aspect, the teachings described in this description may also include reversing insulin resistance in the subject. In another aspect, the teachings described in this description may also include increasing insulin sensitivity in the subject. The teachings described in this description may, in another broad aspect, relate to a pharmaceutical composition comprising a therapeutically effective amount of avocatin B and a carrier. The therapeutically effective amount may range from approximately 25 mg to approximately 200 mg. The therapeutically effective amount may comprise around 50 mg. Therefore, the present inventors have developed a novel therapy for DIO and associated pathologies. By harnessing fatty acid metabolism, this therapy can be used to improve the metabolic complications associated with DIO and lipotoxicity. In particular, this treatment can be administered after DIO to improve glucose tolerance, glucose utilization, and insulin sensitivity. This therapeutic use may provide a well-tolerated treatment option with a clinically acceptable safety profile for the treatment of DIO and associated metabolic disorders. Other advantages of the invention will become apparent to those skilled in the art upon reviewing the present description. BRIEF DESCRIPTION OF THE FIGURES The modalities in this description will be described with reference to the accompanying figures, where similar reference numbers indicate similar parts, and where: Figures 1A-1L illustrate the effects of avocatin B (AvoB) in a mouse model of DIO. (1A) Schematic treatment study design. Mice (N=28) were fed either a high-fat diet (HFD) or a standard low-fat diet (STD) for 8 weeks. At the end of week 8, mice were randomly divided into three groups: (i) vehicle-STD (STD); n=10, (ii) vehicle-HFD (HFD); n=9, and (iii) HFD mice treated with 100 mg / kg body weight, AVO twice weekly (HFD+AVO; n=9). STD and HFD mice were treated with vehicle control twice weekly. (IB) Weights of mice at the end of the treatment study (week 13). (IC) The gonadal (left) and mesenteric (right) fat pads (GFP and MFP) were removed and weighed at the end of the study. (ID) Effect of AVO on glucose tolerance. For GTT, a glucose injection was administered ip(1E) Area under the curve (AUC) calculated for the glucose excursion curve of the GTT. (1F) Effect of AVO on whole-body insulin sensitivity. For ITT, an intraperitoneal (ip) insulin injection was administered, after which blood glucose levels were monitored at regular intervals for 60 min. (1G) AUC calculated for the glucose excursion curve of the ITT. (1H) The HOMA-IR index, a measure of insulin resistance, was calculated as described in the Methods section. (II) Plasma free fatty acids and (1J) plasma triacylglycerols (TAGs) were measured at the endpoint in ad libitum animals. (1K) Native pyruvate dehydrogenase (PDH) activity was measured in whole gastrocnemius muscle lysates in the presence of phosphatase inhibitors.(1L) Phosphorylation of Akt-Ser473 (a marker of insulin signaling) and CPT-1A enzyme protein levels were assessed by Western blot in gastrocnemius muscle lysates from ad libitum animals. The figure shows representative Western blots for pAkt-Ser473, total AKT, CPT-1A, and GAPDH (loading control); the histograms (right) represent the change in times in pAkt-Ser473 and CPT-1A levels relative to STD mice. For data (IB - CI) presented as mean ± SD, *p < 0.05, **p < 0.01, two-tailed, unpaired Student's t-test. For (1E, 1G, 1H, and 1K) data represent mean ± SD, N = 8–10 / group. *p < 0.05, **p < 0.01; *** p < 0.001, one-way ANOVA, Bonferroni post hoc test. For (II-1J), data represent mean + SD, N = 9-10 / group for I and n = 5 for J, *p < 0.05, **p < 0.01; *** p < 0.001, Mann-Whitney U test. For (1L), data represent mean + SD, N = 5-6 / group, **p < 0.01; *** p < 0.001, one-way ANOVA, Bonferroni post hoc test;. Figures 2A-2K illustrate the effects of AvoB on fatty acid oxidation in INS-1 (832 / 13) cells, which enhances lipotoxicity and glucose-stimulated insulin secretion. (2A) INS-1 (832 / 13) cells were treated for 24 h with either 0.5% BSA / 1 mM L-carnitine (Control), 0.5 mM palmitate-BSA / 1 mM L-carnitine (PA), PA + 25 mM AVO (AVO+PA), or PA + 100 pM etomoxir (ETO+PA) in low glucose (5.5 mM) medium. After 24 h, fatty acid oxidation (FAO) was determined by measuring the incorporation of [14C] into CO2 and acid-soluble metabolites (ASM), representing complete and incomplete oxidation, respectively. (2B) The palmitate-supported oxygen consumption rate (OCR) was measured using high-resolution respirometry (HRR) on INS-1 (832 / 13) cells treated as described in (2A) except with palmitate-BSA 0.25 mM / L-carnitine 1 mM; basal, uncoupled (oligomycin addition), and maximal uncoupled (FCCP + pyruvate addition) respiration were assessed. (2C) INS-1 (832 / 13) cells were treated for 24 h as described in (2A), and glucose oxidation was determined by measuring the incorporation of [14C] into CO2, which represents complete glucose oxidation. (2D) To indirectly assess metabolic flexibility toward glucose utilization, oxygen consumption rate (OCR) was measured using high-resolution respirometry (HRR) in INS-1 (832 / 13) cells treated as described in (2A) except with 0.25 mM palmitate-BSA / 1 mM L-carnitine for 24 h. After treatment, the cells were trypsinized, collected and subjected to the addition of D-glucose (10 mM) for 15 minutes before HRR measurements; basal, uncoupled and maximum uncoupled respiration were assessed as described in (2C).(2E) INS-1 cells (832 / 13) were treated for 24 h as described in (2A) and a GSIS assay was performed in which insulin secreted into the media (KRB) was quantified using an ELISA kit. Insulin in KRB was normalized to total protein content as described in the Methods section. (2F) Data from (2E) are presented as the glucose-dependent insulin secretory index (GSIS 16 / 3), defined as the ratio of insulin secretion at 3 mM (stimulatory, surrogate for postprandial glucose levels) to 3 mM (basal, surrogate for fasting glucose levels). (2G) Mitochondrial ROS were measured in INS-1 cells (832 / 13) treated as described in (2A) using MitoSOX® Red. (2H) Mitochondrial membrane potential was measured in INS-1 cells (832 / 13) treated as described in (2A) using JC-1 staining.(21) Total cellular ATP content was measured in INS-1 (832 / 13) cells treated as described in (2A). (2J) Cytoplasmic Ca2+ was measured in INS-1 (832 / 13) cells using Fluo-3AM staining. The cells were treated as described in (2A) and with either 10 mM TMB-8 (endoplasmic reticulum calcium blocker) or 25 nM cyclosporine (mitochondrial calcium blocker) in the presence of PA. (2K) AMPKa (Thr-172) phosphorylation and PGC-α protein levels were assessed by Western blot in INS-1 (832 / 13) cells treated as described in (2A) and also with 2.5 pM rosiglitazone (RGZ). The figure shows representative Western transfers for pAMPKa-Thrl72, total AMPKa, PGC-la and GAPDH (load control); the histograms (right) represent the change in times in the levels of pAMPK and PGC-la with respect to vehicle control.For (2A), the data represent the means + SEM of three independent experiments conducted in triplicate. ***p < 0.001; ****p < 0.0001 vs. vehicle control for the CO2 group or ASM group, two-way ANOVA, Sidak post hoc test. For (2B), the data represent the means ± SEM of three independent experiments, ****p < 0.0001 vs. vehicle control, two-way ANOVA, Sidak post hoc test. For (2C), the data represent the means ± SEM of three independent experiments conducted in triplicate, **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. PA group, one-way ANOVA, Sidak post hoc test. For (2D), the data represent the means + SEM of three independent experiments, *p < 0.05; **p < 0.01; compared to the control group, Sidak post hoc test. For (3E), data were presented as mean ± SEM (N=4), **p < 0.01; ****p < 0.0001 vs. vehicle control for the 16 mM glucose group, two-way ANOVA, Dunnett's post hoc test. For (2F), data are presented as mean ± SEM (N=4) *p<0.05 vs. vehicle, one-way ANOVA, Dunnett's post hoc test. For (2G-2H), data are presented as mean + SEM (N=3), *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. CTL group (PA- or PA+), two-way ANOVA, Dunnett's post hoc test. For (21), data represent mean ± SEM (N=3), **p<0.01; ****p<0.0001 vs. control, one-way ANOVA, Dunnett's post hoc test; For (2J) the data represent the mean ± SEM (N=3), **p<0.01; ***p<0.001; ****p<0.0001 versus the PA group, one-way ANOVA, Dunnett's post hoc test. For (2K), the data represent the mean ± SEM (N=3), *p<0.05; **p<0.01; ***p<0.001 versus the Control group, one-way ANOVA, Dunnett's post hoc test. Figures 3A-3K illustrate the effects of AvoB on FAO in C2C12 myotubes, which enhances lipotoxicity and insulin signaling. (3A) C2C12 myotubes were treated for 24 h with 0.5% BSA / 1 mM L-carnitine (Control) or 0.5 mM palmitate-BSA / 1 mM L-carnitine (PA) or PA + 25 mM AVO (AVO+PA) or PA + 100 mM etomoxir (ETO+PA) in low-glucose media. After 24 h, FAO was determined by measuring the incorporation of [14C] into CO2 and acid-soluble metabolites (ASM), representing complete and incomplete oxidation, respectively. (3B) Palmitate-supported oxygen consumption rate (OCR) was measured using high-resolution respirometry (HRR) on C2C12 myotubes treated as described in (3A) except with palmitate-BSA 0.25 mM / L7 carnitine 1 rnM; basal, uncoupled (oligomycin addition) and maximum uncoupled (FCCP + pyruvate addition) respiration were assessed.(3C) C2C12 myotubes were treated as described in (3A). After 24 h, glucose oxidation was determined under insulin-stimulated and non-stimulated (basal) conditions by measuring the incorporation of [l4C] into CO2, which represents complete glucose oxidation. (3D) Metabolic flexibility toward glucose utilization was measured using HRR in C2C12 myotubes treated as described in (3A) except with 0.25 mM palmitate-BSA / 1 mM L-carnitine for 24 h. After treatment, the cells were trypsinized, harvested, and subjected to the addition of D-glucose (10 mM) for 15 minutes before HRR measurements; basal, uncoupled, and maximal capacities for electron flow (by FCCP uncoupling) were then assessed.(3E) C2C12 myotubes were treated as described in (3A), after which the myotubes were deprived of food and stimulated with 100 nM insulin for 30 min before lysis for Western blot. Insulin signaling was measured by assessing Akt-Ser473 phosphorylation. Inflammatory stress was assessed by ERK1 / 2 phosphorylation and p38 phosphorylation. The figure shows representative Western blots for pAkt-Ser473, total AKT, pERK1 / 2, total ERK, pP38, total p38, and GAPDH (load control); the histograms (right) represent the change in times in pAkt-Ser473, pERK1 / 2, and pP38 relative to the vehicle control (baseline). (3F) Insulin-stimulated glucose uptake was measured using the fluorescent glucose analog 2-NDBG by flow cytometry in C2C12 myotubes treated as described in (3A). Data are presented as % uptake of 2-NDBG relative to control.(3G) Mitochondrial ROS were measured using MitoSOX® Red in C2C12 myotubes treated as described in (3A). (3H) Mitochondrial membrane potential was measured in C2C12 myotubes treated as described in (3A) using JC-1 staining. (31) Total cellular ATP content was measured in C2C12 myotubes treated as described in (3A), as well as with two other metabolic modulators: 1 mM dichloroacetate (DCA) and 25 mM trimetazidine (TRI) in the presence or absence of PA. (3J) The effect of DCA and TRI on insulin signaling (pAkt-Ser473) in C2C12 myotubes was also assessed by Western blot as described for (3E). (3K) AMPKa (Thr-172) phosphorylation and PGC-la protein levels were assessed by Western blot on C2C12 myotubes treated as described in (3A) and also with 1 mM metformin (MET).The figure shows representative Western blots for pAMPK-Thrl72, total AMPK, PGC-la, and GAPDH (load control); the histograms (right) 8 represent the change in times in pAMPK and PGC-la levels relative to vehicle control. For (3A), data represent the means ± SEM of three independent experiments performed in triplicate. **p<0.01; ****p<0.0001 vs. vehicle control for the CO2 or ASM group, two-way ANOVA, Sidak post hoc test. For (3B), data represent the means ± SEM of three independent experiments, **p<0.01; ****p<0.0001 vs. vehicle control, two-way ANOVA, Sidak post hoc test. For (3C), data represent the means ± SEM of three independent experiments performed in triplicate. *p<0.05; **p<0.01; ****p<0.0001 vs. baseline PA group, and #p<0.0001 vs. insulin-stimulated PA group, two-way ANOVA, Sidak post hoc test.For (3D), data represent the means ± SEM of three independent experiments, **p<0.01; ****p<0.0001; versus the control group, Sidak post hoc test. For (3E), data are expressed as mean ± SEM (N=4), *p<0.05; **p<0.01; ****p<0.0001 versus the basal or insulin-stimulated BP group (as indicated), two-way ANOVA, Sidak post hoc test. For (3F), data were presented as the mean ± SEM of three independent experiments. *p<0.05, **p<0.01; ***p<0.001 versus the control, one-way ANOVA, Dunnett post hoc test. For (3G-3H), data are presented as mean ± SEM (N=3), *p < 0.05; **p < 0.01; versus the CTL group (PA- or PA+), two-way ANOVA, Dunnett's post hoc test. For (31), data represent the mean + SEM (N=3), **p < 0.01;****p < 0.0001 versus the control, one-way ANOVA, Dunnett's post hoc test; for (3J), data represent the mean ± SEM (N=3), ***p < 0.001; ****p < 0.0001 vs. Control (baseline), one-way ANOVA, Dunnett's post hoc test. For (3K), data represent mean ± SEM (N=3), *p < 0.05 vs. Control group, one-way ANOVA, Dunnett's post hoc test;. Figures 4A–4H illustrate the safety profile of avocatin B following human oral consumption. (4A) Schematic of the clinical study design. Healthy human participants consumed placebo, low-dose avocatin B supplement, or high-dose avocatin B once daily for 60 days. Participants dropped out of the study at different time points, as indicated by the N value on day 0, 30, or 60 of the clinical trial. Mean changes between day 30–day 0 and day 60–day 0 are presented for (4B) total bilirubin, (4C) alanine aminotransferase (ALT), (4D) creatinine, (4E) creatine phosphokinase, (4F) body weight, (4G) body mass index (BMI), and (4H) glycosylated hemoglobin (HbA1c). Data were expressed as mean ± SD, One-way Kruskal-Wallis range analysis of variance; Figures 5A-5I illustrate the complete blood count with differential (CBC w / dif) in whole blood collected from 3-5 animals per group at the endpoint of the treatment-DIO study. (5A) Hemoglobin. (5B) Hematocrit. (5C) Red blood cells (RBCs). (5D) White blood cells. (5E) Neutrophils. (5F) Lymphocytes. (5G) Eosinophils. (5H) Basophils. (5I) Platelets. N=3-5 / group, one-way ANOVA with Tukey's post hoc test; Figures 6A-6D illustrate the cell viability assessments. (6A) INS-1 (832 / 13) cells or day 5 C2C12 myotubes were treated for 24 h with 0.5% BSA / 1 mM L-carnitine (Control) or 0.1-0.5 mM palmitate-BSA / 1 mM L-carnitine in low glucose (5.5 mM) media, after which cell viability was measured by flow cytometry using propidium iodide. (6B) INS-1 (832 / 13) cells were treated for 24 h with 25 or 50 mM avocatin B in the absence or presence of 0.25 or 0.5 mM palmitate-BSA / 1 mM L-carnitine, after which cell viability was measured by flow cytometry using propidium iodide. (6C) C2C12 cells were differentiated in differentiation media for 5-6 days, used and collected for Western blot analysis of myogenin (skeletal muscle specific protein).(6D) On day 5, C2C12 myotubes were treated for 24 h with 25 or 50 pM avocatin B in the absence or presence of 0.25 or 0.5 mM palmitate-BSA / 1 mM L-carnitine, after which cell viability was measured by flow cytometry using propidium iodide. All data represent the mean ± SD N=2;. Figures 7A-7D illustrate example high-resolution respirometry (HRR) oxigraphs. (7A-7B) PA-BSA-assisted respiration was measured in INS-1 (832 / 13) cells (7A) or C2C12 myotubes (7B) on day 5 by treatment with FAO inhibitors (avocatin B or etomoxir) in the presence of 0.25 mM PA-BSA for 24 hours, after which the cells were trypsinized, collected, and injected into the respirometer. (7C-7D) To assess metabolic flexibility, INS-1 (832 / 13) (7C) cells or C2C12 (7D) myotubes from day 5 were treated with FAO and PA-BSA inhibitors as described above, after which the cells were trypsinized, collected and subjected to a 15 min incubation with 10 mM Dglucose before being washed and injected into the respirometer; Figures 8A–8B illustrate the determination of mitochondrial mass. INS-1 (832 / 13) (8A) or C2C12 myotubes (8B) were treated for 24 hours with either 0.5% BSA / 1 mM L-carnitine (Control), 0.5 mM palmitate-BSA / 1 mM L-carnitine (PA), PA + 25 mM AVO (AVO + PA), PA + 100 pM etomoxir (ETO + PA), or AVO or ETO alone, in low-glucose medium (5.5 mM). Cells were then collected and stained with NAO stain to determine mitochondrial mass. Data represent the means ± SEM of three independent experiments, *p < 10 0.05; compared to the control group PA- or PA+, Dunnett's post hoc test; Figure 9 illustrates a method of preparation for clinical trial formulations of avocatin B; Figure 10 illustrates the CONSORT participant flowchart; and Figures 11A-11N illustrate the complete blood count with differential (CBC w / dif) in whole blood collected from all clinical trial participants on day 0, day 30, and day 60 (endpoint). (HA) Hemoglobin. (11B) Hematocrit. (11C) Red blood cells (RBCs). (11D) Mean corpuscular volume. (HE) Mean corpuscular hemoglobin. (11F) Mean corpuscular hemoglobin concentration. (11G) Red blood cell distribution width. N=3-10 / group, One-way Kruskal-Wallis range analysis of variance. (11H) White blood cells. (11I) Neutrophils. (11J) Lymphocytes. (11K) Monocytes. (11L) Eosinophils. (11M) Basophils. (UN) Platelets. DETAILED DESCRIPTION OF THE INVENTION Several uses or methods are described below to provide an example of one embodiment of each claimed invention. No embodiment described below limits any claimed invention, and any claimed invention may cover uses or methods that differ from those described below. The claimed inventions are not limited to uses or methods that have all the features of any use or method described below or to features common to multiple or all of the uses or methods described below. A use or method described below may not be an embodiment of any claimed invention.Any invention described in a use or method described below that is not claimed herein may be the subject of another instrument of protection, e.g., a continuation patent application, and the applicants, inventors, or owners do not intend to abandon, waive, or dedicate to the public any such invention by describing it herein. Diet-induced obesity (DIO) is a central risk factor for the development of metabolic complications such as insulin resistance, type 2 diabetes (T2D), and cardiovascular disease.

[0042] The mechanisms by which lipotoxicity (i.e., elevated free fatty acids, FFAs) leads to metabolic dysfunction remain a subject of debate. For example, Randle and colleagues reported that metabolic flexibility, which is the ability of tissues to adapt fuel oxidation to substrate availability, may be impaired in obese and diabetic patients. Based on this, Randle proposed that insulin resistance may be driven by elevated rates of skeletal muscle fatty acid oxidation (FAO; or β-oxidation).In support of this, it has been reported that transgenic mice engineered to increase flow through the FAO develop insulin resistance, whereas transgenic mice with limited FAO capacity challenged with high-fat diets (HFDs) have normal insulin sensitivity.|l2,38, j91. Furthermore, HFDs have been reported to cause insulin resistance in rodents while increasing skeletal muscle mitochondrial biogenesis and β-oxidation|33, 461, where the induced alterations in skeletal muscle mitochondrial structure and function occur only after the onset of insulin resistance|471. Other studies have indicated that nutrient overload causes pathologies as a result of high rates of incomplete FAO (which produce lipotoxic acylcamins) and / or increases in oxidative stress (i.e., excessive ROS production)[1,121. Incomplete mitochondrial FAO has been reported to be a factor in skeletal muscle insulin resistance, with excess β-oxidation in the postprandial state leading to incomplete FAO, which produces lipotoxic acylcamins that impair carbohydrate utilization, deplete organic intermediates of the tricarboxylic acid (TCA) cycle, and generate reactive oxygen species (ROS)|12151. Others have questioned the link between insulin resistance and elevated FAO rates.17-11 For example, accelerating FAO as a means of processing excess FFA has been proposed as a therapeutic strategy to overcome nutrient overload.140-45 These studies have challenged the traditional view of the Randle cycle in skeletal muscle and have suggested that impaired glucose oxidation may be a direct result of defective insulin-stimulated glucose uptake, neither of which is related to increased FAO. In the pancreas, the site of insulin synthesis and secretion, lipotoxicity has been reported to disrupt the glucose-fatty acid cycle in β cells (16, 17), which has been reported to affect glucose-stimulated insulin secretion (GSIS), resulting in hyperinsulinemia in vitro and in vivo (16, 18-20). Furthermore, the oxidative capacity of skeletal muscle has been reported to exceed the energy demands of resting muscle,1481 and despite reductions in mitochondrial content observed in obese and diabetic patients, insulin-resistant skeletal muscle has been reported to have normal mitochondrial function (49, 50). Despite the progress made to date in the development of treatment for DIO and associated pathologies, there is room for improvement in addressing the problems and shortcomings mentioned above. It is an objective of this description to overcome or mitigate at least one of the disadvantages mentioned above and to provide a new therapy for DIO and associated pathologies. The FAO pathway has been reported to be modifiable through targeted drugs, and pharmacological agents that directly or indirectly inhibit the FAO pathway have demonstrated a wide range of protective benefits in DIO.[24, 25, 34, 53] As such, FAO inhibition may be a potential therapeutic strategy in DIO;[21, 27] however, to date, safe and well-tolerated small molecules that effectively target this metabolic pathway are lacking. For example, although ETO has been used as an FAO inhibitor, it can impart dose-limiting toxicities.[51, 52] These dose-limiting toxicities preclude the clinical use of most FAO inhibitors.Ranolazine, a partial FAO inhibitor used to treat angina pectoris, was recently approved for use in combination therapies for obesity and diabetes; however, its beneficial activity is primarily in the liver through a mechanism unrelated to FAO.27,34,541 Furthermore, these pharmacological agents have no activity in pancreatic tissue, the site of insulin secretion. Lee et al. reported that the avocado-derived lipid avocatin B (AvoB) is an FAO inhibitor that can accumulate in mitochondria to induce apoptosis in leukemic cells without imparting toxicity to normal cells.1281 However, there are differences between leukemic cells and other normal cells that can affect their behavior. For example, leukemia cells may have a larger mitochondrial mass (see PMID: 22094260) and altered metabolic characteristics that distinguish them from normal cells, such as a greater reliance on oxidative phosphorylation (22094260), amino acid metabolism (30753831), and fatty acid oxidation (20038799).Therefore, targeting these metabolic disturbances can have different effects on different cell types and can, for example, cause the death of leukemic cells and not the death of normal cells (22094260; 30420752; 30753831; 23333149; 31287994; 29892070). EXPERIMENTAL EXAMPLES The preferred embodiments of the present description will be described with reference to the following example information, which should not be used to limit or interpret the invention. Methods Preclinical studies in mice For the treatment study, twelve-week-old male C57BL / 6J mice were acquired from the Jackson Laboratory (Bar Harbor, ME) and allowed to acclimate for 1 week. After acclimation, the mice received either a high-fat diet (HFD) (60% kcal from lard; Research Diet DI2492; Research Diets, USA) or a standard diet (10–13% kcal from fat; Teklad 2014; Envigo, USA) for eight weeks. At the end of the eighth week, the animals continued their respective diets but were administered AvoB (100 mg / kg body weight) or vehicle via gavage twice weekly for 5 weeks (see Figure 1A). AvoB was formulated in a self-emulsifying drug delivery system (SEDDS) as previously described1291 where the oil phase (composed of surfactant and oil) was less than 10% (v / v) of the final oil-in-water emulsion. Animal weights were monitored twice weekly.At the end of the study, the animals were euthanized (10-12 h in their dark cycle) via CO2 followed by exsanguination, after which tissue and blood were collected. Glucose and insulin tolerance tests were performed. Intraperitoneal glucose and insulin tolerance tests (GTT and ITT) were performed 8 hours after food withdrawal. For GTT, a glucose dose of 1.5 g / kg was used for mice on the high-fat diet (HFD) and standard diets. For ITT, insulin doses (Humulin, Eli Lilly, USA) of 1 U / kg and 0.25 U / kg were used for mice on the HFD and standard diets, respectively. Blood glucose levels were determined at 0, 10, 20, 30, 60, and 90 minutes after glucose / insulin administration by tail bleeding using OneTouch Blood Ultra 2 glucose meters (LifeScan Europe, Switzerland). Measurement of complete blood count in plasma and tissue biochemical markers The University of Guelph Animal Health Laboratory performed a complete blood count with differential (CBC with differential) on 500 ml of whole blood. Plasma insulin, free fatty acids (FFAs), and triacylglycerols (TAGs) were measured using a rat / mouse insulin ELISA kit (Millipore, Rat / Mouse Insulin Detection kit), a fluorometric FFA assay kit (Cayman, MI, USA), and a colorimetric TAG assay kit (Cayman, MI, USA), respectively. Manufacturer protocols were followed for all kits. Calculations of the insulin resistance index (HOMA-IR) The homeostasis model assessment was used to calculate the insulin resistance index (HOMA-IR)60 using fasting plasma glucose (FPG) and fasting plasma insulin (FPI) values ​​as follows: with FPG expressed as mg / dl and FPI as mU / 1; HOMA-IR=FPGxFPI / 22.5, with FPG expressed as mmol / 1 and FPI as mlU / 1. Pyruvate dehydrogenase (PDH) activity assay After sacrificing the mice, the muscle tissues were freeze-held and flash-frozen in liquid nitrogen. PDH activity from flash-frozen whole gastrocnemius muscle lysates was measured using a colorimetric microplate assay kit (ab 109902, Abeam, Cambridge, MA, USA) following the manufacturer's protocol and as previously described (see reference number 12). Briefly, PDH proteins from whole gastrocnemius muscle lysates (lysed in the presence of 10 mM sodium fluoride (NaF) to preserve the phosphorylated state of PDH or to determine native PDH activity as a result of HFD feeding) were immunocaptured onto a microplate, with equal amounts of protein loaded for each sample.Next, PDH activity was determined spectrophotometrically by monitoring the reduction of NAD+ to NADH, along with the reduction of a reporter stain to an absorbance of 450 nm. All animal studies were conducted in accordance with the standards of the Canadian Council for the Care of Animals (CCAC) and with the approval of the University of Guelph's Animal Care Committee. In vitro studies Cell culture The cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C. The INS-1 (832 / 13) rat pancreatic beta cell line was cultured in RPMI 1640 medium containing 11.1 mM glucose and supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 mM beta-mercaptoethanol. INS-1 (832 / 13) cells are a robust culture model of beta cells that is routinely used due to their glucose responsiveness and endogenous insulin production. C2C12 mouse skeletal myoblasts were cultured in growth media consisting of Dulbecco's modified low glucose Eagles medium (DMEM; Hyclone, ThermoFisher) supplemented with 10% FBS and 1% penicillin / streptomycin.The differentiation of C2C12 myoblasts into myotubes was induced by transferring 90% of the confluent cells to differentiation media consisting of low-glucose DMEM supplemented with 2% horse serum and 1% penicillin / streptomycin. The differentiation media were changed every 24 hours for up to 5 days before all experimental treatments. By day 5 of the differentiation process, the myoblasts were fully differentiated into myotubes, as determined by morphological assessment and immunoblotting for myogenin, a skeletal muscle-specific protein (Figure 6C). Cell viability. Quantitative analysis of cell death was assessed using flow cytometry with propidium iodide staining (Biovision, Mountainview, CA), where specified, according to the manufacturer's protocol and as described above28. Viable cells were identified as PI negative (PI-). High-resolution respirometry Cells treated with the test compounds for 24 h were trypsinized, counted, and resuspended in phosphate-buffered saline (PBS). High-resolution O2 consumption measurements were performed in 2 mL of respiration medium (PBS, pH 7.5, stirring speed 750 rpm) using the Oroboros Oxygraph-2k (Oroboros Instruments, Corp., Innsbruck, Austria) set at 37 °C with a gain of 2 in both chambers. Briefly, after the injection of 5 million treated INS-1 (832 / 13) cells or C2C12 myotubes per chamber, basal respiration was measured once steady-state respiratory flow was achieved; this represents coupled respiration fueled by available substrates from the incubation with the test compounds. Next, uncoupled respiration was measured after the addition of the ATP synthase inhibitor oligomycin (0.25 mM).This was followed by measurement of the maximum respiration rate by stepwise titration of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; within 0.1–0.25 mM) in the presence of 1 mmol / L of pyruvate. Finally, respiration was inhibited by the addition of rotenone (0.5 mM; complex I inhibitor) to obtain the residual extramitochondrial oxygen consumption. Data were recorded using DatLab software (Oroboros Instruments, Innsbruck, Austria). Oxygen consumption at each stage of the protocol was normalized to the vehicle control and illustrated in histograms. For all HRR protocols, cell viability was determined after trypsinization and also after the end of the HRR runs by staining the cells with trypan blue and counting them using a hemocytometer. No significant differences in cell viability were observed between the control and treated cells. C2C12 myoblasts or INS-1 cells (832 / 13) were treated in low glucose media with AvoB (25 mM) or etomoxir (100 mM) in the presence of PA 0.25 mM / carnitine 1 mM for 24 h before collection and injection into the respirometer to assess PA-assisted respiration. Additionally, to indirectly assess metabolic flexibility, cells were treated in the same manner as described, collected, and subjected to a 15 min treatment with D-glucose 10 mM before washing and injection into the respirometer; these experiments were performed to assess whether cells incubated under low glucose / high fat conditions for 24 h can readily switch to glucose utilization following acute incubation with D-glucose. Fatty acid oxidation The FAO in cell lines was determined using established methods. Briefly, cells were pretreated with AvoB (25 mM) or etomoxir (100 mM) in the presence of 0.5 mM PA / 1 mM L-carnitine for 24 h, after which they were washed with PBS and subjected to [1-14C]-palmitate radiolabeling for 3 h. The [1-14C]-palmitate was prepared and applied to the cells as follows: purchased stock supplied in ethanol (PerkinElmer, MA, USA) was vacuum dried and then resuspended in a solution containing an unlabeled vehicle in BSA to produce a mixture of 7% [w / v] BSA, 2.5 mM palmitate with [1-14C]-palmitate at a final concentration of 10 pCi / ml.After a 16-hour incubation at 37°C, the solution containing radiolabeled palmitate was diluted with the respective serum-free cell culture media (RPMI low glucose medium for INS-1 cells (832 / 13) and DMEM low glucose medium for C2C12 myotubes) containing 1 mM L-carnitine to a final concentration of 0.3% BSA, 100 mM palmitate, and 0.4 pCi / ml [l-14C]-palmitate. After incubation of the [1-I4C]-palmitate-treated cells, 400 pl of well medium were transferred to acidification vials containing perchloric acid and capped with CO2 filter traps impregnated with 1 M NaOH. The vials were then incubated at room temperature for one hour to allow CO2 capture and precipitation of undigested palmitate.The CO2 trap was transferred to scintillation vials containing 4 mL of scintillation fluid, and the complete FAO was quantified using a Tri-Carb 2910 TR liquid scintillation analyzer (PerkinElmer). The amount of radioactivity in the medium was also determined after cell and debris sedimentation (14,000 x g, 10 min); these quantified acid-soluble metabolites (ASMs) are an indirect measure of incomplete FAO. Oxidation and uptake of glucose C2C12 myotubes and INS-1 (832 / 13) cells were treated as in the FAO assays described above and then incubated with D-[14C(U)]-glucose (0.58 pC / ml) and unlabeled glucose (final glucose concentration 200 mM) for 4 h, after which [14C]CO2 (representing complete glucose oxidation) and ASM (representing incomplete glucose oxidation) were captured and quantified as described in the FAO method. For C2C12 myotubes, glucose oxidation and uptake were measured with and without a 30 min pre-incubation with 100 nM insulin to determine basal and insulin-mediated glucose oxidation or uptake, respectively. To measure glucose uptake in C2C12 myotubes, the fluorescent D-glucose analog 2(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy-D-glucose (2-NBDG) was used. Briefly, myotubes were treated for 24 hours with the test compound, after which they were washed twice with PBS and starved for 1 hour in serum and glucose-free DMEM. After starvation, 100 nM insulin was added over 30 minutes, followed by 60 mM 2-NBDG added to each well and incubated for 30 minutes. The uptake reaction of 2-NBDG was then stopped by removing the incubation medium and washing the cells twice with PBS, after which the cells were trypsinized and collected for flow cytometry analysis (Guava 8HT; EMD Millipore, Billerica, MA). Glucose-stimulated insulin secretion (GSIS) in INS-1 cells (832 / 13) To measure glucose-stimulated insulin secretion (GSIS) in INS-1 (832 / 13) cells, a standard protocol was adopted. Briefly, cells were cultured to 80% confluence in 6-well plates and treated with either AvoB or etomoxir with or without palmitate, as described for the FAO assay. After treatment was completed, the cell medium was replaced with KRB (Krebs-Ringer bicarbonate) buffer. Cells were then exposed to 3 mM glucose for 2 h in KRB buffer, after which 500 mL of KRB buffer were collected and frozen at -80 °C until analysis. For a further 2 h, cells were exposed to 16 mM glucose, after which 500 mL of KRB buffer were collected and frozen at -80 °C until analysis. The amount of insulin released in the KRB buffer was determined using an ELISA kit (Millipore, Rat / Mouse Insulin Detection kit).Data were normalized for cellular protein content, as determined by the MicroBCA protein assay kit. The glucose-dependent insulin secretory index (GSIS 16 / 3), defined as the ratio of insulin secretion at 16 mM (stimulatory, surrogate for postprandial glucose levels) to 3 mM (basal, surrogate for fasting glucose levels), was also calculated. Reactive oxygen species, mitochondrial membrane potential, and determination of mitochondrial mass For all ROS studies, INS-1 (832 / 13) cells and C2C12 myotubes were treated with or without 18 μL palmitate and FAO inhibitors for 24 h in low-glucose cell culture media as described for the FAO assay. After treatment, cells were trypsinized and resuspended in PBS containing 5 mM of the fluorescent dye MitoSOX® (Molecular Probes, Invitrogen) and incubated for 5–15 min in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were then washed in PBS, loaded into a 96-well plate, and fluorescence was measured by flow cytometry. ROS were quantified by normalizing the mean fluorescence values ​​of live, activated cells to the vehicle control. The mitochondrial membrane potential (MMP) in INS-1 (832 / 13) and C2C12 myotubes was measured using the JC-1 cationic fluorescent stain (Molecular Probes).JC-1 accumulates in the mitochondrial matrix in an MMP-dependent manner. JC-1 fluoresces green in the cytosol where it exists as a monomer. After entering the mitochondria, JC-1 monomers form J aggregates and fluoresce red; therefore, the red / green fluorescence ratio is used to determine MMP. After all treatments, cells were washed, trypsinized, and resuspended in PBS containing 1 mM JC-1 and incubated for 5–10 minutes in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were then washed in PBS, loaded into a 96-well plate, and fluorescence was measured by flow cytometry. MMP was quantified by normalizing the mean fluorescence values ​​of live, activated cells to the vehicle control.Mitochondrial mass was measured using 10-N-nonyl acridine orange (NAO; Enzo Life Sciences, ENZ-52306), which accumulates in mitochondria independently of MMPs, where it binds to cardiolipin in the inner mitochondrial membrane. Cells were treated, washed, and collected as described and incubated with 0.35 mM NAO. Green fluorescence was measured using flow cytometry. In all analyses (ROS, MMPs, and mitochondrial mass), front-versus-side scatter plots were blocked to exclude debris (e.g., dead cells), as cell death is associated with increased ROS and altered membrane potentials. Cellular ATP content Total cellular ATP content was determined using the ATP Bioluminescent Assay Kit (Calbiochem, 119107). Briefly, cells were seeded into 96-well white-walled plates (1.2 x 10⁴ cells / well for INS-1 and 2 x 10⁴ cells / well for C2C12) and treated for 24 hours (C2C12 cells were differentiated for 5 days prior to treatment, while INS-1 cells were treated 24 hours after seeding). After 19 hours of treatment, the culture media were removed, and the cells were lysed with nucleotide release buffer by gentle shaking for 5 min. The ATP tracking enzyme was then added to the lysates and cell plates, and the luminescence was read using a Biotek Synergy HT spectrophotometer (Biotek; Winooski, VT). Determination of cytosolic calcium INS-1 (832 / 13) cells were treated as described for FAO assays but also with the metabolic modulators dichloroacetate (DCA) (1 mM) and trimetazidine (25 pM), as well as 10 pM of the endoplasmic reticulum calcium blocker 8-(N,N-diethylamino)-octyl3,4,5-trimethoxybenzoate HCI (TMB-8) (Sigma Aldrich) or 25 nM cyclosporine (Sigma Aldrich) in the presence of 0.5 mM palmitate / 1 mM L-carnitine. Cytosolic calcium was measured in INS-1 (832 / 13) cells using a fluorescent stain loaded with fluo-3AM (Invitrogen, F1241) that accumulates in the cytosol and emits green fluorescence after binding to calcium ions. A 10 mM fluo-3AM stock solution was diluted to 5 pM in fluo-3AM loading buffer (10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 137 mM NaCl, 5 nM KCl, 1 mM Na2HPO4, 5 mM glucose, and 0.5 mM MgCl2 (pH 7.4)).After treatment, the cells were incubated in fluo-3AM loading buffer for 30 min at 37 °C with gentle shaking, after which the cells were washed twice with PBS, trypsinized, and collected for flow cytometry analysis. Cytosolic calcium was quantified by taking the mean green fluorescence values ​​of live, activated cells and normalizing them to the vehicle control. Immunoblotting analysis For all cell culture experiments, after drug treatments, cells were washed with chilled phosphate-buffered saline (PBS) and lysed in chilled lysis buffer containing protease inhibitor cocktail Ix (Sigma). RIPA lysis buffer (Sigma-Aldrich) was used for INS-1 cells (832 / 13), and muscle lysis buffer was used for C2C12 myotubes (20 mM HEPES, 10 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 20% glycerol, and 0.1% Triton-X100). Cells were scraped, mechanically homogenized using a syringe, centrifuged at 14,000 x g for 20 min at 4 °C, and the supernatants were collected. For the preparation of all gastrocnemius muscle lysates, 10 mg of frozen tissue (wet weight) were washed in chilled PBS, placed in a Dounce homogenizer, lysed in muscle lysis buffer, and centrifuged at 14,000 x g for 20 min at 4 °C, and the supernatant was collected.The protein content for cell and tissue lysates was measured using the BCA protein assay kit (ThermoFisher) according to the manufacturer's protocol. Thirty pg of protein lysates were prepared in loading buffer and immunoblotted by heating the lysates for 5 minutes at 95 °C and subjecting them to gel electrophoresis on 10% SDS-polyacrylamide gels at 150 V for 75 minutes. Using a semi-dry transfer apparatus (Bio-Rad), the gels were transferred at 25 V for 45 minutes to a PVDF membrane and blocked with 5% BSA (Sigma) in Tris-buffered saline (TBS-T) for 1 hour. The membrane was incubated overnight with the target primary antibody and a loading control primary antibody (GAPDH or α-tubulin) (1:15,000 or 1:1,000; ThermoFisher) at 4 °C.The following primary antibodies were used at the specified dilutions: pAKTSer473 (9271, 1:500, 60 kDa), AKT (9272, 1:1000, 60 kDa), pAMPKa-Thrl72 (2531, 1:500, 62 kDa), AMPKa (2532, 1:1000, 62 kDa), pERKl / 2-Thr202 / Tyr204 (4370, 1:500, 42 and 44 kDa), Erkl / 2 (4695, 1:1000, 42 and 44 kDa), pP38Thrl80 / Tyrl82 (4511, 1:500, 43 kDa) and P38 (8690, 1:1000, 40 kDa) from Cell Signaling Myogenin (F5D, 1:200, 34 kDa) from Developmental Studies Hybridoma Bank, PGCla (ab54481, 1:500, 105 kDa), and CPT1A (abl28568, 1:500, 88 kDa) were acquired from Abeam. PVDF membranes were then washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) for 1 hour at room temperature. Bands were visualized using enhanced clarity chemiluminescence (ECL) substrates (Bio-Rad) and the ChemiGenius 2 bioimaging system (Syngene).The approximate molecular weight of each protein was estimated using the Precision Plus Protein WesternC marker (Bio-Rad) which was subjected to electrophoresis and transferred to the membrane. Statistical analysis Unless otherwise stated, in vitro results are presented as mean + SEM, while in vivo results are presented as mean ± SD. Data were analyzed using GraphPad Prism 6.0 (GraphPad Software, USA) with one-way or two-way ANOVA and Bonferroni or Dunnett post hoc analysis for between-group comparisons. Standard Student's t-tests were also used where appropriate. P < 0.05 was considered statistically significant. The normality of all datasets was verified using the Shapiro-Wilk normality test in GraphPad Prism 6.0, and nonparametric tests were used to analyze some data, as specified in the Figure legends. Pilot clinical study of oral consumption of avocatin B A single-center, randomized, double-blind, placebo-controlled, pilot clinical study was conducted to determine the safety of consuming either 50 mg (equivalent to consuming a quarter of a Hass avocado pulp) or 200 mg (equivalent to consuming one Hass avocado pulp) of AvoB per day for 60 days in healthy human participants (NCT03898505). Preparation of the AvoB and placebo supplements The investigational product was approved by Health Canada (Natural Product Number (NPN) 80074296). To create the product, freeze-dried avocado pulp powder was obtained from Avocado Oil New Zealand Ltd. (Tauranga, New Zealand) and analyzed for absolute amounts of AvoB per gram of powder using a validated analytical method.61 The clinical trial material was then standardized to contain either low (50 mg) or high (200 mg) doses of AvoB (Figure 9). The placebo product was formulated similarly to the investigational product, except that it contained only the non-medicinal, food-grade ingredients used in the test product and other excipients used to simulate the appearance, odor, texture, and taste of the investigational product.Participants in each group consumed 40 g of the material per day for 60 days by dissolving / mixing the product into 12–16 ounces of a shake as a dilute (e.g., milk (with or without lactose), soy milk, coconut milk, or fruit juice of the participant's choice). The three products were approximately equal in total calories per serving. Table 1 summarizes the nutritional composition of each product and establishes the macro and micronutrient composition of the AvoB clinical trial formulations. Component Placebo Low Dose High Dose Calories (kcal) 83 130 147 Protein (g) 3.2 4.4 2.9 Total Fat (g) 4.2 7.1 9.4 Saturates (g) 0.8 1.3 1.4 Carbohydrates (g) 5.5 8.2 7.8 Dietary Fiber (g) 2.3 3.8 4.8 Sodium (mg) 153 179 23.5 Cholesterol (g) 0 0 0 Potassium (mg) 306 563 869 1.3. 2. Study participants and study objectives Healthy volunteers (13 men and 17 women; age range 20-54 years, BMI range 19.1-29.9) were enrolled in the study. Exclusion criteria included the presence of active clinical disease and a history of diabetes, hypertension, dyslipidemia, major depressive disorder, chronic liver disease, kidney disease, or blood disorders. Volunteers who were pregnant, had a history of bariatric surgery, or were actively using prescription or nonprescription medications that impact weight gain or loss were also excluded. Baseline participant characteristics are highlighted in Table 1. All participants provided written informed consent for enrollment in the study and for participation in all study-related protocols. The primary outcome was the analysis of adverse experiences (AEs) measured throughout the study.Secondary outcomes were safety and tolerability, assessed through clinically relevant changes in standard laboratory tests (blood chemistry and hematology) measured at days 0, 30, and 60. Body mass index (BMI) and glycated hemoglobin (HbA1c) were also measured as secondary outcomes. Laboratory tests included serum alanine aminotransferase (ALT) to assess liver function; serum creatinine to assess kidney function; serum creatine phosphokinase to assess muscle injury; total serum bilirubin to assess blood cell lysis; complete blood count to assess overall health; and HbA1c as a secondary outcome.All AEs were rated by the study investigators according to their severity and relationship to the study drug, as described in the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. All blood collection and analysis was completed at a private diagnostic laboratory (LifeLabs, Canada). 1.3.3. Statistical analysis The mean change from baseline on days 30 and 60 for all laboratory assessments and body weight was analyzed using a non-parametric one-way Kruskal-Wallis ANOVA using GraphPad Prism 6.0 (GraphPad Software, USA), where differences with p<0.05 were considered statistically significant. Antibiotic effects (AAs) were tabulated (for all randomized patients who received at least one dose of the study supplements) and analyzed using Fisher's exact test of independence in the University Edition of the Statistical Analysis System (SAS), where differences with p<0.05 were considered statistically significant. All study protocols were approved by the University of Guelph's Research Ethics Board (REB) and Health Canada's Natural and Nonprescription Health Products Directorate (NNHPD). Results AvoB inhibits FAO and improves glucose tolerance and insulin sensitivity after DIO is established in mice AvoB was first evaluated in vivo in a conventional treatment model of DIO. C57BL / 6J mice received either a standard diet (STD; 10–13% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) for a total of 12 weeks. At week 8, AvoB (100 mg / kg body weight) or the control vehicle was administered via oral gavage twice weekly for a total of 5 weeks (Figure 1A). At the end of the study, HFD mice treated with AvoB weighed significantly less than control HFD mice (Figure IB; t(16) = 3.84, p < 0.01), demonstrating reductions in gonadal and mesenteric fat pad (GFP and MFP) weights (Figure 1C: GFP: t(16) = 3.34; p < 0.01; MFP: t(16) = 2.50; p < 0.05). AvoB had no effect on blood markers of toxicity (Figures 5A–5I). HFD mice treated with AvoB had significantly improved glucose tolerance compared to control HFD mice (Figures ID and 1E; F(2.25) = 51).27; p < 0.0001) and also had increased insulin sensitivity (Figure 1F and 1G; F(2.23) = 6.37, p < 0.01). The homeostasis model assessment index for insulin resistance (HOMA-IR; an index of insulin action) was increased in control HFD mice (Figure 1H), whereas AvoB-treated HFD mice had HOMA-IR index values ​​comparable to those of lean, STD mice (Figure 1H; F(2.25) = 17.22; p < 0.0001). AvoB treatment also increased plasma levels of FEA (Figures 11A–11N; U = 0, p < 0.001) and triacylglycerols (TAGs) (Figure 1J; U = 0, p < 0.001).001) in ad libitum mice compared to ad libitum HFD control, which together are indirect indicators of whole-body decreased FAO and increased glucose utilization, as previously reported [24, 25, 31]. Plasma levels of FFA and triacylglycerol in ad libitum lean mice on standard diets were found to be higher than those in HFD mice due to significant differences in the carbohydrate and fat content of the two diets. Muscle pyruvate dehydrogenase (PDH; a mitochondrial enzyme that facilitates the oxidation of pyruvate to acetyl-CoA) activity is suppressed in DIO due to an excess of FAO that disrupts the balance between fatty acid and glucose oxidation

[30] . Given its role in measuring metabolic flexibility in obese and diabetic patients [31, 32] and in mouse models of DIO

[33] , native PDH activity was quantified in skeletal muscle removed from ad libitum mice at the endpoint. HFD control mice had decreased gastrocnemius muscle PDH activity consistent with elevated FAO. Conversely, PDH activity in HFD mice treated with AvoB was restored to that of lean STD mice (Figure 1K; F(2.25) = 9.235, p<0.001) suggesting greater glucose oxidation in HFD mice treated with AvoB.In addition to increased PDH activity, HFD mice treated with AvoB also had increased phosphorylation of AKTSer473, a key marker of insulin signaling, in the gastrocnemius muscle compared to HFD control mice (Figure 1L). Furthermore, HFD control mice had higher postprandial glucose utilization and higher levels of CPT-1A protein in the gastrocnemius muscle compared to Avo-treated HFD mice, which circumvented higher rates of fatty acid uptake (FAO) in HFD control mice (Figure 1L). Taken together, these results suggest that AvoB inhibited whole-body FAO and altered the substrate preference of skeletal muscle from fatty acids to glucose, thereby reversing insulin resistance in a DIO treatment model. AvoB inhibits FAO during lipotoxicity in pancreatic islet cells, thereby enhancing glucose-stimulated insulin secretion. To determine the mechanism by which AvoB enhances mitochondrial lipid overload (i.e., lipotoxicity), the ability of AvoB to inhibit FAO in pancreatic B cells (INS-1(832 / 13)) was examined. This was done in the presence of excess lipids that cause mitochondrial dysfunction but not cell death (i.e., >250 mM palmitate-BSA (PA-BSA) with 1 mM L-carnitine for 24 hours; referred to herein as lipotoxic conditions) (Figures 6A-6D). In B cells, AvoB inhibited complete FAO, as determined by measuring the oxidation of [l-14C]-palmitate to CO2 (Figure 2A; F(3.16) = 46.76; p<0.0001), and did not increase levels of acid-soluble metabolites (ASMs), an indicator of incomplete FAO. (Figure 2A; F(1.16)= 55.59; p < 0.0001).FAO inhibition under lipotoxic conditions was also tested using high-resolution respirometry (HRR), where AvoB inhibited basal and maximal uncoupled palmitate-supported respiration in INS-1 (832 / 13) cells (Figure 2B; F(6.24)=14.44; p < 0.0001; see Figure 7A for example of HRR oxigraphs). AvoB imparted this activity at concentrations four times lower than etomoxir (ETO), a conventional FAO inhibitor used as a positive control, and had no effect on cell viability (Figure 6A). To determine whether AvoB-induced FAO inhibition could increase glucose utilization in INS-1 cells, the oxidation of D-[14C(U)]-glucose to CO2 was measured. Under lipotoxic conditions, AvoB resulted in greater glucose oxidation compared to cells treated with palmitate alone (Figure 2C; F(3.8) = 77.25, p < 0.0001). We confirmed this effect using HRR, where B cells were incubated under high-fat / low-glucose conditions in the presence or absence of AvoB for 24 hours and then challenged with 10 mM glucose for 15 minutes before injection into the respirometer. The addition of glucose, in the presence of AvoB, increased basal and peak respiration compared with palmitate-only control (Figure 2D; F(2.24) = 10.81; p < 0.0004; see 7C for HRR oxigraphs). These results demonstrate that AvoB inhibits FAO and increases glucose utilization in pancreatic tissue. Lipotoxicity desensitizes islet cells to glucose, resulting in reduced GSIS and hyperinsulinemia; features of obesity and insulin resistance.16,18-201 To better understand the mechanism by which AvoB-induced FAO inhibition improves insulin sensitivity in vivo, we determined its effect on GSIS, mitochondrial superoxide generation, cellular ATP levels, and cytosolic calcium levels in INS-1 cells. Lipotoxicity reduced GSIS (Figure 2E; p < 0.0001); a phenotype that was rescued in the presence of AvoB (Figure 2E; p < 0.001; Figure 2F; p < 0.001). AvoB also reduces palmitate-induced increases in mitochondrial superoxide levels (Figure 2G; F(2.12) = 11.69, p = 0.0015); and mitochondrial membrane potential (MMP) (Figure 2H; F(1.12) = 109.9, p = 0.001).AvoB restores total cellular ATP content in palmitate-challenged cells compared to control levels (Figure 21; F(9.20) = 8.52, p < 0.0001). Together, these results are indicative of AvoB's ability to restore oxidative metabolism and improve the efficiency of the electron transport system. Chronic exposure to excess palmitate can disrupt calcium homeostasis and lead to calcium leakage from the endoplasmic reticulum into the cytosol, thereby disrupting GSIS1361. Consistent with these findings, palmitate-treated cells exhibited increased cytosolic Ca2+ levels under non-stimulatory glucose conditions, where AvoB, ETO, and the endoplasmic reticulum calcium blocker TMB-8 were able to reduce cytosolic Ca2+ (Figure 2J; F(7.16) = 23.08, p < 0.0001). These results directly support the observation that GSIS lipotoxicity was increased in the presence of FAO inhibitors (i.e., AvoB 26 and ETO). Finally, it was determined that the effects of AvoB on INS-1 cells were independent of AMP-activated protein kinase (AMPK) activation (Figure 2K) and mitochondrial biogenesis, which was assessed through PGC-α protein levels (Figure 2K) and / or the fluorescent dye NAO (Figure 8A).Taken together, these results demonstrate that AVO acts as a metabolic modulator to restore GSIS in pancreatic islet cells under lipotoxic conditions by inhibiting FAO, which enhances glucose utilization, mitochondrial oxidative stress, and calcium homeostasis. AvoB inhibits FAO during lipotoxicity in C2C12 myotubes, thereby enhancing insulin signaling. Skeletal muscle has been reported to account for approximately 70% of whole-body glucose clearance

[371] ; therefore, the interaction between pancreatic insulin secretion and skeletal muscle glucose utilization is a key factor in whole-body insulin sensitivity. Similar to INS-1 cells, AvoB inhibited complete FAO in C2C12 myotubes (Figure 3A; F(3.20) = 24.64; p < 0.0001) and did not increase incomplete FAO as assessed by ASM levels (Figure 3A; F(1.20) = 61.69; p < 0.0001). FAO inhibition in C2C12 myotubes was further confirmed with HRR where AvoB inhibited basal and maximal palmitate-supported uncoupled respiration (Figure 3B; F(6.24) = 6.074; p < 0.001; see Figure 7B for HRR oxigraphs).Consistent with known FAO inhibition effects, AvoB increased basal and insulin-stimulated glucose oxidation in C2C12 myotubes in the presence of excess palmitate, as measured by the oxidation of D-[14C(U)]glucose to CO2 (Figure 3C; F(3.16) = 5.284, p < 0.01) and HRR (Figure 3D; F(2.24) = 6.978, p < 0.004; see Figure 7D for HRR oxigraphs). Incomplete FAO products have been shown to directly inhibit insulin signaling in skeletal muscle|12, 381, therefore, we evaluated whether AvoB-induced FAO inhibition under lipotoxic conditions in C2C12 myotubes would restore or enhance insulin signaling. AvoB was able to restore the lipotoxicity-suppressing effects on insulin-stimulated AKTSer473 phosphorylation (Figure 3E; F(3.16)= 11.58; p<0.0003), and also reduced palmitate-induced phosphorylation of the mitogen-activated protein kinases (MAPKs): ERK1 / 2 and P38 (Figure 3E).Consistent with the improvement in glucose oxidation and the reduction in the attenuation of insulin signaling under lipotoxic conditions, AVO also increased glucose uptake compared to palmitate-treated cells alone, as measured by using the fluorescent glucose analogue, 2-NDBG, 27 which accumulates via the dominant glucose transporter of skeletal muscle, GLUT 41391 (Figure 3F; F(2, 17) = 16.03; p < 0.001). Similar to observations in INS-1 cells, AvoB reduces palmitate-induced increases in mitochondrial superoxide levels (Figure 3G; F(2.12) = 4.644, p < 0.05) and MMP (Figure 3H; F(1.12) = 83.56, p < 0.0001) in C2C12 myotubes. AVO also restored cellular ATP content to that of control cells (Figure 31; F(9, 20) = 2.951, p = 0.021), suggesting the restoration of mitochondrial function under lipotoxic conditions. According to previous literature on metabolic modulation in DIO, the pyruvate dehydrogenase kinase inhibitor, dichloroacetate (DCA)1401, restored cellular ATP content (Figure 31) and insulin signaling (Figure 3J), while the partial FAO inhibitor trimetazidine (TRI) did not exert such effects13. Finally, the effects of AvoB on C2C12 myotubes were determined to be independent of AMPK activation (Figure 3K) or mitochondrial biogenesis (Figure 3K; Figure 8B). Overall, AvoB reduces the negative impact of lipotoxicity on insulin signaling in C2C12 myotubes by improving mitochondrial function and increasing glucose uptake and oxidation. AvoB was well tolerated in a pilot clinical study A Phase I pilot study (NCT03898505) was conducted to determine the safety of general use of AvoB. In this study, 50 or 200 mg (equivalent to consuming a quarter or one portion of Hass avocado pulp) of AvoB / day or placebo was consumed for 60 days, and serological and physiological assessments were measured at baseline (day 0) and on days 30 and 60 (see Figure 4A for the study scheme and Figure 9 for the supplement description). Table 2 summarizes the baseline characteristics of participants in the placebo, low dose, and high dose groups at baseline. Table 2 Baseline characteristics Placebo Low-dose avocatin B (50 mg / day) High-dose avocatin B (200 mg / day) 5 Number of participants 10 10 10 Male-to-female ratio (%) 60:40 40:60 30:70 Mean age (years) 30.3 ± 10.1 24.4 ± 3.9 30.5 ± 11.4 Mean body weight (kg) 74.4 ± 14.8 73.4 ± 15.0 69.8 ± 11.2 Mean BMI (kg / m2) 24.3 ± 3.8 25.0 ± 3.6 22.3 ± 1.4 Average hours of exercise / week (h) 4.6 ± 2.9 5.4 ± 3.8 4.6 ± 2.4 Table 3 summarizes the adverse events (AEs) in all groups. Analysis of adverse events (AEs) revealed that AvoB was generally well tolerated, with 15 minor gastrointestinal problems observed in all groups. Table 3 Adverse Events Placebo Low-dose avocatin B (50 mg / day) High-dose avocatin B (200 mg / day) 20 Number of Participants 10 10 10 Abdominal distension / bloating (Grade G) 4 (40%) 5 (50%) 4 (40%) Diarrhea (Grade G) 1 (10%) 1 (10%) 1 (10%) Nausea (Grade G) 2 (20%) 2 (0%) 3 (30%) 25 Hand and foot rash (Grade I) 0 (0%) 0 (0%) 3 (30%)* *p <u.u5 trente a placeboThree participants in the high-dose group developed a skin rash two weeks after supplementation. While this resolved after a one-week interruption of supplementation, one participant resumed supplementation until day 30, while two withdrew from the study before day 30 (see Figure 10 for the CONSORT diagram of trial participant flow). AvoB supplementation had no effect on several 29 blood markers of renal, hepatic, and muscular toxicity (Figures 4B–4E) or complete blood counts (Figures 11A–11IN) compared with placebo. Levels of all safety markers were within normal reference ranges as reported by the external diagnostic laboratory (LifeLabs Canada) that performed the blood analysis.A trend was also observed in which decreases in body weight were seen in both AvoB-supplemented groups between baseline and day 30 compared to placebo (Figure 4F). Although the trend in weight change was not statistically significant due to the small sample size (p=0.10), future, larger Phase 11 efficacy studies may link human supplementation to the observed preclinical activity of AvoB on body weight. No clinically relevant changes from baseline were observed in body mass index or glycated hemoglobin (HbA1c) in either AvoB-treated group compared to placebo (Figures 4G-4H). Overall, the results of this pilot clinical study highlight the translational relevance, potential bioactivity, and favorable safety profile of AvoB. Discussion High dietary fat intake has been associated with insulin resistance; however, the mechanism by which excess lipids cause this condition is still debated. Some studies have pointed to mitochondria as a potential therapeutic target for combating insulin resistance. While we do not wish to be bound to any particular theory or mode of action, the results described herein suggest a shift in metabolic substrate preference as an important mediator of insulin sensitivity. Furthermore, the results described herein support the idea that excess FAO contributes to the pathophysiology of HFD-induced insulin resistance and that FAO inhibition is a strategy in the management of DIO-induced insulin resistance. The results described herein demonstrate that FAO inhibition following DIO-induced mitochondrial dysregulation is a viable therapeutic strategy for improving, and even restoring, insulin sensitivity. AvoB's ability to inhibit FAO, reduce ROS, and enhance glucose oxidation under lipotoxic conditions highlights the Randle cycle as an important mediator of insulin resistance. The interaction between insulin secretion from pancreatic beta cells and glucose uptake by skeletal muscle is often overlooked in the scientific literature, likely due to the more prominent role of skeletal muscle in systemic glucose absorption. ma / Using the examples described herein, we examined the effects of small-molecule-induced FAO inhibition on glucose oxidation or insulin secretion in pancreatic tissue. In human subjects, AvoB was found to accumulate in both skeletal muscle (soleus and gastrocnemius) and the pancreas following oral administration. While we do not wish to limit ourselves to any particular theory or mode of action, these results suggest that AvoB inhibits excess FAO in beta cells, thereby enhancing mitochondrial glucose oxidation and restoring GSIS (i.e., through improved mitochondrial function and reduced ER calcium leakage). These improvements in pancreatic beta cell function, along with the enhanced insulin sensitivity in skeletal muscle, likely combine to contribute to the beneficial whole-body insulin response and glucose tolerance observed in vivo.AvoB did not affect incomplete FAO in C2C12 and INS-1 myotubes. AvoB also blocked palmitate-induced mitochondrial-derived ROS and did not affect incomplete FAO. This further supports the favorable mechanism of action of AvoB reported here, given previous work suggesting that ROS reduction or incomplete FAO may be relevant for reversing insulin resistance in DIO Y12· '5|. Collectively, inhibition, rather than acceleration, of FAO through AvoB treatment resulted in increased glucose utilization and reversed insulin resistance. AvoB was well tolerated in the mouse and human studies described herein. For example, in the Phase I study, where healthy human participants were administered the maximum permitted dose, AvoB did not exhibit toxicity but did demonstrate bioactivity. AvoB was found to impart activity in both skeletal muscle and pancreatic tissue and provides a link between FAO inhibition and insulin sensitivity through increased glucose utilization and ROS modulation. These results support the use of AvoB as a clinically relevant alternative to conventional FAO inhibitors that impart dose-limiting toxicities, such as ETO, or to pharmacological agents that have little or no reported activity in pancreatic tissue and / or have a mechanism of action unrelated to FAO, such as ranolazine. The pharmaceutical compositions of the invention can be formulated for oral administration. Therefore, the pharmaceutical compositions of the invention can be formulated, for example, as tablets, capsules, powders, granules, or liquid preparations, such as oral solutions or suspensions. Such pharmaceutical formulations can be prepared by any method known in the pharmaceutical art, for example, by combining the active ingredient with the carrier(s) or excipient(s). Tablets and capsules for oral administration may be in unit-dose presentations and may contain conventional excipients such as binding agents, for example, syrup, gum arabic, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tablet lubricants, for example, magnesium stearate, talc, polyethylene glycol, or silica; disintegrants, for example, potato starch; or acceptable wetting agents such as sodium lauryl sulfate. Tablets may be coated according to methods well known in normal pharmaceutical practice. Liquid oral preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle prior to use.Liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan, monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oil esters such as glycerin, propylene glycol or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring agents or colorings. It should be understood that, in addition to the ingredients specifically mentioned above, formulations may include other conventional agents in the technique, depending on the type of formulation in question. Although this invention has been described with reference to illustrative embodiments and examples, this description is not intended to be restrictive. Therefore, various modifications of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to those skilled in the art upon reference to the description. It is therefore contemplated that the appended claims will cover any such modifications or embodiments. 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[53] . Ussher, JR, Fillmore, N., Keung, W., Zhang, L., Morí, J., Sidhu, VK, Fukushima, A., Gopal, K., Lopaschuk, DG, Wagg, CS, Jaswal, JS, Dyck, JRB, Lopaschuk, GD, Diabetes 2016, 1855, 83.

[54] , Batran, R. Al, Gopal, K., Aburasayn, H., Eshreif, A., Almutairi, M., Greenwell, AA, Campbell, SA, Sáleme, B., Court, EA, Eaton, F., Light, PE, Sutendra, G., Ussher, JR, JCI Insight, 2019, 2019. 11. The method of claim 1, further comprising increasing glucose oxidation in the pancreatic tissue in the subject. 12. The method of claim 1, further comprising increasing glucose oxidation in skeletal muscle tissue in the subject. 13. The method of claim 1, further comprising reversing insulin resistance in the subject. 14. The method of claim 1, further comprising increasing insulin sensitivity in the subject. 15. The method according to any one of claims 1 to 14, wherein the therapeutically effective amount of avocatin B is administered in at least one daily dose. 16. The method according to claim 15, wherein the at least one daily dose comprises from approximately 25 mg to approximately 200 mg of avocatin B. 17. The method according to claim 15, wherein the at least one daily dose comprises approximately 50 mg of avocatin B. 18. Use of a therapeutically effective amount of avocatin B to treat a disease or condition characterized by a metabolic disorder in a subject who needs it. 19. The use of claim 18, wherein the metabolic disorder is characterized by dysregulation of glucose-stimulated insulin secretion (GSIS) in the subject. 20. The use of claim 18, wherein the metabolic disorder is characterized by insulin resistance in the subject. 21. The use of claim 18, wherein the metabolic disorder is characterized by a reduced sensitivity to insulin in the subject. 22. The use of any one of claims 18 to 21, wherein the disease or condition is diet-induced obesity. 23. The use of claim 18, wherein the metabolic disorder is characterized by obesity-associated lipotoxicity in the subject and the method further comprises rescuing glucose-stimulated insulin secretion (GS1S) in the subject. 24. The use of claim 18, further comprising increasing glucose utilization in the subject. 25. The use of claim 18, further comprising modulating reactive oxygen species in the subject. 26. The use of claim 18, further comprising increasing glucose uptake in pancreatic tissue in the subject. 27. The use of claim 18, further comprising increasing glucose uptake in skeletal muscle tissue in the subject. 28. The use of claim 18, further comprising increasing glucose oxidation in pancreatic tissue in the subject. 29. The use of claim 18, further comprising increasing glucose oxidation in skeletal muscle tissue in the subject. 30. The use of claim 18, further comprising reversing insulin resistance in the subject. 31. The use of claim 18, further comprising increasing insulin sensitivity in the subject. 32. Use according to any one of claims 18 to 31, wherein the therapeutically effective amount of avocatin B is administered in at least one daily dose. 33. Use according to claim 32, wherein the at least one daily dose comprises from approximately 25 mg to approximately 200 mg of avocatin B. 34. Use according to claim 32, wherein the at least one daily dose comprises approximately 50 mg of avocatin B. 35. A pharmaceutical composition comprising: a therapeutically effective amount of avocatin B and a carrier. 36. The composition of claim 35, wherein the therapeutically effective amount comprises from approximately 25 mg to approximately 200 mg. 37. The composition of claim 35, wherein the therapeutically effective amount comprises approximately 50 mg. SUMMARY A method for treating a disease or condition may be characterized by a metabolic disorder in a subject who needs it, which may include administering to the subject a therapeutically effective amount of avocatin B. 2 / 12 ML / t / ZUZZ / UjyZÓZ Figure 2K AMFh PGC-lo GAFO 1 3 / 12 4 / 12 Figure 4A Placebo Avocatina B [Xais Baja Avocatin B High Dose Time (day): N = 10 ► N-'O » NzQ N=10 ► N='O • N-'CN = 10 ► N-8 ·· N-6 0 1 30 60 * * * *tndua the conipiubaciún I* punish lina the* pci ñuños and scvundai ios Figure 4B Figure 4C Figure 4D Figure 4E - Figure 4F Figure 4G Figure 4H 5 / 12 Figure 5A Figure 5B Figure 5C HFD HFD*AVO Figure 5D8Figure 5E 12-, u ' Έ04"2-□5,. έ02o-1---------1----------1----------------------1--------- 0 0-11— STD HFD HFD*AVO gTD Figure 5F HFD HFD*AVO HFD*AvO Figure 5H Figure 51 HFD HFDMVO O 07„ 0 06°C5X. 0.04· £ 0.035 0 025 001ce 00· 0.01 STD 750ra or __ g. 500- O 250—i------------------1--------- 0-1---------1------------------1------------------1------HFD HFD-AVO STD HFD HFD'AVO 6 / 12 Figure 6A Figure 6B o · PA-BSA o * PA-BSA (250uM) a ♦ PA-BSA (500 uM) Figure 6C Day 0 5 6 Myogenin (33-__ kD) Figure 6D α-Tubulin (50 kD) o - PA-BSA o + PA-BSA (250uM) a + PA-BSA (500uM) Μ Λ / E / ZUZZ / UO3ZOZ 8 / 12 Figure 8A Figure 8B Mean change in fluorescence with respect to control (PA-)

Claims

1. A method for treating a disease or condition characterized by a metabolic disorder in a subject in need, the method comprising administering to the subject a therapeutically effective amount of avocatin B.

2. The method of claim 1, wherein the metabolic disorder is characterized by dysregulation of glucose-stimulated insulin secretion (GSIS) in the subject.

3. The method of claim 1, wherein the metabolic disorder is characterized by insulin resistance in the subject.

4. The method of claim 1, wherein the metabolic disorder is characterized by reduced insulin sensitivity in the subject.

5. The method according to any one of claims 1 to 4, wherein the disease or condition is diet-induced obesity.

6. The method of claim 1, wherein the metabolic disorder is characterized by obesity-associated lipotoxicity in the subject and the method further comprises rescuing glucose-stimulated insulin secretion (GSIS) in the subject.

7. The method of claim 1, further comprising increasing glucose utilization in the subject.

8. The method of claim 1, further comprising modulating reactive oxygen species in the subject.

9. The method of claim 1, further comprising increasing glucose uptake in the pancreatic tissue in the subject.

10. The method of claim 1, further comprising increasing glucose uptake in skeletal muscle tissue in the subject.

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