Autoreactive T‐cell receptor (Vβ/D/Jβ) sequences in diabetes are homologous to insulin,glucagon, the insulin receptor,and the glucagon receptor

The hypervariable (Vβ/D/Jβ) regions of T‐cell receptors (TCR) have been sequenced in a variety of autoimmune diseases by various investigators. An analysis of some of these sequences shows that TCR from both human diabetics and NOD mice mimic insulin, glucagon, the insulin receptor, and the glucagon receptor. Such similarities are not found in the TCR produced in other human autoimmune diseases. These data may explain how insulin, glucagon, and their receptors are targets of autoimmunity in diabetes and also suggest that TCR mimicking insulin and its receptor may be targets of anti‐insulin autoantibodies. Such intra‐systemic mimicry of self‐proteins also raises complex questions about how “self” and “nonself” are regulated during TCR production, especially in light of the complementarity of insulin for its receptor and glucagon for its receptor. The data presented here suggest that some TCR may be complementary to other TCR in autoimmune diseases, a possibility that is experimentally testable. Such complementarity, if it exists, could either serve to down‐regulate the clones bearing such TCR or, alternatively, trigger an intra‐immune system civil war between them. Copyright © 2008 John Wiley & Sons, Ltd.     

Involvement of glycoconjugates in insulin-receptor interactions Studies in liver plasma membranes of control and diabetic mice

The involvement of glycoconjugates in the insulin-receptor interactions in mouse liver is tested by digestions of membranes with various enzymes. Trypsin decreased the binding of [¹²⁵I]insulin to liver membranes. After digestion with β-galactosidase no “high affinity” receptor sites could be detected. The effects observed with plant lectins confirm the involvement of galactoconjugates in the insulin binding process. Sophora japonica and Ricinus communis lectins (with galactose specificity) and concanavalin A largely inhibit the binding process of insulin and those effects concern the “high affinity” receptor sites. Other lectins (wheat germ agglutinin, Dolichos) and enzymes (α-l-fucosidase, $\text{β-N-}\text{acetyl-hexosaminidase and neuraminidase}$) are without effect on insulin binding.Comparative studies performed on diabetic mouse liver membrane (KK mice), previously characterized by decreased number of insulin receptors, are in good agreement with qualitatively similar receptor sites in both non-diabetic (control) and diabetic mice. Effects of enzymes and lectins yielded same results as compared to control membranes. Plasma membrane proteins and glycoproteins in both types of mouse are indistinguishable with respect to enzymic and chemical analysis. Sodium dodecyl sulphate acrylamide gel electrophoresis shows identical patterns. Moreover, the decrease in the number of insulin receptors is easily reversed with diet restriction. These data are consistent with the similarity of receptor sites in control and diabetic liver membrane.     

The mobile receptor hypothesis and “cooperativity” of hormone binding. Application to insulin

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone.A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from “negative cooperativity”. (2) Receptor occupancy and biological response may be coupled in a non-linear fashion.By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of “cooperatively.” These considerations may also be pertinent to intepretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

ANALYSIS OF LIGAND–RECEPTOR INTERACTIONS IN CELLS BY ATOMIC FORCE MICROSCOPY

ABSTRACT

Atomic force microscopy (AFM) increasingly has been used to analyse “receptor” function, either by using purified proteins (“molecular recognition microscopy”) or, more recently, in situ in living cells. The latter approach has been enabled by the use of a modified commercial AFM, linked to a confocal microscope, which has allowed adhesion forces between ligands and receptors in cells to be measured and mapped, and downstream cellular responses analysed. We review the application of AFM to cell biology and, in particular, to the study of ligand–receptor interactions and draw examples from our own work and that of others to show the utility of AFM, including for the exploration of cell surface functionalities. We also identify shortcomings of AFM in comparison to “standard” methods, such as receptor auto-radiography or immuno-detection, that are widely applied in cell biology and pharmacological analysis.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.     

Characterization of Insulin-Like Growth Factor Binding to Human Granulosa Cells Obtained During in Vitro Fertilizationd

Abstract

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I.

Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 ± 0.30%/10⁵ cells (SEM, n=9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (αIR-3) inhibited ¹²⁵I-IGF-I but not ¹²⁵I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the αsubunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays.

IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.     

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Insulin receptor recycling in vascular endothelial cells. Regulation by insulin and phorbol ester

Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t1/2 = 18 +/- 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of 125I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by 125I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.     

Syndromic Insulin Resistance: Models for the Therapeutic Basis of the Metabolic Syndrome and Other Targets of Insulin Resistance

ObjectiveTo investigate the link between insulin resistance and the metabolic syndrome how to develop treatment approaches.MethodsWe present 3 cases of extreme syndromic insulin resistance: lipodystrophy, autoantibodies to the insulin receptor, and mutations in the insulin receptor gene, with accompanying discussion of pathophysiology and treatment.ResultsIn lipodystrophy, insulin resistance is a direct consequence of leptin deficiency, and thus leptin replacement reverses metabolic syndrome abnormalities, including diabetes and hypertriglyceridemia. The insulin “receptoropathies,” including autoantibodies to the insulin receptor and insulin receptor gene mutations, are characterized by extreme insulin resistance and ovarian hyperandrogenism, without dyslipidemia or fatty liver disease. Autoantibodies to the insulin receptor can be treated using an immunosuppressive paradigm adapted from treatment of other autoimmune and neoplastic conditions. Leptin treatment has shown some success in treating hyperglycemia in patients with insulin receptor gene mutations. Treatment for this condition remains inadequate, and novel therapies that bypass insulin receptor signaling, such as enhancers of brown adipose tissue, are needed.ConclusionsWe present a clinical approach to the treatment of syndromic insulin resistance. The study of rare diseases that replicate the metabolic syndrome, with clear-cut pathophysiology, promotes understanding of novel physiology and development of targeted therapies that may be applicable to the broader population with obesity, insulin resistance, and diabetes. (Endocr Pract. 2012; 18:763-771)     

Differences in time course of internalization of receptors of insulin and insulin-like growth factor (IGF-1) in isolated rat hepatocytes

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of ¹²⁵I-insulin and ¹²⁵I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular ¹²⁵I-IGF-I correlated with a decrease in the ¹²⁵I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.     

The effects of insulin and tunicamycin on insulin receptors of cultured fibroblasts

The effect of down-regulation on the intracellular pool of insulin receptors and the role of glycosylation in recovery from down-regulation have been studied in fibroblastic cultures from the skin of non-diabetic mice. In control cultures, 55% of the total specific [¹²⁵I]insulin-binding activity was in the intracellular compartment. Insulin caused a time- and concentration-dependent decrease in the number of cell surface insulin receptors, with no significant change in total insulin receptors. This decrease in surface receptors was accompanied by an increase in the specific binding of [¹²⁵I]insulin in the intracellular compartment. Removal of insulin from down-regulated cells resulted in a time-dependent increase in the binding of [¹²⁵I]insulin to surface receptors, reaching 90% of that in controls by 12 h. The recovery of surface insulin receptors after removal of insulin was blocked by incubation of cultures with tunicamycin, but not by cycloheximide. These results indicate that down-regulation of surface insulin receptors by insulin is associated with translocation of receptors into the intracellular pool and suggest that protein glycosylation is important in insulin receptor recycling and externalization.     

Differential signalling potential of insulin- and IGF-1-receptor cytoplasmic domains.

The human receptors for insulin-like growth factor 1 (IGF-1) and insulin, and two chimeric receptors consisting of ligand-binding, extracellular insulin receptor and intracellular IGF-1 receptor structures, have been expressed in NIH-3T3 fibroblasts. All four receptor types were synthesized, processed and transported to the cell surface to form high-affinity binding sites. All normal and chimeric receptors had an active tyrosine kinase which was regulated by homologous or heterologous ligands respectively. In addition, cell surface receptors were internalized efficiently and subjected to accelerated degradation in the presence of ligand. While all four types of receptor stimulated glucose transport with similar efficiency, they displayed significant differences in their mitogenic signalling potentials. Receptors with an IGF-1 receptor cytoplasmic domain were 10 times more active in stimulating DNA synthesis than the insulin receptor. In NIH-3T3 cells overexpressing wild-type and chimeric receptors, maximal growth responses obtained with IGF-1 or insulin alone were equivalent to those obtained with 10% fetal calf serum. We conclude that in the cell system employed the receptors for IGF-1 and insulin mediate short-term responses similarly, but display distinct characteristics in their long-term mitogenic signalling potentials.     

Antibody Bingling Boyding to the Juxtamembr are Ahch of the Insulin Receptor Alters Reotr Affinity

Abstract

The effect of three antibodies that interact with distinct regions of the insulin receptor (the a subunit (83-7), the juxtamembrane region near tyrosine 960 (960) or the carboxy terminal region of the I3 subunit (CT-1)) on insulin binding was examined. Detergent-solubilized insulin receptors from IM-9 cells immobilized on Sepharose beads by 960 antisera bound 2-3 times more IWinsulin tracer (25-60 pM) than receptors immobilized with either 83-7 or CT-1. &-incubation of solubilized receptors with either 83-7 or 960 resulted in equivalent depletion (90%) of insulin binding activity from solubilized IM-9 cell extracts, suggesting that both antibodies were in excess and capable of binding a similar population of receptors. Antibody 960, but not CT-1 or 83-7, also increased insulin binding 2 fold to solubilized receptors precipitated with polyethylene glycol. To determine whether the altered binding observed with antibody 960 was due to increased affinity of the receptor for insulin or appearance of more insulin binding sites, binding studies were performed over a wide range of insulin concentrations. Analysis of the resulting binding curves indicated that 960 increased the affinity of the receptor for insulin 3 fold over control (b= 0.3 nM for 960, and 0.9 nM for 83-7, respectively). The antibody 960 also specifically increased insulin binding to intact, saponin-permeabilized IM-9 cell membranes. These results indicate that binding of 960 antibody to the juxtamembrane region of the insulin receptor alters the affmity of the receptor for insulin. Since tyrosine 960 in the juxtamembrane region has been suggested to play a role in receptor signalling, changes in receptor conformation in this region that are likely to account for the change in affinity may play a role in signal transduction.     

Modeling of Sequestration and Down Regulation in Cells Containing Beta2-Adrenergic Receptors

Abstract

Desensitization of G-protein coupled receptors following agonist occupancy is accompanied by two temporally distinguishable cellular trafficking phenomena of the receptors referred to as sequestration and down regulation. For the β₂-adrenergic receptor, sequestration occurs within minutes of agonist binding and results in a reversible internalization and loss of cell surface receptor binding. With longer occupancy, greater than 1 hour, down regulation results in a variable loss of the complement of cellular receptors. Here we compare the two methods that have been used to monitor these receptor changes, competition of whole cell hydrophobic ligand binding (¹²⁵I-pindolol) with a hydrophilic ligand (CGP-12177) and now cytometry quantification of immunologically tagged β₂-adrenergic receptor. While both methods give reliable results, we show that because of a 1:500 partitioning of the hydrophilic ligand into cells, slightly different conditions should be used to assess basally or agonist stimulated sequestered receptor levels. Using a sequestration defective β₂-adrenergic receptor mutant we demonstrate that even though sequestration and down regulation behave as independent processes, sequestration can significantly affect the rate at which receptors are lost by the down regulatory process by removing receptors from the pool of down regulating receptors. A mathematical model expressing these relationships is provided.     

Characterization of insulin receptors in human promyelocytic leukemia cell HL60

Highly specific insulin receptors have been identified on human promyelocytic leukemia cells HL60. Insulin binding increased progressively with time to reach a maximum at 2 h at 22° and was proportional to the number of cells in the incubation mixture. Insulin degradation as assessed by TCA precipitation and reincubation studies was negligible. Scatchard analysis of the binding data was curvilinear and the total number of insulin receptor sites per cell was around 45,000. The average affinity profile gave an “unoccupied site” affinity constant of 3.5 × 10⁸ M^?1. The promyelocytic cells HL60, thus, have specific binding sites and binding characteristics similar to more mature human myeloid cells.     

Use Of Incretin-Based Therapy in Hospitalized Patients with Hyperglycemia

ObjectiveHyperglycemia is common in hospitalized patients with and without prior history of diabetes and is an independent marker of morbidity and mortality in critically and noncritically ill patients. Tight glycemic control using insulin has been shown to reduce cardiac morbidity and mortality in hospitalized patients, but it also results in hypoglycemic episodes, which have been linked to poor outcomes. Thus, alternative treatment options that can normalize blood glucose levels without undue hypoglycemia are being sought. Incretin-based therapies, such as glucagon-like peptide (GLP)-1 receptor agonists (RAs) and dipeptidyl peptidase (DPP)-4 inhibitors, may have this potential.MethodsA PubMed database was searched to find literature describing the use of incretins in hospital settings. Title searches included the terms “diabetes” (care, management, treatment), “hospital,” “inpatient,” “hypoglycemia,” “hyperglycemia,” “glycemic,” “incretin,” “dipeptidyl peptidase-4 inhibitor,” “glucagon-like peptide-1,” and “glucagon-like peptide-1 receptor agonist.”ResultsThe preliminary research experience with native GLP-1 therapy has shown promise, achieving improved glycemic control with a low risk of hypoglycemia, counteracting the hyperglycemic effects of stress hormones, and improving cardiac function in patients with heart failure and acute ischemia. Large, randomized controlled clinical trials are necessary to determine whether these favorable results will extend to the use of GLP-1 RAs and DPP-4 inhibitors.ConclusionsThis review offers hospitalist physicians and healthcare providers involved in inpatient diabetes care a pathophysiologic-based approach for the use of incretin agents in patients with hyperglycemia and diabetes, as well as a summary of benefits and concerns of insulin and incretin-based therapy in the hospital setting. (Endocr Pract. 2014;20:933-944)     

Obese mice have higher insulin receptor levels in the hepatocyte cell nucleus following insulin stimulation in vivo with an oral glucose meal.

Internalization of the insulin receptor occurs following insulin binding at the cell surface, which serves to attenuate the insulin signal as well as modulate the number of surface insulin receptors. Obese animals exhibit decreased cell surface insulin receptor number as well as defects in insulin receptor internalization and processing. The insulin receptor may also translocates to the nucleus of hepatocytes and adipocytes following stimulation of cells with insulin. The objective of this study was to determine if insulin receptor trafficking to the hepatocyte cell nucleus could be observed in vivo and whether this process was altered in obese compared to lean mice. Mice were fasted for 12 h to reduce serum insulin to basal levels. Animals were then given an oral meal of glucose to stimulate the binding of insulin to receptor in vivo. Hepatocyte plasma membrane and nuclei were fractionated to purity following the glucose meal. Levels of insulin receptor were determined using insulin binding assays and a Western blotting assay using anti-insulin receptor antibody. As the amount of serum insulin increased following the glucose meal, a corresponding increase in nuclear insulin binding occurred in lean animals but not obese animals (P<0.05). Following the glucose meal, insulin receptor detected in the cell nucleus was increased in obese compared to lean mice (P<0.05). Thus insulin receptor translocation to the nucleus was demonstrated in vivo following a glucose meal in hepatocytes of both lean and obese animals. It is suggested that serum hyperglycemia and hyperinsulinemia in obese mice increased translocation of the insulin receptor to the nucleus.     

Hormonal Steroids Indirectly Influence Insulin Binding in Rat Testis

Abstract

Pituitary LH is a key factor in controlling Leydig cell activity. In order to verify how steroids produced by the male gonads could influence testicular insulin binding, different steroids at varying doses were administered. Testosterone propionate (Tp) as well as estradiol-17β (E₂-17β) and 5α-dihydrotestosterone (DHT) led to a 50% decrease of insulin binding in testis while no effect was noted when HCG was administered concomitantly with DHT. LH receptors were parallely measured: DHT and E₂ -17β significantly depressed testis LH binding. It is concluded that steroids play a role in the regulation of testis membrane insulin receptor via their feedback mechanism on pituitary LH.     

Vitamin D and Prebiotics may benefit The Intestinal Microbacteria and improve Glucose Homeostasis in Prediabetes and Type 2 Diabetes

ObjectiveTo review the role of human large bowel microbacteria (microbiota) in the glucose homeostasis, to address vitamin D (VD) and prebiotics interactions with microbiota, and to summarize recent randomized clinical trials (RCTs) of VD and prebiotics supplementation in prediabetes (PreDM) and type 2 diabetes mellitus (T2DM).MethodsPrimary literature was reviewed in the following areas: composition and activity of human microbiota associated with PreDM and T2DM, interactions between microbiota and glucose homeostasis, the interaction of microbiota with VD/prebiotics, and RCTs of VD/prebiotics in subjects with PreDM or T2DM.ResultsThe human microbiota is comprised of 100 trillion bacteria with an aggregate genome that is 150-fold larger than the human genome. Data from the animal models and human studies reveal that an “obesogenic” diet results into the initial event of microbiota transformation from symbiosis to dysbiosis. The microbial antigens, such as Gram(-) bacteria and lipopolysaccharide (LPS), translocate to the host interior and trigger increased energy harvesting and Toll-like receptor (TLR) activation with subsequent inflammatory pathways signaling. The “double hit” of steatosis (ectopic fat accumulation) and “—itis” (inflammation) and contribution of “corisks” (e.g., vitamin D deficiency [VDD]) are required to activate molecular signaling, including impaired insulin signaling and secretion, that ends with T2DM and associated diseases. Dietary changes (e.g., prebiotics, VD supplementation) may ameliorate this process if initiated prior to the process becoming irreversible.ConclusionEmerging evidence suggests an important role of microbiota in glucose homeostasis. VD supplementation and prebiotics may be useful in managing PreDM and T2DM. (Endocr Pract. 2013;19:497-510)