Composition for reducing the level of triglycerides in the liver and / or improving metabolism

Abstract

FIELD: medicine.SUBSTANCE: use of a dairy composition containing branched-chain amino acid metabolites selected from the group consisting of alpha-hydroxyisocaproic acid (HICA), alpha-hydroxyisovaleric acid (HIVA), 2-hydroxy-3-methylvaleric acid (HMVA) and mixtures thereof, for reducing liver triglyceride levels and / or improving glucose metabolism in a subject, wherein said branched-chain amino acid metabolites are metabolites of lactic acid bacteria formed as a result of milk fermentation.EFFECT: reduction in liver triglyceride levels and / or improvement in glucose metabolism in a subject.4 cl, 2 dwg

Description

[0001] FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to a composition for lowering the level of triglycerides in the liver and / or improving glucose metabolism in a subject.

[0003] STATE OF THE ART

[0004] Type 2 diabetes mellitus (T2DM) is a disease characterized by insulin's failure to properly metabolize glucose due to abnormal glucose uptake by skeletal muscle and impaired suppression of blood sugar production by the liver, which paradoxically promotes the accumulation of triglycerides in the liver. This combination leads to numerous health risks and disorders, including high blood sugar and non-alcoholic fatty liver disease (NAFLD).

[0005] The incidence of T2DM and NAFLD is increasing to epidemic proportions, with the latest figures reaching over 463 million and 62 million people worldwide, respectively, and it is well known that the main causes of their development are poor diet and lifestyle.

[0006] Therefore, there is a need to find means to reduce liver triglyceride levels and / or improve glucose metabolism, particularly for the prevention or treatment of NAFLD and T2DM.

[0007] SUMMARY OF THE INVENTION

[0008] The present invention is a result of the inventors' unexpected discovery that oral administration of yogurt containing branched-chain amino acid (BCAA) metabolites correlates with a reduction in liver triglyceride levels and an improvement in glucose metabolism and insulin sensitivity.

[0009] Specifically, the authors demonstrated that yogurt consumption affects liver metabolism, significantly increasing levels of branched-chain hydroxy acids (h-BCAA or BCHA), which correlates with improved metabolic parameters. These BCAA metabolites are formed during milk fermentation and are concentrated in yogurt. Interestingly, diet-induced obesity reduces h-BCAA levels in plasma and tissues, a reduction partially prevented by diet yogurt consumption. Furthermore, the authors demonstrated that h-BCAAs improve insulin action on glucose metabolism in liver and muscle cells, identifying h-BCAAs as cell-autonomous regulators of metabolism and potential mediators of yogurt's health effects.

[0010] Thus, the present invention relates to branched-chain amino acid metabolites or a composition containing branched-chain amino acid metabolites for use in lowering liver triglyceride levels and / or improving glucose metabolism.

[0011] In embodiments, the present invention provides branched-chain amino acid metabolites or a composition comprising branched-chain amino acid metabolites for use in lowering liver triglyceride levels, glycemia (preferably fasting glucose) and / or insulinemia (preferably fasting insulin).

[0012] The present invention also relates to the use of a composition containing branched-chain amino acid metabolites for reducing liver triglyceride levels and / or improving glucose metabolism.

[0013] In embodiments, the invention provides branched-chain amino acid metabolites or a composition comprising branched-chain amino acid metabolites for use in the treatment or prevention of insulin resistance, diabetes mellitus, in particular T2DM, hepatic steatosis and / or (NAFLD).

[0014] In another aspect, the present invention also relates to a method for reducing the level of triglycerides in the liver and / or improving glucose metabolism by administering to a subject in need thereof branched-chain amino acid metabolites or a composition comprising branched-chain amino acid metabolites.

[0015] In another aspect, the present invention also relates to the use of branched-chain amino acid metabolites or a composition containing branched-chain amino acid metabolites for the production of a food supplement or a medicament for lowering liver triglyceride levels and / or improving glucose metabolism.

[0016] The term "dietary supplement" as used herein shall be understood to mean a product that is intended to be consumed in addition to the normal diet of a subject for nutritional purposes and / or to provide a health benefit such as the prevention or treatment of diseases or disorders.

[0017] The term "probiotic supplement" used herein shall be understood to mean a food supplement containing probiotic varieties for supplementing the diet with probiotics.

[0018] As assumed here, the expression "x% (w / w)" is considered equivalent to "x g per 100 g".

[0019] The term "at least" as used herein also includes the initial value of the open range. For example, the amount "at least 95.00% w / w" means any amount equal to or greater than 95.00 percent by weight.

[0020] The term "about" used herein defines a range of plus or minus 10% of the stated value. For example, a quantity of "about 20% by weight" means any quantity between 18% and 22% by weight.

[0021] As intended herein, the term "milk composition" refers to a milk-based composition suitable for animal consumption, in particular for human consumption.

[0022] The term "HICA" as used herein should be understood to refer to alpha-hydroxyisocaproic acid.

[0023] The term "HIVA" as used herein should be understood to refer to alpha-hydroxyisovaleric acid.

[0024] The term "HMVA" as used herein should be understood to refer to 2-hydroxy-3-methylvaleric acid.

[0025] The term "h-BCAA" as used herein refers to the hydroxy metabolites of branched-chain amino acids (i.e., branched-chain hydroxy acids or "BCHA") and should be considered collectively as referring to HICA, HIVA, and HMVA, along with the enantiomers, racemic mixtures, salts, esters, or hydrates thereof.

[0026] "Diet-induced weight gain" and "diet-induced insulin resistance" are defined herein as weight gain and insulin resistance resulting from excess food intake, including excess dietary fat intake, in particular unsaturated fat, and, optionally, excess dietary sugar intake, including sucrose and fructose. For a given subject, excess food intake, in particular fat and, optionally, simple sugars, refers to the consumption of an amount of food, in particular fat and, optionally, simple sugars, in excess of the amount necessary to meet the physiological needs and maintain energy balance of said subject.The effect of treatment on reducing or preventing diet-induced weight gain and insulin resistance in a subject can be assessed by comparing the weight gain and insulin resistance observed in a subject receiving treatment with the weight gain and insulin resistance observed in the same untreated subject consuming the same diet and having the same level of physical activity.

[0027] The term "reducing body weight gain" as used herein means limiting, reducing or decreasing the body weight increase induced by a given diet, as defined above, in a subject compared to the body weight increase induced by said diet in said subject, but who will not consume h-BCAA according to the invention.

[0028] The term "improving insulin resistance" as used herein means normalizing or reducing the insulin resistance index induced by a given diet, as defined above, in a subject compared to the insulin resistance index induced by said diet in said subject, but who will not consume h-BCAA according to the invention.

[0029] Tests for assessing insulin resistance in a subject are known in the art. A subject's insulin resistance index can be measured using any insulin resistance test known in the art, such as the homeostatic model assessment of insulin resistance (HOMA-IR) or, more classically, the gold standard euglycemic hyperinsulinemic clamp.

[0030] In a preferred embodiment of the present invention, weight gain and insulin resistance are induced by (i.e., associated with) a high-fat diet (HFD) in said subject.

[0031] In a preferred embodiment of the present invention, weight gain and insulin resistance are induced by (i.e., associated with) a high sucrose (HS) diet in said subject.

[0032] In a preferred embodiment of the present invention, weight gain and insulin resistance are induced by (i.e., associated with) a high-fat, high-sucrose (HFHS) diet in said subject.

[0033] The term "glucose metabolism" as used herein should be understood to include glucose homeostasis, glucose regulation, or insulin sensitivity.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides branched-chain amino acid metabolites for use in lowering liver triglyceride levels and / or improving glucose metabolism.

[0036] Preferably, said branched-chain amino acid metabolites are h-BCAA. Preferably, said h-BCAA is selected from the group consisting of HICA, HIVA, HMVA, and combinations thereof. In the most preferred embodiment, said h-BCAA is a combination of HICA, HIVA, and HMVA. In the uses or methods of the present invention, said h-BCAA is a metabolite of lactic acid bacteria during the fermentation of a dairy product.

[0037] In embodiments, the invention provides branched chain amino acid metabolites for use in lowering liver triglyceride levels, glycemia (preferably fasting glucose), and insulinemia (preferably fasting insulin).

[0038] Accordingly, in one embodiment, the present invention provides a composition comprising branched-chain amino acid metabolites for use in lowering liver triglyceride levels and / or improving glucose metabolism, preferably glycemia (preferably fasting glucose), insulinemia (preferably fasting insulin).

[0039] In embodiments, the invention provides branched-chain amino acid metabolites for use in the treatment or prevention of insulin resistance, diabetes mellitus, in particular T2DM, hepatic steatosis and / or non-alcoholic fatty liver disease.

[0040] Subject

[0041] A "subject" according to the invention is a mammal, preferably a human. The subject according to the invention may be obese or overweight and / or suffer from a disease or disorder such as insulin resistance, prediabetes, diabetes mellitus, in particular type 2 diabetes, hepatic steatosis (i.e., fat accumulation in the liver), and / or non-alcoholic fatty liver disease. In some embodiments, the subject exhibits diet-induced weight gain and / or diet-induced insulin resistance.

[0042] In embodiments, the subject exhibits high-fat diet-induced weight gain and / or high-fat diet-induced insulin resistance. In embodiments, the subject exhibits high-sucrose diet-induced weight gain and / or high-sucrose diet-induced insulin resistance. In embodiments, the subject exhibits HFHS diet-induced weight gain and / or HFHS diet-induced insulin resistance.

[0043] In embodiments, the invention provides branched chain amino acid metabolites for use in lowering liver triglyceride levels and / or improving glucose metabolism.

[0044] In embodiments, the invention provides branched chain amino acid metabolites for use in lowering liver triglyceride levels, glycemia (preferably fasting glucose), and insulinemia (preferably fasting insulin).

[0045] In embodiments, the invention provides branched-chain amino acid metabolites for use in the treatment or prevention of insulin resistance, diabetes mellitus, in particular T2DM, hepatic steatosis and / or non-alcoholic fatty liver disease.

[0046] Preferably, said branched-chain amino acid metabolites are h-BCAA. Preferably, said h-BCAA is selected from the group consisting of HICA, HIVA, HMVA, and combinations thereof. In the most preferred embodiment, said h-BCAA is a combination of HICA, HIVA, and HMVA. In the uses or methods of the present invention, said h-BCAA is a metabolite of lactic acid bacteria during the fermentation of a dairy product.

[0047] In embodiments, the branched chain amino acid metabolites are administered to or consumed by a subject in the form of a food composition, a nutraceutical composition, a food supplement, a probiotic supplement, or a nutritional composition.

[0048] In embodiments, the branched chain amino acid metabolites are administered to or consumed by the subject in the form of a dairy composition, more preferably milk or yogurt.

[0049] In embodiments, the branched chain amino acid metabolites are present in the composition at a total concentration of 0.1 μM to 1 mM, preferably 0.1 μM to 10 μM, more preferably 0.1 μM to 1 μM.

[0050] In embodiments, the branched chain amino acid metabolites are present in the composition at a total concentration of at least 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, or 1 mM.

[0051] In embodiments, HICA is present in the composition at a concentration of at least 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, or 1 mM.

[0052] In embodiments, HIVA is present in the composition at a concentration of at least 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, or 1 mM.

[0053] In embodiments, HMVA is present in the composition at a concentration of at least 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM, or 1 mM.

[0054] In embodiments, the branched chain amino acid metabolites are present in the composition as a mixture of HICA, HIVA and HMVA in a total concentration of at least 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 500 μM or 1 mM.

[0055] In embodiments, the branched chain amino acid metabolites are present in the composition as a mixture of HICA, HIVA and HMVA at a concentration of 0.1 μM to 1 mM, preferably 0.1 μM to 10 μM, more preferably 0.1 μM to 1 μM.

[0056] In embodiments, the branched chain amino acid metabolites are administered to or consumed by the subject weekly, preferably daily.

[0057] In embodiments, the branched chain amino acid metabolites are administered to or consumed by the subject at the same time.

[0058] Composition

[0059] The present invention provides a composition comprising branched-chain amino acid metabolites for use in lowering liver triglyceride levels and / or improving glucose metabolism in a subject.

[0060] Preferably, said branched-chain amino acid metabolites are h-BCAA. Preferably, said h-BCAA is selected from the group consisting of HICA, HIVA, HMVA, and combinations thereof. In the most preferred embodiment, said h-BCAA is a combination of HICA, HIVA, and HMVA. In the uses or methods of the present invention, said h-BCAA is a metabolite of lactic acid bacteria during the fermentation of a dairy product.

[0061] In embodiments, the invention provides a composition comprising branched-chain amino acid metabolites for use in reducing liver triglyceride levels, glycemia (preferably fasting glucose), insulinemia (preferably fasting insulin).

[0062] The composition of the invention is suitable for consumption or ingestion, preferably orally. Accordingly, the composition comprises or essentially consists of an edible substance. It is particularly preferred that the compositions of the invention be substantially free of pathogenic or toxigenic substances.

[0063] The composition of the invention may be a pharmaceutical composition, a food composition, a nutraceutical composition, a food supplement, a probiotic supplement and / or a nutritional composition.

[0064] In some embodiments, the supplement is in the form of tablets, powder, capsules, or any other form not typically associated with food products. If the composition of the invention is a probiotic or food supplement, it may also contain suitable fillers, flavorings, sweeteners, preservatives, and / or emulsifiers.

[0065] If the composition of the invention is a pharmaceutical composition or a food supplement, it may also contain at least one pharmaceutically acceptable excipient or carrier.

[0066] Nutritional compositions that can be used according to the invention include dairy products, preferably fermented dairy products. Fermented products can be in the form of a liquid or in the form of a dry powder obtained by drying a fermented liquid. Examples of dairy products include fermented milk and / or fermented whey in a thickened, broken-curd, or drinkable form, cheese, and yogurt. The fermented product can also be a fermented plant product, such as fermented soybeans, cereals, and / or fruits in a thickened, broken-curd, or drinkable form. Nutritional compositions that can be used according to the invention also include infant formula, first-stage infant formula, and second-stage infant formula. In a preferred embodiment, the fermented product is a fresh product.A fresh product that has not undergone intensive heat treatment has the advantage that the bacterial strains present are alive.

[0067] Milk composition

[0068] Preferably, the composition of the invention is a dairy composition, in particular a fermented dairy composition, containing a branched-chain amino acid metabolite.

[0069] Preferably, said branched-chain amino acid metabolites are h-BCAA. Preferably, said h-BCAA is selected from the group consisting of HICA, HIVA, HMVA, and combinations thereof. In the most preferred embodiment, said h-BCAA is a combination of HICA, HIVA, and HMVA. In the uses or methods of the present invention, said h-BCAA is a metabolite of lactic acid bacteria during the fermentation of a dairy product.

[0070] Preferably, the milk composition of the invention comprises or is obtained (in particular by fermentation) from a composition comprising from 30% to 100% (w / w) milk, more preferably from 50% to 100% (w / w) milk, and even more preferably from 70% to 100% (w / w) milk.

[0071] Also preferably, the dairy composition of the invention comprises or is obtained (in particular by fermentation) from a composition consisting essentially of milk or consisting only of milk. As intended herein, "milk" preferably refers to milk of plant or animal origin.

[0072] Preferably, the dairy composition of the invention comprises or is obtained (in particular by fermentation) from a composition comprising one or both types of skim or full-fat milk. Preferably, said milk or milks may be in liquid, powdered and / or concentrated form. In one embodiment, said milk or milks may be enhanced or enriched with additional milk components or other nutrients, such as, but not limited to, vitamins, minerals, trace elements or other micronutrients. Preferably, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition such as, but not limited to, yogurt.

[0073] As intended herein, a "fermented dairy composition" is obtained from a dairy composition of the invention by the acidifying action of at least one lactic acid bacterium, which may be contained in a ferment, inoculum, culture, or starter. More preferably, said dairy composition of the invention is obtained by the acidifying action of at least one, two, three, four, five, six, seven, or more strains of lactic acid bacteria. Accordingly, the "fermented dairy composition" contains at least one, two, three, four, five, six, seven, or more strains of lactic acid bacteria.

[0074] The lactic acid bacterium of the invention preferably belongs to the families Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae or Bifidobacteriaceae, and more preferably to the genera Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, Weissella or Bifidobacterium. More preferably, the lactic acid bacterium of the invention refers to Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckei, in particular L. delbruckeisupsb.bulgaricus or lactis, Lactobacillus diolivorans, Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus jensenii, Lactobacillus kunkeei, Lactobacillus mali, Lactobacillus nagelii, Lactobacillus paracasei, in particular:

[0075] K species L. paracaseisupsb.paracasei,Lactobacillus plantarum,Lactobacillus vini,Lactobacillus rhamnosus,Streptococcus thermophilus,Streptococcus lactis,Streptococcus raffinolactis,Streptococcus cremoris,Bifidobacterium adolescentis,Bifidobacterium animalis,Bifidobacteriumbifidum,Bifidobacterium breve,Bifidobacterium infantis,Bifidobacterium lactis or Bifidobacterium longum.

[0076] One or more lactic acid bacteria can be used to produce the fermented milk composition of the invention. Thus, in a preferred embodiment, multiple species of lactic acid bacteria, including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, are used to produce the fermented milk composition of the invention. In another embodiment, bacteria including Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium, and Lactococcus are used to produce the fermented milk composition of the invention. Accordingly, in one embodiment, the invention provides a fermented milk composition comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, which in another embodiment may further comprise Bifidobacterium and Lactococcus bacteria.

[0077] In a preferred embodiment, the lactic acid bacterium is a probiotic bacterium.

[0078] The expressions "fermented milk" and "yogurt" have the ordinary meanings defined herein and may in appropriate circumstances be used interchangeably, for example, a fermented milk composition containing Lactobacillus bulgaricus, Streptococcus thermophilus and other additional bacteria (e.g., probiotic strains) may be referred to as "fermented milk" or alternatively as "yogurt".

[0079] Methods for producing fermented dairy products, such as yogurts or their equivalents, are well known in the art. Typically, a fermented dairy product is obtained by culturing heat-treated (e.g., pasteurized) skim and / or full-fat milk with suitable microorganisms to provide a lower pH. The selection of suitable microorganisms (e.g., thermophilic lactic acid bacteria) is within the skill of the art and, for yogurt production, will typically include Lactobacillus bulgaricus (also known as Lactobacillus delbruckei subsp. bulgaricus) and Streptococcus thermophilus, optionally with additional microorganisms, such as, but not limited to, probiotic species or other species that can impart the desired organoleptic properties to the composition.

[0080] The dairy composition, in particular the fermented dairy composition, according to the invention may optionally further comprise secondary components such as fruits, vegetables, nutritive and non-nutritive sweeteners, cereals, flavorings, starch, thickeners, preservatives or stabilizers. Preferably, the dairy composition, in particular the fermented dairy composition, according to the invention should contain up to about 30% (w / w) of the said secondary components, for example up to about 10%, 15%, 20%, 25% (w / w).

[0081] Preferably, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition that contains, contains essentially or consists of milk that has been subjected to heat treatment at least equivalent to pasteurization, preferably said heat treatment is carried out before the preparation of the dairy composition or fermented milk composition.

[0082] Preferably, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition containing more than about 0.3 g per 100 g by weight of free lactic acid, more preferably, the invention provides a fermented dairy composition containing more than about 0.7 g or 0.6 g per 100 g by weight of free lactic acid. Preferably, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition that has a protein content at least equivalent to the protein content of the milk or milks from which it is obtained.

[0083] Preferably, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition that has a pH value equal to or lower than 5, more preferably from about 3.5 to about 4.5.

[0084] Preferably, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition that has a viscosity of below 200 mPa⋅s, more preferably below 100 mPa⋅s, and most preferably below 60 mPa⋅s at 10°C, at a shear rate of 64 s -1 In one embodiment, the dairy composition of the invention is a drinkable fermented dairy composition, more preferably a fermented dairy beverage, such as, but not limited to, a yogurt drink, kefir, and the like. In an alternative embodiment, the dairy composition of the invention is a fermented dairy composition, more preferably a fermented dairy composition, which is viscous. The term "viscous" as used herein should be understood to mean a solid or semi-solid product that can be consumed with a spoon or other utensil.

[0085] Preferably, the dairy composition, in particular the fermented dairy composition, according to the invention or the product according to the invention can also be stored at a temperature of from 1°C to 10°C.

[0086] A single serving of a dairy composition, in particular a fermented dairy composition according to the invention, more preferably a fermented dairy composition or product according to the invention, is preferably about 50 g, 60 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, 100 g, 105 g, 110 g, 115 g, 120 g, 125 g, 130 g, 135 g, 140 g, 145 g, 150 g, 200 g, 300 g or 320 g, or alternatively about 1 ounce, 2 ounces, 3 ounces, 4 ounces, 5 ounces, 6 ounces or 12 ounces by weight.

[0087] Preferably, the dairy composition, in particular the fermented dairy composition, of the invention, more preferably, the fermented dairy composition of the invention contains at least 10 6 , more preferably at least 10 7and most preferably at least 10 8 colony forming units (CFU) of the bacteria Lactobacillus bulgaricus (also called Lactobacillus delbruckei subsp. bulgaricus) and Streptococcus thermophilus according to the invention per gram (g) of the composition according to the invention, for example at least 5×10 6 Lactobacillus bulgaricus 5×10 6 Streptococcus thermophilus. Preferably, the composition of the invention also contains up to about 10 11 , more preferably at least 10 10 and most preferably at least 10 9 colony forming units (CFU) of the bacteria Lactobacillus bulgaricus (also called Lactobacillus delbruckei subsp. bulgaricus) and Streptococcus thermophilus per gram (g) of the composition of the invention.

[0088] Methods for producing fermented dairy products, such as yogurts or their equivalents, are well known in the art. Typically, a fermented dairy product is obtained by culturing heat-treated (e.g., pasteurized) skim and / or full-fat milk with suitable microorganisms to provide a lower pH. The selection of suitable microorganisms (e.g., thermophilic lactic acid bacteria) is within the skill of the art and, for yogurt production, will typically include Lactobacillus bulgaricus (also known as Lactobacillus delbruckei subsp. bulgaricus) and Streptococcus thermophilus, optionally with additional microorganisms, such as, but not limited to, probiotic species and / or other species that can impart the desired organoleptic properties to the composition. Fermented dairy products can be either thickened or broken-curd after fermentation.

[0089] Methods

[0090] The present invention also relates to a method for reducing liver triglyceride levels and / or improving glucose metabolism in a subject in need thereof, comprising administering to the subject an effective amount of a composition containing branched-chain amino acid metabolites.

[0091] In embodiments, the invention provides methods for treating or preventing insulin resistance, diabetes mellitus, in particular type 2 diabetes, hepatic steatosis and / or non-alcoholic fatty liver disease in subjects in need thereof.

[0092] Preferably, said branched-chain amino acid metabolites are h-BCAA. Preferably, said h-BCAA is selected from the group consisting of HICA, HIVA, HMVA, and combinations thereof. In the most preferred embodiment, said h-BCAA is a combination of HICA, HIVA, and HMVA. In the uses or methods of the present invention, said h-BCAA is a metabolite of lactic acid bacteria during the fermentation of a dairy product.

[0093] DESCRIPTION OF FIGURES

[0094] Fig. 1. The correlation between each liver BCHA level and metabolic parameters, i.e., fasting glucose (trend curve highlighted by solid gray line) and liver triglycerides (trend curve highlighted by dashed gray line). n=4-8. C1: low-fat, low-sucrose control diet; H1: high-fat, high-sucrose diet with casein substitute protein mixture; Y1: freeze-dried yogurt included in diet H.

[0095] Fig. 2. (A) HGP of FAO cells treated with 1 mM HICA:HIVA:HMVA mixture at a molar ratio of 1:1:0.5 under basal condition. (B) HGP of FAO cells treated with 1 mM HICA:HIVA:HMVA mixture at a molar ratio of 1:1:0.5 under insulin condition. (C) HGP of FAO cells treated with 0.5 and 1 mM HICA, leucine, or a combination of them under basal condition. (D) HGP of FAO cells treated with 0.5 and 1 mM HICA, leucine, or a combination of them under insulin condition. (E) Glucose uptake by L6 muscle cells treated with 0.1 or 1 μM HICA:HIVA:HMVA mixture at a molar ratio of 1:1:0.5 under basal condition. (F) Glucose uptake by L6 cells treated with 0.1 or 1 μM HICA:HIVA:HMVA mixture at a molar ratio of 1:1:0.5 under insulin condition. (G) Glucose uptake by L6 cells treated with 0.01, 0.1, or 1 μM HICA under basal condition. (H) Glucose uptake by L6 cells treated with 0.01, 0.1, or 1 μM HICA under insulin condition.*p˂0.05 compared to control, **p˂0.01 compared to control, ***p˂0.001 compared to control, kp˂0.05 compared to 0.5 mM Leu. $ p˂0.05 compared to Leu 1 mM, $$ p˂0.01 compared to 1 mM Leu, $$$ p˂0.001 compared to Leu 1 mM. For groups C, D, G, H, expressed as a multiple ratio, standard and basal insulin are considered as a reference and are indicated by a dotted line.

[0096] EXAMPLES

[0097] Background and objectives: Epidemiological studies show that yogurt consumption is associated with a reduced incidence of diabetes mellitus (T2DM). However, the mechanism by which yogurt consumption may prevent T2DM is unclear. The authors investigated the effect and mechanism of yogurt consumption in reducing the development of insulin resistance and type 2 diabetes in mice fed a high-fat, high-sucrose diet containing a protein mixture representative of the U.S. diet (HFHS-PM).

[0099] Materials and methods:

[0100] Yogurt was freeze-dried and included in the HFHS-PM diet (HFHS-PM+LYP; 4.8 kcal / g), accounting for 8% of daily energy intake. The control group was maintained 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 diet reference. Three independent experiments were conducted to measure glucose homeostasis and insulin resistance using either oral glucose tolerance tests and glucose-stimulated insulin response, or hyperinsulinemic euglycemic clamps associated with the indicator to determine whole-body insulin sensitivity and insulin action in the liver and peripheral tissues.

[0101] Cell culture and hepatic glucose production

[0102] FAO rat hepatocytes were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS. The cells were maintained in this medium for 48 h before treatment. They were then serum-separated overnight for 16 h with or without insulin (1 nM) and with HICA, HIVA, or HMVA product at concentrations of 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 treatments. Cell supernatant was collected, and glucose concentration was measured using an Amplex-Red glucose assay kit according 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.

[0103] Results:

[0104] We found that 12 weeks of yogurt consumption improved glucose homeostasis in HFHS-PM-fed mice, as evidenced by a reduction in fasting glucose (234 mice, mean difference -0.7 mmol / L, 95% CI: -1.05 to -0.28, two-tailed Student's t-test p<0.001) and insulin (235 mice, mean log-transformed data ratio 0.79, 95% CI: 0.69 to 0.93, p=0.006, pooled analysis of three experiments) levels. A single clamp study also confirmed that yogurt improved insulin sensitivity (52 mice; glucose infusion rate +3.6 mg / min / kg, p=0.02) and that the liver was the primary site of improvement in glucose metabolism (52 ​​mice; endogenous glucose production -1.9 mg / min / kg, p=0.06). Yogurt consumption also reduced hepatic steatosis.

[0105] Interestingly, the authors then discovered the effect of yogurt consumption on liver metabolism, revealing a new inverse correlation between the levels of many branched-chain amino acid metabolites and triglycerides in the liver, fasting insulin, and fasting glucose. BCAAs were inversely associated with fasting glucose, fasting insulin, and triglycerides in the liver. These BCAA metabolites were present in yogurt and formed during milk fermentation.

[0106] To determine the biological role of h-BCAA in the preventive effect of yogurt, the correlation of their content in the liver with the corresponding metabolic parameters was analyzed.

[0107] An inverse correlation was found between liver BCHA levels and fasting glucose levels (HICA r 2 =0.237, p=0.035; HMVA r 2 =0.370, p=0.016; HIVA r 2 =0.327, p=0.016), as well as the content of triglycerides in the liver (HICA r 2 =0.210, p=0.042; HMVA r 2 =0.485, p=0.002; HIVA r2 =0.451; p=0.002) (Fig. 1). In addition, there was a tendency for an inverse relationship between HMVA and HIVA in the liver and fasting insulin (HMVA r 2 =0.177 p=0.097; HIVA r 2 =0.255, p=0.070). Interestingly, HICA, HMVA, and HIVA are involved in the metabolism of branched-chain amino acids (BCAAs), as they are derived from leucine, isoleucine, and valine, respectively.

[0108] The inventors found that BCHA levels were decreased in H-fed obese mice and increased by yogurt treatment in the liver and, to a lesser extent, in the skeletal muscle of H-fed animals. We set out to test the hypothesis that BCHA may have a direct effect on the corresponding cells. First, using FAO liver cells and L6 muscle cells, we examined the effects of BCHA on hepatic glucose production (HGP) and glucose uptake, respectively. We found that a HICA:HIVA:HMVA mixture, used at a concentration of 1 mM and at a relative ratio similar to that in the freeze-dried yogurt product, simultaneously reduced basal glucose production and enhanced the suppressive effect of insulin in FAO cells (Fig. 2A and B). Furthermore, HICA dose-dependently inhibited basal and insulin-suppressed HGP and competed with the action of its BCAA precursor leucine on this metabolic process (Fig.2C and D). Furthermore, we found that the BCHA mixture, used at concentrations of 0.1-1 μM to match their plasma levels in yogurt-fed mice, also enhanced glucose uptake by L6 myocytes (Fig. 2E and F), and that these effects appeared to be predominantly explained by HICA, as determined in studies using individual BCHAs (Fig. 2G and H).

[0109] Overall, in vitro studies provide mechanistic evidence that BCHA and HICA in particular are cell-autonomous modulators of hepatic glucose production and muscle glucose uptake.

Claims

1. The use of a dairy composition containing branched-chain amino acid metabolites selected from the group consisting of alpha-hydroxyisocaproic acid (HICA), alpha-hydroxyisovaleric acid (HIVA), 2-hydroxy-3-methylvaleric acid (HMVA) and mixtures thereof, for reducing liver triglyceride levels and / or improving glucose metabolism in a subject, wherein said branched-chain amino acid metabolites are metabolites of lactic acid bacteria formed as a result of milk fermentation.

2. The use according to claim 1, wherein said subject is obese or overweight.

3. The use according to claim 1 or 2, wherein said composition is a nutraceutical composition, a food supplement, a probiotic supplement and / or a nutritional composition.

4. The use according to one of paragraphs 1-3, wherein said composition is a food composition.

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