WO2023076144A1 - Methods and compositions for improving insulin production and secretion - Google Patents

Abstract

A method of improving insulin production in a subject suffering from impaired β-cell function comprises administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject. A method of restoring and/or preserving β-cell mass in a subject suffering from impaired insulin production comprises administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject. A method of delaying diabetes progression in a subject suffering from impaired insulin production comprises administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject.

Description

METHODS AND COMPOSITIONS FOR IMPROVING INSULIN PRODUCTION AND SECRETION

FIELD OF THE INVENTION

[0001] The present invention relates to methods of improving insulin production in a subject suffering from impaired p-cell function, to methods of restoring and/or preserving p-cell mass in a subject suffering from reduced insulin production, and to methods of delaying the progression of diabetes in a subject suffering from reduced insulin production.

BACKGROUND OF THE INVENTION

[0002] Pancreatic p-cells produce and secrete insulin, which is the hormone that regulates blood glucose concentration by promoting absorption of glucose from the blood into liver, fat and skeletal muscle cells, p-cells must be able to produce, store, and secrete insulin in sufficient concentrations to maintain normal levels of glucose in the blood, i.e., euglycemia. Physiologically, euglycemia is governed by the balance between peripheral insulin sensitivity, which is how readily body cells in the periphery tissue can absorb glucose, and insulin secretion: when insulin sensitivity is reduced, insulin secretion is increased. Since p-cells are very sensitive to blood glucose concentrations, changes in the body’s ability to maintain equilibrium in blood glucose concentration greatly influences p-cell function. When elevated glucose levels and insulin production are prolonged, the pancreas loses the ability to adapt p-cell mass to insulin demand over time, thereby leading to a decrease in functional p-cell mass. Eventually, as diabetes progresses, dysfunctional p-cells no longer produce enough insulin to meet the body’s insulin demand, thus resulting in persistent hyperglycemia. Dysregulation of p-cell functional mass thus represents a key mechanistic factor linked to the onset and progression of diabetes.

[0003] Although there has been continuous progress in treating diabetes through pharmaceutical therapy, handling diabetes progression is a critical issue because the etiology and mechanisms of diabetes development are still not fully understood. Two principal problems exist: how to cure diabetes and how to lower the prevalence of diabetes by primary and secondary prevention.

[0004] Insulin resistance and p-cell dysfunction have important roles in the pathogenesis and evolution of diabetes. Insulin resistance, often present years before diabetes is diagnosed, reflects a diminished response to insulin in key target tissues, such as muscle, liver and adipose tissue, and has been shown to predict the development of the disease, p-cell function is already reduced in subjects with impaired glucose tolerance (e.g., prediabetic patients) and is even more reduced in subjects with type 2 diabetes mellitus (T2DM), often referred to simply as type 2 diabetes.

[0005] According to a LIKPDS study, p-cell function in subjects with T2DM might be reduced by 50% at diagnosis with a 5% decline per each subsequent year, suggesting the beginning of deterioration several years before disease onset. Lancet, 352 (1998): pp. 837-853 and pp 854- 865; Br Med J, 317 (1998), pp. 703-713 and pp. 713-720. Since the deficit of p-cell functional mass is a necessary and early condition for the development of T2DM, methods providing for improved p-cell function, p-cell restoration and/or p-cell preservation, and preserved functional islet integrity are desirable for T2DM prevention, delay of progression, treatment, remission, and potentially a cure.

[0006] Currently, the use of antidiabetic medication is widespread. Available pharmaceutical therapies for treating diabetes have been developed as “symptomatic” medications since they primarily act to reduce elevated blood glucose levels. However, it has been described that monotherapy with antidiabetic medications (e.g., metformin, rosiglitazone, and glyburide) fails over time, albeit with differences in the rates of decline. In addition, current therapies do not completely abolish the progressive loss of p-cell function, and their use is also associated with hypoglycemia and weight gain. In order to be capable of preventing the onset and progression of T2DM, treatment should also stop p-cell dysfunction and promote the restoration of fully functional P-cell mass, independently of reducing hyperglycemia. Management of T2DM should ideally involve early and simultaneous treatment of insulin resistance and p-cell dysfunction.

[0007] In addition to pharmaceutical therapies, as mentioned above, major effort has been made in understanding the effective preventative management of both prediabetes and diabetes through exercise and nutritional intervention. Preventative strategies for the management of diabetes progression, for example the progression from prediabetes to T2DM, or the progression from gestational diabetes to diabetes following pregnancy, should focus on minimizing the factors associated with diabetes, preventing hyperglycemia, and reestablishing muscle structure and functions.

[0008] Accordingly, methods of improving insulin production and methods for restoring and/or preserving p-cell mass and/or p-cell functional mass are desirable, particularly for patients suffering from metabolic syndrome, prediabetes, and diabetes. It is also desirable to provide methods of improving insulin production, restoring p-cell mass and/or functional p-cell mass, and/or preserving p-cell mass and/or functional p-cell mass in order to help delay progression of, prevent, or reverse diabetes. A nutritional intervention that can help address the above limitations associated with existing diabetes treatments is also desirable.

SUMMARY OF THE INVENTION

[0009] In one embodiment, the invention is directed to a method of improving insulin production in a subject suffering from impaired p-cell function, comprising administering an exosome- enriched product comprising intact bovine milk-derived exosomes to the subject.

[0010] In an additional embodiment, the present invention is directed to a method of restoring and/or preserving p-cell mass in a subject suffering from impaired insulin production, comprising administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject. [0011] In a further additional embodiment, the present invention is directed to a method of delaying diabetes progression in a subject suffering from impaired insulin production, comprising administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject.

[0012] The methods of improving insulin production, restoring and/or preserving p-cell mass, and delaying diabetes progression are advantageous in that they may contribute to improved regulation of blood glucose concentration, reduced p-cell deterioration and/or improved p-cell functionality. The methods of the invention can thus help delay or prevent the onset of diabetes, delay or prevent diabetes progression, provide treatment, and/or promote remission. 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

[0013] 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:

[0014] FIG. 1 illustrates p-cell proliferation and viability in INS-1 cells incubated with palmitate for 24 hours and 48 hours in the presence, or absence of, respectively, (1) an exosome-enriched product comprising intact bovine milk-derived exosomes, and (2) an exosome-enriched product comprising sonicated bovine milk-derived exosomes, as described in Example 2.

[0015] FIG. 2 illustrates the acute response of insulin secretion to extracellular glucose concentration in INS-1 cells incubated for 2 hours with, or in the absence of, respectively, (1) an exosome-enriched product comprising intact bovine milk-derived exosomes, and (2) an exosome- enriched product comprising sonicated bovine milk-derived exosomes, as described in Example

3. [0016] FIG. 3 illustrates the effects on insulin production of INS-1 cells incubated for 24 hours with, or in the absence of, respectively, (1) an exosome-enriched product comprising intact bovine milk-derived exosomes, and (2) an exosome-enriched product comprising sonicated bovine milk- derived exosomes, followed by 2 hours of incubation with 10 mM glucose, as described in Example 3.

[0017] FIG. 4 illustrates the effects on insulin secretion and insulin production of INS-1 cells incubated for 24 hours with, or in the absence of, respectively, (1) an exosome-enriched product comprising intact bovine milk-derived exosomes, and (2) an exosome-enriched product comprising sonicated bovine milk-derived exosomes, and subsequently lysed, as described in Example 3.

DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] All percentages are percentages by weight unless otherwise indicated.

[0025] The term “diabetes” as used herein, unless otherwise specified, refers to T2DM and gestational diabetes. T2DM is a chronic condition that affects the way the body metabolizes sugar. In patients with T2DM, the body either resists the effects of insulin, resulting in reduced glucose uptake by cells, or does not produce enough insulin to maintain normal glucose levels. T2DM often starts as insulin resistance, meaning that the body is not capable of using insulin efficiently. Gestational diabetes occurs only during pregnancy and is a result of insulin-blocking hormones that are produced during pregnancy. Patients diagnosed with gestational diabetes are at a higher risk of developing diabetes after pregnancy. [0026] The term “prediabetes” as used herein, unless otherwise specified, refers to a condition wherein a patient has a higher than normal blood sugar level, but this higher blood sugar level is not yet high enough to be considered diabetes. More specifically, patients suffering from prediabetes typically have impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT). A prediabetic patient will normally have a fasting blood glucose level of 100 to 125 mg/dL, a blood glucose level of 140-199 mg/dL following a glucose tolerance test, and an HbA1c value of 5.7-6.4%. A prediabetic patient is considered to be at an elevated risk for developing diabetes. [0027] The methods of the present invention employ an exosome-enriched product comprising intact bovine milk-derived exosomes. As mentioned above, the present inventors have surprisingly discovered that the administration of an exosome-enriched product comprising intact bovine milk-derived exosomes increases insulin production, restores and/or preserves - cell mass and/or p-cell functional mass, and can be used to delay diabetes progression.

[0028] Bovine milk contains exosomes which are extracellular membrane vesicles of approximately 20-200 nm in diameter. These nanosized structures contain several bioactive agents, including, but not limited to, enzymatic and non-enzymatic proteins (e.g., CD9, CD63, MHC-class II, lactadherin, TSG101 and Hsc70), nucleic acids (including high amounts of microRNA (miRNA) and messenger RNA (mRNA)) and lipids (e.g., phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin).

[0029] As will be described in detail below, bovine milk exosomes can be isolated from the milk whey fraction or from other dairy streams, such as a cheese whey fraction. Procedures for the isolation of bovine milk exosomes include physical methods (e.g., ultracentrifugation at increasing speeds, membrane ultrafiltration or size exclusion chromatography) and chemical methods (e.g., the use of polymers to precipitate bovine milk exosomes by an incubation step). Remarkably, most of these procedures tend to co-purify exosomes and other dairy constituents (i.e., caseins and other whey proteins). The isolation methods yield a fraction enriched in bovine milk exosomes that may undergo further processing to produce a powder suitable for its end use application.

[0030] The term “exosome-enriched product comprising intact bovine milk-derived exosomes” as used herein, unless otherwise specified, refers to a product in which intact exosomes have been substantially separated from other bovine milk components such as lipids, cells, and debris, and are concentrated in an amount higher than that found in bovine milk. Exosomes are small, extracellular vesicles and account for a minor percentage of milk’s total content. In specific embodiments, the exosome-enriched product is provided in a liquid form or a powdered form and also contains co-isolated milk solids.

[0031] The term “intact exosomes” as used herein refers to exosomes in which the vesicle membrane is not ruptured and/or otherwise degraded and the endogenous cargo, i.e. , the bioactive agents, therapeutics (e.g. miRNA), and/or other biomolecules which are inherently present in a bovine milk-derived exosome, are retained therein in active form.

[0032] The term “impaired p-cell function” or “P-cell dysfunction” as used herein, unless otherwise specified, refers to an impaired ability of the pancreatic p-cells to produce and/or secrete insulin.

[0033] The term “diabetes progression” as used herein, unless otherwise specified, refers to the gradual worsening of p-cell function and/or p-cell mass. Diabetes progression may be categorized into stages, with each stage being defined by various physiological changes within the body, e.g., changes in p-cell mass, phenotype, and function.

[0034] With regard to the staging of T2DM, stage 1 is normally defined as insulin resistance. The pancreatic p-cells are able to compensate for insulin resistance by producing more insulin, thereby maintaining normal blood sugar levels. During stage 2, which is often referred to as prediabetes, insulin resistance increases and the production of extra insulin is not enough to maintain euglycemia. At this stage, fasting blood glucose levels reach around 100 to 125 mg/dL, blood glucose levels are around 140-199 mg/dL following a glucose tolerance test, and HbA1c values are between 5.7-6.4%. In stage 3, a patient is diagnosed as diabetic, and will exhibit increased insulin resistance and/or further reduced p-cell mass and/or further impaired p-cell functional mass. Blood sugar levels are abnormally high, i.e. , fasting blood sugar levels are above 126 mg/dL and blood sugar levels are above 200 mg/dL following a glucose tolerance test. HbA1c values are typically 6.5% or higher. Stage 4 normally coincides with “end-stage diabetes” and is linked to a number of diabetic complications, including albuminuria, chronic kidney disease, coronary artery disease, heart failure, peripheral neuropathy, and/or stroke. Fortunately, it is possible for some patients suffering from T2DM to improve their condition through appropriate medication and lifestyle modifications, including improved diet and exercise.

[0035] The term “P-cell functional mass” as used herein, unless otherwise specified, refers to the amount of pancreatic p-cells which are able to produce and/or secrete appropriate amounts of insulin. An assessment of p-cell functional mass is essential for the diagnosis and staging of diabetes.

[0036] The term “P-cell mass” as used herein, unless otherwise specified, refers to the number of existing pancreatic p-cells. p-cell mass is determined as the sum of replication, neogenesis and hypertrophy minus the rate of apoptosis.

[0037] The term “insulin production” as used herein, unless otherwise specified, refers to the process wherein insulin is synthesized by pancreatic p-cells. Insulin is responsible for regulating the level of glucose in the blood. Insulin production involves the biologically inactive precursor, preproinsulin, being cleaved by signal peptidase to form proinsulin. Proinsulin is then converted to insulin by removal of C-peptide by a specialized set of endoproteases and a carboxypeptidase activity. Preproinsulin, proinsulin, and insulin are synthesized in the p-cells of the pancreas.

[0038] The term “insulin secretion” as used herein, unless otherwise specified, refers to the process wherein insulin is released by pancreatic p-cells. Insulin secretion is primarily regulated by the glucose level in the extracellular fluid surrounding the p-cells, although insulin secretion is also stimulated by, inter alia, growth hormone, cortisol, prolactin, melatonin, estrogen, leptin, and glucagon.

[0039] In one embodiment, a method of improving insulin production in a subject suffering from impaired p-cell function is provided. The method comprises administering an exosome- enriched product comprising intact bovine milk-derived exosomes to the subject. In another embodiment of the invention, a method of restoring and/or preserving p-cell mass in a subject suffering from impaired insulin production is provided. The method comprises administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject. In yet another embodiment of the invention, a method of delaying diabetes progression in a subject suffering from impaired insulin production is provided. The method comprises administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject. In specific embodiments of these methods, the subject is suffering from diabetes. In further specific embodiments of these methods, the subject is suffering from metabolic syndrome or prediabetes.

[0040] The term “metabolic syndrome” as used herein, unless otherwise specified, refers to a combination of conditions including high blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels. This grouping of conditions increase the risk of heart disease, stroke and T2DM. According to the National Cholesterol Education Program (NCEP) (Adult Treatment Panel) ATP III definition, metabolic syndrome is present if three or more of the following five criteria are met: waist circumference over 40 inches (men) or 35 inches (women), blood pressure over 130/85 mmHg, fasting triglyceride (TG) level over 150 mg/dl, fasting high-density lipoprotein (HDL) cholesterol level less than 40 mg/dl (men) or 50 mg/dl (women), and fasting blood sugar over 100 mg/dl.

[0041] In specific embodiments of the methods of the invention, the exosome-enriched product comprises at least about 0.001 wt % exosomes. In other specific embodiments, the exosome-enriched product comprises at least about 0.01 wt %, 1 wt %, 5 wt %, 10 wt %, 15 wt %, 20 wt %, 25 wt %, 30 wt %, 35 wt %, 40 wt %, 45 wt %, or 50 wt % exosomes. In additional specific embodiments of the invention, the exosome-enriched product comprising intact bovine milk-derived exosomes comprises at least 10 wt % exosomes. In further embodiments, the exosome-enriched product comprises at least about 10⁸ exosomes per gram of the exosome- enriched product as measured by a nanotracking procedure. Briefly, nanoparticle tracking analysis (NTA) can be used to determine exosome diameter and concentration. The principle of NTA is based on the characteristic movement of nanosized particles in solution according to the Brownian motion. The trajectory of the particles in a defined volume is recorded by a camera that is used to capture the scatter light upon illumination of the particles with a laser. The Stokes-Einstein equation is used to determine the size of each tracked particle. In addition to particle size, this technique also allows determination of particle concentration.

[0042] In a more specific embodiment, the exosome-enriched product comprises from about 10⁸ to about 10¹⁴ exosomes per gram of the exosome-enriched product. In yet a more specific embodiment, the exosome-enriched product comprises from about 10⁹ to about 10¹³ exosomes per gram of the exosome-enriched product. In another specific embodiment, the exosome- enriched product contains at least about a three-fold increase in the number of exosomes, as compared to a raw whey-containing bovine milk fraction. In a more specific embodiment, the exosome-enriched product contains a 3-fold to 50-fold increase in the number of exosomes, as compared to a raw whey-containing bovine milk fraction, for example cheese whey.

[0043] In specific embodiments of the invention, at least about 50 wt % of the exosomes in the exosome-enriched product are intact. In further specific embodiments of the invention, at least about 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt % of the exosomes in the exosome-enriched product are intact.

[0044] In specific embodiments of the methods of the invention, the intact bovine milk-derived exosomes are sourced from a whey-containing bovine milk fraction. In further specific embodiments of the methods of the invention, the whey-containing bovine milk fraction is cheese whey.

[0045] Generally, in one embodiment, the exosomes are obtained from a whey-containing bovine milk fraction using gentle procedures which do not disrupt the exosome vesicle membrane, thereby leaving the exosomes intact and active bioactive agents contained within the exosome structure.

[0046] Various methods may be employed to isolate exosomes with care being exercised to avoid disruption of the lipid membrane. Fresh bovine milk, refrigerated bovine milk, thawed frozen bovine milk, or otherwise preserved bovine milk, or any bovine milk fraction containing exosomes, for example, cheese whey, may be employed as a source of exosomes. Isolating the exosomes may comprise performing the isolation immediately upon obtaining milk from a bovine. By way of example, isolating the exosomes may comprise performing the isolation within about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days or about 6 days, or about 7 days from the time of obtaining the milk from a bovine. The exosomes may be isolated within about 10 days, or within about 14 days from the time of obtaining milk from a bovine. Additionally, the bovine milk may be frozen and then thawed for processing for isolating exosomes, with the bovine milk preferably having been frozen within about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days or about 6 days, or about 7 days from the time of obtaining the milk from a bovine. Thawed milk is preferably processed immediately upon thawing. In specific embodiments, the fresh bovine milk may be subjected to the processing within about 5 days of obtaining the milk from a bovine, or thawed bovine milk which is subjected to processing is thawed from bovine milk that was frozen within about 5 days of obtaining the milk from a bovine.

[0047] As mentioned above, a whey-containing bovine milk fraction or, specifically, cheese whey, may serve as a source of exosomes. Cheese whey is the liquid by-product of milk after the formation of curd during the cheese-making or casein manufacturing process. Since cheese whey has already been separated from the casein fraction during the cheese manufacture process, cheese whey has very low casein content. Furthermore, cheese whey advantageously retains more than 50% of milk nutrients, including lactose, fat, proteins, mineral salts, and, surprisingly, a significant number of exosomes that were originally present in the milk in intact form. In addition to these benefits, cheese whey is less expensive than raw milk, and thus using cheese whey as a starting material significantly reduces costs for production of an exosome- enriched product. As such, cheese whey is a novel and promising source for isolating milk exosomes and producing exosome-enriched products.

[0048] In a specific embodiment, the cheese whey is obtained by applying an enzyme or enzyme mixture, and more specifically a protease enzyme, for example chymosin, to milk to hydrolyze casein peptide bonds, thus allowing for enzymatic coagulation of casein in the milk. Thus, when the protease enzyme cleaves the protein, it causes the casein in the milk to coagulate and form a gel structure. The casein protein gel network and milk fat then contract together and form curd. The resulting liquid that is separated from the curd is often referred to as sweet whey or cheese whey, typically has a pH from about 6.0 to about 6.5, and comprises whey proteins, lactose, minerals, water, fat and other low level components.

[0049] As indicated above, the enzyme or enzyme mixture is capable of destabilizing the casein protein in the milk fraction by cleaving peptides which stabilize the casein protein in the milk. Therefore, any proteolytic enzyme suitable for this purpose may be used to obtain cheese whey. In a preferred embodiment, however, the cheese whey is provided by adding rennet enzyme to bovine milk, resulting in enzymatic coagulation of casein. Rennet enzyme is commonly used in the cheese making process and comprises a set of enzymes which are produced in the stomachs of ruminant mammals. These enzymes normally include chymosin, pepsin, and lipase. The rennet enzyme mix destabilizes the casein protein in the bovine milk fraction by proteolytically cleaving peptides which stabilize the protein in the milk. As indicated above, the casein in the milk coagulates and contracts with milk fat to form the cheese curd. The remaining liquid, i.e., the sweet cheese whey, comprises whey proteins, lactose, minerals, water, fat, and other low level components.

[0050] By way of example, a gentle procedure of obtaining an exosome-enriched product containing intact bovine milk-derived exosomes may comprise physical methods and/or chemical methods. In one embodiment, an exosome-enriched product is obtained by cascade membrane filtration. In a specific embodiment, the exosome-enriched product is lactose-free. In a specific embodiment, sweet cheese whey, which may be obtained as described in the preceding paragraph, is processed using tandem multiple ceramic filtration steps. In a specific embodiment, a multiple filtration process employs, successively, membranes with cut offs which gradually decrease in size. In a specific embodiment, the method of processing sweet cheese whey is subjected to microfiltration (MF), ultrafiltration (UF) and diafiltration (DF). In a more specific embodiment, the process employs, successively, MF, UF and DF membranes with cut offs of about 1.4 pm, 0.14 pm and 10 kDa, respectively, to provide an exosome-enriched product. For example, a first MF step employs a first membrane with a molecular weight cut off of, for example, about 1.4 pm and yields a first retentate R1 and a first permeate P1. The first permeate P1 is then subjected to a an UF step employing a second membrane with a molecular weight cut off of, for example, about 0.14 pm, which yields a second retentate R2 and second permeate P2. The second retentate R2 may be re-suspended in water and again passed through the second membrane to remove additional lactose, minerals and the like, if desired. For example, in one embodiment, about 5 volumes of water may be added to one volume of the second retentate R2 and the resulting suspension is then passed through the 0.14 pm MF membrane. The resulting third retentate R3 is then subjected to a DF step using a 10 kDa membrane. In a specific embodiment, the third retentate is first diluted with an approximately equal volume of water and diafiltered to obtain a fourth retentate R4, and then the fourth retentate R4 is again diluted with water, for example with a volume of water five times that of the fourth retentate R4 and then diafiltered to yield a concentrated retentate R5. This exosome- enriched product may be used in the form of the concentrated retentate R5, or the concentrated retentate R5 may be further processed.

[0051] In a specific embodiment, the exosome-enriched product resulting from successive filtration steps may be pasteurized to provide storage stability. For example, the exosome- enriched product may be heated, for example, at about 70°C for about 15 seconds, to ensure microbiological stability in order to yield a pasteurized fraction, R6. Other pasteurization conditions will be apparent to those skilled in the art and may be employed.

[0052] With or without pasteurization, the exosome-enriched product may be used as is or subjected to additional processing steps to provide a desired physical form. In one embodiment, the exosome-enriched product, optionally pasteurized, may be converted to a powder form. In more specific embodiments, the exosome-enriched product can be spray-dried, freeze dried, or otherwise converted to powder form. In one specific embodiment, the exosome-enriched product may be spray dried, for example, at 185°C/85°C, to obtain an exosome-enriched product in the form of a spray-dried powder (SP). Prior to spray drying, the exosome-enriched product may be subjected to an optional evaporation step to increase the solids content of the product and therefore reduce the time and/or energy demand for the spray drying process. Other spray drying conditions will be apparent to those skilled in the art and may be employed. Alternatively, the exosome-enriched product may be freeze-dried, for example at -50°C and 0.5 mbar vacuum to obtain an exosome-enriched freeze-dried powder (FP). Other freeze drying conditions will be apparent to those skilled in the art and may be employed.

[0053] In specific embodiments of the methods of the invention, the exosome-enriched product is administered in the form of an exosome-enriched powder. In another specific embodiment, the exosome-enriched product is administered in the form of an exosome- enriched liquid. The exosome enriched product can be administered to the subject in either form. In a further specific embodiment, the exosome-enriched product is an exosome-enriched powder comprising spray-dried intact exosomes. [0054] In specific embodiments of the methods of the invention, the exosome-enriched product comprising intact bovine milk-derived exosomes is administered to the subject orally. In a more specific embodiment, the exosome-enriched product comprising intact bovine milk- derived exosomes is provided in a nutritional composition, which can be administered to the subject orally. In further specific embodiments, the nutritional composition is in the form of a powder. In another specific embodiment, the nutritional composition is in the form of a liquid. [0055] 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 or otherwise mixed with a liquid in order to form a nutritional liquid, and are suitable for oral consumption by a human.

[0056] 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. In specific embodiments, the nutritional compositions comprise water, and in more specific embodiments, comprise emulsions.

[0057] 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.

[0058] The methods described herein employ amounts of an exosome-enriched product comprising intact bovine milk-derived exosomes that are effective to improve insulin production, restore and/or preserve p-cell mass and/or p-cell functional mass, and/or delay diabetes progression. In specific embodiments of the methods of the invention, the subject is administered a dose of about 0.01 to about 50 g, about 0.5 to about 40 g, about 1 to about 40 g, about 1 to about 30 g, about 1 to about 20 g, about 1 to about 10 g, or about 1 to about 5 g of the exosome-enriched product comprising intact bovine milk-derived exosomes per day.

[0059] In further specific embodiments of the invention, the nutritional composition comprises about 0.001 to about 30 wt %, about 0.01 to about 30 wt %, about 0.01 to about 20 wt %, about 0.01 to about 10 wt %, about 0.01 to about 5 wt %, about 0.1 to about 30 wt %, about 0.1 to about 20 wt %, about 0.1 to about 10 wt %, about 0.1 to about 5 wt %, about 1 to about 30 wt %, about 1 to about 20 wt %, about 1 to about 10 wt %, or about 1 to about 5 wt % of the exosome-enriched product comprising intact bovine milk-derived exosomes, based on the weight of the nutritional composition. In a specific embodiment, the nutritional composition comprises about 0.01 to about 10 wt % of the exosome-enriched product comprising intact bovine milk-derived exosomes, based on the weight of the nutritional composition.

[0060] 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.

[0061] 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.

[0062] In specific embodiments, the nutritional composition may comprise protein in an amount of about 0.01 wt % to about 90 wt % of the nutritional composition. More specifically, the protein may be present in an amount of about 1 wt% to about 80 wt %, including about 1 wt% to about 70 wt%, about 1 wt% to about 60 wt%, about 1 wt% to about 50 wt%, about 1 wt% to about 40 wt %, about 1 wt% to about 30 wt %, about 1 wt% to about 25 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 4 wt % to about 80 wt %, about 4 wt % to about 70 wt %, about 4 wt % to about 60 wt %, about 4 wt% to about 50 wt%, about 4 wt% to about 40 wt%, about 4 wt% to about 30 wt%, about 4 wt% to about 20 wt%, about 4 wt% to about 10 wt %, about 9 wt % to about 80 wt %, about 9 wt % to about 70 wt %, about 9 wt % to about 60 wt %, about 9 wt% to about 50 wt%, about 9 wt % to about 40 wt%, about 9 wt% to about 30 wt%, about 9 wt% to about 20 wt%, about 10 wt % to about 90 wt %, about 10 wt % to about 60 wt %, about 10 wt % to about 30 wt %, about 10 wt% to about 20 wt %, about 40 wt % to about 80 wt %, or about 60 wt % to about 75 wt % of the nutritional composition. In a specific embodiment, the nutritional composition is a powder and the protein comprises about 10 wt % to about 90 wt %, about 10 wt % to about 70 wt %, about 10 wt % to about 50 wt %, about 10 wt % to about 30 wt %, about 20 wt % to about 80 wt %, about 30 wt % to about 80 wt %, about 40 wt % to about 80 wt %, about 50 wt % to about 80 wt % or about 60 wt % to about 75 wt %, of the nutritional composition. In a further specific embodiment, the nutritional composition is a liquid and the protein comprises about 0.01 wt % to about 15 wt %, about 1 wt % to about 10 wt %, or about 2 wt % to about 6 wt %, of the nutritional composition.

[0063] In other specific embodiments, the carbohydrate in the nutritional composition comprises fiber, human milk oligosaccharides (HMOs), maltodextrin, corn maltodextrin, corn syrup, organic corn starch, corn syrup, corn syrup solids, soluble corn fiber, sucralose, cellulose gel, cellulose gum, gellan gum, carrageenan, fructooligosaccharides (FOS), inositol, hydrolyzed starch, glucose polymers, rice-derived carbohydrates, sucrose, glucose, glycerin, 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, or 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, resistant maltodextrin (e.g., Fibersol or Nutriose), FOS, inulin, oat fiber, soy fiber, or a combination of two or more thereof.

[0064] In specific embodiments, the nutritional composition may comprise carbohydrate in an amount of about 0.01 wt % to about 75 wt %, including about 0.01 wt % to about 60 wt %, about 0.01 wt % to about 50 wt %, about 0.01 wt % to about 40 wt %, about 0.01 to about 30 wt %, about 0.01 to about 20 wt %, about 0.01 to about 10 wt %, about 1 wt % to about 70 wt %, 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 10 wt % to about 65 wt %, about 20 wt % to about 65 wt %, about 30 wt % to about 65 wt %, about 40 wt % to about 65 wt %, about 40 wt % to about 70 wt %, or about 15 wt % to about 25 wt %, of the nutritional composition. More specifically, the carbohydrate may be present in an amount about 1 wt % to about 10 wt % of the nutritional composition, including about 1 wt % to about 9 wt %, about 1 wt % to about 8 wt %, about 1 wt % to about 7 wt %, about 1 wt % to about 6 wt %, about 2 wt % to about 10 wt %, about 2 wt % to about 9 wt %, about 2 wt % to about 8 wt %, about 2 wt % to about 7 wt %, or about 2 wt % to about 6 wt %, of the nutritional composition. In a specific embodiment, the nutritional composition is a powder and the carbohydrate comprises about 0.01 wt % to about 15 wt %, about 1 wt % to about 12 wt %, or about 8 wt % to about 10 wt %, of the nutritional composition. In a further specific embodiment, the nutritional composition is a liquid and the carbohydrate comprises about 0.01 wt % to about 10 wt %, about 2 wt % to about 8 wt %, or about 4 wt % to about 7 wt %, of the nutritional composition.

[0065] 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, alpha-linolenic acid, fractionated coconut oil, soy oil, corn oil, olive oil, safflower 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.

[0066] 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.

[0067] In specific embodiments, the nutritional composition may comprise fat in an amount of about 0.01 wt % to about 30 wt % of the nutritional composition. More specifically, the fat may be present in an amount of about 1 wt % to about 30 wt%, including 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 9 wt %, about 1 wt % to about 8 wt %, about 1 wt % to about 7 wt %, about 1 wt % to about 6 wt %, about 1 wt% to about 5 wt%, , about 2 wt % to about 10 wt %, about 2 wt % to about 9 wt %, about 2 wt % to about 8 wt %, about 2 wt % to about 7 wt %, about 2 wt % to about 6 wt %, about 2 wt % to about 5 wt %, or about 2 wt % to about 4 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. In a specific embodiment, the nutritional composition is a powder and the fat comprises about 0.01 wt % to about 15 wt %, about 1 wt % to about 10 wt %, about 1 wt % to about 5 wt %, or about 6 wt % to about 8.5 wt %, of the nutritional composition. In a further specific embodiment, the nutritional composition is a liquid and the fat comprises about 0.01 wt % to about 10 wt %, about 1 wt % to about 5 wt %, or about 2 wt % to about 4 wt %, of the nutritional composition. [0068] The concentration and relative amounts of the sources of protein, carbohydrate, and fat in the exemplary nutritional compositions can vary considerably depending upon, for example, the specific dietary needs of the intended user.

[0069] In one embodiment, the nutritional composition is a nutritional liquid composition and comprises about 0.01 to about 15 wt % of protein, about 0.01 to about 10 wt % fat, and about 0.01 to about 10 wt % carbohydrate, based on the weight of the nutritional composition.

[0070] In another embodiment, the nutritional composition is a nutritional powder composition and comprises about 10 to about 90 wt % of protein, about 0.01 to about 15 wt % fat, and about 0.01 wt % to about 15 wt % carbohydrate, based on the weight of the nutritional composition. [0071] 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, digestion resistant maltodextrin, sucrose, and/or short-chain fructooligosaccharide. [0072] 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. [0073] 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.

[0074] 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 oilsoluble components; and (c) mixing together the aqueous solution and the oil blend to form an emulsified liquid nutritional composition. The exosome-enriched product can be added at any point in the formation of the nutritional composition.

[0075] As indicated above, the nutritional composition can be administered in the form of a powder or in the form of a liquid.

[0076] When the nutritional composition is a powder, for example, a serving size is about 10 g to about 70 g, about 20 g to about 45 g, or about 45 g to about 70 g, to be administered as a powder or to be reconstituted in about 1 mL to about 500 mL of liquid. In a specific embodiment, a serving size is about 31 g to be reconstituted in about 237 mL of liquid.

[0077] 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 230 mL to about 475 mL, about 230 mL to about 350 mL, about 230 mL to about 300 mL, about 290 mL to about 475 mL, about 290 mL to about 350 mL, about 290 mL to about 300 mL, about 300 mL to about 475 mL, about 300 mL to about 350 mL, and about 330 mL to about 475 mL. In specific embodiments, the serving is about 237 mL, or about 296 mL, or about 330 mL, or about 473 mL.

[0078] In specific embodiments, the nutritional composition comprising an exosome-enriched product comprising intact bovine milk-derived exosomes 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.

[0079] 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 B12, 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.

[0080] 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, iodine, selenium, chloride, and combinations thereof.

[0081] 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.

EXAMPLES

[0082] Example 1 : Preparation and Characterization of Exosome-enriched Products

[0083] This example describes a method of preparing an exosome-enriched product from cheese whey. The cheese whey was provided by adding rennet enzyme to bovine milk, resulting in enzymatic coagulation of casein and production of sweet cheese whey, as described above. [0084] An exosome-enriched product containing from about 10⁸ to about 10¹⁴ intact bovine milk- derived exosomes per gram of the exosome-enriched product was prepared by cascade membrane filtration. First, 1 ,000 L of sweet cheese whey was processed using tandem multiple ceramic filtration steps. The first microfiltration MF step employed a membrane with a molecular weight cut off of 1.4 pm, which yielded a first retentate R1 and a first permeate P1. The first permeate P1 was then subjected to a ultrafiltration step UF with a molecular weight cut off of 0.14 pm, which yielded a second retentate R2 and second permeate P2. About 5 volumes of water was added to one volume of the second retentate R2, and the diluted retentate was then passed through the 0.14 pm UF membrane again to remove at least part of the lactose and minerals. The resulting retentate R3 was then combined with an equal volume of water and diafiltered using a 10 kDa membrane to produce a fourth retentate R4. The retentate R4 was diluted with a volume of water five times that of the fourth retentate R4 and diafiltered a second time using the 10 kDa membrane to yield a concentrated retentate, R5. The lactose-free exosome-enriched product R5 was pasteurized at 70°C for 15 seconds to ensure microbiological stability in order to yield a pasteurized exosome-enriched product R6. The pasteurized exosome-enriched product R6 was subjected to evaporation at about 65°C to increase the solids content up to 17-18% and then spray-dried at 185°C/85°C to obtain a exosome-enriched spray-dried product, SP.

[0085] Example 2: MTT Assay for p-cell Proliferation and Viability

[0086] This example demonstrates that a spray-dried exosome-enriched product comprising intact bovine milk-derived exosomes (SDEx) increased p-cell proliferation of pancreatic p-cells subjected to prolonged exposure (24 hours and 48 hours) of palmitate, as compared to an exosome-enriched product comprising sonicated bovine milk-derived exosomes (SDEx-S), and as compared to the absence of an exosome-enriched product comprising bovine milk-derived exosomes (C). This was shown by evaluating p-cell proliferation through MTT assay of the rat INS-1 cell line (clone 832/13). MTT assay is a colorimetric method which measures the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) to formazan, an insoluble crystalline product with a deep purple color. Only metabolically active cells are able to convert MTT to formazan. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means. Absorbance values greater than the control indicate cell proliferation and increased viability, while lower values suggest cell death or inhibition of proliferation.

[0087] More specifically, the rat INS-1 cell line is a cell line capable of insulin release in response to glucose stimulation and is therefore used as a p-cell model for diabetes research. The cells were maintained in RPMI 1640 medium with 11.1 mmol/L D-glucose supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 pg/mL streptomycin, 10 mmol/L HEPES, 2mmol/L glutamine, 1 mmol/L sodium pyruvate, and 50 pmol/L p-mercaptoethanol at 37°C/5% CO2 in a humidified atmosphere.

[0088] Pre-confluent pancreatic cells were employed. In one set of experiments, the cells were treated with palmitate at a concentration of 250 pM for 24 hours in complete medium in order to induce pancreatic p-cell lipotoxicity. Lipotoxicity, which is defined as the accumulation of excess lipids in non-adipose tissues, has roles in both insulin resistance and pancreatic beta cell dysfunction. Saturated fatty acids, such as palmitate, are known to promote insulin resistance in peripheral tissues and prolonged exposure to palmitate induces changes in p-cell protein lysine acetylation, thereby resulting in p-cell damage. In another set of experiments, the cells were not treated with palmitate.

[0089] In each of the palmitate-treated and non-palmitate treated sets of experiments, an exosome-enriched product comprising intact bovine milk-derived exosomes was added to a first subset of cells, and an exosome-enriched product comprising sonicated bovine milk-derived exosomes was separately added to a second subset of the cells, both exosome products being added at a concentration of 15 ug/mL, three hours before incubation with palmitate. A third subset of cells in each set of experiments, Control cells, had no exosome addition. A cell viability assay was performed by the indirect measurement of cell metabolic activity using MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After 24 hours of incubation, the MTT solution (0.5 mg/mL) was added for 30 minutes. The supernatant was removed, 100 pl MTT solvent was added to each well, and the culture plate was shaken for 10 minutes before the optical density (OD) values were recorded at 570 nm.

[0090] As evidenced by the results in FIG. 1 , compared with Control (C) cells, both nonpalmitate treated and palmitate-treated, and as compared with cells incubated with the sonicated bovine milk-derived exosomes (SDEx-S), both non-palmitate treated and palmitate-treated, the cells incubated with an exosome-enriched product comprising intact bovine milk-derived exosomes (SDEx), both non-palmitate treated and palmitate-treated, respectively, showed increased proliferation capacity at both 24 and 48 hours. With regard to FIG.1 , a two-way ANOVA test was used to analyze the statistical significance between groups, with (*) representing palmitate treated cells vs untreated cells (p<0.05), (&) representing untreated cells vs an exosome-enriched product comprising intact bovine milk-derived exosomes (p<0.05), and (#) representing palmitate treated cells vs an exosome-enriched product comprising intact bovine milk-derived exosomes (p<0.05).

[0091] More specifically, non-palmitate treated INS-1 cells incubated with the exosome- enriched product comprising intact bovine milk-derived exosomes increased p-cell proliferation at 24 and 48 hours by almost 31% and 30%, respectively, as compared to non-palmitate treated Control. Palmitate-treated INS-1 cells incubated with the exosome-enriched product comprising intact bovine milk-derived exosomes increased p-cell proliferation at 24 and 48 hours by almost 42% and 140%, respectively, as compared to palmitate-treated Control.

[0092] Further, non-palmitate treated INS-1 cells incubated with the exosome-enriched product comprising intact bovine milk-derived exosomes increased p-cell proliferation at 24 and 48 hours by almost 42% and 33%, respectively, as compared to non-palmitate treated INS-1 cells incubated with the exosome-enriched product comprising sonicated intact bovine milk-derived exosomes. Palmitate-treated INS-1 cells incubated with the exosome-enriched product comprising intact bovine milk-derived exosomes increased p-cell proliferation at 24 and 48 hours by almost 35% and 32%, respectively, as compared to palmitate-treated INS-1 cells incubated with the exosome-enriched product comprising sonicated intact bovine milk-derived exosomes.

[0093] Surprisingly, palmitate-treated INS-1 cells incubated with an exosome-enriched product comprising intact bovine milk-derived exosomes exerted a significant positive effect, partially preserving p-cell viability of palmitate-treated Control. These results indicate that an exosome- enriched product comprising intact bovine milk-derived exosomes overcomes p-cell lipotoxicity, to a statistically significant degree. These results also indicate that an exosome-enriched product comprising intact bovine milk-derived exosomes was able to improve p-cell viability, and consequently preserve p-cell mass, both of which are critical for improving and/or maintaining p- cell functionality.

[0094] Example 3: Insulin Secretion and Production

[0095] This example demonstrates that an exosome-enriched product comprising intact bovine milk-derived exosomes (SDEx) increased both insulin secretion and insulin production of pancreatic p-cells exposed to extracellular glucose concentration, as compared to an exosome- enriched product comprising sonicated bovine milk-derived exosomes (SDEx-S), or the absence of any exosome-enriched product comprising bovine milk-derived exosomes (C). This was shown by an insulin secretion assay. Experiments were performed in 48-well plates. Acute exposures (2 hours) to extracellular glucose concentration (10 mM) were performed during the last phase of the insulin secretion assay. Insulin secretion and content were expressed as pmol/L insulin per mg protein. All results were obtained from six independent experiments.

[0096] Specifically, INS-1 cells were maintained in RPMI 1640 medium with 11.1 mmol/L D- glucose supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 pg/mL streptomycin, 10 mmol/L HEPES, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, and 50 pmol/L P-mercaptoethanol at 37°C/5% CO2 in a humidified atmosphere. [0097] In assessing insulin secretion, the cells were washed twice with 300 pL glucose-free Krebs-Ringer Bicarbonate buffer (KRB) (116 mM NaCI, 1.8 mM CaCl2'2(H2O), 0.8 Mm MgSO₄'7(H₂O), 5.4 mM KCI, 1 mM NaH₂PO4-2(H₂O), 26 mM NaHCO₃, and 0.5% BSA, pH 7.4), followed by pre-incubation for 1 hour at 37°C in 200 pL 2 mM glucose KRB. Thereafter, cells were washed two times with 200 pL glucose-free KRB, prior to a 2 hour incubation in 200 pL KRB under 10 mM glucose and the different effectors, i.e., an exosome-enriched product comprising intact bovine milk-derived exosomes and an exosome-enriched product comprising sonicated bovine milk-derived exosomes. After 2 hours, medium was collected and stored at -80°C.

[0098] As evidenced by the results in FIG. 2, compared with control, INS-1 cells incubated with an exosome-enriched product comprising intact bovine milk-derived exosomes (20 pg/mL) increased insulin secretion of pancreatic p-cells. The acute response (2 hour incubation) of insulin secretion to extracellular glucose concentration in the presence of an exosome-enriched product comprising intact bovine milk-derived exosomes showed a significant difference (*p<0.05 vs untreated cells (C) and #p<0.05 vs SDEx-S treated cells). Interestingly, no effects on insulin secretion were observed with the exosome enriched product comprising sonicated bovine milk- derived exosomes.

[0099] Chronic experiments were also conducted to assess insulin production and secretion in vitro. The cells were incubated for 24 hours with effectors, i.e., an exosome-enriched product comprising intact bovine milk-derived exosomes and an exosome-enriched product comprising sonicated bovine milk-derived exosomes. Cells were then subjected to an insulin secretion assay as described above. The cells were washed twice with 300 pL glucose-free Krebs-Ringer Bicarbonate buffer (KRB) (116 mM NaCI, 1.8 mM CaCl2'2(H2O), 0.8 Mm MgSO4'7(H2O), 5.4 mM KCI, 1 mM NaH2PO4'2(H2O), 26 mM NaHCO₃, and 0.5% BSA, pH 7.4), followed by pre-incubation for 1 hour at 37°C in 200 pL 2 mM glucose KRB. Thereafter, cells were washed two times with 200 pL glucose-free KRB, prior to a 2 hour incubation in 200 pL KRB under 10 mM glucose and the different effectors, i.e., an exosome-enriched product comprising intact bovine milk-derived exosomes and an exosome-enriched product comprising sonicated bovine milk-derived exosomes. After 2 hours, medium was collected and stored at -80°C.

[00100] As evidenced by the results in FIG. 3, compared with control C, INS-1 cells incubated with an exosome-enriched product comprising intact bovine milk-derived exosomes (SDEx, 15 pg/mL) significantly increased insulin production of pancreatic p-cells. No effects on insulin production were observed with the exosome enriched product comprising sonicated bovine milk- derived exosomes (SDEx-S). The exosome-enriched product comprising intact bovine milk- derived exosomes (15 pg/mL) increased insulin production by almost 111 % as compared to Control, and almost 102% as compared to the exosome-enriched product comprising sonicated bovine milk-derived exosomes. These results indicate that the exosome-enriched product containing intact bovine milk-derived exosomes was able to stimulate and thus increase insulin production. Sonicated exosomes did not provide any significant improvement on insulin production.

[00101] In order to confirm the results set forth above, total insulin production in response to effectors was again measured. The cells were incubated for 24 hours in the presence or absence of an exosome-enriched product comprising intact bovine milk-derived exosomes and an exosome-enriched product comprising sonicated bovine milk-derived exosomes. Insulin concentration in INS-1 cell lysates was measured using the Rat Insulin Enzyme Immunoassay Kit according to manufacturer’s instructions (Alpco Diagnostics, Salem, NH).

[00102] INS-1 cells were maintained in RPMI 1640 medium with 11.1 mmol/L D-glucose supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 pg/mL streptomycin, 10 mmol/L HEPES, 2mmol/L glutamine, 1 mmol/L sodium pyruvate, and 50 pmol/L - mercaptoethanol at 37°C/5% CO2 in a humidified atmosphere.

[00103] The cells were incubated for 24 hours in the presence and the absence of an exosome- enriched product comprising intact bovine milk-derived exosomes and an exosome-enriched product comprising sonicated bovine milk-derived exosomes. Cells were washed twice with PBS, scraped from the plate, sonicated and the insulin concentration was measured in the cell lysates. [00104] As illustrated in FIG. 4, the results confirm that pancreatic p-cells incubated with an exosome-enriched product comprising intact bovine milk-derived exosomes exhibited enhanced insulin production.

[00105] In summary, an exosome-enriched product comprising intact bovine milk-derived exosomes increases insulin secretion, insulin production, and p-cell proliferation and viability. The increased insulin secretion, insulin production, and p-cell proliferation and viability leads to improved p-cell functionality, which has a significant application in the prevention or treatment of diabetes.

[00106] The in vivo consumption of an effective dose of an exosome-enriched product comprising intact bovine milk-derived exosomes can thus promote increased insulin production and increased insulin secretion and thereby improve diabetes treatments and clinical outcomes. [00107] While the present application has been illustrated by the description of embodiments and examples thereof, and while the embodiments and examples 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 methods or compositions, 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 improving insulin production in a subject suffering from impaired p-cell function, comprising administering an exosome-enriched product comprising intact bovine milk- derived exosomes to the subject.

2. A method of restoring and/or preserving p-cell mass in a subject suffering from impaired insulin production, comprising administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject.

3. A method of delaying diabetes progression in a subject suffering from impaired insulin production, comprising administering an exosome-enriched product comprising intact bovine milk-derived exosomes to the subject.

4. The method of any one of claims 1-3, wherein the subject is suffering from diabetes.

5. The method of claim 1 or 2, wherein the subject is suffering from metabolic syndrome or prediabetes.

6. The method of any one of claims 1-5, wherein the intact bovine milk-derived exosomes are sourced from a whey-containing bovine milk fraction.

7. The method of claim 6, wherein the whey-containing bovine milk fraction is cheese whey.

8. The method of any one of claims 1-7, wherein the exosome-enriched product comprises at least about 0.001 wt %, 0.01 wt %, 1 wt %, 5 wt %, 10 wt %, 15 wt %, 20 wt %, 25 wt %, 30 wt %, 35 wt %, 40 wt %, 45 wt %, or 50 wt % exosomes. The method of any one of claims 1-8, wherein at least about 50 wt % of the exosomes in the exosome-enriched product are intact. The method of claim 9, wherein at least about 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt % of the exosomes in the exosome-enriched product are intact. The method of any one of claims 1-10, wherein the exosome-enriched product is an exosome-enriched powder. The method of any one of claim 11 , wherein the exosome-enriched powder comprises spray-dried intact exosomes. The method of any one of claims 1-10, wherein the exosome-enriched product is an exosome-enriched liquid. The method of any one of claims 1-13, wherein the exosome-enriched product comprising intact bovine milk-derived exosomes is administered to the subject orally. The method of any one of claims 1-14, wherein the exosome-enriched product comprising intact bovine milk-derived exosomes is administered to the subject at a dose of about 0.01 to about 50 g, about 0.5 to about 40 g, about 1 to about 40 g, about 1 to about 30 g, about 1 to about 20 g, about 1 to about 10 g, or about 1 to about 5 g per day. The method of any one of claims 1-15, wherein the exosome-enriched product comprising intact bovine milk-derived exosomes is administered to the subject in a nutritional composition. The method of claim 16, wherein the nutritional composition comprises protein, carbohydrate and/or fat. The method of claim 17, 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, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, fava bean protein, or combinations of two or more thereof. The method of any one of claims 17 or 18, wherein the nutritional composition comprises carbohydrate and the carbohydrate comprises fiber, human milk oligosaccharides (HMOs), maltodextrin, digestion resistant maltodextrin, corn maltodextrin, corn syrup, organic corn starch, corn syrup, corn syrup solids, soluble corn fiber, sucralose, cellulose gel, cellulose gum, gellan gum, carrageenan, fructooligosaccharides (FOS), inositol, hydrolyzed starch, glucose polymers, rice-derived carbohydrates, sucrose, glucose, glycerin, 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, or combinations of two or more thereof. The method of any one of claims 17-19, wherein the nutritional composition comprises fat and the fat comprises canola oil, coconut oil, fractionated coconut oil, soy oil, soy lecithin, corn oil, safflower oil, sunflower oil, palm kernel oil, palm olein, canola oil monoglycerides, lecithin, omega-3 fatty acid, anola oil, medium chain triglycerides, cocoa butter, fatty acid, linoleic acid, alpha-linolenic acid, or combinations of two or more thereof. The method of claim 20, wherein the nutritional composition comprises omega-3 fatty acid and the omega-3 fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), alpha-linolenic acid (ALA), and combinations of two or more thereof. The method of any one of claims 16-21 , wherein the nutritional composition comprises about 0.001 wt % to about 30 wt%, or about 0.01 wt % to about 10 wt % of the exosome- enriched product comprising intact bovine milk-derived exosomes, based on the weight of the nutritional composition. The method of any one of claims 16-22 wherein the nutritional composition is in the form of a powder. The method of claim 23, wherein the nutritional composition comprises about 0.01 to about 15 wt %, about 1 to about 10 wt %, about 1 wt % to about 5 wt %, or about 6 to about 8.5 wt % fat, based on the weight of the nutritional composition. The method of claim 23 or claim 24, wherein the nutritional composition comprises about 0.01 wt % to about 90 wt %, about 1 wt % to about 50 wt %, about 1 wt % to about 25 wt %, about 1 wt % to about 10 wt %, about 1 wt % to about 5 wt %, about 10 wt % to about 90 wt

%, about 10 wt % to about 60 wt %, about 10 wt % to about 30 wt %, about 20 wt % to about 80 wt %, about 40 wt % to about 80 wt %, or about 60 wt % to about 75 wt % protein, based on the weight of the nutritional composition. The method of any one of claims 23-25, wherein the nutritional composition comprises about 0.01 wt % to about 40 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 12 wt %, or about 8 wt % to about 10 wt % carbohydrate, based on the weight of the nutritional composition. The method of any one of claims 16-22, wherein the nutritional composition is in the form of a liquid. The method of claim 27, wherein the nutritional composition comprises about 0.01 to about 10 wt %, about 1 to about 5 wt %, or about 2 to about 4 wt % fat, based on the weight of the nutritional composition. The method of claim 27 or claim 28, wherein the nutritional composition comprises about 0.01 to about 15 wt %, about 1 to about 10 wt %, or about 2 to about 6 wt % protein, based on the weight of the nutritional composition. The method of any one of claims 27-29, wherein the nutritional composition comprises about 0.01 to about 10 wt %, about 2 to about 8 wt %, or about 4 to about 7 wt % carbohydrate, based on the weight of the nutritional composition.

31. The method of any one of claims 16-30, wherein the nutritional composition further comprises one or more vitamins and/or minerals.