bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Structurally Distinct CD101 Molecule Encoded in the 950-kbIdd10Region of NOD Mice
Authors: Penha-Gonçalves Carlos , Moule Carolyn , Smink Luc J. , Howson Joanna , Gregory Simon , Rogers Jane , Lyons Paul A. , Suttie Joseph J. , Lord Christopher J. , Peterson Laurence B. , Todd John A. , Wicker Linda S.
Published: March 2007

Genes affecting autoimmune type 1 diabetes susceptibility in the nonobese diabetic (NOD) mouse (Idd loci) have been mapped using a congenic strain breeding strategy. In the present study, we used a combination of BAC clone contig construction, polymorphism analysis of DNA from congenic strains, and sequence mining of the human orthologous region to generate an integrated map of the Idd10 region on mouse chromosome 3. We found seven genes and one pseudogene in the 950-kb Idd10 region. Although all seven genes in the interval are Idd10 candidates, we suggest the gene encoding the EWI immunoglobulin subfamily member EWI-101 (Cd101) as the most likely Idd10 candidate because of the previously reported immune-associated properties of the human CD101 molecule. Additional support for the candidacy of Cd101 is the presence of 17 exonic single-neucleotide polymorphisms that differ between the NOD and B6 sequences, 10 causing amino acid substitutions in the predicted CD101 protein. Four of these 10 substitutions are nonconservative, 2 of which could potentially alter N-linked glycosylation. Considering our results together with those previous reports that antibodies recognizing human CD101 modulate human T-cell and dendritic cell function, there is now justification to test whether the alteration of CD101 function affects autoimmune islet destruction.
A Polymorphism in the TCF7 Gene, C883A, Is Associated With Type 1 Diabetes
Authors: Noble Janelle A. , White Amy M. , Lazzeroni Laura C. , Valdes Ana M. , Mirel Daniel B. , Reynolds Rebecca , Grupe Andrew , Aud Dee , Peltz Gary , Erlich Henry A.
Published: March 2007

Type 1 diabetes is an autoimmune disease with a Th1 phenotype in which insulin-producing β-cells in the pancreas are destroyed. The T-cell–specific transcription factor TCF7 activates genes involved in immune regulation and is a candidate locus for genetic susceptibility to type 1 diabetes. A nonsynonymous single nucleotide polymorphism (SNP) (Pro to Thr) in the TCF7 gene, C883A, was examined in samples from 282 Caucasian multiplex type 1 diabetic families. HLA-DRB1 and -DQB1 genotypes were previously determined for these samples, allowing data stratification based on HLA-associated risk. The transmission disequilibrium test showed significant overtransmission of the A allele from fathers (64.1%, P < 0.007) and nonsignificant overtransmission (57.4%, P < 0.06) of the A allele to patients who do not carry the highest-risk HLA-DR3/DR4 genotype. Elliptical sib pair analysis showed significant associations of the A allele with type 1 diabetes in paternal transmissions (P < 0.03), transmissions to male children (P < 0.04), and in the non-DR3/DR4 group (P < 0.04). These data also suggest that TCF7 C883A may affect age of disease onset. Analysis of genotype data from surrounding SNPs suggests that this TCF7 polymorphism may itself represent a risk factor for type 1 diabetes.
Dynamics of Calcium Clearance in Mouse Pancreatic β-Cells
Authors: Chen Liangyi , Koh Duk-Su , Hille Bertil
Published: March 2007

Pancreatic β-cells maintain glucose homeostasis by their regulated Ca2+-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca2+ levels, but their relative significance in mouse β-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca2+ ([Ca2+]i) clearance after brief, depolarization-induced Ca2+ entry. Treatment with thapsigargin or cyclopiazonic acid, inhibitors of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps, nearly doubled the peak and slowed the decay of the depolarization-induced Ca2+ transients. The remaining thapsigargin-insensitive decay was slowed further by inhibition of the plasma membrane Ca2+-ATPase (PMCA) and plasma membrane Na+/Ca2+ exchanger (NCX) via alkalization of the bath solution, by adding lanthanum, or by substitution of Na+ with Li+. Mitochondrial Ca2+ uptake contributed little to clearance in thapsigargin-pretreated cells. Together, the SERCA, PMCA, and NCX transport mechanisms accounted for 89 to 97% of clearance in normal solutions. We developed a quantitative model for the dynamic role of removal mechanisms over a wide range of [Ca2+]i. According to our model, 50 to 64% of initial Ca2+ removal is via the SERCA pump, whereas the NCX contributes 21–30% of the extrusion at high [Ca2+]i, and the PMCA contributes 21–27% at low [Ca2+]i.
β-Cell Capacity and Insulin Sensitivity in Prepubertal Children Born Small for Gestational Age
Authors: Veening Margreet A. , van Weissenbruch Mirjam M. , Heine Robert J. , Delemarre-van de Waal Henriette A.
Published: March 2007

Insulin secretion and sensitivity was studied in 28 prepubertal children born small for gestational age (SGA) and in 22 prepubertal children born appropriate for gestational age (AGA). The effect of body size during childhood was also assessed. Insulin sensitivity was measured using the hyperinsulinemic-euglycemic clamp (M value), and β-cell function was studied with the hyperglycemic clamp plus arginine. First-phase insulin response (FIR) was used to calculate the disposition index (FIR × M value). Arginine-stimulated second-phase insulin response was used as a measure of β-cell capacity. ΔBMI0–1 year, ΔBMI0–2 years, and ΔBMI2–9 years were classified in tertiles. SGA children were less insulin sensitive than AGA children (P = 0.009). β-Cell capacity and disposition indexes were similar in the two groups. In SGA children, M values were lower in the tertile with the highest ΔBMI2–9 years than in the tertile with the lowest ΔBMI2–9 years (P = 0.01). No association between ΔBMI0–2 years and decreased insulin sensitivity was found. In conclusion, prepubertal SGA children show decreased insulin sensitivity rather than decreased β-cell capacity. Interventions to improve fetal growth and prevent overweight after the second year of life appear to be important factors in the prevention of type 2 diabetes in children born SGA.
Chronic Diabetes Increases Advanced Glycation End Products on Cardiac Ryanodine Receptors/Calcium-Release Channels
Authors: Bidasee Keshore R. , Nallani Karuna , Yu Yongqi , Cocklin Ross R. , Zhang Yinong , Wang Mu , Dincer Ü. Deniz , Besch Henry R.
Published: March 2007

Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M+H+) ≥500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M+H+ peaks corresponding to theoretical RyR2 peptides with single Nε-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetes-induced RyR2 dysfunction.
Activation of Protein Kinase C-ζ by Insulin and Phosphatidylinositol-3,4,5-(PO4)3 Is Defective in Muscle in Type 2 Diabetes and Impaired Glucose Tolerance
Authors: Beeson Mary , Sajan Mini P. , Dizon Michelle , Grebenev Dmitry , Gomez-Daspet Joaquin , Miura Atsushi , Kanoh Yoshinori , Powe Jennifer , Bandyopadhyay Gautam , Standaert Mary L. , Farese Robert V.
Published: March 2007

Insulin resistance in type 2 diabetes is partly due to impaired glucose transport in skeletal muscle. Atypical protein kinase C (aPKC) and protein kinase B (PKB), operating downstream of phosphatidylinositol (PI) 3-kinase and its lipid product, PI-3,4,5-(PO4)3 (PIP3), apparently mediate insulin effects on glucose transport. We examined these signaling factors during hyperinsulinemic-euglycemic clamp studies in nondiabetic subjects, subjects with impaired glucose tolerance (IGT), and type 2 diabetic subjects. In nondiabetic control subjects, insulin provoked twofold increases in muscle aPKC activity. In both IGT and diabetes, aPKC activation was markedly (70–80%) diminished, most likely reflecting impaired activation of insulin receptor substrate (IRS)-1-dependent PI 3-kinase and decreased ability of PIP3 to directly activate aPKCs; additionally, muscle PKC-ζ levels were diminished by 40%. PKB activation was diminished in patients with IGT but not significantly in diabetic patients. The insulin sensitizer rosiglitazone improved insulin-stimulated IRS-1-dependent PI 3-kinase and aPKC activation, as well as glucose disposal rates. Bicycle exercise, which activates aPKCs and stimulates glucose transport independently of PI 3-kinase, activated aPKCs comparably to insulin in nondiabetic subjects and better than insulin in diabetic patients. Defective aPKC activation contributes to skeletal muscle insulin resistance in IGT and type 2 diabetes, rosiglitazone improves insulin-stimulated aPKC activation, and exercise directly activates aPKCs in diabetic muscle.
Insulin-Stimulated Protein Kinase C λ/ζ Activity Is Reduced in Skeletal Muscle of Humans With Obesity and Type 2 Diabetes
Authors: Kim Young-Bum , Kotani Ko , Ciaraldi Theodore P. , Henry Robert R. , Kahn Barbara B.
Published: March 2007

In humans with obesity or type 2 diabetes, insulin target tissues are resistant to many actions of insulin. The atypical protein kinase C (PKC) isoforms λ and ζ are downstream of phosphatidylinositol-3 kinase (PI3K) and are required for maximal insulin stimulation of glucose uptake. Phosphoinositide-dependent protein kinase-1 (PDK-1), also downstream of PI3K, mediates activation of atypical PKC isoforms and Akt. To determine whether impaired PKCλ/ζ or PDK-1 activation plays a role in the pathogenesis of insulin resistance, we measured the activities of PKCλ/ζ and PDK-1 in vastus lateralis muscle of lean, obese, and obese/type 2 diabetic humans. Biopsies were taken after an overnight fast and after a 3-h hyperinsulinemic-euglycemic clamp. Obese subjects were also studied after weight loss on a very-low-calorie diet. Insulin-stimulated glucose disposal rate is reduced 26% in obese subjects and 62% in diabetic subjects (both comparisons P < 0.001). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and PI3K activity are impaired 40–50% in diabetic subjects compared with lean or obese subjects. Insulin stimulates PKCλ/ζ activity ∼2.3-fold in lean subjects; the increment above basal is reduced 57% in obese and 65% in diabetic subjects. PKCλ/ζ protein amount is decreased 46% in diabetic subjects but is normal in obese nondiabetic subjects, indicating impaired insulin action on PKCλ/ζ. Importantly, weight loss in obese subjects normalizes PKCλ/ζ activation and increases IRS-1 phosphorylation and PI3K activity. Insulin also stimulates PDK-1 activity approximately twofold with no impairment in obese or diabetic subjects. In contrast to our previous data on Akt, reduced insulin-stimulated PKCλ/ζ activity could play a role in the pathogenesis of insulin resistance in muscle of obese and type 2 diabetic subjects.
Vesicular Inhibitory Amino Acid Transporter Is Present in Glucagon-Containing Secretory Granules in αTC6 Cells, Mouse Clonal α-Cells, and α-Cells of Islets of Langerhans
Authors: Hayashi Mitsuko , Otsuka Masato , Morimoto Riyo , Muroyama Akiko , Uehara Shunsuke , Yamamoto Akitsugu , Moriyama Yoshinori
Published: March 2007

Islets of Langerhans contain γ-aminobutyrate (GABA) and may use it as an intercellular transmitter. In β-cells, GABA is stored in synaptic-like microvesicles and secreted through Ca2+-dependent exocytosis. Vesicular inhibitory amino acid transporter (VIAAT), which is responsible for the storage of GABA and glycine in neuronal synaptic vesicles, is believed to be responsible for the storage and secretion of GABA in β-cells. However, a recent study by Chessler et al. indicated that VIAAT is expressed in the mantle region of islets. In the present study, we investigated the precise localization of VIAAT in rat islets of Langerhans and clonal islet cells and found that it is present in α-cells, a minor population of F-cells and αTC6 cells, and clonal α-cells but not in β-cells, δ-cells, or MIN6 m9-cells (clonal β-cells). Combined biochemical, immunohistochemical, and electronmicroscopical evidence indicated that VIAAT is specifically localized with glucagon-containing secretory granules in α-cells. ATP-dependent uptake of radiolabeled GABA, which is energetically coupled with a vacuolar proton pump, was detected in digitonin-permeabilized αTC6 cells as well as in MIN6 m9 cells. These results demonstrate that functional neuronal VIAAT is present in glucagon-containing secretory granules in α-cells and suggest that the ATP-dependent GABA transporter in β-cells is at least immunologically distinct from VIAAT. Because glucagon-containing secretory granules also contain vesicular glutamate transporter and store l-glutamate, as demonstrated by Hayashi et al., the present results suggest more complex features of the GABAergic phenotype of islets than previously supposed.
Inflammation-Sensitive Plasma Proteins Are Associated With Future Weight Gain
Authors: Engström Gunnar , Hedblad Bo , Stavenow Lars , Lind Peter , Janzon Lars , Lindgärde Folke
Published: March 2007

Cross-sectional studies have associated obesity and other components of the so-called metabolic syndrome with low-grade inflammation. The temporal and causal relations of this association have not been fully explored. This study explored whether elevated levels of inflammation-sensitive plasma proteins (ISPs) (fibrinogen, orosomucoid, α1-antitrypsin, haptoglobin, and ceruloplasmin) are associated with future weight gain. Five ISPs were measured in 2,821 nondiabetic healthy men (38–50 years of age) who were reexamined after a mean follow-up of 6.1 years. Future weight gain was studied in relation to the number of elevated ISPs (i.e., in the top quartile). The proportion with a large weight gain (75th percentile ≥3.8 kg) was 21.0, 25.9, 26.8, and 28.3%, respectively, among men with none, one, two, and three or more ISPs in the top quartile (P for trend 0.0005). This relation remained significant after adjustments for weight at baseline, follow-up time, height (at baseline and follow-up), physical inactivity (at baseline and follow-up), smoking (at baseline and follow-up), high alcohol consumption, and insulin resistance. The relations were largely similar for all individual ISPs. Elevated ISP levels predict a large weight gain in middle-aged men. This relation could contribute to the relation between inflammation, the metabolic syndrome, and cardiovascular disease.
Cortisol Acts Through Central Mechanisms to Blunt Counterregulatory Responses to Hypoglycemia in Conscious Rats
Authors: Sandoval Darleen A. , Ping Ling , Neill Anthony Ray , Morrey Sachiko , Davis Stephen N.
Published: March 2007

Physiological levels of cortisol have been found to blunt neuroendocrine and metabolic responses to subsequent hypoglycemia in humans. The aim of this study was to determine whether cortisol acts directly on the brain to elicit this effect. A total of 41 conscious unrestrained Sprague-Dawley rats were studied during 2-day experiments. Day 1 consisted of two episodes of clamped 2-h hyperinsulinemic (30 pmol · kg−1 · min−1) hypoglycemia (2.8 ± 0.1 mmol/l; n = 12; ANTE HYPO), euglycemia (6.2 ± 0.1 mmol/l; n = 12; ANTE EUG), or euglycemia (6.2 ± 0.1 mmol/l) plus simultaneous intracerebroventricular (ICV) infusion of cortisol (25 μg/h; n = 9; ANTE EUG+Cort) or saline (24 μl/h; n = 8; ANTE EUG+Sal). For all groups, day 2 consisted of a 2-h hyperinsulinemic (30 pmol · kg−1 · min−1) hypoglycemic (2.9 ± 0.2 mmol/l) clamp. Plasma epinephrine and glucagon incremental area under the curve (ΔAUC) responses were significantly less in ANTE EUG+Cort and ANTE HYPO versus both ANTE EUG and ANTE EUG+Sal (P < 0.05). The ΔAUC responses of plasma norepinephrine were significantly lower in ANTE EUG+Cort versus both ANTE EUG and ANTE EUG+Sal (P < 0.05). Endogenous glucose production was significantly less in ANTE HYPO and ANTE EUG+Cort versus the other groups (P < 0.05). Lastly, the glucose infusion rate to maintain the desired hypoglycemia was significantly greater in ANTE EUG+Cort and ANTE HYPO versus the other two groups (P < 0.05). In summary, ICV infusion of cortisol significantly blunted norepinephrine, epinephrine, glucagon, and endogenous glucose production responses to next-day hypoglycemia. We conclude that cortisol can act directly on the central nervous system to blunt counterregulatory responses to subsequent hypoglycemia in the conscious rat.
DcR3/TR6 Effectively Prevents Islet Primary Nonfunction After Transplantation
Authors: Wu Yulian , Han Bing , Luo Hongyu , Roduit Raphael , Salcedo Theodora W. , Moore Paul A. , Zhang Jun , Wu Jiangping
Published: March 2007

Islet primary nonfunction (PNF) is defined as the loss of islet function after transplantation for reasons other than graft rejection. It is a major obstacle to successful and efficient islet transplantation. DcR3/TR6 is a soluble death decoy receptor belonging to the tumor necrosis factor (TNF) receptor family, and it can block apoptosis mediated by several TNF receptor family members such as Fas and LTβR. In this study, we used TR6 to protect islets from PNF after transplantation. Untreated isogeneic or allogeneic islet transplantation had PNF incidence of 25 and 26.5%, respectively. Administration of TR6 totally prevented PNF in allogeneic islet transplantation. In vitro experiments showed an increased apoptosis among islets that were treated with FasL and γ-interferon (IFN-γ) in combination. TR6 significantly reduced such apoptosis. Functional study showed that insulin release was compromised after FasL and IFN-γ treatment, and the compromise could be prevented with TR6-Fc. This indicates that TR6 indeed protected β-cells from damage caused by FasL and IFN-γ. Further in vivo experiments showed that syngeneic islet transplantation between lpr/lpr and gld/gld mice was significantly more efficacious than that conducted between wild-type mice. These results suggest that Fas-mediated apoptosis plays an important role in PNF, and use of TR6 may be a novel strategy to prevent PNF in clinical islet transplantation.
Two Phases of Nitrergic Neuropathy in Streptozotocin-Induced Diabetic Rats
Authors: Cellek Selim , Foxwell Neale A. , Moncada Salvador
Published: March 2007

The distinction between metabolic and structural changes occurring in autonomic neurons during diabetes has not been fully clarified. Here we demonstrate that nitric oxide synthase-containing (nitrergic) neurons innervating the penis and gastric pylorus of streptozotocin-induced diabetic rats undergo a selective degenerative process in two phases. In the first phase, nitrergic nerve fibers lose some of their neuronal nitric oxide synthase content and function. In the second phase, nitrergic degeneration takes place in the cell bodies in the ganglia, leading to complete loss of nitrergic function. The changes in the first phase are reversible with insulin replacement; however, the neurodegeneration in the second phase is irreversible. Neurodegeneration is due to apoptotic cell death in the ganglia, which is selective for the nitrergic neurones.
Sensory Neurons With Activated Caspase-3 Survive Long-Term Experimental Diabetes
Authors: Cheng Chu , Zochodne Douglas W.
Published: March 2007

Long-term experimental diabetes may best model the prominent and irreversible sensory deficits of chronic human diabetic polyneuropathy. Whereas irretrievable loss of sensory neurons, if present, would be an unfortunate feature of the disease, systematic unbiased counting has indicated that sensory neurons survive long-term experimental diabetes. In this study, we examined whether incipient cell loss from apoptosis in chronic experimental diabetes might nonetheless be in process, or whether neurons somehow adapt to their chronic insults. We examined sensory neurons in L4 and L5 dorsal root ganglia of long-term experimental streptozotocin-induced diabetic rats using transferase-mediated dUTP nick-end labeling (TUNEL), 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclear morphology, and electron microscopic appraisal of cell morphology. None provided any evidence for ongoing apoptosis. Despite this confirmation that sensory neurons survive, neurons had elevated expression of activated caspase-3 in unique patterns that included their nuclei, cytoplasm, and proximal axonal segments. Bcl-2 expression, a marker of antiapoptosis signaling, was observed in similar numbers of diabetic and nondiabetic neurons. In contrast, diabetic sensory neurons had elevated expression of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) in their nuclei, cytoplasm, and proximal axonal segments not overlapping with caspase-3 localization. Diabetic sensory neurons also had an apparent rise in cytoplasmic labeling of nitrotyrosine, a marker of peroxynitrite toxicity reported to activate PARP.
B-Type Natriuretic Peptide Prevents Acute Hypertrophic Responses in the Diabetic Rat Heart
Authors: Rosenkranz Anke C. , Hood Sally G. , Woods Robyn L. , Dusting Gregory J. , Ritchie Rebecca H.
Published: March 2007

Stimulation of cardiomyocyte guanosine 3′,5′-cyclic monophosphate (cyclic GMP) via endothelial-derived nitric oxide (NO) is an important mechanism by which bradykinin and ACE inhibitors prevent hypertrophy. Endothelial NO dysfunction and cardiac hypertrophy are morbid features of diabetes not entirely prevented by ACE inhibitors. In cardiomyocyte/endothelial cell cocultures, bradykinin efficacy is abolished by high-glucose–induced endothelial NO dysfunction. We now demonstrate that antihypertrophic actions of natriuretic peptides, which stimulate cyclic GMP independently of NO, are preserved in cardiomyocytes despite high-glucose–induced endothelial dysfunction. Further, streptozotocin-induced diabetes significantly impairs the effectiveness of acute antihypertrophic strategies in isolated rat hearts. In hearts from citrate-treated control rats, angiotensin II–stimulated [3H]phenylalanine incorporation and atrial natriuretic peptide and β-myosin heavy chain mRNA expression were prevented by B-type natriuretic peptide (BNP), bradykinin, the ACE inhibitor ramiprilat, and the neutral endopeptidase inhibitor candoxatrilat. These antihypertrophic effects were accompanied by increased left ventricular cyclic GMP. In age-matched diabetic hearts, the antihypertrophic and cyclic GMP stimulatory actions of bradykinin, ramiprilat, and candoxatrilat were absent. However, the blunting of hypertrophic markers and accompanying increases in cyclic GMP stimulated by BNP were preserved in diabetes. Thus BNP, which increases cyclic GMP independently of NO, is an important approach to prevent growth in the diabetic myocardium, where endothelium-dependent mechanisms are compromised.
Impaired Cathepsin L Gene Expression in Skeletal Muscle Is Associated With Type 2 Diabetes
Authors: Huang Xudong , Vaag Allan , Carlsson Emma , Hansson Mona , Ahrén Bo , Groop Leif
Published: March 2007

To identify abnormally expressed genes associated with muscle insulin resistance or type 2 diabetes, we screened the mRNA populations using cDNA differential display combined with relative RT-PCR analysis from muscle biopsies of diabetes-prone C57BL/6J and diabetes-resistant NMRI mice fed with a high-fat or normal diet for 3 or 15 months. Six abnormally expressed genes were isolated from the mice after a 3-month fat feeding; one of them was cathepsin L. No significant difference in mRNA levels of these genes was observed between fat- and normal-diet conditions in either strains. However, cathepsin L mRNA levels in muscle were higher in normal diet–fed C57BL/6J mice compared with normal diet–fed NMRI mice at 3 months (0.72 ± 0.04 vs. 0.51 ± 0.04 relative units, P < 0.01, n = 8–10) and at 15 months (0.41 ± 0.05 vs. 0.27 ± 0.04 relative units, P = 0.01, n = 9–10). Further, cathepsin L mRNA levels in muscle correlated inversely with plasma glucose in both strains regardless of diets at 3 (r = −0.49, P < 0.01, n = 31) and 15 (r = −0.42, P = 0.007, n = 39) months. To study whether cathepsin L plays a role in human diabetes, we measured cathepsin L mRNA levels in muscle biopsies taken before and after an insulin clamp from 12 monozygotic twin pairs discordant for type 2 diabetes and from 12 control subjects. Basal cathepsin L mRNA levels were not significantly different between the study groups. Insulin infusion increased cathepsin L mRNA levels in control subjects from 1.03 ± 0.30 to 1.90 ± 0.32 relative units (P = 0.03). Postclamp cathepsin L mRNA levels were lower in diabetic twins but similar in nondiabetic twins compared with control subjects (0.66 ± 0.22, 1.16 ± 0.18 vs. 1.38 ± 0.21 relative units, P < 0.02, NS, respectively). Further, postclamp cathepsin L mRNA levels were correlated with insulin-mediated glucose uptake (r = 0.37, P = 0.03), particularly, with glucose oxidation (r = 0.37, P = 0.03), and fasting glucose concentrations (r = −0.45, P < 0.01) across all three study groups. In conclusion, muscle cathepsin L gene expression is increased in diabetes-prone mice and related to glucose tolerance. In humans, insulin-stimulated cathepsin L expression in skeletal muscle is impaired in diabetic but not in nondiabetic monozygotic twins, suggesting that the changes may be secondary to impaired glucose metabolism.
Insights Into the Biochemical and Genetic Basis of Glucokinase Activation From Naturally Occurring Hypoglycemia Mutations
Authors: Gloyn Anna L. , Noordam Kees , Willemsen Michèl A.A.P. , Ellard Sian , Lam Wayne W.K. , Campbell Ian W. , Midgley Paula , Shiota Chyio , Buettger Carol , Magnuson Mark A. , Matschinsky Franz M. , Hattersley Andrew T.
Published: March 2007

Glucokinase (GCK) is a key regulatory enzyme in the pancreatic β-cell and catalyzes the rate-limiting step for β-cell glucose metabolism. We report two novel GCK mutations (T65I and W99R) that have arisen de novo in two families with familial hypoglycemia. Insulin levels, although inappropriately high for the degree of hypoglycemia, remain regulated by fluctuations in glycemia, and pancreatic histology was normal. These mutations are within the recently identified heterotropic allosteric activator site in the theoretical model of human β-cell glucokinase. Functional analysis of the purified recombinant glutathionyl S-transferase fusion proteins of T65I and W99R GCK revealed that the kinetic changes result in a relative increased activity index (a measure of the enzyme’s phosphorylating potential) of 9.81 and 6.36, respectively, compared with wild-type. The predicted thresholds for glucose-stimulated insulin release using mathematical modeling were 3.1 (T65I) and 2.8 (W99R) mmol/l, which were in line with the patients’ fasting glucose. In conclusion, we have identified two novel spontaneous GCK-activating mutations whose clinical phenotype clearly differs from mutations in ATP-sensitive K+ channel genes. In vitro studies confirm the validity of structural and functional models of GCK and the putative allosteric activator site, which is a potential drug target for the treatment of type 2 diabetes.
Elevated Mitochondrial Cytochrome P450 2E1 and Glutathione S-Transferase A4-4 in Streptozotocin-Induced Diabetic Rats
Authors: Raza Haider , Prabu Subbuswamy K. , Robin Mari-Anne , Avadhani Narayan G.
Published: March 2007

Oxidative stress is an important factor in the etiology and pathogenesis of diabetes. We investigated changes in mitochondrial production of reactive oxygen species (ROS) and mitochondrial antioxidant defense systems in different tissues of streptozotocin (STZ)-induced diabetic rats. Our results show that increased ROS production and oxidative stress differentially affect mitochondrial and cytosolic glutathione (GSH) metabolism. Of the four tissues investigated, the pancreas, kidney, and brain appear to be affected more severely than the liver. We show a five- to eightfold increase of cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase (GST) A4-4 levels in mitochondria from STZ-treated rat tissues compared with those in nondiabetic rat tissues, suggesting possible roles in the disease process. Transient transfection of COS cells with CYP2E1 cDNA caused a similar accumulation of CYP2E1 and GST A4-4 in mitochondria and increased production of mitochondrial ROS. Our results also show an increase in steady-state levels of Hsp70 in the mitochondrial and cytosolic fractions of different tissues of diabetic rats. These results indicate, for the first time, a marked increase in mitochondrial oxidative stress in target tissues of STZ-treated rats and implicate a direct role for mitochondrial CYP2E1 in the generation of intramitochondrial ROS.
Peroxisome Proliferator-Activated Receptor-γ Ligands Inhibit TGF-β1-Induced Fibronectin Expression in Glomerular Mesangial Cells
Authors: Guo Baoliang , Koya Daisuke , Isono Motohide , Sugimoto Toshiro , Kashiwagi Atsunori , Haneda Masakazu
Published: March 2007

The thiazolidinedione (TZD) class of antidiabetic drugs, which are ligands for peroxisome proliferator-activated receptor (PPAR)-γ, has been shown to possess potent anti-inflammatory and antineoplastic actions. Here, we show in mesangial cells that PPAR-γ agonists inhibit fibronectin expression by transforming growth factor (TGF)-β1. TGF-β1 enhanced fibronectin mRNA expression, and this enhancement was abrogated by pretreatment with pioglitazone. Electrophoretic mobility shift assay identified that pioglitazone inhibited TGF-β1-induced DNA binding of activator protein-1 (AP-1). Pioglitazone inhibited AP-1 reporter activity but not Smad binding elements reporter activity without affecting TGF-β1-induced activation of mitogen-activated protein kinases (MAPKs) or Smad2. PPAR-γ overexpression inhibited TGF-β1-induced fibronectin expression as well as the activation of AP-1. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a natural PPAR-γ ligand, also inhibited TGF-β1-induced fibronectin expression by suppressing AP-1 activation by TGF-β1. 15d-PGJ2 inhibited the TGF-β1-induced MAPK activation. Dominant-negative PPAR-γ (ΔPPAR-γ) completely abrogated the inhibitory effect of pioglitazone and incompletely blocked its effect of 15d-PGJ2 on TGF-β1-induced AP-1 reporter activity. ΔPPAR-γ overexpression did not affect the inhibitory effect of 15d-PGJ2 on TGF-β1-induced MAPK activation. In conclusion, pioglitazone inhibits TGF-β1-induced fibronectin expression by inhibiting AP-1 activation dependent on PPAR-γ, while 15d-PGJ2 acts through a dual mechanism independent of and dependent on PPAR-γ activation in mouse mesangial cells.
Genetic Variation at the Adiponectin Locus and Risk of Type 2 Diabetes in Women
Authors: Hu Frank B. , Doria Alessandro , Li Tricia , Meigs James B. , Liu Simin , Memisoglu Asli , Hunter David , Manson JoAnn E.
Published: March 2007

Previous data suggesting that polymorphisms in the adiponectin gene were associated with insulin resistance or type 2 diabetes have been inconsistent. We assessed the relationship between five common haplotype-tagging single nucleotide polymorphisms (SNPs) in the adiponectin gene (−11365C>G, −4034A>C, −3964A>G, +45T>G, and +276G>T), haplotypes defined by these SNPs, and the risk of type 2 diabetes by conducting a nested case-control study of 642 incident cases of type 2 diabetes and 995 matching control subjects in the Nurses’ Health Study. Overall, we did not observe significant differences in genotype or allele frequencies for the five SNPs between the case and control subjects. After adjustment for diabetes risk factors, the −4034 C/C genotype was associated with a reduced risk of diabetes (odds ratio [OR] compared with the A/A genotype = 0.70, 95% CI 0.50–0.99, P = 0.04). In subgroup analyses, the +276 genotype was significantly associated with diabetes risk only among subjects with peroxisome proliferator-activated receptor-γ (PPARγ) variant 12Ala allele (OR comparing +276 T alleles with the G/G genotype = 1.69, 1.04–2.75, P = 0.035) or among obese subjects (1.46, 1.03–2.08, P = 0.03). These data suggest a potential interaction between the adiponectin genotype and PPARγ genotype or obesity, but these analyses should be considered exploratory and require further investigation in larger studies.
Lipoprotein Lipase Is a Gene for Insulin Resistance in Mexican Americans
Authors: Goodarzi Mark O. , Guo Xiuqing , Taylor Kent D. , Quiñones Manuel J. , Saad Mohammad F. , Yang Huiying , Hsueh Willa A. , Rotter Jerome I.
Published: March 2007

The insulin resistance syndrome is increasingly recognized as a risk factor for cardiovascular disease. Lipoprotein lipase (LPL) is a candidate gene for components of the syndrome. A small number of studies have demonstrated association of single nucleotide polymorphisms within LPL and indirect or surrogate measures of insulin resistance, largely based on glucose and insulin values obtained in the fasting state or during an oral glucose tolerance test. To test directly whether LPL is an insulin resistance gene, we performed the hyperinsulinemic-euglycemic clamp in a large family-based population of Mexican Americans who were genotyped at six polymorphisms in LPL that define the most common haplotypes in the population. LPL haplotypes showed linkage to the glucose infusion rate (GINF), a direct physiologic measurement of insulin sensitivity (P = 0.034). In addition, significant associations with GINF were demonstrated for the most common haplotype (P = 0.031) and the fourth most common haplotype (P = 0.007). Haplotype 1 was associated with insulin sensitivity (mean GINF for haplotype 1 carriers = 383.0 mg/min) and haplotype 4 with insulin resistance (mean GINF for haplotype 4 carriers = 344.3 mg/min). This haplotype-based genetic analysis provides compelling evidence that variation in the LPL gene plays a role in determining insulin resistance in this ethnic group with a high prevalence of the insulin resistance syndrome.
The Common −866 G/A Polymorphism in the Promoter of Uncoupling Protein 2 Is Associated With Increased Carbohydrate and Decreased Lipid Oxidation in Juvenile Obesity
Authors: Le Fur Sophie , Le Stunff Catherine , Dos Santos Christine , Bougnères Pierre
Published: March 2007

Uncoupling protein (UCP) 2 is a member of the mitochondrial transporter superfamily that uncouples proton entry in the mitochondrial matrix from ATP synthesis. Although its physiological role remains to be established, UCP2 is considered a candidate gene for association with energy metabolism and obesity. A common promoter polymorphism, −866 G/A, has been associated with increased UCP2 gene expression and middle-aged adult obesity. In fact, our analysis of 296 juvenile obese and 568 nonobese control subjects revealed no difference in the prevalence of this polymorphism. Insulin and glucose response to oral glucose was comparable across the −866 genotypes. Metabolic studies in 147 of these juvenile obese subjects showed that homozygosity for the UCP2 promoter variant A was associated with important changes in energy metabolism compared with other genotypes, i.e., a 34% increase of carbohydrate oxidation (94 ± 10 vs. 70 ± 3 mg · min−1 · m−2, P = 0.004) and a 23% decrease of lipid oxidation (26 ± 3 vs. 34 ± 1 mg · min−1 · m−2, P = 0.03). Therefore, the juvenile obese subjects who are homozygous for the A variant have an increased ratio (3.6 ± 1.2) of calories derived from carbohydrates to those from lipids compared with G/A or G/G obese children (1.4 ± 0.2, P = 0.003), suggesting a role for UCP2 in the partitioning of metabolic fuels.
C-Peptide Is the Appropriate Outcome Measure for Type 1 Diabetes Clinical Trials to Preserve β-Cell Function
Authors: Palmer Jerry P. , Fleming G. Alexander , Greenbaum Carla J. , Herold Kevan C. , Jansa Lisa D. , Kolb Hubert , Lachin John M. , Polonsky Kenneth S. , Pozzilli Paolo , Skyler Jay S. , Steffes Michael W.
Published: March 2007

The underlying cause of type 1 diabetes, loss of β-cell function, has become the therapeutic target for a number of interventions in patients with type 1 diabetes. Even though insulin therapies continue to improve, it remains difficult to achieve normal glycemic control in type 1 diabetes, especially long term. The associated risks of hypoglycemia and end-organ diabetic complications remain. Retention of β-cell function in patients with type 1 diabetes is known to result in improved glycemic control and reduced hypoglycemia, retinopathy, and nephropathy. To facilitate the development of therapies aimed at altering the type 1 diabetes disease process, an American Diabetes Association workshop was convened to identify appropriate efficacy outcome measures in type 1 diabetes clinical trials. The following consensus emerged: While measurements of immune responses to islet cells are important in elucidating pathogenesis, none of these measures have directly correlated with the decline in endogenous insulin secretion. HbA1c is a highly valuable clinical measure of glycemic control, but it is an insensitive measure of β-cell function, particularly with the currently accepted standard of near-normal glycemic control. Rates of severe hypoglycemia and diabetic complications ultimately will be improved by therapies that are effective at preserving β-cell function but as primary outcomes require inordinately large and protracted trials. Endogenous insulin secretion is assessed best by measurement of C-peptide, which is cosecreted with insulin in a one-to-one molar ratio but unlike insulin experiences little first pass clearance by the liver. Measurement of C-peptide under standardized conditions provides a sensitive, well accepted, and clinically validated assessment of β-cell function. C-peptide measurement is the most suitable primary outcome for clinical trials of therapies aimed at preserving or improving endogenous insulin secretion in type 1 diabetes patients. Available data demonstrate that even relatively modest treatment effects on C-peptide will result in clinically meaningful benefits. The development of therapies for addressing this important unmet clinical need will be facilitated by trials that are carefully designed with β-cell function as determined by C-peptide measurement as the primary efficacy outcome.
Nonesterified Fatty Acids and Hepatic Glucose Metabolism in the Conscious Dog
Authors: Moore Mary Courtney , Satake Shosuke , Lautz Margaret , Soleimanpour Scott A. , Neal Doss W. , Smith Marta , Cherrington Alan D.
Published: March 2007

We used tracer and arteriovenous difference techniques in conscious dogs to determine the effect of nonesterified fatty acids (NEFAs) on net hepatic glucose uptake (NHGU). The protocol included equilibration ([3-3H]glucose), basal, and two experimental periods (−120 to −30, −30 to 0, 0–120 [period 1], and 120–240 min [period 2], respectively). During periods 1 and 2, somatostatin, basal intraportal insulin and glucagon, portal glucose (21.3 μmol · kg−1 · min−1), peripheral glucose (to double the hepatic glucose load), and peripheral nicotinic acid (1.5 mg · kg−1 · min−1) were infused. During period 2, saline (nicotinic acid [NA], n = 7), lipid emulsion (NA plus lipid emulsion [NAL], n = 8), or glycerol (NA plus glycerol [NAG], n = 3) was infused peripherally. During period 2, the NA and NAL groups differed (P < 0.05) in rates of NHGU (10.5 ± 2.08 and 4.7 ± 1.9 μmol · kg−1 · min−1), respectively, endogenous glucose Ra (2.3 ± 1.4 and 10.6 ± 1.0 μmol · kg−1 · min−1), net hepatic NEFA uptakes (0.1 ± 0.1 and 1.8 ± 0.2 μmol · kg−1 · min−1), net hepatic β-hydroxybutyrate output (0.1 ± 0.0 and 0.4 ± 0.1 μmol · kg−1 · min−1), and net hepatic lactate output (6.5 ± 1.7 vs. −2.3 ± 1.2 μmol · kg−1 · min−1). Hepatic glucose uptake and release were 2.6 μmol · kg−1 · min−1 less and 3.5 μmol · kg−1 · min−1 greater, respectively, in the NAL than NA group (NS). The NAG group did not differ significantly from the NA group in any of the parameters listed above. In the presence of hyperglycemia and relative insulin deficiency, elevated NEFAs reduce NHGU by stimulating hepatic glucose release and suppressing hepatic glucose uptake.
Epac: A New cAMP-Binding Protein in Support of Glucagon-Like Peptide-1 Receptor-Mediated Signal Transduction in the Pancreatic β-Cell
Authors: Holz George G.
Published: March 2007

Recently published studies of islet cell function reveal unexpected features of glucagon-like peptide-1 (GLP-1) receptor-mediated signal transduction in the pancreatic β-cell. Although GLP-1 is established to be a cAMP-elevating agent, these studies demonstrate that protein kinase A (PKA) is not the only cAMP-binding protein by which GLP-1 acts. Instead, an alternative cAMP signaling mechanism has been described, one in which GLP-1 activates cAMP-binding proteins designated as cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac). Two variants of Epac (Epac1 and Epac2) are expressed in β-cells, and downregulation of Epac function diminishes stimulatory effects of GLP-1 on β-cell Ca2+ signaling and insulin secretion. Of particular note are new reports demonstrating that Epac couples β-cell cAMP production to the stimulation of fast Ca2+-dependent exocytosis. It is also reported that Epac mediates the cAMP-dependent mobilization of Ca2+ from intracellular Ca2+ stores. This is a process of Ca2+-induced Ca2+ release (CICR), and it generates an increase of [Ca2+]i that may serve as a direct stimulus for mitochondrial ATP production and secretory granule exocytosis. This article summarizes new findings concerning GLP-1 receptor-mediated signal transduction and seeks to define the relative importance of Epac and PKA to β-cell stimulus-secretion coupling.
Neuronal Glucosensing
Authors: Levin Barry E. , Routh Vanessa H. , Kang Ling , Sanders Nicole M. , Dunn-Meynell Ambrose A.
Published: March 2007

Glucosensing neurons are specialized cells that use glucose as a signaling molecule to alter their action potential frequency in response to variations in ambient glucose levels. Glucokinase (GK) appears to be the primary regulator of most neuronal glucosensing, but other regulators almost certainly exist. Glucose-excited neurons increase their activity when glucose levels rise, and most use GK and an ATP-sensitive K+ channel as the ultimate effector of glucose-induced signaling. Glucose-inhibited (GI) neurons increase their activity at low glucose levels. Although many use GK, it is unclear what the final pathway of GI neuronal glucosensing is. Glucosensing neurons are located in brain sites and respond to and integrate a variety of hormonal, metabolic, transmitter, and peptide signals involved in the regulation of energy homeostasis and other biological functions. Although it is still uncertain whether daily fluctuations in blood glucose play a specific regulatory role in these physiological functions, it is clear that large decreases in glucose availability stimulate food intake and counterregulatory responses that restore glucose levels to sustain cerebral function. Finally, glucosensing is altered in obesity and after recurrent bouts of hypoglycemia, and this altered sensing may contribute to the adverse outcomes of these conditions. Thus, although much is known, much remains to be learned about the physiological function of brain glucosensing neurons.
Local Activation of Dendritic Cells Leads to Insulitis and Development of Insulin-Dependent Diabetes in Transgenic Mice Expressing CD154 on the Pancreatic β-Cells
Authors: Haase Claus , Skak Kresten , Michelsen Birgitte K. , Markholst Helle
Published: March 2007

The initial events leading to activation of the immune system in type 1 diabetes are still largely unknown. In vivo, dendritic cells (DCs) are thought to be the only antigen-presenting cells (APCs) capable of activating naïve T-cells and are therefore important for the initiation of the autoimmune response. To test the effect of activating islet-associated APCs in situ, we generated transgenic mice expressing CD154 (CD40 ligand) under control of the rat insulin promoter (RIP). RIP-CD154 mice developed both insulitis and diabetes, although with different incidence in independent lines. We show that activated DCs could be detected both in the pancreas and in the draining pancreatic lymph nodes. Furthermore, diabetes development was dependent on the presence of T- and B-cells since recombination-activating gene (RAG)-deficient RIP-CD154 mice did not develop diabetes. Finally, we show that the activation of immune cells was confined to the pancreas because transplantation of nontransgenic islets to diabetic recipients restored normoglycemia. Together, these data suggest that expression of CD154 on the β-cells can lead to activation of islet-associated APCs that will travel to the lymph nodes and activate the immune system, leading to insulitis and diabetes.
Gene Therapy Using Replication-Defective Herpes Simplex Virus Vectors Expressing Nerve Growth Factor in a Rat Model of Diabetic Cystopathy
Authors: Sasaki Katsumi , Chancellor Michael B. , Goins William F. , Phelan Michael W. , Glorioso Joseph C. , de Groat William C. , Yoshimura Naoki
Published: March 2007

Diabetic cystopathy is one of the common complications of diabetes and current therapy is limited. In the present study, the effects of gene therapy, using replication-defective herpes simplex virus type 1 (HSV-1) vectors to deliver and express the nerve growth factor (NGF) gene (HSV-NGF) on tissue NGF levels and bladder function, were evaluated in streptozotocin (STZ)-induced diabetic rats. Diabetic rats exhibited a significant decrease in NGF levels in the bladder and lumbosacral dorsal root ganglia (DRG) detected by enzyme-linked immunosorbent assay and displayed marked bladder dysfunction 12 weeks after STZ injection. In contrast, rats with bladder wall injection of the NGF expression vector 8 weeks after STZ treatment exhibited a significant increase of NGF levels in the bladder and L6 DRG 4 weeks after HSV-NGF injection. Along with the restoration of tissue NGF expression, in metabolic cage studies and cystometry, HSV-NGF–injected rats also showed significantly reduced bladder capacity and postvoid residual volume than diabetic rats injected with the control vector (HSV-lacZ), indicating that voiding function was improved after HSV vector–mediated NGF gene delivery. Thus, HSV vector–mediated NGF gene therapy may prove useful to restore decreased NGF expression in the bladder and bladder afferent pathways, thereby improving hypoactive bladder function in diabetes.