WO2008115390A2 - Methods of using defensins to treat diabetes - Google Patents

Abstract

The present invention provides methods of regulating metabolic disorders including administering to a subject in need thereof an effective amount of a defensin. Metabolic disorders that can be regulated according to the present invention include diabetes, hyperglycemia, insulin resistance, hyperinsulinemia, obesity, hyperlipidemia and hyperlipoproteinemia.

Description

Methods of Using Defensins to Treat Diabetes

Related Application Data

This application claims the benefit of U.S. Patent Application Serial No. 60/895,216, filed March 16, 2007, the contents of which are incorporated herein by reference in its entirety.

Field of the Invention

The present invention relates to methods of using defensins to regulate metabolic disorders such as diabetes and hyperglycemia.

Background of the Invention

The World Health Organization (WHO) estimates that more than 180 million people worldwide have diabetes. Diabetes can be characterized as a chronic condition that occurs when the pancreas does not produce sufficient insulin or when the body cannot effectively use the insulin it produces. The cause of diabetes is generally unknown, however, both genetics and environmental factors such as obesity and lack of exercise appear to be contributory factors.

In general, there are two basic forms of diabetes: type I which results from the body's failure to produce insulin such that people with this type of diabetes produce very little or no insulin, and type Il which results from insulin resistance (a condition in which the body fails to properly use insulin), combined with relative insulin deficiency. A third type of diabetes, gestational diabetes mellitus (GDM), develops during some cases of pregnancy, but usually disappears after pregnancy. Pre-diabetes is a condition that occurs when a person's blood glucose levels are higher than normal but not high enough for a diagnosis of type Il diabetes. It is estimated that 5-10% of Americans who are diagnosed with diabetes have type I diabetes, while most Americans who are diagnosed with diabetes have type Il diabetes.

Currently, people with type I diabetes may require daily injections of insulin to survive. People with type Il diabetes can sometimes manage their condition with lifestyle measures alone, but oral drugs are often required, and less frequently insulin, in order to achieve satisfactory metabolic control.

Diabetes may result in numerous life-threatening complications. For example, diabetes may lead to various microvascular diseases, such as retinopathy, nephropathy, and neuropathy. Diabetic individuals also have a higher likelihood of developing life-threatening macrovascular diseases, such as heart disease and stroke. Other complications arising from long-standing diabetes include blindness, kidney failure, and limb amputations.

In general, the goal of diabetes treatment is to control glucose level in the blood and maintain glucose levels in a range that mimics that of a non-diabetic individual, namely one that reproduces natural physiological glucose homeostasis. To date, this goal has not been fully effectively achieved. Moreover, virtually everyone with type I diabetes (and generally at least one in three people with type II) must inject insulin to correct the deficiency.

In view of the foregoing, there is a need in the art for methods of treating diabetes that better mimic the natural delivery of insulin to the liver resulting in improved glucose homeostasis.

Summary of the Invention

Embodiments of the present invention provide methods of regulating metabolic disorders. In particular, embodiments of the present invention relate to regulating glucose and lipid metabolism, generally to reduce insulin resistance, hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia, hyperlipoproteinemia (such as chylomicrons, VLDL and LDL), and to regulate body fat and more generally lipid stores, and, more generally, for the improvement of metabolism disorders, especially those associated with diabetes and obesity and/or atherosclerosis.

According to some embodiments of the present invention, defensins can be used to treat diabetes by substituting for insulin in type I diabetes. In other embodiments of the present invention, defensins can bypass the intracellular signaling pathway of insulin in type Il diabetes. Embodiments of the present invention provide methods of enhancing the immune system in a diabetic subject including administering to the subject an effective amount of a defensin.

Embodiments of the present invention further provide methods of treating infections in a subject including administering to the subject afflicted with or at risk of developing an infection an effective amount of a defensin. In particular embodiments, the subject is diabetic. In other embodiments, the infection is a microbial infection, septicemia, bacterial meningitis, urinary tract infection or infection-induced hypoglycemia.

Embodiments of the present invention also provide methods of inhibiting glucose production including administering an effective amount of a defensin to a subject.

Embodiments of the present invention further provide methods of inhibiting expression of a gluconeogenic nucleic acid sequence including administering an effective amount of a defensin to a subject.

Embodiments of the present invention further provide methods as described herein including administering defensins in combination with other agents useful for the treatment, management and control of metabolic disorders such as diabetes and hyperglycemia.

Brief Description of the Drawings

Figure 1 illustrates that HNP-1 decreases fasting plasma glucose levels in mice through inhibition of hepatic gluconeogenesis. HNP-1 (0.4 mg/kg body weight) dissolved in 100 μ\ PBS was administered to C57BL/6 mice that were fasted for 24 h via tail vein injection. Control mice received equal volume of the vehicle solution PBS. (A) Plasma levels of glucose were measured with blood from tail veins at ^ΛA h, 3 h, and 8 h after the injection. (B) Transcripts of PEPCK, G6Pase and PGC-1σ genes in liver were quantified by Taqman Real-time RT- PCR. (C) Phospho-c-Src^Tyr416 in liver was detected by immunoblotting.

Figure 2 illustrates that HNP-1 inhibits glucose production via gluconeogenesis in isolated perfused liver. Livers from Sprague Dawley rats that were fasted for 24 h were isolated, cannulated, and perfused as detailed in "Methods". (A) Glucose production from liver was quantified in the presence or absence of gluconeogenic substrate sodium lactate (2 mM). HNP-1 (250 nM) or the vehicle solution was infused over a 12 min span as noted. (S) Bile was collected every 5-10 min, and the bile flow was presented as μl/min/100g body weight (BW). (C and D) Levels of G6Pase and PEPCK transcripts along with phosphorylated c-Src and Akt in liver were measured by TaqMan real-time PCR and immunoblotting, respectively. Results represent mean ± SD of 5 livers perfused with the vehicle solution and 7 livers perfused with HNP-1.

Figure 3 illustrates that HNP-1 inhibits hepatic gluconeogenesis in cultured hepatocytes. (A) Mouse primary hepatocytes in 24-well plates were prepared as previously described (Cao, W.H. et al. p38 mitogen-activated protein kinase plays a stimulatory role in hepatic gluconeogenesis. J Biol Chem 280, 42731-7 (2005); Collins, Q.F., Xiong, Y., Lupo, J., E.J., Liu, H.Y. & Cao, W. p38 mitogen-activated protein kinase mediates free fatty acid-induced gluconeogenesis in hepatocytes. J. Biol. Chem. 281 , 24336-44 (2006)), and pre- treated with HNP-1 or insulin at indicated concentrations for 4 h, followed by further treatment with dexamethasome (Dex, 500 nM) and N⁶,2'-O- Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (cAMP) (0.1 mM) in the presence of sodium pyruvate (2 mM) for 3 h in serum- and glucose-free media. Glucose production via gluconeogenesis was quantified and normalized to protein concentrations, (β) Levels of LDH in the media of the cultures were measured. (C) Hepatocytes were pre-treated with HNP-1 or insulin for 4 h, and then stimulated by forskolin for 2 h as noted. Levels of PEPCK and G6Pase transcripts were quantified by Taqman real-time PCR and normalized to GAPDH. (D) The PEPCK promoter was introduced into Hep1c1c7 cells via transient transfection, and was stimulated by forskolin for 4 h in the presence or absence of a pre-treatment with HNP-1 or insulin as noted. Activity of the promoter was measured by luciferase assays and normalized to protein concentrations. All results represent means ± SEM of 3 independent experiments.

Figure 4 illustrates that HNPs stimulate phosphorylation of Akt and FoxO1 in hepatocytes. (A-E) Hepatoma cells (HepG2 and Hepa1c1c7) and isolated mouse hepatocytes were treated with HNP-1 , HNP-2, or insulin for 15 min as noted. Levels of phosphorylated Akt and FoxO1 were then measured by immunoblotting with specific antibodies as indicated. Levels of total Akt, FoxO1 , or β-actin in the same blots were also measured as loading controls. (F) The constitutively nuclear form of FoxO1 (Ad-FoxO1-ADA) or GFP encoded by adenoviruses (10⁸ plaque-forming units/well of 6-well plate) were introduced into isolated hepatocytes 24 h before cells were treated with forskolin for 2 h in the presence of HNP-1 or insulin as noted. The expression of GδPase and PEPCK genes was quantified by TaqMan real-time PCR. All results represent 3 independent experiments.

Figure 5 illustrates that HNPs suppress hepatic gluconeogenesis through c-Src tyrosine kinase. (A and S) Primary mouse hepatocytes were isolated and treated with HNP-1 , HNP-2, or insulin for 15 min as noted. Levels of c-Src phosphorylation, phosphorylation of IRS1 at tyrosine⁸⁹⁶, and β-actin were measured by immunoblotting with specific antibodies as indicated. (C) Hepatocytes were pre-treated with a c-Src kinase inhibitor, PP2, or an inactive analog, PP3, for 30 min prior to treatment with HNP-1 or insulin for 4 h. Cells were then stimulated with forskolin for another 2 h, followed by measurements of GδPase gene transcripts by TaqMan real-time PCR. (D) CSK, a suppressor of c- Src activation, was overexpressed in primary hepatocytes by transient transfection for 36 h. Glucose production from these cells was quantified as detailed in "Methods" after the cells were treated with cAMP/Dex (0.1 mM/500 nM) for 3 h in the presence or absence of a 30 min-preincubation with either HNP-1 or insulin. Results represent means ± SD of 2 independent experiments, each in triplicate.

Figure 6 illustrates that HNP-1 reduces blood glucose levels in ZDF diabetic rats primarily through suppression of endogenous glucose production (EGP). (A) HNP-1 (2 μmol) was administrated via jugular vein catheter, followed by measurements of blood glucose levels. Hyperinsulinemic-euglycemic clamps were performed as detailed in "Materials and Methods", (β) Basal blood glucose right before clamp studies, but 30 min after treatment with HNP-1 or the vehicle solution, (C) basal plasma insulin, (D) clamped blood glucose, (E) clamped plasma insulin, (F) glucose infusion rate, (G) 2-[14C]DG disappearance from blood, (H) glucose uptake in gastronemius, (/) glycolysis in gastrocnemius, (J) EGP, and (K) plasma SGOT during the clamps were measured. All data represent the mean ± SEM (n = 5 for the vehicle group and n = 6 for the HNP-1 group).

Figure 7 illustrates that HNP-1 inhibits hepatic glucose production in primary hepatocytes. (A-C) Primary hepatocytes from mice were seeded in 24- well plates and pre-treated with HNP-1 or insulin at indicated concentrations for 4 h. Cells were then treated with dexamethasome (Dex) and cAMP or forskolin alone in the presence or absence of sodium lactate for 3 h in serum- and glucose-free media. Total glucose production and glucose production via glycogenosis were quantified and normalized to protein concentrations. Glucose production via gluconeogenesis was calculated as detailed in "Methods". *: P < 0.05 vs. Dex/cAMP. (D) Primary hepatocytes were pre-treated with HNP-1 or insulin for 4 h, and then stimulated by forskolin for 2 h as noted. Levels of PEPCK and GΘPase transcripts were quantified by TaqMan^® real-time PCR and normalized to GAPDH. (E) The PEPCK promoter was introduced into Hep1c1c7 cells via transient transfection, and stimulated by forskolin for 4 h in the presence or absence of a pre-treatment with HNP-1 or insulin as noted. Activity of the promoter was measured by luciferase assays and normalized to protein concentrations. (F) Levels of LDH in the culture media were measured. All results represent means ± SEM of 3 independent experiments.

Figure 8 illustrates that HNPs stimulate phosphorylation of Akt and FoxO1 in hepatocytes. (A-F) Hepatoma cells (HepG2 and Hepa1c1c7) and isolated mouse hepatocytes were treated with HNP-1 (100 nM or 300 nM), HNP-2 (100 nM or 300 nM), or insulin (10 nM) for 15 min as noted. Levels of phosphorylated Akt, FoxO1 , and AMPK were then measured by immunoblotting with specific antibodies as indicated. Levels of total Akt, FoxO1 , and AMPKα in the same blots were also measured as loading controls. (G) The constitutively nuclear form of FoxO1 (Ad-FoxO1-ADA) or GFP encoded by adenoviruses (10⁸ plaqueforming units/well of 6-well plate) were introduced into isolated hepatocytes 24 h before cells were treated with forskolin for 2 h in the presence of HNP-1 or insulin as noted. Transcripts of G6Pase and PEPCK genes were quantified by TaqMan^® real-time PCR. All results represent 3 independent experiments.

Figure 9 illustrates that HNPs suppress hepatic gluconeogenesis through c-Src tyrosine kinase. (A and S) Primary mouse hepatocytes were treated with HNP-1 (100 nM or 300 nM), HNP-2 (100 nM or 300 nM), or insulin (10 nM in (A) and 10 nM or 100 nM in (^β)) for 15 min as noted. Levels of c-Src phosphorylation, IRS1 phosphorylation at tyrosine896, total c-Src, and total IRS1 were measured by immunoblotting with specific antibodies as indicated and quantified. (C) Primary hepatocytes were pre-treated with a c-Src kinase inhibitor, PP2, or an inactive analog, PP3, for 30 min prior to treatment with HNP-1 or insulin for 4 h. Cells were then stimulated with forskolin for another 2 h, followed by measurements of GδPase gene transcripts by TaqMan^® real-time PCR. (D) CSK₁ a suppressor of c-Src activation, was overexpressed in H411 E hepatoma cells by transient transfection for 36 h. Glucose production via gluconeogenesis from these cells was quantified as detailed in "Methods" after the cells were treated with cAMP/Dex for 3 h in the presence or absence of a 30 min- preincubation with either HNP-1 or insulin. Results represent means ± SEM of 2 independent experiments, each in triplicate.

Detailed Description

The present invention will now be described with reference to the following embodiments. As is apparent by these descriptions, this invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Except as otherwise indicated, standard methods can be used for manipulation of nucleic acid sequences and the like according to the present invention. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention described herein.

As used herein, "a" or "an" or "the" can mean one or more than one. Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

"Metabolic disorder" as used herein refers to diabetes, insulin resistance, glucose intolerance, hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia, or hyperlipoproteinemia. The terms "diabetes" and "diabetes mellitus" are intended to encompass both insulin dependent and non-insulin dependent (Type I and Type II, respectively) diabetes mellitus, gestational diabetes, as well as prediabetes, unless one condition or the other is specifically indicated.

"Hyperglycemia" or "hyperglycaemia", also known as high blood sugar, as used herein refers to elevated blood glucose concentration and can result when an excessive amount of glucose circulates in the blood plasma.

"Defensin" as used herein refers to an antimicrobial peptide wherein mammalian defensins are classified as three subfamilies: α-, β-, and θ-defensins, with several members in each; and their sequences and molecular structures are conserved among species (Selsted, M. E. & Ouellette, A.J. Mammalian defensins in the antimicrobial immune response. Nat Immunol 6, 551-7 (2005)). Defensins, as used in the present invention, may be derived from any species such as rabbit, rat, mouse, insect, amphibian or human or may be chemically synthesized. Defensins are produced by neutrophils and intestinal epithelial cells. Human α-defensins produced by neutrophils are natural peptide antibiotics and called human neutrophil peptides, α-defensins according to embodiments of the present include, but are not limited to, HNP-1 , HNP-2, HNP-3, HNP-4, HNP-5 and HNP-6 variants and isoforms thereof. β-defensins according to embodiments of the present include, but are not limited to, BD1, BD2, BD4, EP2E and variants and isoforms thereof, and in some embodiments, hBD1 , hBD4 and variants and isoforms thereof, θ-defensin according to embodiments of the present include, but are not limited to, RTD-1 and variants and isoforms thereof.

"Immune system" as used herein refers to the complex system that serves its host by providing natural resistance and recovery against both pathogens of an external source as well as aberrant "self cells. Generally, the immune system provides both "innate", i.e. inborn and unchanging, or "adaptive", i.e., acquired immune response and includes both humoral immune responses (mediated by B lymphocytes) and cellular immune responses (mediated by T lymphocytes).

"Nucleic acid" or "nucleic acid sequence" as used herein encompasses both RNA and DNA, including cDNA, genomic DNA₁ synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be a sense strand or an antisense strand. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

By the term "express," "expresses" or "expression" of a nucleic acid sequence, in particular a gluconeogenic nucleic acid sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, transcription and translation of the coding sequence will result in production of a gluconeogenic polypeptide or active fragment thereof.

"Fragment" as used herein is one that substantially retains at least one biological activity normally associated with that protein or polypeptide. In particular embodiments, the "fragment" substantially retains all of the activities possessed by the unmodified protein. By "substantially retains" biological activity, it is meant that the protein retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native protein (and can even have a higher level of activity than the native protein).

"Isoform" as used herein refers to a different form of a protein, regardless of whether it originates from a different gene or splice variant or by modification of a single gene product. Thus, "isoform" as used herein refers to a form of a protein that migrates differently from another form of that protein on a two- dimensional gel.

"Variant" as used herein refers to nucleic acid molecules described herein that encode portions, analogs or derivatives of the gluconeogenic proteins described herein or the resulting protein product therefrom. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells, et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)). Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both.

"Microbial infection" as used herein refers to the pathological state resulting from the invasion of the body by pathogenic microorganisms and includes viral infections, bacterial infections, fungal, protozoal and parasitic infections, etc. See, e.g., U.S. Patent No. 6,902,743. Examples include, but are not limited to, sepsis, toxic shock syndrome (Staphylococcus, Streptococcus), meningitis (both bacterial and viral/ Group B Streptococcus, Escherichia coli, Listeria monocytogenes, Streptococcus pneumoniae (pneumococcus) and Neisseria meningitides and septicemia. An increased incidence of urinary tract infections in diabetes has been suggested to be associated with decreased expression of certain types of defensins in kidney and bladder. (Hiratsuka et al. Nucleotide sequence and expression of rat beta-defensin-1: its significance in diabetic rodent models. Nephron 88, 65-70 (2001); Froy et al. Differential effect of insulin treatment on decreased levels of beta-defensins and Toll-like receptors in diabetic rats. MoI Immunol (2006)).

"Septicemia" or "Septicaemia" as used herein refers to a microbe-induced condition in which the subject generally experiences an exaggerated inflammatory response. This response can lead to varying degrees of hypotension (possibly shock, i.e., septic shock), and hypoxemic and edema- related organ failure called multiple organ dysfunction syndrome (MODS). Bacterial infections account for many cases of septicemia. Septicemia is usually categorized by the particular group of microorganism involved, i.e., bacterial, Gram negative or Gram positive, and fungal.

"Subjects" as used herein are generally human subjects and include, but are not limited to, "patients." The subjects may be male or female and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. Subjects may also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates (including non-human primates), etc., for treatment of metabolic disorders as well as veterinary medicine and/or pharmaceutical drug development purposes. Subjects further include, but are not limited to, those who are afflicted with or at risk for a metabolic disorder such as diabetes, insulin resistance, glucose intolerance, hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia, or hyperlipoproteinemia. Risk factors for type Il diabetes include, but are not limited to, obesity, apple-shaped figure, increased age, sedentary lifestyle, family history, history of diabetes in pregnancy, impaired glucose tolerance, ethnic ancestry, in particular, being of Aboriginal, African, Latin American or Asian ethnic ancestry increases the risk of developing of type Il diabetes, high blood pressure and high cholesterol or other fats in the blood.

An "effective" amount as used herein is an amount of the compound or composition of this invention that provides some improvement or benefit to the subject. Alternatively stated, an "effective" amount is an amount that provides some alleviation, mitigation, or decrease in at least one clinical symptom of glucose intolerance or diabetes in the subject (e.g., improved glucose tolerance, enhanced glucose-stimulated insulin secretion, and the like) or in at least one clinical symptom of a disorder associated with hypersecretion of insulin or hyperproliferation of pancreatic islet beta cells (e.g., more normalized insulin levels, etc.) as is well-known in the art. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

The term "regulate" as used herein refers to the ability to affect a method, process, state of being, disorder or the like. The effect may be that of prevention, treatment or modulation. "Modulate," "modulates" or "modulation" refers to enhancement or inhibition in the specified activity. "Inhibition" or "inhibiting" refers to the prevention, reduction, decrease or cessation of the specified activity or process.

"Treat," "treating" and "treatment" as used herein refer to an action resulting in a reduction in the severity of the subject's condition or at least the condition is partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition and/or prevention or delay of the onset of the condition. Thus, the term "treat" refers to both prophylactic and therapeutic treatment regimes.

According to embodiments of the present invention, methods of regulating a metabolic disorder include administering to a subject in need thereof an effective amount of a defensin. The metabolic disorder can be at least one of diabetes, hyperglycemia, insulin resistance, hyperinsulinemia, obesity, hyperlipidemia or hyperlipoproteinemia. In some embodiments, the metabolic disorder is diabetes. In some embodiments, the metabolic disorder is type I diabetes, type Il diabetes, gestational diabetes or pre-diabetes. In other embodiments, the metabolic disorder is hyperglycemia.

In further embodiments of the present invention, the defensin is a mammalian defensin, i.e., derived from canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, etc. The mammalian defensin can be an α-, β-, and θ-defensin. α- defensins according to embodiments of the present invention include, but are not limited to, HNP-1 , HNP-2, HNP-3, HNP-4, HNP-5 and HNP-6 variants and isoforms thereof, β-defensins according to some embodiments of the present invention include, but are not limited to, BD1 , BD2, BD4 and variants and isoforms thereof, and also hBD1 , hBD2, hBD4, EP2E and variants and isoforms thereof. Further, θ-defensins according to embodiments of the present invention include, but are not limited to, RTD-1 and variants and isoforms thereof. In particular embodiments, any defensin now known or later discovered can be used in the methods of the present invention.

According to other embodiments of the present invention, methods of enhancing the immune system in a diabetic subject include administering to the subject an effective amount of a defensin such as those described herein. In particular embodiments, defensins can directly kill microorganisms such as bacteria, fungi, protozoa, and enveloped viruses or indirectly kill microorganisms by stimulating host cells (Selsted et al. Mammalian defensins in the antimicrobial immune response. Nat Immunol 6, 551-7 (2005); Klotman et al. Defensins in innate antiviral immunity. Nat Rev Immunol 6, 447-56 (2006)). In such embodiments, the activity of defensins is not compromised by the diabetic condition of the subject and/or does not interfere with the ability of defensins to regulate metabolic processes associated with diabetes as described herein.

According to some embodiments, the present invention provides methods of treating microbial infections in a diabetic subject including administering to the diabetic subject afflicted with or at risk of developing a microbial infection an effective amount of a defensin such as those described herein. In some embodiments, the microbial infection includes viral infections, bacterial infections, fungal, protozoal and parasitic infections, etc. Examples include, but are not limited to, sepsis, toxic shock syndrome (Staphylococcus, Streptococcus), meningitis (both bacterial and viral/ Group B Streptococcus, Escherichia coli, Listeria monocytogenes, Streptococcus pneumoniae (pneumococcus) and Neisseria meningitides and septicemia. In further embodiments, the infection may be a urinary tract infection.

The methods according to the present invention further include inhibiting glucose production including administering an effective amount of a defensin, such as those described herein, to a subject. Glucose production can be inhibited via hepatic gluconeogenesis, which can contribute to hyperglycemia.

According to further embodiments, the present invention provides methods of inhibiting expression of a gluconeogenic nucleic acid sequence including administering an effective amount of a defensin, such as those described herein, to a subject. In particular embodiments, the gluconeogenic nucleic acid sequence encodes phosphoenolpyruvate carboxykinase (PEPCK) or glucose-6- phosphatase (GδPase). In particular embodiments, the defensin activates Akt also known as "protein kinase B (PKB)".

Embodiments of the present invention further provide pharmaceutical formulations including defensins for regulating the metabolic disorders described herein. The metabolic disorder can be at least one of diabetes, hyperglycemia, insulin resistance, hyperinsulinemia, obesity, hyperlipidemia or hyperlipoproteinemia. In some embodiments, the metabolic disorder is diabetes. In some embodiments, the metabolic disorder is type I diabetes, type Il diabetes, gestational diabetes or pre-diabetes. In other embodiments, the metabolic disorder is hyperglycemia.

The active agents can be formulated for administration in accordance with known pharmacy techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical composition according to the present invention, the active agents (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier can be a solid or a liquid, or both, and can be formulated with the active agent as a unit-dose formulation, for example, a tablet, which can contain from 0.01% or 0.5% to 95% or 99%, or any value between 0.01% and 99%, by weight of the active agent. One or more active agents can be incorporated in the compositions of the invention, which can be prepared by any of the well-known techniques of pharmacy, comprising admixing the components, optionally including one or more accessory ingredients. Moreover, the carrier can be preservative free, as described herein above.

The pharmaceutical compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalational (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, topical (i.e., skin, ocular and mucosal surfaces, including airway surfaces), intraoperative, transdermal administration and parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intraarterial, or intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used.

For example, formulations suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations can be prepared by any suitable method of pharmacy which includes bringing into association the active compound and a suitable carrier (which can contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets can be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

As another example, formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain buffers and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unifλdose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising active compounds, or a salt thereof, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate that is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt. When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. Non-limiting examples of agents that contribute to the pharmaceutical acceptability of the compositions of the present invention include normal saline, phosphatidyl choline, and glucose. In some embodiments, the pharmaceutically acceptable carrier can be normal saline. In other embodiments, the pharmaceutically acceptable carrier can be normal saline with up to 0.0, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, and 20%, and any value between 0.01 % and 20%, glucose.

The effective amount of the composition will vary somewhat from subject to subject, and will depend upon factors such as the age and condition of the subject and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art. For example, the active agents of the present invention can be administered to the subject in an amount ranging from a lower limit of about 0.01 , 0.02. 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mg to an upper limit of about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg in a single dose; in an amount ranging from a lower limit of about 0.01 , 0.02. 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mg to an upper limit of about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg in a 24 hour period. In some embodiments, the dosage can be in a range from about 0.1 mg/kg to about 100 mg/kg of total body weight of said individual. The frequency of administration can be one, two, three, four, five times or more per day or as necessary to control the condition. The duration of therapy depends on the type of condition being treated and can be for as long as the life of the subject. In some embodiments, the dosage regimen is 0.1 to 1000 mg/kg/day.

Embodiments of the present invention further provide methods as described herein including administering defensins in combination with other agents useful for the treatment, management and control of metabolic disorders such as diabetes and hyperglycemia. Such agents include, but are not limited to, all forms of insulin; sulfonylurea drugs, including but not limited to, second- generation sulfonylureas such as glipizide (Glucotrol, Glucotrol XL), glyburide (DiaBeta, Glynase PresTab, Micronase) and glimepiride (Amaryl); meglitinides, including, but not limited to, repaglinide (Prandin); biguanides, including but not limited to, metformin (Glucophage, Glucophage XR); alpha-glucosidase inhibitors, including but not limited to, acarbose (Precose) and miglitol (Glyset), thiazolidinediones, including but not limited to, rosiglitazone (Avandia) and pioglitazone hydrochloride (Actos); and drug combinations.

The present invention is explained in greater detail in the following non- limiting examples.

Examples Materials and Methods

Reagents. HNP-1 and HNP-2 peptides with 1-6, 2-4, and 3-5 specific disulfide bonds were purchased from Sigma. Antibodies against Akt, phospho- _Akts^er⁴⁷³ _FoχOi _f FoxO1^Ser256, phospho-c-Src^1"^⁴¹⁶, phospho-AMPK and AMPKα were from Cell Signalling Technology, and antibodies against IRS1^pY896 or β-actin were from Sigma. Antibodies against G6Pase and PEPCK and the siRNA against AMPKα were from Santa Cruz Biotechnology, Inc. The PEPCK promoter reporter construct was a kind gift from Dr. Jianhua Shao. The adenoviruses encoding a constitutively nuclear FoxO1 mutant (Ad-Fox-O1 -ADFA) was a kind gift from Dr. Domenico Accili. Animals. All animal studies were approved by the Animal Care and Use Committee of the Hamner Institutes for Health Sciences and fully complied with the guidelines from the US National Institutes for Health.

Cell cultures and glucose production via gluconeogenesis in hepatocytes. Hepatoma cell lines including Hepa1c1c7 cells, H411E and HepG2 were cultured and maintained in DMEM media supplemented with 10% FBS. Primary hepatocytes were isolated as previously described (Cao et al. (2005) J. Biol. Chem. 280, 42731-42737; Collins et al. (2006) J. Biol. Chem. 281 , 24336-24344; Xiong et al. (2006) J. Biol. Chem. 282, 4975-4982; Liu et al. (2007) J. Biol. Chem. 282, 14205-14212; Collins et al. (2007) J. Biol. Chem. Ahead of print) (Note: Mice were not fasted before hepatocyte isolations.) Hepatocytes in 24-well plates in William's Medium E were washed with PBS, and pre-treated in serum-free media with HNP-1 or insulin for 4 h. They were then treated with 0.1 mM N⁶,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (cAMP) and 500 nM dexamethasome for 3 h in the presence or absence of gluconeogenic substrates (2 mM sodium lactate) in serum- and glucose-free media. The culture media were subsequently collected for measuring glucose and lactate dehydrogenase (LDH) and glucose with an YSI immobilized-enzyme glucose analyzer (YSI, Yellow Springs, OH). The glucose production via hepatic gluconeogenesis was calculated as previously described (Collins et al. (2006) J. Biol. Chem. 281 , 24336-24344). Specifically, glucose production in the presence of the gluconeogenic substrate (sodium lactate) was considered as total glucose production; glucose production in the absence of sodium lactate was defined as glycogenosis. Glucose production via gluconeogenesis = total glucose production - glycogenosis.

Measurements of hepatic glucose production via gluconeogenesis in isolated perfused liver. Rat liver perfusion was performed using a non- recirculating method as previously described (Adams et al. (1998) Biochem. Pharmacol. 55, 1915-1920). Briefly, livers from male rats (300-400 g) that had been fasted for 24 h were cannulated and isolated under anaesthesia with pentobarbital (45 mg/kg BW). Perfusion was through a portal vein cannula with hemoglobin-free Krebs-Ringer buffer, which was saturated with 95% oxygen/5% carbon dioxide at 37°C. The perfusion rate was 3 ml/min/g liver. Livers were flushed and equilibrated with the perfusion buffer for 20 min before further experimentation. Perfusion fractions were collected every minute for 80 min. Bile was collected every 5-10 min. Glucose production was quantified in the presence or absence of gluconeogenic substrate (2 mM lactate) as noted. HNP-1 (250 nM) or vehicle solution was infused over a 12 min span as indicated. Immediately after the perfusion, livers were stored at -80°C, and phosphorylation of c-Src and Akt along levels of G6Pase and PEPCK transcripts in the liver were measured by immunoblotting and real-time PCR, respectively.

Treatment of mice with HNP-1. HNP-1 (0.4 mg/kg body weight) dissolved in 100 μl PBS or the vehicle solution PBS was administered to C57BU6 mice that were fasted for 24 h via tail vein injection. Control mice received the vehicle solution PBS. Plasma or blood glucose levels were measured with blood from tail veins at the time points as noted. At the end of the experiments, all blood from sacrificed mice was collected for measurements of plasma insulin, and/or serum glutamicoxaloacetic transaminase (SGOT) and lactate dehydrogenase (LDH). Liver samples were collected and stored at -80⁰C. Transcripts of PEPCK, G6Pase and PGC-1α genes were measured by TaqMan^® Realtime RT-PCR. Levels of GβPase and PEPCK proteins were determined by immunoblotting with the specific antibodies and quantified by densitometry.

Hyperinsulinemic-euglycemic clamp study. All clamp studies were performed in male 11-week-old Zucker diabetic fatty rats (ZDF/Cr\-Leprfa, strain code 370), purchased from Charles River Laboratories (Wilmington, MA). The ZDF rats were fed with Purina 5008 diet. Indwelling catheters were placed into the right carotid artery and the left jugular vein with a standard procedure, and animals were allowed to recover from the surgery for 3 days before clamp studies. All clamp studies were performed after a 16 h fast as previously described (Kim et al.. (2001) J. CHn. Invest. 108, 437-446; Jin et al. (2007) J. Nutr. 137, 339-344). The synthesized HNP-1 peptide (2 μmol per kg body weight) or same volume of saline solution (vehicle) was administered through the catheter in the left jugular vein 30 minutes prior to the clamp studies. During the 120-min clamps, hyperinsulinemia was achieved by a continuous infusion of human insulin (60 pmol/kg/min) while the blood glucose level was maintained at euglycemia via infusion of 20% glucose at various rates. Blood glucose levels were evaluated every 10 min. At the end of the clamps, additional 100 μl of blood was collected for measurements of insulin, SGOT and LDH. Levels of endogenous glucose production (EGP) during the clamp studies were measured as previously described (Kim et al.. (2001) J. CHn. Invest. 108, 437-446; Jin et al. (2007) J. Nutr. 137, 339-344). Specifically, a prime-continuous infusion of [3-³H] glucose (8 μCi bolus, 0.11 μCi/min; CAT#: TRK239, GE Healthcare) was performed during the clamps. Blood samples were collected at 0, 60, 90, and 120 min time points for measurements of blood glucose with a glucose meter (Ascensia Breeze 2, Bayer) and plasma ³H by scintillation counting. To examine glucose uptake by skeletal muscle, a solution of 2- deoxy- D-[I-¹⁴C] glucose (2-[¹⁴C]DG₁ Cat#: CFA-728, GE Healthcare) was administered as a bolus (10 μCi) at 75 min through the clamps. Blood samples (20 μl) were collected at 80, 85, 90, 100, 110, and 120 min time points of the clamps for the determination of plasma [³H] glucose, 3H2O, and 2-[¹⁴C]DG concentrations. Uptake of 2- [¹⁴C]DG by gastrocnemius muscle was measured as previously described (Kim et al. (2003) Diabetes 52, 1311-1318).

RNA extraction and real-time PCR. Total RNAs from liver samples or cells were extracted by an RNase mini kit from Qiagen and reverse-transcribed into cDNA. The target cDNAs were further quantified by a TaqMan^® real-time PCR with specific probes from Applied Biosciences, and normalized to levels of GAPDH. The assay identification numbers for the probes and primers used in this study were Mm00440636_m1 (PEPCK), Mm00839363_m1 (G6Pase), Mm00447183_m1 (PGC-1α), and Mm99999915_g1 (GAPDH). lmmunoblotting and measurements of plasma insulin. As previously described (31-33), tissue or cell lysates were prepared with lysis buffer (20 mM Tris-HCI (pH 7.5), 137 mM NaCI, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 2 μg/ml leupeptin and 10 μg/ml aprotinin, supplemented with 1 mM PMSF before use), resolved in 4-20% Trisglycine gels, and transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% skim milk, the membranes were incubated overnight with primary antibodies (1 :1000 dilution). After extensive washes, the membranes were then incubated in 5% skim milk containing a 1 :5000 dilution of the second antibody against rabbit IgG coupled to alkaline phosphatase (Sigma). Fluorescent bands were visualized with Typhoon 9410 variable mode Imager from GE Healthcare (Piscataway, NJ), and then quantified by densitometry using ImageQuant 5.2 software from Molecular Dynamics (Piscataway, NJ).

Plasma levels of human and rat/mouse insulin were determined by Linco ELISA kits (Linco Research Inc., St. Charles, MO).

DNA transfection, luciferase assay and adenoviral infection. DNA plasmids were introduced into the indicated cells by Lipofectamine²⁰⁰⁰ transfection agents. Promoter activity was detected by a luciferase assay system (Promega) with a Wallac 1420 Multilabel Counter (Perkin- Elmer) and normalized to the protein level. Adenoviruses encoding the nuclear form of FoxO1 (Ad- FoxO1-ADA), amplified in HEK-293 cells, were applied to infect primary hepatocytes at 10⁸ plaque-forming units/well in 6-well plates as described before (Cao et al. (2005) J. Biol. Chem. 280, 42731^2737).

Statistical Analysis. All data are presented as the mean ± SEM. Data were evaluated for statistical significance by Student f-tests using GraphPad Prism version 5.0 for Windows (San Diego, CA). Differences at values of p < 0.05 were considered significant.

Example 1 HNP-1 inhibits hepatic gluconeogenesis in animals

To test the hypothesis that HNPs can inhibit hepatic gluconeogenesis, HNP-1 was administered to mice that were fasted for 24 h via tail vein injection for V2-8 h,. As shown in Fig. 1A₁ HNP-1 reduced fasting plasma levels of glucose. Hepatic transcripts of PEPCK and G6Pase genes (P < 0.05 in all cases) were also decreased with no effect on PGC-1α mRNA (Fig. 1B). Application of HNP-1 also activated c-Src through phosphorylation at c-Src^Tyr416 in liver (Fig. 1C). These results support an inhibitory role for HNPs in hepatic gluconeogenesis.

Example 2 HNP-1 suppresses hepatic gluconeogenesis in isolated liver

To directly examine the effect of HNP-1 on hepatic gluconeogenesis, isolated rat livers were infused with HNP-1 , followed by measurements of hepatic glucose production via gluconeogenesis, expression of key gluconeogenic genes, and phosphorylation of Akt and c-Src. Application of HNP-1 promptly inhibited hepatic glucose production via gluconeogenesis with no effect on bile production and release of SGOT and LDH from liver (Fig. 2A and 2B₁ and data not shown), indicating no negative impact on viability of the liver. The inhibition of hepatic glucose production by HNP-1 was coincident with activation of c-Src and Akt along with decreased expression of G6Pase and PEPCK genes (Fig. 2C and 2D).

Example 3

HNPs can suppress hepatic glucose production via gluconeogenesis in isolated hepatocytes

To test the hypothesis that HNPs can inhibit hepatic gluconeogenesis, isolated mouse hepatocytes were stimulated by cAMP/dexamethasome (Dex) in the presence or absence of HNP-1. Glucose production via gluconeogenesis was measured as previously described (Collins et al. (2006) J. Biol. Chem. 281 , 24336-24344). HNP-1 significantly inhibited glucose production via gluconeogenesis (Fig. 3A) although to a lesser extent than equimolar amounts of insulin. Similar results were observed when HNP-2 was used (data not shown). To control for the possibility that the inhibition of hepatic gluconeogenesis by HNP-1 was caused by some non-specific cellular damage, levels of lactate dehydrogenase (LDH) in the media were measured. As shown in Fig. 3B, HNP-1 at concentrations effective at suppressing gluconeogenesis did not evoke release of LDH from hepatocytes, while HNP-1 at 2 μM caused a significant release of LDH.

Example 4 HNP-1 suppresses transcription of gluconeogenic genes

Since hepatic gluconeogenesis is tightly controlled by transcription of the rate-limiting genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6- phosphatase (G6Pase) Hanson et al. (1997) Ann u. Rev. Biochem. 66, 581-611), the effect of HNP-1 on their expression was examined. As shown in Fig. 3C, HNP-1 significantly decreased the expression of GδPase and PEPCK genes induced by a cAMP elevating agent, forskolin. Again, the inhibition by HNP-1 was not as strong as that induced by insulin. The effect of HNP-1 on activation of the PEPCK promoter was examined. As shown in Fig. 3D, activation of the PEPCK promoter by forskolin was significantly suppressed by HNP-1 in a concentration- dependent manner. Together, these results demonstrate that HNP-1 can inhibit transcription of the rate-limiting genes of hepatic gluconeogenesis.

Example 5 HNPs stimulate phosphorylation of Akt and FoxO1 in hepatocytes

Since phosphorylation of Akt is a necessary event in the signalling cascade for insulin to suppress transcription of hepatic gluconeogenic genes (Whiteman et al. (2002) Trends Endocrinol. Metab. 13, 444-451), levels of phosphorylated Akt in hepatoma cells (HepG2 and Hep1c1c7 cell lines) and primary mouse hepatocytes that had been treated with HNP-1 , HNP-2 or insulin were measured. As shown in Fig. 4A-C, both HNP-1 and -2 increased Akt phosphorylation to an extent similar to insulin suggesting that HNPs might suppress transcription of gluconeogenic genes through Akt.

Akt-mediated phosphorylation and the consequent exclusion of FoxO1 from the nucleus are necessary for insulin to inhibit hepatic gluconeogenesis (Puigserver et al. (2003) Nature 423, 550-555; Matsumoto et al. (2006) J. Clin. Invest. (2006) 116, 2464-2472). Levels of phosphorylated FoxO1 in hepatocytes that had been treated with HNPs or insulin were examined. Both HNP-1 and -2 could stimulate phosphorylation of FoxO1 similar to insulin in Hepa1c1c7 cells and primary hepatocytes (Fig. 4D and E). These results together support a notion that HNPs suppress gluconeogenesis through Akt and FoxO1.

To further examine the role of FoxO1 in the mechanism by which HNPs inhibit gluconeogenesis, a mutant FoxO1 (Ad-FoxO1-ADA) that cannot be excluded from the nucleus (Frescas et al. (2005) J. Biol. Chem. 280, 20589- 20595; Kitamura and Forkhed (2006) Nat. Med. 12, 534-540), was overexpressed in hepatocytes. The cells were then treated with forskolin in the presence or absence of HNP-1 or insulin. As shown in Fig. 4F, the presence of constitutively nuclear FoxO1 prevented the ability of both HNP-1 and insulin to suppress the expression of G6Pase and PEPCK genes. Since hepatic gluconeogenesis can also be inhibited by the 5'-AMP- activated protein kinase (AMPK) in an insulin-independent manner (Berasi (2006) J. Biol. Chem. 281 , 27167-27177), the possibility that AMPK was involved in the HNP effect was examined. Levels of AMPK in primary hepatocytes that had been treated with HNP-1 , HNP-2, or insulin were measured. It was noted that AMPK was not activated by either HNPs or insulin (Fig. 4E).

Example 6

HNPs inhibit hepatic gluconeogenesis through a c-Src-dependent signalling pathway

To further define the intracellular signalling pathway of HNPs in hepatocytes, we measured levels of IRS1 tyrosine phosphorylation, which is essential for insulin-induced phosphorylation of Akt (reviewed in Thirone et al. (2006) Trends Endocrinol. Metab.), and phosphorylation of c-Src, another known activator of Akt (Warmuth et al. (2003) Curr. Farm. Des. 9, 2043-2059; Choudhury et al. (2006) Cell. Signal. 18, 1854-1864). As shown in Fig. 5A and B, in contrast to insulin, which led to IRS1 tyrosine phosphorylation without significantly activating c-Src, HNP-1 and -2 failed to stimulate tyrosine phosphorylation of IRS1 , but clearly stimulated phosphorylation of c-Src. To define the role of c-Src in HNP-mediated suppression of hepatic gluconeogenesis, a c-Src inhibitor PP2 prior to the treatment with HNP-1 was used, followed by measurements of G6Pase gene transcripts. As shown in Fig. 5C, both HNP-1 and insulin significantly inhibited expression of the G6Pase gene induced by forskolin. Interestingly, the blockade of c-Src function prevented the HNP-1 -induced inhibition of G6Pase gene expression without affecting the insulin-induced suppression. To further examine the role of c-Src activation in the HNP-dependent inhibition of hepatic gluconeogenesis by a separate approach, CSK, a suppressor of c-Src kinase activation (Chong et al. (2005) Growth Factors 23, 233-244), was overexpressed in hepatocytes. As shown in Fig. 5D, CSK prevented the inhibitory effect of HNP-1 on glucose production via gluconeogenesis with no effect on insulin. Together, these results indicate that HNP defensins inhibit hepatic gluconeogenesis through a c-Src-dependent signalling pathway. In summary, results from this study demonstrate an interesting role for HNPα-defensins in hepatic gluconeogenesis, and this role is mediated through a pathway distinct from insulin signalling. Under normal physiological conditions, it is unlikely that HNPs and other defensins play a significant role in regulation of hepatic gluconeogenesis because plasma levels of HNPs in healthy subjects vary between 13.2-42 ng/ml (0.0038-0.012 μM) (Shiomi et al. (1993) Biochem. Biophys. Res. Commun. 195, 1336-1344; Panyutich et al. (1993) J. Lab. CHn. Med. 122, 202-207), which is well below the effective level (0.1-0.3 μM) of HNPs in suppression of hepatic gluconeogenesis. However, under certain conditions such as severe bacterial infection (septicaemia and meningitis), plasma levels of HNPs in humans are dramatically increased and can reach as high as 900- 170,000 ng/ml (0.257-48.57 μM) (Shiomi et al. (1993) Biochem. Biophys. Res. Commun. 195, 1336-1344; Panyutich et al. (1993) J. Lab. CHn. Med. 122, 202- 207). It has been observed that severe infections such as septicaemia and bacterial meningitis are frequently accompanied by hypoglycaemia (Ardawi et al. (1989) J. Lab. Clin. Med. 144, 579-586; Romijn et al. (1990) J. Endocrinol. Invest. 13, 743-747; Giovambattista et al. (2000) Neuroimmunomodulation 7, 92-98). Furthermore, it is known that severe bacterial infection can suppress expression of hepatic gluconeogenic genes without affecting plasma glucose levels (Ardawi et al. (1989) J. Lab. CHn. Med. 144, 579-586; de Vasconcelos et al. (1987) CHn. Sci. (Lond.) 72, 683-691). This infection-induced inhibition of hepatic gluconeogenesis and the consequent hypoglycaemia are currently believed to be related to TNF-α (Romijn et al. (1990) J. Endocrinol. Invest. 13, 743-747; Kennison et al. (1991) Am. J. Vet. Res. 52, 1320-1326). However, these findings on the inhibitory role of HNP defensins in hepatic gluconeogenesis may provide a new or additional explanation for infection-induced hypoglycaemia.

Some studies have shown that plasma levels of defensins are decreased in diabetic subjects, and this decrease has been suggested to be associated with an increased incidence of certain infections in diabetes (Hiratsuka et al. (2001) Nephron. 88, 65-70; Froy et al. (2006) MoI. Immunol. 44, 796-802). Although it is still unclear whether defensins play any role in the improvement of glycaemic indices, treatment of diabetic subjects with insulin can normalize plasma levels of defensins along with glycaemic indices (Froy et al. (2006) MoI. Immunol. 44, 796-802).

Since hepatic gluconeogenesis is a contributor to hyperglycaemia in diabetes mellitus at least due to a deficiency in either insulin production or intracellular signalling of insulin (Accili (2004) Diabetes 53, 1633-1642), these findings on the inhibitory role of HNPs in hepatic gluconeogenesis may provide a new investigative opportunity in the management of diabetes.

Example 7

HNP-1 reduces blood glucose levels via suppression of hepatic glucose production in both normal mice and Zucker Diabetic fatty (ZDF) rats.

As outlined in Example 1 , to test the hypothesis that HNPs inhibit hepatic glucose production, normal mice were administered HNP-1 by tail vein injection for V2-8 h, followed by measurements of blood glucose and expression of key gluconeogenic genes in the liver. As shown in Fig. 1A, blood glucose levels were significantly reduced by HNP-1 in a time-dependent manner. Coincidently, hepatic levels of PEPCK and G6Pase proteins were decreased (Fig. 1B). Levels of peroxisome proliferator-activated receptor coactivatoMα (PGC-1α) were not altered (Fig. 1B). These results suggest that HNPs can lower blood glucose levels in wild-type normal animals by inhibiting hepatic gluconeogenesis.

To determine whether or not HNPs can similarly diminish blood glucose levels in animals with insulin-resistant and diabetes, HNP-1 was administered to ZDF rats. As shown in Fig. 6A, blood glucose levels were significantly decreased by one hour treatment through infusion indicating that HNPs can reduce blood glucose in diabetic animals with insulin resistance.

To define the mechanism by which HNPs reduce blood glucose in diabetic animals, the effect of HNP-1 on endogenous glucose production (EGP) and glucose uptake in skeletal muscle and epididymal fat was examined using the hyperinsulinemic-euglycemic clamp procedure. When blood insulin was clamped at a relatively stable level, HNP-1 increased the glucose infusion rate dramatically (Fig. 6 B-F), suggesting either an increase in glucose uptake or a decrease of EGP or both. Glucose uptake and utilization (glycolysis) were not altered significantly in skeletal muscle (gastrocnemius) (Fig. 6H and I). Importantly, EGP was strongly suppressed by HNP-1 (Fig. 6J). It is noteworthy that HNP-1 at the concentrations used in this study did not cause detectable liver damage as measured by enzyme release (Fig. 6K). Together, these results support the proposition that HNPs reduce blood glucose levels in insulin resistant, diabetic animals primarily through inhibition of hepatic glucose production.

Example 8 HNPs suppress hepatic glucose production in isolated hepatocytes.

To recapitulate the effect of HNPs on hepatic glucose production at a cellular level, isolated mouse hepatocytes were stimulated by cAMP/dexamethasome (Dex) or forskolin in the presence or absence of HNP-1. Glucose production via gluconeogenesis or glycogenosis was measured as detailed in "Methods" (Collins et al. (2006) J. Biol. Chem. 281 , 24336-24344). As shown in Fig. 7A-C, HNP-1 significantly inhibited glucose production including gluconeogenesis and glycogenosis although to a lesser extent than equimolar amounts of insulin. Similar results were observed with HNP-2 (data not shown).

Example 9 HNP-1 suppresses transcription of gluconeogenic genes.

Since hepatic gluconeogenesis is tightly controlled by the transcription of rate-limiting genes encoding PEPCK and GβPase (Hanson and Reshef (1997) Annu. Rev. Biochem. 66, 581-611), the effect of HNP- 1 on expression of these genes was examined. As shown in Fig. 7D, HNP-1 significantly decreased transcripts for G6Pase and PEPCK genes induced by a cAMP elevating agent, forskolin. The effect of HNP-1 on activation of the PEPCK promoter was examined. As shown in Fig. 7E, activation of the PEPCK promoter by forskolin was significantly suppressed by HNP-1 in a concentration-dependent manner. To control for the possibility that the inhibition of hepatic glucose by HNP-1 was caused by non-specific cellular damage in the hepatocytes, levels of lactate dehydrogenase (LDH) in the media were measured. As shown in Fig. 7F, HNP-1 at concentrations effective at suppressing gluconeogenesis and glycogenosis did not evoke release of LDH from primary hepatocytes, while a higher concentration of HNP-1 (2 μM) caused a significant release of LDH. Together, these results demonstrate that HNP-1 can inhibit transcription of the rate-limiting genes of hepatic gluconeogenesis.

Example 10 HNPs stimulate phosphorylation of Akt and FoxO1 in hepatocytes.

Since phosphorylation of Akt is a prominent event in the signaling cascade of insulin to suppress transcription of hepatic gluconeogenic genes (Whiteman et al. (2002) Trends Endocrinol. Metab. 13, 444-451), levels of phosphorylated Akt in hepatoma cells (HepG2 and Hep1c1c7 cell lines) and primary mouse hepatocytes that had been treated with HNP-1 , HNP-2 or insulin were measured. As shown in Fig. 8A-C, both HNP-1 and -2 increased Akt phosphorylation suggesting that HNPs suppress transcription of gluconeogenic genes through Akt.

Akt-mediated phosphorylation and the consequent exclusion of FoxO1 from the nucleus are implicated in insulin-mediated inhibition of hepatic gluconeogenesis (Puigserver et al. (2003) Nature 423,550-555; Matsumoto et al. (2006) J. CHn. Invest. 116, 2464-2472). Therefore, levels of phosphorylated FoxO1 in the whole cell lysates of hepatocytes that had been treated with HNPs or insulin were examined. Both HNP-1 and -2 stimulated phosphorylation of FoxO1 in Hepa1c1c7 cells and primary hepatocytes (Fig. 8D and E). These results together support the notion that HNPs suppress gluconeogenesis through Akt and FoxOL To further examine the role of FoxO1 in HNP inhibition of gluconeogenesis, a mutant FoxO1 (Ad-FoxO1-ADA) that can not be excluded from the nucleus (Frescas et al. (2005) J. Biol. Chem. 280, 20589-20595; Kitamura et al. (2006) Nat. Med. 12, 534-540) was overexpressed in hepatocytes. Cells were then treated with forskolin in the presence or absence of HNP-1 or insulin. As shown in Fig. 8F, the presence of constitutively nuclear FoxO1 prevented the ability of both HNP-1 and insulin to suppress expression of G6Pase and PEPCK genes.

Since hepatic gluconeogenesis can also be inhibited by AMPK in an insulin independent manner (Berasi et al. (2006) J. Biol. Chem. 281 , 27167- 27177), the possible involvement of AMPK was examined. Levels of AMPK were measured in primary hepatocytes that had been treated with HNP-1 , HNP-2, insulin, or metformin. It was found that AMPK was activated by HNP-2 and metformin, but not by HNP-1. Blockade of AMPKα did not prevent HNP-2 suppression of hepatic glucose production. (Fig.δF)

Example 11

HNPs inhibit hepatic gluconeogenesis through a c-Src-dependent signaling pathway.

To further define the intracellular signaling pathway of HNPs in hepatocytes, levels of IRS1 tyrosine phosphorylation, which is essential for insulin-induced phosphorylation of Akt (reviewed in (Thirone et al. (2006) Trends Endocrinol. Metab. 17, 72-78)), and phosphorylation of c-Src, another known activator of Akt (Warmuth et al. (2003) Curr. Pharm. Des. 9, 2043-2059; Chodhury et al. (2006) Cell. Signal. 18, 1854-1864) were measured. As shown in Fig. 9A-B, in contrast to insulin, which led to IRS1 tyrosine phosphorylation without significantly activating c-Src, HNP-1 and -2 failed to stimulate significant tyrosine phosphorylation of IRS1 , but clearly stimulated phosphorylation of c-Src. These findings implicated a role for c-Src in HNP-1 suppression of hepatic gluconeogenesis. Therefore, the c-Src inhibitor PP2 was used prior to the treatment with HNP-1 , followed by measurements of G6Pase gene transcripts. As shown in Fig. 9C, both HNP-1 and insulin significantly inhibited expression of the G6Pase gene induced by forskolin. Interestingly, blockade of c-Src activation prevented the HNP-1 -induced inhibition of GδPase gene expression without affecting insulin-induced suppression. The role of c-Src activation in the HNP- dependent inhibition of hepatic gluconeogenesis by a separate approach was examined. CSK, a suppressor of c-Src kinase activation (Chong et al. (2005) Growth Factors 23, 233-244), was overexpressed in hepatocytes. As shown in Fig. 9D, CSK largely prevented the inhibitory effect of HNP-1 on glucose production via gluconeogenesis with no effect on insulin function. Together, these results indicate that HNP defensins inhibit hepatic gluconeogenesis through a c- Src-dependent signaling pathway.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That Which is Claimed is:

1. A method of regulating a metabolic disorder comprising administering to a subject in need thereof an effective amount of a defensin, wherein the metabolic disorder is at least one of diabetes, hyperglycemia, insulin resistance, hyperinsulinemia, obesity, hyperlipidemia or hyperlipoproteinemia.

2. The method of claim 1 , wherein the metabolic disorder is diabetes.

3. The method of claim 1 , wherein the defensin is a mammalian defensin.

4. The method of claim 1 , wherein the mammalian defensin is an α- defensin, β-defensin or θ-defensin.

5. The method of claim 4, wherein the α-defensin is HNP1 , HNP2, HNP3, HNP4, HD5, HD6 or a variant or isoform thereof.

6. The method of claim 4, wherein the β-defensin is BD1 , BD2, BD4, EP2E or a variant or isoform thereof.

7. The method of claim 4, wherein the θ-defensin is RTD-1 or a variant or isoform thereof.

8. The method of claim 2, wherein the diabetes is type I diabetes, type Il diabetes, gestational diabetes or pre-diabetes.

9. The method of claim 1 , wherein the metabolic disorder is hyperglycemia.

10. A method of enhancing the immune system in a diabetic subject comprising administering to said subject an effective amount of a defensin.

11. A method of treating an infection in a subject comprising administering to said subject afflicted with or at risk of developing an infection an effective amount of a defensin.

12. The method of claim 11 , wherein the subject is diabetic.

13. The method of claim 11 , wherein the infection is microbial, septicemia, bacterial meningitis, a urinary tract infection or infection-induced hypoglycemia.

14. The method of claim 11 , wherein the defensin is a mammalian defensin.

15. The method of claim 14, wherein the mammalian defensin is an α- defensin, β-defensin or θ-defensin.

16. The method of claim 15, wherein the α-defensin is HNP1 , HNP3, HD5, HD6 or a variant or isoform thereof.

17. The method of claim 15, wherein the β-defensin is hBD1 , hBD4 or a variant or isoform thereof.

18. The method of claim 15, wherein the θ-defensin is RTD-1 or a variant or isoform thereof.

19. A method of inhibiting glucose production comprising administering an effective amount of a defensin to a subject.

20. A method of inhibiting expression of a gluconeogenic nucleic acid sequence comprising administering an effective amount of a defensin to a subject.

21. The method of claim 20, wherein the gluconeogenic nucleic acid sequence encodes phosphoenolpyruvate carboxykinase (PEPCK) or glucose-6- phosphatase (GδPase).