Showing papers in "Diabetes in 2008"

Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice

Abstract: OBJECTIVE— Diabetes and obesity are characterized by a low-grade inflammation whose molecular origin is unknown. We previously determined, first, that metabolic endotoxemia controls the inflammatory tone, body weight gain, and diabetes, and second, that high-fat feeding modulates gut microbiota and the plasma concentration of lipopolysaccharide (LPS), i.e., metabolic endotoxemia. Therefore, it remained to demonstrate whether changes in gut microbiota control the occurrence of metabolic diseases. RESEARCH DESIGN AND METHODS— We changed gut microbiota by means of antibiotic treatment to demonstrate, first, that changes in gut microbiota could be responsible for the control of metabolic endotoxemia, the low-grade inflammation, obesity, and type 2 diabetes and, second, to provide some mechanisms responsible for such effect. RESULTS— We found that changes of gut microbiota induced by an antibiotic treatment reduced metabolic endotoxemia and the cecal content of LPS in both high-fat–fed and ob/ob mice. This effect was correlated with reduced glucose intolerance, body weight gain, fat mass development, lower inflammation, oxidative stress, and macrophage infiltration marker mRNA expression in visceral adipose tissue. Importantly, high-fat feeding strongly increased intestinal permeability and reduced the expression of genes coding for proteins of the tight junctions. Furthermore, the absence of CD14 in ob/ob CD14 − / − mutant mice mimicked the metabolic and inflammatory effects of antibiotics. CONCLUSIONS— This new finding demonstrates that changes in gut microbiota controls metabolic endotoxemia, inflammation, and associated disorders by a mechanism that could increase intestinal permeability. It would thus be useful to develop strategies for changing gut microbiota to control, intestinal permeability, metabolic endotoxemia, and associated disorders.

Oxidative Stress as a Major Culprit in Kidney Disease in Diabetes

Abstract: It is postulated that localized tissue oxidative stress is a key component in the development of diabetic nephropathy. There remains controversy, however, as to whether this is an early link between hyperglycemia and renal disease or develops as a consequence of other primary pathogenic mechanisms. In the kidney, a number of pathways that generate reactive oxygen species (ROS) such as glycolysis, specific defects in the polyol pathway, uncoupling of nitric oxide synthase, xanthine oxidase, NAD(P)H oxidase, and advanced glycation have been identified as potentially major contributors to the pathogenesis of diabetic kidney disease. In addition, a unifying hypothesis has been proposed whereby mitochondrial production of ROS in response to chronic hyperglycemia may be the key initiator for each of these pathogenic pathways. This postulate emphasizes the importance of mitochondrial dysfunction in the progression and development of diabetes complications including nephropathy. A mystery remains, however, as to why antioxidants per se have demonstrated minimal renoprotection in humans despite positive preclinical research findings. It is likely that the utility of current study approaches, such as vitamin use, may not be the ideal antioxidant strategy in human diabetic nephropathy. There is now an increasing body of data to suggest that strategies involving a more targeted antioxidant approach, using agents that penetrate specific cellular compartments, may be the elusive additive therapy required to further optimize renoprotection in diabetes.

Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients

Abstract: OBJECTIVE— To explore the possibility that oscillating glucose may outweigh A1C levels in determining the risk for cardiovascular diabetes complications. RESEARCH DESIGN AND METHODS— A euinsulinemic hyperglycemic clamp at 5, 10, and 15 mmol/l glucose was given in increasing steps as a single “spike” or oscillating between basal and high levels over 24 h in normal subjects and type 2 diabetic patients. Flow-mediated dilatation, a marker of endothelial function, and plasma 3-nitrotyrosine and 24-h urinary excretion rates of free 8-iso PGF2α, two markers of oxidative stress, were measured over 48 h postclamp. RESULTS— Glucose at two different levels (10 and 15 mmol/l) resulted in a concentration-dependent fasting blood glucose–independent induction of both endothelial dysfunction and oxidative stress in both normal and type 2 diabetic patients. Oscillating glucose between 5 and 15 mmol/l every 6 h for 24 h resulted in further significant increases in endothelial dysfunction and oxidative stress compared with either continuous 10 or 15 mmol/l glucose. CONCLUSIONS— These data suggest that oscillating glucose can have more deleterious effects than constant high glucose on endothelial function and oxidative stress, two key players in favoring cardiovascular complications in diabetes. Concomitant vitamin C infusion can reverse this impairment.

Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study: Associations with Neonatal Anthropometrics

Abstract: Objective: To examine associations of neonatal adiposity with maternal glucose levels and cord serum C-peptide in a multicenter multinational study, the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study, thereby assessing the Pederson hypothesis linking maternal glycemia and fetal hyperinsulinemia to neonatal adiposity. Research Design and Methods: Eligible pregnant women underwent a standard 75 gm OGTT between 24 and 32 weeks gestation (as close to 28 weeks as possible). Neonatal anthropometrics and cord serum C-peptide were measured. Associations of maternal glucose and cord serum C-peptide with neonatal adiposity (sum of skinfolds > 90th percentile or percent body fat > 90th percentile) were assessed using multiple logistic regression analyses, with adjustment for potential confounders, including maternal age, parity, BMI, mean arterial pressure, height, gestational age at delivery, and the baby's gender. Results: Among 23,316 HAPO study participants with glucose levels blinded to caregivers, cord serum C-peptide results were available for 19,885 babies and skin fold measurements for 19,389. For measures of neonatal adiposity there were strong statistically significant gradients across increasing levels of maternal glucose and cord serum C-peptide, which persisted after adjustment for potential confounders. In fully adjusted continuous variable models, odds ratios ranged from 1.35 to 1.44 for the two measures of adiposity for fasting, 1-hour, and 2-hour plasma glucose higher by one standard deviation. Conclusions: These findings confirm the link between maternal glucose and neonatal adiposity, and suggest that the relationship is mediated by fetal insulin production and that the Pedersen hypothesis describes a basic biologic relationship influencing fetal growth.

Phenotypic Switching of Adipose Tissue Macrophages With Obesity Is Generated by Spatiotemporal Differences in Macrophage Subtypes

Abstract: OBJECTIVE—To establish the mechanism of the phenotypic switch of adipose tissue macrophages (ATMs) from an alternatively activated (M2a) to a classically activated (M1) phenotype with obesity. RESEARCH DESIGN AND METHODS—ATMs from lean and obese (high-fat diet–fed) C57Bl/6 mice were analyzed by a combination of flow cytometry, immunofluorescence, and expression analysis for M2a and M1 genes. Pulse labeling of ATMs with PKH26 assessed the recruitment rate of ATMs to spatially distinct regions. RESULTS—Resident ATMs in lean mice express the M2a marker macrophage galactose N-acetyl-galactosamine specific lectin 1 (MGL1) and localize to interstitial spaces between adipocytes independent of CCR2 and CCL2. With diet-induced obesity, MGL1+ ATMs remain in interstitial spaces, whereas a population of MGL1−CCR2+ ATMs with high M1 and low M2a gene expression is recruited to clusters surrounding necrotic adipocytes. Pulse labeling showed that the rate of recruitment of new macrophages to MGL1− ATM clusters is significantly faster than that of interstitial MGL1+ ATMs. This recruitment is attenuated in Ccr2−/− mice. M2a- and M1-polarized macrophages produced different effects on adipogenesis and adipocyte insulin sensitivity in vitro. CONCLUSIONS—The shift in the M2a/M1 ATM balance is generated by spatial and temporal differences in the recruitment of distinct ATM subtypes. The obesity-induced switch in ATM activation state is coupled to the localized recruitment of an inflammatory ATM subtype to macrophage clusters from the circulation and not to the conversion of resident M2a macrophages to M1 ATMs in situ.

Serum FGF21 Levels Are Increased in Obesity and Are Independently Associated With the Metabolic Syndrome in Humans

Abstract: Objective: Fibroblast growth factor 21 (FGF21) is a metabolic regulator with multiple beneficial effects on glucose homeostasis and insulin sensitivity in animal models. This study aimed to investigate the relationship between its serum levels and various cardio-metabolic parameters in humans. Research Design And Methods: A newly developed immunoassay was used to measure serum FGF21 levels in 232 Chinese subjects recruited from our previous cross-sectional studies. The mRNA expression levels of FGF21 in the liver and adipose tissues were quantified by real-time PCR. RESULTS: Serum FGF21 levels in overweight/obese subjects were significantly higher than in lean individuals. Serum FGF21 correlated positively with adiposity, fasting insulin and triglycerides, but negatively with HDL-cholesterol, after adjusting for age and BMI. Logistic regression analysis demonstrated an independent association betweenserum FGF21 and the metabolic syndrome (MetS). Furthermore, the increased risk of the MetS associated with high serum FGF21 was over and above the effects of individual components of the MetS. Our in vitro study detected a differentiation-dependent expression of FGF21 in 3T3-L1 adipocytes and human adipocytes. In db/db obese mice, FGF21 mRNA expression was markedly increased in both the liver and adipose tissue comparing with their lean littermates. Furthermore, FGF21 expression in subcutaneous fat correlated well with its circulating concentrations in humans. CONCLUSIONS: FGF21 is a novel adipokine associated with obesity-related metabolic complications in humans. The “paradoxical” increase of serum FGF21 in obese individuals, which may be explained by a compensatory response or resistance to FGF21, warrants further investigation.

HLA DR-DQ Haplotypes and Genotypes and Type 1 Diabetes Risk: Analysis of the Type 1 Diabetes Genetics Consortium Families

Abstract: OBJECTIVE— The Type 1 Diabetes Genetics Consortium has collected type 1 diabetic families worldwide for genetic analysis. The major genetic determinants of type 1 diabetes are alleles at the HLA-DRB1 and DQB1 loci, with both susceptible and protective DR-DQ haplotypes present in all human populations. The aim of this study is to estimate the risk conferred by specific DR-DQ haplotypes and genotypes. RESEARCH DESIGN AND METHODS:— Six hundred and seven Caucasian families and 38 Asian families were typed at high resolution for the DRB1, DQA1, and DQB1 loci. The association analysis was performed by comparing the frequency of DR-DQ haplotypes among the chromosomes transmitted to an affected child with the frequency of chromosomes not transmitted to any affected child. RESULTS— A number of susceptible, neutral, and protective DR-DQ haplotypes have been identified, and a statistically significant hierarchy of type 1 diabetes risk has been established. The most susceptible haplotypes are the DRB1\*0301-DQA1\*0501-DQB1*0201 (odds ratio [OR] 3.64) and the DRB1\*0405-DQA1\*0301-DQB1*0302, DRB1\*0401-DQA1\*0301-DQB*0302, and DRB1\*0402-DQA1\*0301-DQB1*0302 haplotypes (ORs 11.37, 8.39, and 3.63), followed by the DRB1\*0404-DQA1\*0301-DQB1*0302 (OR 1.59) and the DRB1\*0801-DQB1\*0401-DQB1*0402 (OR 1.25) haplotypes. The most protective haplotypes are DRB1\*1501-DQA1\*0102-DQB1*0602 (OR 0.03), DRB1\*1401-DQA1\*0101-DQB1*0503 (OR 0.02), and DRB1\*0701-DQA1\*0201-DQB1*0303 (OR 0.02). CONCLUSIONS— Specific combinations of alleles at the DRB1, DQA1, and DQB1 loci determine the extent of haplotypic risk. The comparison of closely related DR-DQ haplotype pairs with different type 1 diabetes risks allowed identification of specific amino acid positions critical in determining disease susceptibility. These data also indicate that the risk associated with specific HLA haplotypes can be influenced by the genotype context and that the trans- complementing heterodimer encoded by DQA1*0501 and DQB1*0302 confers very high risk.

β-Cell Replication Is the Primary Mechanism Subserving the Postnatal Expansion of β-Cell Mass in Humans

Abstract: OBJECTIVE— Little is known about the capacity, mechanisms, or timing of growth in β-cell mass in humans. We sought to establish if the predominant expansion of β-cell mass in humans occurs in early childhood and if, as in rodents, this coincides with relatively abundant β-cell replication. We also sought to establish if there is a secondary growth in β-cell mass coincident with the accelerated somatic growth in adolescence. RESEARCH DESIGN AND METHODS— To address these questions, pancreas volume was determined from abdominal computer tomographies in 135 children aged 4 weeks to 20 years, and morphometric analyses were performed in human pancreatic tissue obtained at autopsy from 46 children aged 2 weeks to 21 years. RESULTS— We report that 1 ) β-cell mass expands by severalfold from birth to adulthood, 2 ) islets grow in size rather than in number during this transition, 3 ) the relative rate of β-cell growth is highest in infancy and gradually declines thereafter to adulthood with no secondary accelerated growth phase during adolescence, 4 ) β-cell mass (and presumably growth) is highly variable between individuals, and 5 ) a high rate of β-cell replication is coincident with the major postnatal expansion of β-cell mass. CONCLUSIONS— These data imply that regulation of β-cell replication during infancy plays a major role in β-cell mass in adult humans.

The Barrier of Hypoglycemia in Diabetes

Abstract: Glycemic control, a worthwhile goal for people with diabetes, is limited by the barrier of iatrogenic hypoglycemia (1). Iatrogenic hypoglycemia 1 ) causes recurrent morbidity in most people with type 1 diabetes and many with advanced type 2 diabetes and is sometimes fatal, 2 ) compromises physiological and behavioral defenses against subsequent falling plasma glucose concentrations and thus causes a vicious cycle of recurrent hypoglycemia, and 3 ) precludes maintenance of euglycemia over a lifetime of diabetes and therefore full realization of the vascular benefits of glycemic control. The premise of this “Perspective in Diabetes” is that insight into the pathophysiology of glucose counterregulation in diabetes leads to understanding of the frequency and impact of, risk factors for, and prevention of iatrogenic hypoglycemia. Partial glycemic control reduces, but does not eliminate, the development of microvascular complications of diabetes (retinopathy, nephropathy, and neuropathy) in type 1 (2) and type 2 (3,4) diabetes. Extrapolation of the Diabetes Control and Complications Trial (DCCT) retinopathy data suggests that long-term maintenance of euglycemia might eliminate those complications (5). Follow-up of the DCCT patients seemingly indicates that a period of earlier partial glycemic control reduces the development of macrovascular complications in type 1 diabetes (6). Aside from the metformin subset of the U.K. Prospective Diabetes Study (4), randomized controlled trials of intensive glycemic therapy have not documented a cardiovascular mortality benefit in type 2 diabetes (3,7,8). However, those trials do not exclude a cardiovascular benefit if glycemic control, or even partial glycemic control, could be maintained over a longer period of time. In any event, given its documented microvascular benefit, maintenance of euglycemia over a lifetime of diabetes would be in the best interest of people with diabetes if that could be accomplished safely. Unfortunately, maintenance of euglycemia over a lifetime of diabetes …

Baseline Serum 25-Hydroxy Vitamin D Is Predictive of Future Glycemic Status and Insulin Resistance: The Medical Research Council Ely Prospective Study 1990–2000

Abstract: OBJECTIVE— Accumulating epidemiological evidence suggests that hypovitaminosis D may be associated with type 2 diabetes and related metabolic risks. However, prospective data using the biomarker serum 25-hydroxyvitamin D [25(OH)D] are limited and therefore examined in the present study. RESEARCH DESIGN AND METHODS— A total of 524 randomly selected nondiabetic men and women, aged 40–69 years at baseline, with measurements for serum 25(OH)D and IGF-1 in the population-based Ely Study, had glycemic status (oral glucose tolerance), lipids, insulin, anthropometry, and blood pressure measured and metabolic syndrome risk (metabolic syndrome z score) derived at baseline and at 10 years of follow-up. RESULTS— Age-adjusted baseline mean serum 25(OH)D was greater in men (64.5 nmol/l [95% CI 61.2–67.9]) than women (57.2 nmol/l [54.4,60.0]) and varied with season (highest late summer). Baseline 25(OH)D was associated inversely with 10-year risk of hyperglycemia (fasting glucose: β = −0.0023, P = 0.019; 2-h glucose: β = −0.0097, P = 0.006), insulin resistance (fasting insulin β = −0.1467, P = 0.010; homeostasis model assessment of insulin resistance [HOMA-IR]: β = −0.0059, P = 0.005), and metabolic syndrome z score (β = −0.0016, P = 0.048) after adjustment for age, sex, smoking, BMI, season, and baseline value of each metabolic outcome variable. Associations with 2-h glucose, insulin, and HOMA-IR remained significant after further adjustment for IGF-1, parathyroid hormone, calcium, physical activity, and social class. CONCLUSIONS— This prospective study reports inverse associations between baseline serum 25(OH)D and future glycemia and insulin resistance. These associations are potentially important in understanding the etiology of abnormal glucose metabolism and warrant investigation in larger, specifically designed prospective studies and randomized controlled trials of supplementation.

Gpr40 Is Expressed in Enteroendocrine Cells and Mediates Free Fatty Acid Stimulation of Incretin Secretion

Abstract: OBJECTIVE: The G-protein-coupled receptor Gpr40 is expressed in beta-cells where it contributes to free fatty acid (FFA) enhancement of glucose-stimulated insulin secretion. However, other sites of ...

Endurance Exercise as a Countermeasure for Aging

Abstract: OBJECTIVE— We determined whether reduced insulin sensitivity, mitochondrial dysfunction, and other age-related dysfunctions are inevitable consequences of aging or secondary to physical inactivity. RESEARCH DESIGN AND METHODS— Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and ATP production in mitochondria isolated from vastus lateralis biopsies of 42 healthy sedentary and endurance-trained young (18–30 years old) and older (59–76 years old) subjects. Expression of proteins involved in fuel metabolism was measured by mass spectrometry. Citrate synthase activity, mitochondrial DNA (mtDNA) abundance, and expression of nuclear-encoded transcription factors for mitochondrial biogenesis were measured. SIRT3, a mitochondrial sirtuin linked to lifespan-enhancing effects of caloric restriction, was measured by immunoblot. RESULTS— Insulin-induced glucose disposal and suppression of endogenous glucose production were higher in the trained young and older subjects, but no age effect was noted. Age-related decline in mitochondrial oxidative capacity was absent in endurance-trained individuals. Although endurance-trained individuals exhibited higher expression of mitochondrial proteins, mtDNA, and mitochondrial transcription factors, there were persisting effects of age. SIRT3 expression was lower with age in sedentary but equally elevated regardless of age in endurance-trained individuals. CONCLUSIONS— The results demonstrate that reduced insulin sensitivity is likely related to changes in adiposity and to physical inactivity rather than being an inevitable consequence of aging. The results also show that regular endurance exercise partly normalizes age-related mitochondrial dysfunction, although there are persisting effects of age on mtDNA abundance and expression of nuclear transcription factors and mitochondrial protein. Furthermore, exercise may promote longevity through pathways common to effects of caloric restriction.

Low physical activity accentuates the effect of the FTO rs9939609 polymorphism on body fat accumulation

Abstract: OBJECTIVE— Three independent studies have shown that variation in the fat mass and obesity-associated ( FTO ) gene associates with BMI and obesity. In the present study, the effect of FTO variation on metabolic traits including obesity, type 2 diabetes, and related quantitative phenotypes was examined. RESEARCH DESIGN AND METHODS— The FTO rs9939609 polymorphism was genotyped in a total of 17,508 Danes from five different study groups. RESULTS— In studies of 3,856 type 2 diabetic case subjects and 4,861 normal glucose-tolerant control subjects, the minor A-allele of rs9939609 associated with type 2 diabetes (odds ratio 1.13 [95% CI 1.06–1.20], P = 9 × 10−5). This association was abolished when adjusting for BMI (1.06 [0.97–1.16], P = 0.2). Among 17,162 middle-aged Danes, the A-allele associated with overweight (1.19 [1.13–1.24], P = 1 × 10−12) and obesity (1.27 [1.20–1.34], P = 2 × 10−16). Furthermore, obesity-related quantitative traits such as body weight, waist circumference, fat mass, and fasting serum leptin levels were significantly elevated in A-allele carriers. An interaction between the FTO rs9939609 genotype and physical activity ( P = 0.007) was found, where physically inactive homozygous risk A-allele carriers had a 1.95 ± 0.3 kg/m2 increase in BMI compared with homozygous T-allele carriers. CONCLUSIONS— We validate that variation in FTO is associated with type 2 diabetes when not adjusted for BMI and with an overall increase in body fat mass. Furthermore, low physical activity seems to accentuate the effect of FTO rs9939609 on body fat accumulation.

The “Perfect Storm” for Type 1 Diabetes: The Complex Interplay Between Intestinal Microbiota, Gut Permeability, and Mucosal Immunity

Abstract: It is often stated that type 1 diabetes results from a complex interplay between varying degrees of genetic susceptibility and environmental factors. While agreeing with this principal, our desire is that this Perspectives article will highlight another complex interplay potentially associated with this disease involving facets related to the gut, one where individual factors that, upon their interaction with each another, form a “perfect storm” critical to the development of type 1 diabetes. This trio of factors includes an aberrant intestinal microbiota, a “leaky” intestinal mucosal barrier, and altered intestinal immune responsiveness. Studies examining the microecology of the gastrointestinal tract have identified specific microorganisms whose presence appears related (either quantitatively or qualitatively) to disease; in type 1 diabetes, a role for microflora in the pathogenesis of disease has recently been suggested. Increased intestinal permeability has also been observed in animal models of type 1 diabetes as well as in humans with or at increased-risk for the disease. Finally, an altered mucosal immune system has been associated with the disease and is likely a major contributor to the failure to form tolerance, resulting in the autoimmunity that underlies type 1 diabetes. Herein, we discuss the complex interplay between these factors and raise testable hypotheses that form a fertile area for future investigations as to the role of the gut in the pathogenesis and prevention of type 1 diabetes.

Berberine and Its More Biologically Available Derivative, Dihydroberberine, Inhibit Mitochondrial Respiratory Complex I: A Mechanism for the Action of Berberine to Activate AMP-Activated Protein Kinase and Improve Insulin Action

Abstract: OBJECTIVE— Berberine (BBR) activates AMP-activated protein kinase (AMPK) and improves insulin sensitivity in rodent models of insulin resistance. We investigated the mechanism of activation of AMPK by BBR and explored whether derivatization of BBR could improve its in vivo efficacy. RESEARCH DESIGN AND METHODS— AMPK phosphorylation was examined in L6 myotubes and LKB1 −/− cells, with or without the Ca 2+ /calmodulin-dependent protein kinase kinase (CAMKK) inhibitor STO-609. Oxygen consumption was measured in L6 myotubes and isolated muscle mitochondria. The effect of a BBR derivative, dihydroberberine (dhBBR), on adiposity and glucose metabolism was examined in rodents fed a high-fat diet. RESULTS— We have made the following novel observations: 1 ) BBR dose-dependently inhibited respiration in L6 myotubes and muscle mitochondria, through a specific effect on respiratory complex I, similar to that observed with metformin and rosiglitazone; 2 ) activation of AMPK by BBR did not rely on the activity of either LKB1 or CAMKKβ, consistent with major regulation at the level of the AMPK phosphatase; and 3 ) a novel BBR derivative, dhBBR, was identified that displayed improved in vivo efficacy in terms of counteracting increased adiposity, tissue triglyceride accumulation, and insulin resistance in high-fat–fed rodents. This effect is likely due to enhanced oral bioavailability. CONCLUSIONS— Complex I of the respiratory chain represents a major target for compounds that improve whole-body insulin sensitivity through increased AMPK activity. The identification of a novel derivative of BBR with improved in vivo efficacy highlights the potential importance of BBR as a novel therapy for the treatment of type 2 diabetes.

Effect of Glycemic Exposure on the Risk of Microvascular Complications in the Diabetes Control and Complications Trial—Revisited

Abstract: OBJECTIVE— The Diabetes Control and Complications Trial ( Diabetes 44:968–983, 1995) presented statistical models suggesting that subjects with similar A1C levels had a higher risk of retinopathy progression in the conventional treatment group than in the intensive treatment group. That analysis has been cited to support the hypothesis that specific patterns of glucose variation, in particular postprandial hyperglycemia, contribute uniquely to an increased risk of microvascular complications above and beyond that explained by the A1C level. RESEARCH DESIGN AND METHODS— We performed statistical evaluations of these models and additional analyses to assess whether the original analyses were flawed. RESULTS— Statistically, we show that the original results are an artifact of the assumptions of the statistical model used. Additional analyses show that virtually all (96%) of the beneficial effect of intensive versus conventional therapy on progression of retinopathy is explained by the reductions in the mean A1C levels, similarly for other outcomes. Furthermore, subjects within the intensive and conventional treatment groups with similar A1C levels over time have similar risks of retinopathy progression, especially after adjusting for factors in which they differ. CONCLUSIONS— A1C explains virtually all of the difference in risk of complications between the intensive and conventional groups, and a given A1C level has similar effects within the two treatment groups. While other components of hyperglycemia, such as glucose variation, may contribute to the risk of complications, such factors can only explain a small part of the differences in risk between intensive and conventional therapy over time.

Immunomodulation by Mesenchymal Stem Cells A Potential Therapeutic Strategy for Type 1 Diabetes

Abstract: Mesenchymal stem cells (MSCs) are pluripotent stromal cells that have the potential to give rise to cells of diverse lineages. Interestingly, MSCs can be found in virtually all postnatal tissues. The main criteria currently used to characterize and identify these cells are the capacity for self-renewal and differentiation into tissues of mesodermal origin, combined with a lack in expression of certain hematopoietic molecules. Because of their developmental plasticity, the notion of MSC-based therapeutic intervention has become an emerging strategy for the replacement of injured tissues. MSCs have also been noted to possess the ability to impart profound immunomodulatory effects in vivo. Indeed, some of the initial observations regarding MSC protection from tissue injury once thought mediated by tissue regeneration may, in reality, result from immunomodulation. Whereas the exact mechanisms underlying the immunomodulatory functions of MSC remain largely unknown, these cells have been exploited in a variety of clinical trials aimed at reducing the burden of immune-mediated disease. This article focuses on recent advances that have broadened our understanding of the immunomodulatory properties of MSC and provides insight as to their potential for clinical use as a cell-based therapy for immune-mediated disorders and, in particular, type 1 diabetes.

Increase in Endoplasmic Reticulum Stress–Related Proteins and Genes in Adipose Tissue of Obese, Insulin-Resistant Individuals

Abstract: OBJECTIVE—To examine fat biopsy samples from lean insulin-sensitive and obese insulin-resistant nondiabetic individuals for evidence of endoplasmic reticulum (ER) stress. RESEARCH DESIGN AND METHODS—Subcutaneous fat biopsies were obtained from the upper thighs of six lean and six obese nondiabetic subjects. Fat homogenates were used for proteomic (two-dimensional gel and MALDI-TOF/TOF), Western blot, and RT-PCR analysis. RESULTS—Proteomic analysis revealed 19 differentially upregulated proteins in fat of obese subjects. Three of these proteins were the ER stress–related unfolded protein response (UPR) proteins calreticulin, protein disulfide-isomerase A3, and glutathione-S-transferase P. Western blotting revealed upregulation of several other UPR stress–related proteins, including calnexin, a membrane-bound chaperone, and phospho c-jun NH2-terminal kinase (JNK)-1, a downstream effector protein of ER stress. RT-PCR analysis revealed upregulation of the spliced form of X-box binding protein-1s, a potent transcription factor and part of the proximal ER stress sensor inositol-requiring enzyme-1 pathway. CONCLUSIONS—These findings represent the first demonstration of UPR activation in subcutaneous adipose tissue of obese human subjects. As JNK can inhibit insulin action and activate proinflammatory pathways, ER stress activation of JNK may be a link between obesity, insulin resistance, and inflammation.

miR-375 Targets 3′-Phosphoinositide–Dependent Protein Kinase-1 and Regulates Glucose-Induced Biological Responses in Pancreatic β-Cells

Abstract: OBJECTIVE—MicroRNAs are short, noncoding RNAs that regulate gene expression. We hypothesized that the phosphatidylinositol 3-kinase (PI 3-kinase) cascade known to be important in β-cell physiology could be regulated by microRNAs. Here, we focused on the pancreas-specific miR-375 as a potential regulator of its predicted target 3′-phosphoinositide–dependent protein kinase-1 (PDK1), and we analyzed its implication in the response of insulin-producing cells to elevation of glucose levels. RESEARCH DESIGN AND METHODS—We used insulinoma-1E cells to analyze the effects of miR-375 on PDK1 protein level and downstream signaling using Western blotting, glucose-induced insulin gene expression using quantitative RT-PCR, and DNA synthesis by measuring thymidine incorporation. Moreover, we analyzed the effect of glucose on miR-375 expression in both INS-1E cells and primary rat islets. Finally, miR-375 expression in isolated islets was analyzed in diabetic Goto-Kakizaki (GK) rats. RESULTS—We found that miR-375 directly targets PDK1 and reduces its protein level, resulting in decreased glucose-stimulatory action on insulin gene expression and DNA synthesis. Furthermore, glucose leads to a decrease in miR-375 precursor level and a concomitant increase in PDK1 protein. Importantly, regulation of miR-375 expression by glucose occurs in primary rat islets as well. Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets. CONCLUSIONS—Our findings provide evidence for a role of a pancreatic-specific microRNA, miR-375, in the regulation of PDK1, a key molecule in PI 3-kinase signaling in pancreatic β-cells. The effects of glucose on miR-375 are compatible with the idea that miR-375 is involved in glucose regulation of insulin gene expression and β-cell growth.

Serum Vaspin Concentrations in Human Obesity and Type 2 Diabetes

Abstract: OBJECTIVE— Vaspin was identified as an adipokine with insulin-sensitizing effects, which is predominantly secreted from visceral adipose tissue in a rat model of type 2 diabetes. We have recently shown that vaspin mRNA expression in adipose tissue is related to parameters of obesity and glucose metabolism. However, the regulation of vaspin serum concentrations in human obesity and type 2 diabetes is unknown. RESEARCH DESIGN AND METHODS— For the measurement of vaspin serum concentrations, we developed an enzyme-linked immunosorbent assay (ELISA). Using this ELISA, we assessed circulating vaspin in a cross-sectional study of 187 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance and in 60 individuals with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes before and after a 4-week physical training program. RESULTS— Vaspin serum concentrations were significantly higher in female compared with male subjects. There was no difference in circulating vaspin between individuals with NGT and type 2 diabetes. In the normal glucose-tolerant group, circulating vaspin significantly correlated with BMI and insulin sensitivity. Moreover, physical training for 4 weeks resulted in significantly increased circulating vaspin levels. CONCLUSIONS— We found a sexual dimorphism in circulating vaspin. Elevated vaspin serum concentrations are associated with obesity and impaired insulin sensitivity, whereas type 2 diabetes seems to abrogate the correlation between increased circulating vaspin, higher body weight, and decreased insulin sensitivity. Low circulating vaspin correlates with a high fitness level, whereas physical training in untrained individuals causes increased vaspin serum concentrations.

Metformin Inhibits Hepatic Gluconeogenesis Through AMP-Activated Protein Kinase–Dependent Regulation of the Orphan Nuclear Receptor SHP

Abstract: OBJECTIVE —Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS —We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP, PEPCK, and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes, and metformin–and adenovirus SHP (Ad-SHP)–mediated hepatic glucose production was measured in B6- Lep ob/ ob mice. RESULTS —Hepatic SHP gene expression was induced by metformin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK), as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4α–or FoxA2-mediated promoter activity of PEPCK and G6Pase, and the inhibition was blocked with siRNA SHP. Additionally, SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore, oral administration of metformin increased SHP mRNA levels in B6- Lep ob/ob mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6- Lep ob/ob mice. CONCLUSIONS —We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP.

High glucose induces Toll-like receptor expression in human monocytes: Mechanism of activation

Abstract: OBJECTIVE— Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions. RESEARCH DESIGN AND METHODS— High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner ( P n = 10). RESULTS— Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein ( P P P P CONCLUSIONS— Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-α and PKC-δ, respectively, by stimulating NADPH oxidase in human monocytes.

Acute Metformin Therapy Confers Cardioprotection Against Myocardial Infarction Via AMPK-eNOS–Mediated Signaling

Abstract: OBJECTIVE— Clinical studies have reported that metformin reduces cardiovascular end points of type 2 diabetic subjects by actions that cannot solely be attributed to glucose-lowering effects. The therapeutic effects of metformin have been reported to be mediated by its activation of AMP-activated protein kinase (AMPK), a metabolite sensing protein kinase whose activation following myocardial ischemia has been suggested to be an endogenous protective signaling mechanism. We investigated the potential cardioprotective effects of a single, low-dose metformin treatment (i.e., 286-fold less than the maximum antihyperglycemic dose) in a murine model of myocardial ischemia-reperfusion (I/R) injury. RESEARCH DESIGN AND METHODS— Nondiabetic and diabetic ( db/db ) mice were subjected to transient myocardial ischemia for a period of 30 min followed by reperfusion. Metformin (125 μg/kg) or vehicle (saline) was administered either before ischemia or at the time of reperfusion. RESULTS— Administration of metformin before ischemia or at reperfusion decreased myocardial injury in both nondiabetic and diabetic mice. Importantly, metformin did not alter blood glucose levels. During early reperfusion, treatment with metformin augmented I/R-induced AMPK activation and significantly increased endothelial nitric oxide (eNOS) phosphorylation at residue serine 1177. CONCLUSIONS— These findings provide important information that myocardial AMPK activation by metformin following I/R sets into motion events, including eNOS activation, which ultimately lead to cardioprotection.

Voltage-gated ion channels in human pancreatic beta-cells: electrophysiological characterization and role in insulin secretion.

Abstract: OBJECTIVE— To characterize the voltage-gated ion channels in human β-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS— Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated β-cells. The ion channel complement was determined by quantitative PCR. RESULTS— Human β-cells express two types of voltage-gated K+ currents that flow through delayed rectifying (KV2.1/2.2) and large-conductance Ca2+-activated K+ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of KV2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na+ currents (NaV1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na+ currents with TTX reduced glucose-stimulated (6–20 mmol/l) insulin secretion by 55–70%. Human β-cells are equipped with L- (CaV1.3), P/Q- (CaV2.1), and T- (CaV3.2), but not N- or R-type Ca2+ channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca2+ channels reduced glucose-induced (6 mmol/l) secretion by 60–70%. Membrane potential recordings suggest that L- and T-type Ca2+ channels participate in action potential generation. Blockade of P/Q-type Ca2+ channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca2+ channels only had a minor effect. CONCLUSIONS— Voltage-gated T-type and L-type Ca2+ channels as well as Na+ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca2+-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca2+ influx through P/Q-type Ca2+ channels.

25-Hydroxyvitamin D, IGF-1, and Metabolic Syndrome at 45 Years of Age: A Cross-Sectional Study in the 1958 British Birth Cohort

Abstract: OBJECTIVE —Hypovitaminosis D and reduced IGF-1 are associated, individually, with metabolic syndrome. Physiological interactions between vitamin D and IGF-1 are reported; this is the first study to investigate their combined associations with metabolic syndrome prevalence. RESEARCH DESIGN AND METHODS —Data on 25-hydroxyvitamin D (25(OH)D), IGF-1, and metabolic syndrome abnormalities (abdominal obesity; raised A1C, blood pressure, and triglycerides; and low HDL cholesterol) were collected from 6,810 British white subjects in the 1958 cohort, surveyed during 2002–2004 (age 45 years). RESULTS —IGF-1 concentrations increased with 25(OH)D up to ∼75–85 nmol/l but not thereafter. Both 25(OH)D and IGF-1 were inversely associated with metabolic syndrome. There was an interaction between 25(OH)D and IGF-1 ( P = 0.025) on metabolic syndrome prevalence: IGF-1 was not significantly associated with metabolic syndrome among those with the lowest levels of 25(OH)D ( P > 0.09), whereas higher 25(OH)D was associated with metabolic syndrome at all IGF-1 concentrations ( P ≤ 0.006). Metabolic syndrome prevalence was lowest for participants with the highest concentrations of both 25(OH)D and IGF-1 (odds ratio for highest vs. lowest third of both 0.26 [95% CI 0.17–0.40], P P ≤ 0.004 for all comparisons). CONCLUSIONS —Serum 25(OH)D is inversely associated with metabolic syndrome, whereas the inverse association with IGF-1 was found only among those without hypovitaminosis D. These results suggest that metabolic syndrome prevalence is the lowest when both 25(OH)D and IGF-1 are high.

Amp-activated protein kinase regulates glut4 transcription by phosphorylating histone deacetylase 5

Abstract: OBJECTIVE— Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms. RESEARCH DESIGN AND METHODS AND RESULTS— Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription. CONCLUSIONS— These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Receptor for Advanced Glycation End Products (RAGE) Deficiency Attenuates the Development of Atherosclerosis in Diabetes

Abstract: OBJECTIVE— Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE−/− model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS— ApoE−/− and RAGE−/−/apoE−/− double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS— Although diabetic apoE−/− mice showed increased plaque accumulation (14.9 ± 1.7%), diabetic RAGE−/−/apoE−/− mice had significantly reduced atherosclerotic plaque area (4.9 ± 0.4%) to levels not significantly different from control apoE−/− mice (4.3 ± 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-κB subunit p65 , VCAM-1 , and MCP-1 ; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox , p47phox , and rac-1 . Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE−/− mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE−/−/apoE−/− mice. CONCLUSIONS— This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.

Separate Impact of Obesity and Glucose Tolerance on the Incretin Effect in Normal Subjects and Type 2 Diabetic Patients

Abstract: OBJECTIVE— To quantitate the separate impact of obesity and hyperlycemia on the incretin effect (i.e., the gain in β-cell function after oral glucose versus intravenous glucose). RESEARCH DESIGN AND METHODS— Isoglycemic oral (75 g) and intravenous glucose administration was performed in 51 subjects (24 with normal glucose tolerance [NGT], 17 with impaired glucose tolerance [IGT], and 10 with type 2 diabetes) with a wide range of BMI (20–61 kg/m 2 ). C-peptide deconvolution was used to reconstruct insulin secretion rates, and β-cell glucose sensitivity (slope of the insulin secretion/glucose concentration dose-response curve) was determined by mathematical modeling. The incretin effect was defined as the oral-to-intravenous ratio of responses. In 8 subjects with NGT and 10 with diabetes, oral glucose appearance was measured by the double-tracer technique. RESULTS— The incretin effect on total insulin secretion and β-cell glucose sensitivity and the GLP-1 response to oral glucose were significantly reduced in diabetes compared with NGT or IGT ( P ≤ 0.05). The results were similar when subjects were stratified by BMI tertile ( P ≤ 0.05). In the whole dataset, each manifestation of the incretin effect was inversely related to both glucose tolerance (2-h plasma glucose levels) and BMI (partial r = 0.27–0.59, P ≤ 0.05) in an independent, additive manner. Oral glucose appearance did not differ between diabetes and NGT and was positively related to the GLP-1 response ( r = 0.53, P CONCLUSIONS— Potentiation of insulin secretion, glucose sensing, glucagon-like peptide-1 release, and glucagon suppression are physiological manifestations of the incretin effect. Glucose tolerance and obesity impair the incretin effect independently of one another.

Dapagliflozin, a Selective SGLT2 Inhibitor, Improves Glucose Homeostasis in Normal and Diabetic Rats

Abstract: OBJECTIVE— The inhibition of gut and renal sodium-glucose cotransporters (SGLTs) has been proposed as a novel therapeutic approach to the treatment of diabetes. We have identified dapagliflozin as a potent and selective inhibitor of the renal sodium-glucose cotransporter SGLT2 in vitro and characterized its in vitro and in vivo pharmacology. RESEARCH DESIGN AND METHODS— Cell-based assays measuring glucose analog uptake were used to assess dapagliflozin's ability to inhibit sodium-dependent and facilitative glucose transport activity. Acute and multi-dose studies in normal and diabetic rats were performed to assess the ability of dapagliflozin to improve fed and fasting plasma glucose levels. A hyperinsulinemic-euglycemic clamp study was performed to assess the ability of dapagliflozin to improve glucose utilization after multi-dose treatment. RESULTS— Dapagliflozin potently and selectively inhibited human SGLT2 versus human SGLT1, the major cotransporter of glucose in the gut, and did not significantly inhibit facilitative glucose transport in human adipocytes. In vivo, dapagliflozin acutely induced renal glucose excretion in normal and diabetic rats, improved glucose tolerance in normal rats, and reduced hyperglycemia in Zucker diabetic fatty (ZDF) rats after single oral doses ranging from 0.1 to 1.0 mg/kg. Once-daily dapagliflozin treatment over 2 weeks significantly lowered fasting and fed glucose levels at doses ranging from 0.1 to 1.0 mg/kg and resulted in a significant increase in glucose utilization rate accompanied by a significant reduction in glucose production. CONCLUSIONS— These data suggest that dapagliflozin has the potential to be an efficacious treatment for type 2 diabetes.

mTOR Inhibition by Rapamycin Prevents β-Cell Adaptation to Hyperglycemia and Exacerbates the Metabolic State in Type 2 Diabetes

Abstract: OBJECTIVE— Mammalian target of rapamycin (mTOR) and its downstream target S6 kinase 1 (S6K1) mediate nutrient-induced insulin resistance by downregulating insulin receptor substrate proteins with subsequent reduced Akt phosphorylation. Therefore, mTOR/S6K1 inhibition could become a therapeutic strategy in insulin-resistant states, including type 2 diabetes. We tested this hypothesis in the Psammomys obesus ( P. obesus ) model of nutrition-dependent type 2 diabetes, using the mTOR inhibitor rapamycin. RESEARCH DESIGN AND METHODS— Normoglycemic and diabetic P. obesus were treated with 0.2 mg · kg −1 · day −1 i.p. rapamycin or vehicle, and the effects on insulin signaling in muscle, liver and islets, and on different metabolic parameters were analyzed. RESULTS— Unexpectedly, rapamycin worsened hyperglycemia in diabetic P. obesus without affecting glycemia in normoglycemic controls. There was a 10-fold increase of serum insulin in diabetic P. obesus compared with controls; rapamycin completely abolished this increase. This was accompanied by weight loss and a robust increase of serum lipids and ketone bodies. Rapamycin decreased muscle insulin sensitivity paralleled by increased glycogen synthase kinase 3β activity. In diabetic animals, rapamycin reduced β-cell mass by 50% through increased apoptosis. Rapamycin increased the stress-responsive c-Jun NH 2 -terminal kinase pathway in muscle and islets, which could account for its effect on insulin resistance and β-cell apoptosis. Moreover, glucose-stimulated insulin secretion and biosynthesis were impaired in islets treated with rapamycin. CONCLUSIONS— Rapamycin induces fulminant diabetes by increasing insulin resistance and reducing β-cell function and mass. These findings emphasize the essential role of mTOR/S6K1 in orchestrating β-cell adaptation to hyperglycemia in type 2 diabetes. It is likely that treatments based on mTOR inhibition will cause exacerbation of diabetes.