COMPOSITION FOR REDUCING HEPATIC TRIGLYCERIDES AND/OR IMPROVING GLUCOSE METABOLISM

Abstract

The present invention relates to a composition comprising branched chain amino acid metabolites for use in reducing hepatic triglycerides and/or improving glucose metabolism in an individual.

Claims

1. A method for reducing hepatic triglycerides and/or improving glucose metabolism in an individual, comprising administering branched chain amino acid metabolites or a composition comprising branched chain amino acid metabolites to the individual.

2. The method according to claim 1, wherein said branched chain amino acid metabolites are branched chain hydroxy acids.

3. The method according to claim 1, wherein said individual is obese or overweight.

4. The method according to claim 1, wherein said composition is a pharmaceutical composition, a nutraceutical composition, dietary supplement, probiotic supplement and/or a nutritional composition.

5. The method according to claim 1, wherein said composition is a food composition.

6. The method according to claim 2, wherein said branched chain hydroxy acids are alpha-hydroxyisocaproic acid (HICA), alpha-hydroxyisovaleric acid (HIVA), 2-hydroxy-3-methylvaleric acid (HMVA), or mixtures thereof.

7. The method according to claim 2, wherein said individual is obese or overweight.

Description

DESCRIPTION OF THE FIGURES

[0088] FIG. 1. Correlations between each hepatic BCHA levels and metabolic parameters, i.e., fasting glucose (trend curve in solid grey line) and hepatic triglycerides (trend curve in dash grey line). n=4-8. C1: low-fat low-sucrose control diet; H1: high-fat high sucrose diet with a protein mixture replacing casein; Y1: lyophilized yogurt incorporated in H diet.

[0089] FIG. 2. (A) HGP of FAO cells treated with a 1 mM mixture of HICA: HIVA: HMVA at molar ratio 1:1:0.5 in basal condition. (B) HGP of FAO cells treated with a 1 mM mixture of HICA: HIVA: HMVA at molar ratio 1:1:0.5 in insulin condition. (C) HGP of FAO cells treated with 0.5 and 1 mM of HICA, leucine or combination of both in basal condition. (D) HGP of FAO cells treated with 0.5 and 1 mM of HICA, leucine or combination of both in insulin condition. (E) Glucose uptake of L6 muscle cells treated with a 0.1 or 1 μM mixture of HICA: HIVA: HMVA at molar ratio 1:1:0.5 in basal condition. (F) Glucose uptake of L6 cells treated with a 0.1 or 1 μM mixture of HICA: HIVA: HMVA at molar ratio 1:1:0.5 in insulin condition. (G) Glucose uptake of L6 cells treated with 0.01, 0.1 or 1 μM of HICA in basal condition. (H) Glucose uptake of L6 cells treated with 0.01, 0.1 or 1 μM of HICA in insulin condition.*p<0.05 versus control, **p<0.01 versus control, ***p<0.001 vs control, kp<0.05 versus Leu 0.5 mM, .sup.$ p<0.05 versus Leu 1 mM, .sup.$$ p<0.01 versus Leu 1 mM, .sup.$$$ p<0.001 versus Leu 1 mM. For panel C, D, G, H expressed in fold, basal and basal insulin are considered as a reference and represented by a dash line.

EXAMPLES

[0090] Background and Aims:

[0091] Epidemiological studies indicate that yogurt intake is associated with reduced incidence of diabetes (T2D). However, the mechanism by which yogurt consumption may prevent T2D is unclear. The Inventors investigated the effect and mode of action of yogurt consumption to reduce the development of insulin resistance and T2D in a mouse model fed high-fat high-sucrose diet that contains a protein mixture representative of US diet (HFHS-PM).

[0092] Materials and Methods:

[0093] Yogurt was lyophilized and incorporated into the HFHS-PM diet (HFHS-PM+LYP; 4.8 kcal/g) representing 8% of daily energy intake. The control group was kept on the HFHS-PM diet (4.8 kcal/g). A group of mice was also fed a low-fat low sucrose diet (LFLS-PM; 3.7 kcal/g) and used as a healthy reference. Three independent experiments were performed to measure glucose homeostasis and insulin resistance using either oral glucose tolerance tests and glucose-stimulated insulin response, or tracer-coupled hyperinsulinemic euglycemic clamps to determine whole-body insulin sensitivity as well as hepatic and peripheral tissue insulin action.

[0094] Cell Culture and Hepatic Glucose Production

[0095] FAO rat hepatocytes were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS. Cells were maintained in this medium 48 h before treatment. They were then serum-deprived overnight for 16 h with or without insulin (1 nM) and with HICA, HIVA or HMVA product at 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM. The cells were washed three times with PBS and incubated with phenol red- and glucose-free DMEM medium supplemented with 20 mM sodium L-lactate and 2 mM sodium pyruvate for 5 h with or without insulin and the indicated study treatment. Cell supernatant was collected, and glucose concentration was measured with the Amplex-Red Glucose assay kit accordingly to the manufacturer's instructions (Invitrogen). Cells were lysed with 50 mM NaOH, and protein concentration was determined using a BCA protein assay kit to normalize glucose production.

[0096] Results:

[0097] The Inventors found that yogurt intake at 12 weeks improves glucose homeostasis in HFHS-PM fed mice, as shown by reduced fasting glucose (234 mice, −0.7 mmol/I mean difference, 95% CI: −1.05 to −0.28, two-sided Student t-test p<0.001) and insulin levels (235 mice, 0.79 mean ratio of log-transformed data, 95% CI: 0.69 to 0.93, p=0.006, pooled summary analysis of three experiments). Single clamp study further validated that yogurt improves insulin sensitivity (52 mice; glucose infusion rate+3.6 mg/min/kg, p=0.02) and that the liver was the main site of improved glucose metabolism (52 mice; endogenous glucose production −1.9 mg/min/kg, p=0.06). Yogurt consumption also reduced hepatic steatosis.

[0098] Interestingly, the Inventors then found an impact of yogurt intake on the hepatic metabolome, revealing a novel inverse correlation between levels of many branched chain amino acid metabolites and hepatic triglycerides, fasting insulin and fasting glucose. BCHA were associated inversely with fasting glucose, fasting insulin and hepatic triglycerides. These BCAA metabolites were present in yogurt and generated upon milk fermentation.

[0099] In order to determine the biological role of h-BCAA in the preventive effect of yogurt, the correlation of their hepatic abundance with relevant metabolic parameters was analyzed.

[0100] An inverse correlation between BCHA hepatic levels and fasting glucose was found (HICA r.sup.2=0.237, p=0.035; HMVA r.sup.2=0.370, p=0.016; HIVA r.sup.2=0.327, p=0.016), as well as hepatic triglyceride content (HICA r.sup.2=0.210, p=0.042; HMVA r.sup.2=0.485, p=0.002; HIVA r.sup.2=0.451; p=0.002) (FIG. 1). In addition, a trend for an inverse association between hepatic HMVA and HIVA and fasting insulin was observed (HMVA r.sup.2=0.177 p=0.097; HIVA r.sup.2=0.255, p=0.070). Interestingly, HICA, HMVA and HIVA fall within the metabolism of branched chain amino acid (BCAA) as they are derived from leucine, isoleucine and valine, respectively.

[0101] The Inventors found BCHA to be reduced in H-fed obese mice and increased by yogurt treatment in the liver and to a lesser extent in skeletal muscle of H-fed animals. The Inventors elected to test the hypothesis that BCHA could exert direct effects in relevant cells. First, using FAO hepatic and L6 muscle cells, the Inventors investigated the effect of BCHA on hepatic glucose production (HGP) and glucose uptake, respectively. The Inventors found that a mixture of HICA:HIVA:HMVA used at 1 mM and at their relative ratio as in the lyophilized yogurt product reduced both basal glucose production and increased the suppressive effect of insulin in FAO cells (FIGS. 2A and B). Furthermore, HICA dose-dependently inhibited basal and insulin-suppressed HGP and competed the effect of its BCAA leucine precursor on this metabolic process (FIGS. 2C and D). Furthermore, the Inventors found that the BCHA mixture used at 0.1-1 μM concentrations to match their plasma levels in yogurt treated mice also increased glucose uptake in L6 myocytes (FIGS. 2E and F), and that these effects appeared to be mostly explained by HICA as determined from studies using individual BCHA (FIGS. 2G and H).

[0102] Overall, the in vitro studies provide mechanistic evidence that BCHA, and especially HICA, are cell-autonomous modulators of liver glucose production and muscle glucose uptake.