WO2023183767A1

WO2023183767A1 - Methods and compositions for increasing insulin sensitivity

Info

Publication number: WO2023183767A1
Application number: PCT/US2023/064696
Authority: WO
Inventors: José María LÓPEZ PEDROSA; Manuel MANZANO; Ricardo Rueda Cabrera
Original assignee: Abbott Laboratories
Priority date: 2022-03-21
Filing date: 2023-03-20
Publication date: 2023-09-28

Abstract

A method of increasing insulin sensitivity in a subject in need thereof comprises administering a nutritional composition comprising inositol, lysine, arginine, and beta-hydroxy-beta-methylbutyrate (HMB) to the subject. A nutritional composition comprises from about 0.01 to about 15 wt % HMB, from about 0.03 to about 40 wt % lysine, from about 0.02 to about 30 wt % arginine, and from about 0.01 to about 20 wt % inositol, all based on the weight of the nutritional composition.

Description

METHODS AND COMPOSITIONS FOR INCREASING INSULIN SENSITIVITY

FIELD OF THE INVENTION

[0001] The present invention relates to methods of increasing insulin sensitivity in a subject and to nutritional compositions which employ inositol, lysine, arginine, and beta-hydroxy-beta- methylbutyrate (HMB).

BACKGROUND OF THE INVENTION

[0002] Insulin is the pivotal hormone regulating cellular energy supply and macronutrient balance, directing anabolic processes of the fed state. Insulin is essential for the intracellular transport of glucose from the bloodstream into insulin-dependent tissues such as adipose tissue and muscle. In healthy individuals, skeletal muscle is essential for glucose clearance and is responsible for about 70-80% of glucose uptake from the bloodstream. Individuals with normal glucose tolerance (a normal ability to clear glucose from the blood) are highly sensitive to insulin in skeletal muscle, whereas obese individuals and individuals with type 2 diabetes are insulin resistance. Insulin resistance can be defined as a condition in which insulin’s target organs are resistant to its action, so that higher concentrations of this hormone are needed to obtain a normal glucose uptake. Therefore, insulin resistance is reflected by reduced glucose uptake into key insulin-sensitive tissues such as skeletal muscle, liver and adipose tissue.

[0003] The predominant consequence of insulin resistance is type 2 diabetes mellitus (T2DM), often referred to more simply as type 2 diabetes. Insulin resistance is thought to precede the development of T2DM by 10 to 15 years, and the development of insulin resistance typically results in a compensatory increase in endogenous insulin production. Elevated levels of endogenous insulin, an anabolic hormone, is associated with insulin resistance and results in weight gain which, in turn, exacerbates insulin resistance. This vicious cycle continues until pancreatic beta cell activity can no longer adequately meet the insulin demand created by insulin resistance, resulting in hyperglycemia. A continued mismatch between insulin demand and insulin production causes glycemic levels to rise to levels consistent with T2DM.

[0004] In addition to development of T2DM, insulin resistance can result in other negative health outcomes. The spectrum of diseases associated with insulin resistance includes obesity (dyslipidemia, visceral adiposity), cardiovascular disease (hypertension, prothrombic state, peripheral neuropathy, retinopathy), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome, polycystic ovary syndrome (PCOS), cancer, and aging.

[0005] Consequently, reducing insulin resistance to increase insulin sensitivity may be an efficient intervention strategy to address multiple risk factors, targeted through one common mechanism. Reduced insulin resistance may lead to reduced risk of disease development, for example, type 2 diabetes, cardiovascular disease, and/or several age-related diseases.

[0006] Lifestyle modification represents the cornerstone of treatment for insulin resistance. Dietary intervention including a combination of caloric reduction and avoidance of carbohydrates that stimulate excessive insulin demand often form a basis for treatment of insulin resistance. In addition, physical activity helps to increase energy expenditure and improve muscle insulin sensitivity. Such interventions are however often difficult to practice on a continuing basis. Antidiabetes drugs, including metformin and thiazolidinediones, are also used to improve insulin response and reduce insulin demand, although the US FDA does not identify their use specifically for reducing insulin resistance.

[0007] Accordingly, methods for reducing insulin resistance, or, specifically, increasing insulin sensitivity, in a subject are desired, and especially desired are such methods which are more easily implemented on a continuous basis than strict diet and/or rigorous exercise regimens and which avoid pharmaceutical medication. A nutritional intervention that can increase insulin sensitivity is also desirable. SUMMARY OF THE INVENTION

[0008] Accordingly, it is an object of the invention to provide improved methods for increasing insulin sensitivity.

[0009] In one embodiment, the invention is directed to a method of increasing insulin sensitivity in a subject in need thereof. The method comprises administering a nutritional composition comprising inositol, lysine, arginine, and beta-hydroxy-beta-methylbutyrate (HMB) to the subject. [0010] In an additional embodiment, the present invention is directed to a nutritional composition comprising from about 0.01 to about 15 wt % HMB, from about 0.03 to about 40 wt % lysine, from about 0.02 to about 30 wt % arginine, and from about 0.1 to about 20 wt % inositol, all based on the weight of the nutritional composition.

[0011] The methods of increasing insulin sensitivity, as well as the nutritional compositions according to the present invention, are advantageous in that they reduce insulin resistance, or delay the development of insulin resistance, and thus may contribute to reduced and/or delayed disease development, including, for example, reduced or delayed development of T2DM and/or cardiovascular diseases associated with insulin resistance. The methods and nutritional compositions of the invention also provide a convenient manner in which to achieve increased insulin sensitivity, and therefore encourage a subject’s ability to increase insulin sensitivity. These and additional objects and advantages of the invention will be more fully apparent in view of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The embodiments set forth in the drawings are illustrative of certain aspects of the invention and exemplary in nature and are not intended to limit the invention defined by the claims, wherein:

[0013] FIG. 1 illustrates the effects of myo-inositol (myo), arginine (Arg), lysine (Lys), HMB, each individually, and the combination of myo-inositol, arginine, lysine, and HMB, on glucose uptake in L6.C11 rat skeletal muscle cell-derived myotubes, as described in the Example. [0014] FIG. 2 illustrates the effects of myo-inositol (myo), arginine (Arg), lysine (Lys), HMB, each individually, and the combination of myo-inositol, arginine, lysine, and HMB on the expression level of the glucose transporter, GLUT4, in L6.C11 rat skeletal muscle cell-derived myotubes, as described in the Example.

DETAILED DESCRIPTION

[0015] Specific embodiments of the invention are described herein. The invention can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to illustrate more specific features of certain aspects of the invention to those skilled in the art.

[0016] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the disclosure as a whole. All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably. Furthermore, as used in the description and the appended claims, the singular forms “a,” “an,” and “the” are inclusive of their plural forms, unless the context clearly indicates otherwise.

[0017] To the extent that the term “includes” or “including” is used in the description or the claims, it is intended to be inclusive of additional elements or steps, in a manner similar to the term “comprising” as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term “or” is employed (e.g., A or B), it is intended to mean “A or B or both.” When the “only A or B but not both” is intended, then the term “only A or B but not both” is employed. Thus, use of the term “or” herein is the inclusive, and not the exclusive use. When the term “and” as well as “or” are used together, as in “A and/or B” this indicates A or B as well as A and B. [0018] The methods and compositions described in the present disclosure can comprise, consist of, or consist essentially of any of the elements and steps as described herein.

[0019] All ranges and parameters, including but not limited to percentages, parts, and ratios disclosed herein are understood to encompass any and all sub-ranges subsumed therein, and every number between the endpoints. For example, a stated range of “1 to 10” should be considered to include any and all sub-ranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less (e.g., 1 to 6.1 , or 2.3 to 9.4), and to each integer (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) contained within the range.

[0020] Any combination of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0021] All percentages are percentages by weight unless otherwise indicated.

[0022] The term “insulin sensitivity” as used herein, unless otherwise specified, refers to how sensitive a subject's cells are in response to insulin. High insulin sensitivity allows the cells of the body to use blood glucose more effectively, reducing blood sugar. Conversely, “insulin resistance” as used herein, unless otherwise indicated, refers to a decreased ability of a subject’s cells to respond normally to insulin, preventing glucose from entering the cells as easily and accumulating in the blood.

[0023] The term “nutritional powder” as used herein, unless otherwise specified, refers to nutritional powders that are generally flowable particulates and that are reconstitutable with an aqueous liquid, and which are suitable for oral administration to a human.

[0024] The term “nutritional liquid” as used herein, unless otherwise specified, refers to nutritional products in ready-to-drink liquid form and to nutritional liquids made by reconstituting the nutritional powders described herein prior to use. [0025] The terms “nutritional product” and “nutritional composition” as used herein, unless otherwise specified, refer to nutritional liquids and nutritional powders, the latter of which may be reconstituted to form a nutritional liquid, and are suitable for oral consumption by a human.

[0026] The methods and compositions of the invention employ inositol, arginine, lysine and HMB to increase insulin sensitivity in a subject in need thereof, i.e. , a subject experiencing or at risk of experiencing insulin resistance. An obese subject may be at risk of developing insulin resistance. Aging subjects may also be at risk of developing insulin resistance. Accordingly, in one embodiment, the subject is greater than 40, 50 or 60 years of age.

[0027] Inositol has the chemical formula CeH^Oe, and occurs as 8 different stereoisomers:

D-pinitol (3-O-methyl-D-chiro-inositol):

is often referred to as a ninth stereoisomer of inositol.

[0028] Myo-inositol is the most common isomer in plants and animal cells, and myo-inositol, as well as several other forms of inositol, have been described as improving insulin resistance, for example in patients with gestational diabetes and diabetic patients. The present inventors have surprisingly discovered that inositol, in combination with HMB, arginine and lysine, synergistically increases insulin sensitivity in a subject. The inositol which is employed in the inventive methods and nutritional compositions may comprise any one or any combination of two or more of the inositol stereoisomers. In one embodiment, the inositol comprises myo-inositol, D-chiro-inositol, D-pinitol, scyllo-inositol, muco-inositol, L-chiro-inositol, epi-inositol or allo-inositol, or a combination of two or more thereof. In a more specific embodiment, the inositol comprises, consists essentially of, or consists of, myo-inositol.

[0029] Beta-hydroxy-beta-methylbutyrate (HMB) is a naturally occurring amino acid metabolite that is known for use in a variety of nutritional products and supplements. HMB is a metabolite of the essential amino acid leucine and has been shown to modulate protein turnover and inhibit proteolysis. While HMB is commonly used in nutritional products to help build or maintain healthy muscle in selected individuals, the present inventors have surprisingly discovered that HMB, in combination with inositol, arginine and lysine, increases insulin sensitivity. The term “HMB” as used herein refers to beta-hydroxy-beta-methylbutyrate (also referred to as beta-hydroxyl-3- methyl butyric acid, beta-hydroxy isovaleric acid) and sources thereof. All weights, percentages, and concentrations as used herein to characterize HMB are based on the weight of HMB, regardless of the source, unless otherwise specified.

[0030] Any source of HMB is suitable for use in the methods and nutritional compositions of the invention. Examples include HMB as the free acid, a salt, including an anhydrous salt or a hydrate salt, an ester, a lactone, or other product forms that otherwise provide a bioavailable form of HMB. In specific embodiments of the methods and compositions of the invention, the source of HMB is selected from the group consisting of alkali metal HMB, alkaline earth metal HMB, HMB free acid, HMB lactone and combinations thereof. In more specific embodiments, the source of HMB is selected from the group consisting of sodium HMB, potassium HMB, magnesium HMB, chromium HMB, calcium HMB and combinations thereof, or the HMB is calcium HMB monohydrate. [0031] Lysine and arginine can be added to the nutritional composition in either inherent or supplemented form. Inherent amino acids are those provided by dietary proteins, whereas supplemented amino acids are the free amino acids in the L- or D- configuration. In specific embodiments of the invention, the nutritional composition employs supplemental lysine and/or arginine. In more specific embodiments of the invention, the nutritional composition employs lysine and/or arginine in the L- form.

[0032] According to the invention, the method of increasing insulin sensitivity in a subject comprises administering a nutritional composition comprising inositol, lysine, arginine, and HMB to the subject. In specific embodiments of these methods, the subject is suffering from insulin resistance or is at risk of developing insulin resistance.

[0033] In an additional embodiment of the invention, a nutritional composition comprising HMB, lysine, arginine and inositol is provided. In one embodiment, the nutritional composition comprises from about 0.01 to about 15 wt % HMB, from about 0.03 to about 40 wt % lysine, from about 0.02 to about 30 wt % arginine, and from about 0.01 to about 20 wt % inositol, all based on the weight of the nutritional composition.

[0034] In more specific embodiments, the nutritional composition comprises from about 0.01 to about 10 wt % of HMB, about 0.01 to about 8 wt % of HMB, about 0.01 to about 5 wt % of HMB, about 0.1 to about 10 wt % of HMB, about 0.1 to about 8 wt % of HMB, about 0.1 to about 5 wt % of HMB, about 0.2 to about 5 wt % of HMB, about 0.2 to about 3 wt % of HMB, about 0.2 to about 2 wt % of HMB, about 0.2 to about 1.5 wt % of HMB, or about 0.2 to about 1 wt %, of HMB, based on the weight of the nutritional composition.

[0035] In further embodiments, the nutritional composition comprises from about 0.03 to about 30 wt % of lysine, about 0.03 to about 20 wt % of lysine, about 0.1 to about 20 wt % of lysine, about 0.1 to about 15 wt % of lysine, about 0.1 to about 10 wt % of lysine, about 0.5 to about 10 wt % of lysine, about 0.5 to about 5 wt % of lysine, or about 0.5 to about 3 wt % of lysine, based on the weight of the nutritional composition. [0036] In further embodiments, the nutritional composition comprises from about 0.02 to about

20 wt % of arginine, about 0.02 to about 10 wt % of arginine, about 0.05 to about 5 wt % of arginine, about 0.1 to about 15 wt % of arginine, about 0.1 to about 10 wt % of arginine, about 0.2 to about 10 wt % of arginine, about 0.2 to about 5 wt % of arginine, or about 0.5 to about 2 wt % of arginine, based on the weight of the nutritional composition.

[0037] In further embodiments, the nutritional composition comprises from about 0.01 to about 15 wt % of inositol, about 0.1 to about 15 wt % of inositol, about 0.1 to about 10 wt % of inositol, about 0.1 to about 5 wt % of inositol, or about 0.5 to about 2 wt % of inositol, based on the weight of the nutritional composition.

[0038] In another embodiment of the methods and nutritional compositions of the invention, the molar ratio of lysine to arginine in the nutritional composition is about 10:1 to about 1 :1 , or about 5:1 to about 1 :1, or about 3:1 to about 1 :1 , the molar ratio of a combination of lysine and arginine to HMB in the nutritional composition is about 15:1 to about 1 :1 , or about 10:1 to about 1 :1 , or about 5:1 to about 1 :1 , or about 3:1 to about 1 :1, and the molar ratio of a combination of lysine and arginine to inositol in the nutritional composition is about 10:1 to about 1:1, or about 5:1 to about 1:1 , or about 3: 1 to about 1 :1.

[0039] According to specific embodiments of the methods, inositol, lysine, arginine and HMB provided in a nutritional composition are administered orally.

[0040] The methods and nutritional compositions as described herein employ amounts of inositol, lysine, arginine, and HMB that are effective to increase insulin sensitivity, and, more specifically, to increase insulin sensitivity to an extent greater than that achieved with inositol, lysine, arginine, or HMB alone.

[0041] In specific embodiments of the methods, the subject is administered from about 0.1 to about 10 g of HMB, about 1 to about 10 g of HMB, about 2 to about 5 g of HMB, about 0.1 to about 5 g of HMB, or about 0.5 to about 3 g of HMB, per day; and/or from about 0.1 to about 30 g of lysine, about 0.5 to about 10 g of lysine, about 1 to about 10 g of lysine, about 1 to about 6 g of lysine, or about 3 to about 6 g of lysine, per day; and/or from about 0.1 to about 20 g of arginine, about 0.5 to about 10 g of arginine, about 1 to about 10 g of arginine, about 1 to about 5 g of arginine, or about 0.5 to about 3 g of arginine, per day; and/or from about 0.1 to about 20 g of inositol, about 0.5 to about 10 g of inositol, about 1 to about 10 g of inositol, about 1 to about 5 g of inositol, or about 0.5 to about 3 g of inositol, per day.

[0042] In a specific embodiment, the nutritional composition is in the form of a powder. In another specific embodiment, the nutritional composition is in the form of a liquid. The nutritional composition may be in the form of a powder or liquid when administered to the subject. A liquid composition may be a ready-made liquid composition or a liquid composition reconstituted form powder.

[0043] In other specific embodiments of the invention, the nutritional composition further comprises protein, carbohydrate, and/or fat, in any amounts as desired. A wide variety of sources and types of protein, carbohydrate, and fat can be used in embodiments of nutritional compositions described herein. In a specific embodiment, the nutritional composition includes protein, carbohydrate and fat.

[0044] In further specific embodiments, the protein in the nutritional composition comprises whey protein concentrate, whey protein isolate, whey protein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, organic milk protein concentrate, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, acid casein, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, nonfat dry milk, condensed skim milk, collagen protein, collagen protein isolate, L-Carnitine, taurine, lutein, rice protein concentrate, rice protein isolate, rice protein hydrolysate, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, meat protein, potato protein, chickpea protein, canola protein, mung protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax seed protein, earthworm protein, insect protein, or combinations of two or more thereof. [0045] In specific embodiments, the nutritional composition may comprise protein in an amount about 1 wt % to about 30 wt % of the nutritional composition. More specifically, the protein may be present in an amount about 1 wt % to about 25 wt % of the nutritional composition, including about 1 wt % to about 20 wt %, about 2 wt % to about 20 wt %, about 1 wt % to about 15 wt %, about 1 wt % to about 10 wt %, about 5 wt % to about 10 wt %, about 10 wt % to about 25 wt %, or about 10 wt % to about 20 wt % of the nutritional composition. Even more specifically, the protein comprises about 1 wt % to about 10 wt % of the nutritional composition, or about 15 wt % to about 30 wt % of the nutritional composition.

[0046] In other specific embodiments, the carbohydrate in the nutritional composition comprises human milk oligosaccharides (HMOs), maltodextrin, for example, from rice, corn, wheat, or potato, resistant maltodextrin, corn syrup, corn syrup solids, sucralose, cellulose gel, cellulose gum, gellan gum, carrageenan, fructooligosaccharides (FOS), hydrolyzed starch, glucose polymers, rice-derived carbohydrates, sucrose, glucose, lactose, honey, sugar alcohols, isomaltulose, sucromalt, pullulan, potato starch, galactooligosaccharides, oat fiber, soy fiber, corn fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinoglactins, glucomannan, xanthan gum, alginate, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucans, psyllium, inulin, and combinations of two or more thereof. In further specific embodiments, the carbohydrate in the nutritional composition comprises a combination of two or more carbohydrates, wherein the carbohydrates have varying rates of absorption. In specific embodiments, the carbohydrate that may be used in the nutritional composition of the invention comprises isomaltulose, sucromalt, maltodextrin, resistant maltodextrin, FOS, inulin, fructose, corn fiber, oat fiber, soy fiber, or a combination of two or more thereof.

[0047] In specific embodiments, the nutritional composition may comprise carbohydrate in an amount about 0.5 wt % to about 75 wt % of the nutritional composition. More specifically, the carbohydrate may be present in an amount about 1 wt % to about 70 wt % of the nutritional composition, including about 5 wt % to about 70 wt %, about 5 wt % to about 65 wt %, about 5 wt % to about 50 wt %, about 5 wt % to about 40 wt %, about 5 wt % to about 30 wt %, about 5 wt % to about 25 wt %, about 5 wt % to about 20 wt %, about 10 wt % to about 65 wt %, about 10 wt % to about 25 wt %, or about 10 wt % to about 20 wt %, about 20 wt % to about 65 wt %, about 30 wt % to about 65 wt %, about 40 wt % to about 65 wt %, or about 40 wt % to about 70 wt %, of the nutritional composition.

[0048] The terms “fat” and “oil” as used herein, unless otherwise specified, are used interchangeably to refer to lipid materials derived or processed from plants or animals. These terms also include synthetic lipid materials so long as such synthetic materials are suitable for oral administration to humans.

[0049] In further specific embodiments, the fat comprises coconut oil, fractionated coconut oil, soy oil, soy lecithin, corn oil, safflower oil, sunflower oil, palm olein, canola oil monoglycerides, lecithin, canola oil, medium chain triglycerides, one or more fatty acids such as linoleic acid, alphalinolenic acid, fractionated coconut oil, soy oil, corn oil, olive oil, medium chain triglyceride oil (MCT oil), high gamma linolenic (GLA) safflower oil, palm oil, palm kernel oil, marine oil, fish oil, algal oil, borage oil, cottonseed oil, fungal oil, interesterified oil, transesterified oil, structured lipids, omega-3 fatty acid, or combinations of two or more thereof. In a specific embodiment, the omega- 3 fatty acid is selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, and alpha-linolenic acid, and combinations of two or more thereof.

[0050] In specific embodiments, the nutritional composition may comprise fat in an amount of about 0.5 wt % to about 30 wt % of the nutritional composition. More specifically, the fat may be present in an amount of about 0.5 wt % to about 10 wt %, about 1 wt % to about 30 wt %, about 1 wt % to about 20 wt %, about 1 wt % to about 15 wt %, about 1 wt % to about 10 wt %, about 1 wt % to about 5 wt %, about 3 wt % to about 30 wt %, about 5 wt % to about 30 wt %, about 5 wt % to about 25 wt %, about 5 wt % to about 20 wt %, about 5 wt % to about 10 wt %, or about 10 wt % to about 20 wt % of the nutritional composition. [0051] The concentration and relative amounts of the sources of protein, carbohydrate, and fat in the nutritional compositions can vary considerably depending upon, for example, the specific dietary needs of the intended user. In one embodiment, the nutritional composition comprises about 1 to about 15 wt % of protein, about 0.5 to about 10 wt % fat, and about 1 to about 20 wt % carbohydrate, based on the weight of the nutritional composition.

[0052] In additional embodiments, the nutritional composition is in liquid form and comprises a source of protein in an amount about 1 wt % to about 20 wt %, a source of carbohydrate in an amount about 5 wt % to about 30 wt %, and a source of fat in an amount about 0.5 wt % to about 10 wt %, based on the weight of the nutritional composition. In a more specific embodiment, the nutritional composition is in liquid form and comprises a source of protein in an amount about 5 wt % to about 15 wt %, a source of carbohydrate in an amount about 5 wt % to about 20 wt %, and a source of fat in an amount about 0.5 wt % to about 10 wt %, based on the weight of the nutritional composition.

[0053] In additional embodiments, the nutritional composition is in powder form and comprises a source of protein in an amount about 10 wt % to about 30 wt %, a source of carbohydrate in an amount about 40 wt % to about 70 wt %, and a source of fat in an amount of about 5 wt % to about 20 wt %, based on the weight of the nutritional composition. In a more specific embodiment, the nutritional composition is in powder form and comprises about 10 to about 25 wt % of protein, about 5 to about 15 wt % fat, and about 40 wt % to about 65 wt % carbohydrate, based on the weight of the nutritional composition.

[0054] In a specific embodiment, the nutritional composition comprises at least one protein comprising milk protein concentrate and/or soy protein isolate, at least one fat comprising canola oil, corn oil, coconut oil and/or marine oil, and at least one carbohydrate comprising maltodextrin, resistant maltodextrin, sucrose, and/or short-chain fructooligosaccharide.

[0055] The nutritional composition may also comprise one or more components to modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition or serve as additional nutritional components. Non-limiting examples of additional components include preservatives, emulsifying agents (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharine, aspartame, acesulfame K, sucralose), colorants, flavorants, thickening agents, stabilizers, and so forth.

[0056] In another embodiment of the invention, the nutritional composition comprises protein, carbohydrate, fat, and one or more nutrients selected from the group consisting of vitamins and minerals. Specific embodiments of the nutritional composition may comprise vitamins and/or related nutrients, non-limiting examples of which include vitamin A, vitamin B 12, vitamin C, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, niacin, folic acid, pantothenic acid, biotin, choline, inositol, and/or salts and derivatives thereof, and combinations thereof.

[0057] Specific embodiments of the nutritional composition comprise minerals, non-limiting examples of which include calcium, phosphorus, magnesium, zinc, manganese, sodium, potassium, molybdenum, chromium, iron, copper, and/or chloride, and combinations thereof.

[0058] In specific embodiments, the nutritional composition has a neutral pH, i.e., a pH of about 6 to 8 or, more specifically, about 6 to 7.5. In more specific embodiments, the nutritional composition has a pH of about 6.5 to 7.2 or, more specifically, about 6.8 to 7.1.

[0059] The nutritional composition may be formed using any techniques known in the art. In one embodiment, the nutritional composition may be formed by (a) preparing an aqueous solution comprising protein and carbohydrate; (b) preparing an oil blend comprising fat and oil-soluble components; and (c) mixing together the aqueous solution and the oil blend to form an emulsified liquid nutritional composition. The inositol, HMB, lysine, and arginine can be added at any point in the formation of the nutritional composition.

[0060] As indicated above, the nutritional composition can be administered in the form of a powder or in the form of a liquid, and the liquid may be a ready-to-drink liquid product or may be a liquid made by reconstituting a nutritional powder as described herein prior to use. [0061] When the nutritional composition is a powder, for example, a serving size is about 40 g to about 60 g, or about 45 g to about 50 g, to be administered as a powder or to be reconstituted in water, for example about 50 ml to about 500 ml of liquid.

[0062] When the nutritional composition is in the form of a liquid, for example, reconstituted from a powder or manufactured as a ready-to-drink product, a serving ranges about 1 ml to about 500 ml, including about 100 ml to about 500 ml, about 100 ml to about 400 ml, about 120 ml to about 500 ml, about 120 ml to about 400 ml, about 150 ml to about 500 ml, about 200 ml to about 300 ml, or about 230 m to about 245 ml. In specific embodiments, the serving is about 100 ml, or about 225 ml, or about 237 ml, or about 500 ml.

[0063] In specific embodiments, the nutritional composition comprising inositol, HMB, lysine and arginine is administered to a subject once or multiple times daily or weekly. In specific embodiments, the nutritional composition is administered to the subject about 1 to about 6 times per day or per week, or about 1 to about 5 times per day or per week, or about 1 to about 4 times per day or per week, or about 1 to about 3 times per day or per week. In specific embodiments, the nutritional composition is administered once or twice daily for a period of at least one week, at least two weeks, at least three weeks, or at least four weeks.

[0064] The following Examples demonstrate aspects of the inventive methods and are provided solely for the purpose of illustration. The Examples are not to be construed as limiting of the general inventive concepts, as many variations thereof are possible without departing from the spirit and scope of the general inventive concepts.

EXAMPLE

[0065] This Examples describes in vitro experiments performed with the L6.C11 rat skeletal muscle myoblast line (ECACC No. 92102119) to demonstrate the synergistic increase in insulin sensitivity provided by the invention.

[0066] More specifically, L6.C11 rat skeletal muscle cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mmol/l glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin in an atmosphere of 5% CO2 and

95% air. Cells were maintained at sub-confluent densities in the growth media. The L6 cells were differentiated into myotubes by culturing them for 5 days in DM EM containing 2% FBS (v/v).

2-Deoxy-[3H]D-glucose (2-DG) uptake

[0067] A 2-Deoxy-[3H]D-glucose (2-DG) assay was used to examine the effect of myo-inositol, arginine, lysine, HMB, each individually and all in combination, on glucose uptake in L6-myotubes. Cells were grown in 48-well plates (Corning, NY, USA). They were differentiated into myotubes as described above, and the different effectors were added: myo-inositol (5 mM), arginine (2.5 mM), lysine (10 mM), HMB (25 pM), each individually, and the combination of myo-inositol, arginine, lysine and HMB (Myo 5 mM + Arg 2.5 mM + Lys 10 mM + HMB 25 pM, Myo:Arg:Lys:HMB). A Control was provided with no added effector. The cells were incubated overnight and then rinsed with HEPES-buffered Krebs-Ringer phosphate (KRPH), consisting of 118 mmol/l NaCI, 5 mmol/l KCI, 1.3 mmol/l CaCh, 1.2 mmol/l MgSC , 1.2 mmol/l KH2PO4, and 30 mmol/l HEPES (pH 7.4). The 10 pmol/l 2-deoxy-[3H]d-glucose (2- DG) (1 pCi/ml) uptake was measured over a 10-min period under conditions in which the uptake was linear. The uptake measurement was made in triplicate. The uptake of 2-DG was terminated after 10 min by rapidly aspirating off the radioactive incubation medium and washing the cells three times in ice-cold phosphate-buffered saline. The radioactivity associated with the cells was determined by cell lysis in 0.5 N NaOH with neutralization by the addition of 0.5 N HCI, followed by liquid scintillation. Aliquots from each well were also used to determine protein concentration using the bicinchoninic acid (BCA) protein assay as discussed below.

[0068] The results of 2-DG uptake are presented in Fig. 1 . Results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance. Multiple comparisons of means were done by the Fisher’s test. A p value <0.05 was considered significant, with (*) versus Control, (#) versus the combination Myo:Arg:Lys:HMB. Fig. 1 shows that a statistically significant higher glucose uptake was seen when myotubes were incubated with lysine with respect to the Control cells, and with the combination Myo:Arg:Lys:HMB with respect to the Control cells. However, the highest glucose uptake was obtained with the combination Myo:Arg:Lys:HMB, which exhibited a statistically significant, synergistic improvement on glucose uptake. The glucose uptake increased by 60% in the presence of the combination Myo:Arg:Lys:HMB with respect to control cells and by 31% respect to the cells incubated with lysine alone.

Protein analysis

[0069] The expression of proteins involved in glucose transport (GLUT4) was also studied. The transport of glucose across plasma membranes into skeletal muscle is facilitated by the glucose transporter 4 (GLUT4), which is stimulated by insulin. In this study, cells were incubated for 24 h in the presence or absence of effectors myo-inositol (5 mM), arginine (2.5 mM), lysine (10 mM), HMB (25 pM), each individually, and the combination of myo-inositol, arginine, lysine and HMB (Myo 5 mM + Arg 2.5 mM + Lys 10 mM + HMB 25 pM, Myo:Arg:Lys:HMB), as described above. After treatment, cells were lysed, and the protein concentration was measured using the BCA assay method. To assay the phosphorylation degree of key kinases, antibodies against phosphorylated protein were used. The expression of GAPDH was used as a load control. Immunoblots were developed by an enhanced chemiluminescence detection method using a Chemidoc-it 810 Imager (UVP, Cambridge, UK). Quantification was performed by densitometry with the NIH Image Software.

[0070] The results are presented in Fig. 2 and again are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance. Multiple comparisons of means were done by the Fisher’s test. A p < 0.05 was considered statistically significant, with (*) versus Control, (#) versus the combination Myo:Arg:Lys:HMB. Fig. 2 shows that of the individual ingredients, lysine was the only one that presented a significant difference with respect to the Control cells in the expression of GLUT4, increasing it by 22%. However, Fig. 2 further shows a statistically significant synergistic effect in the expression of GLUT4 was provided by the combination of myo-inositol, lysine, arginine, and HMB (Myo:Arg:Lys:HMB), wherein the expression of GLUT4 was increased by 45% with respect to the Control cells and 23% in comparison with the cells incubated with lysine alone.

[0071] The results presented herein demonstrate that the combination of myo-inositol, lysine, arginine, and HMB increases insulin sensitivity, as evidenced by increased glucose uptake, and particularly as compared with any of the individual components.

[0072] While the present invention has been illustrated by the description of embodiments thereof and the Example, and while the embodiments and Example have been described in considerable detail, such descriptions are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the invention, in its broader aspects, is not limited to the specific details, the representative compositions and methods, or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.

Claims

WHAT IS CLAIMED IS:

1 . A method of increasing insulin sensitivity in a subject in need thereof, comprising: administering a nutritional composition comprising inositol, lysine, arginine, and beta-hydroxy- beta-methylbutyrate (HMB) to the subject.

2. The method of claim 1, wherein the subject is suffering from insulin resistance.

3. The method of claim 1 or 2, wherein the inositol comprises myo-inositol.

4. The method of any one of claims 1-3, wherein the HMB is selected from the group consisting of alkali metal HMB, alkaline earth metal HMB, HMB free acid, HMB lactone and combinations thereof, or the HMB is selected from the group consisting of sodium HMB, potassium HMB, magnesium HMB, chromium HMB, calcium HMB and combinations thereof, or the HMB is calcium HMB monohydrate.

5. The method of any one of claims 1-4, wherein the molar ratio of lysine to arginine in the nutritional composition is about 10:1 to about 1 :1 , or about 5: 1 to about 1 :1 , or about 3: 1 to about 1 :1 , the molar ratio of a combination of lysine and arginine to HMB in the nutritional composition is about 15: 1 to about 1:1, or about 10:1 to about 1:1, or about 5: 1 to about 1 :1 , or about 3: 1 to about 1 :1 , and the molar ratio of a combination of lysine and arginine to inositol in the nutritional composition is about 10: 1 to about 1:1, or about 5: 1 to about 1 : 1 , or about 3: 1 to about 1 :1.

6. The method of any one of claims 1-5, wherein the nutritional composition is in the form of a powder. The method of any one of claims 1-5, wherein the nutritional composition is in the form of a liquid. The method of any one of claims 1-7, wherein the subject is administered from about 0.1 to about 10 g of HMB, about 1 to about 10 g of HMB, about 2 to about 5 g of HMB, about 0.1 to about 5 g of HMB, or about 0.5 to about 3 g of HMB, per day; and/or from about 0.1 to about 30 g of lysine, about 0.5 to about 10 g of lysine, about 1 to about 10 g of lysine, about 1 to about 6 g of lysine, or about 3 to about 6 g of lysine, per day; and/or from about 0.1 to about 20 g of arginine, about 0.5 to about 10 g of arginine, about 1 to about 10 g of arginine, about 1 to about 5 g of arginine, or about 0.5 to about 3 g of arginine, per day; and/or from about 0.1 to about 20 g of inositol, about 0.5 to about 10 g of inositol, about 1 to about 10 g of inositol, about 1 to about 5 g of inositol, or about 0.5 to about 3 g of inositol, per day. The method of any one of claims 1-8, wherein the nutritional composition further comprises protein, carbohydrate, and/or fat. The method of claim 9, wherein the nutritional composition comprises protein and the protein comprises whey protein concentrate, whey protein isolate, whey protein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, organic milk protein concentrate, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, acid casein, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, nonfat dry milk, condensed skim milk, collagen protein, collagen protein isolate, L-Carnitine, taurine, lutein, rice protein concentrate, rice protein isolate, rice protein hydrolysate, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, meat protein, potato protein, chickpea protein, canola protein, mung protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax seed protein, earthworm protein, insect protein, or combinations of two or more thereof. The method of claim 9 or 10, wherein the nutritional composition comprises carbohydrate and the carbohydrate comprises human milk oligosaccharides (HMOs), maltodextrin, resistant maltodextrin, corn syrup, corn syrup solids, sucralose, cellulose gel, cellulose gum, gellan gum, carrageenan, fructooligosaccharides (FOS), hydrolyzed starch, glucose polymers, rice-derived carbohydrates, sucrose, glucose, fructose, lactose, honey, isomaltulose, sucromalt, pullulan, potato starch, galactooligosaccharides, oat fiber, soy fiber, corn fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinoglactins, glucomannan, xanthan gum, alginate, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucans, psyllium, inulin, and combinations of two or more thereof. The method of claim 11, wherein the carbohydrate comprises isomaltulose, sucromalt, maltodextrin, resistant maltodextrin, FOS, inulin, fructose, corn fiber, oat fiber, soy fiber, or a combination of two or more thereof. The method of any one of claims 9-12, wherein the nutritional composition comprises fat and the fat comprises coconut oil, fractionated coconut oil, soy oil, soy lecithin, corn oil, safflower oil, sunflower oil, palm olein, canola oil monoglycerides, lecithin, canola oil, medium chain triglycerides, linoleic acid, alpha-linolenic acid, fractionated coconut oil, soy oil, corn oil, olive oil, high gamma linolenic (GLA) safflower oil, palm oil, palm kernel oil, marine oil, fish oil, algal oil, borage oil, cottonseed oil, fungal oil, interesterified oil, transesterified oil, structured lipids, omega-3 fatty acid, or combinations of two or more thereof. A nutritional composition comprising: from about 0.01 to about 15 wt % HMB, from about 0.03 to about 40 wt % lysine, from about 0.02 to about 30 wt % arginine, and from about 0.01 to about 20 wt % inositol, all based on the weight of the nutritional composition. The nutritional composition of claim 14, wherein the nutritional composition comprises: about 0.01 to about 10 wt % of HMB, about 0.01 to about 8 wt % of HMB, about 0.01 to about 5 wt % of HMB, about 0.1 to about 10 wt % of HMB, about 0.1 to about 8 wt % of HMB, about 0.1 to about 5 wt % of HMB, about 0.2 to about 5 wt % of HMB, about 0.2 to about 3 wt % of HMB, about 0.2 to about 2 wt % of HMB, about 0.2 to about 1.5 wt % of HMB, or about 0.2 to about 1 wt %, of HMB, about 0.03 to about 30 wt % of lysine, about 0.03 to about 20 wt % of lysine, about 0.1 to about 20 wt % of lysine, about 0.1 to about 15 wt % of lysine, about 0.1 to about 10 wt % of lysine, about 0.5 to about 10 wt % of lysine, about 0.5 to about 5 wt % of lysine, or about 0.5 to about 3 wt % of lysine, about 0.02 to about 20 wt % of arginine, about 0.02 to about 10 wt % of arginine, about

0.05 to about 5 wt % of arginine, about 0.1 to about 15 wt % of arginine, about 0.1 to about 10 wt % of arginine, about 0.2 to about 10 wt % of arginine, about 0.2 to about 5 wt % of arginine, or about 0.5 to about 2 wt % of arginine, and about 0.01 to about 15 wt % of inositol, about 0.1 to about 15 wt % of inositol, about 0.1 to about 10 wt % of inositol, about 0.1 to about 5 wt % of inositol, or about 0.5 to about 2 wt % of inositol, all based on the weight of the nutritional composition. The nutritional composition of claim 14 or 15, wherein the nutritional composition further comprises protein, carbohydrate, and/or fat. The nutritional composition of claim 16, wherein nutritional composition comprises protein and the protein comprises whey protein concentrate, whey protein isolate, whey protein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, organic milk protein concentrate, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, acid casein, sodium caseinate, calcium caseinate, potassium caseinate, casein hydrolysate, nonfat dry milk, condensed skim milk, collagen protein, collagen protein isolate, L-Carnitine, taurine, lutein, rice protein concentrate, rice protein isolate, rice protein hydrolysate, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, meat protein, potato protein, chickpea protein, canola protein, mung protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax seed protein, earthworm protein, insect protein, or combinations of two or more thereof. The nutritional composition of claim 16 or 17, wherein the nutritional composition comprises carbohydrate and the carbohydrate comprises human milk oligosaccharides (HMOs), maltodextrin, resistant maltodextrin, corn syrup, corn syrup solids, sucralose, cellulose gel, cellulose gum, gellan gum, carrageenan, fructooligosaccharides (FOS), hydrolyzed starch, glucose polymers, rice-derived carbohydrates, sucrose, glucose, fructose, lactose, honey, isomaltulose, sucromalt, pullulan, potato starch, galactooligosaccharides, oat fiber, soy fiber, corn fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinoglactins, glucomannan, xanthan gum, alginate, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucans, psyllium, inulin, and combinations of two or more thereof. The nutritional composition of claim 18, wherein the carbohydrate comprises isomaltulose, sucromalt, maltodextrin, resistant maltodextrin, FOS, inulin, fructose, corn fiber, oat fiber, soy fiber, or a combination of two or more thereof. The nutritional composition of any one of claims 14-19, wherein the nutritional composition comprises fat and the fat comprises coconut oil, fractionated coconut oil, soy oil, soy lecithin, corn oil, safflower oil, sunflower oil, palm olein, canola oil monoglycerides, lecithin, canola oil, medium chain triglycerides, linoleic acid, alpha-linolenic acid, fractionated coconut oil, soy oil, corn oil, olive oil, high gamma linolenic (GLA) safflower oil, palm oil, palm kernel oil, marine oil, fish oil, algal oil, borage oil, cottonseed oil, fungal oil, interesterified oil, transesterified oil, structured lipids, omega-3 fatty acid, or combinations of two or more thereof. The nutritional composition of any one of claims 14-20 , wherein the nutritional composition comprises about 1 to about 15 wt % of protein, about 0.5 to about 10 wt % fat, and about 1 to about 20 wt % carbohydrate, based on the weight of the nutritional composition.