bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and role of laminin-1 in mouse pancreatic organogenesis.
Authors: Crisera C A , Kadison A S , Breslow G D , Maldonado T S , Longaker M T , Gittes G K
Published: March 2007

Previous studies have suggested that basement membrane alone may induce ductal differentiation and morphogenesis in the undifferentiated embryonic pancreas. The mechanism by which this induction occurs has not been investigated. Studies of other organ systems such as the lungs and mammary glands, where differentiation has been shown to be induced by basement membrane, have suggested a major role for laminin as a mediator of ductal or tubular morphogenesis and differentiation. We first defined the ontogeny of laminin-1 in the developing mouse pancreas. To determine the specific role of basement membrane laminin in pancreatic ductal morphogenesis and differentiation, we microdissected 11-day mouse embryonic pancreatic epithelium free from its surrounding mesenchyme and then suspended the explants in a 3-dimensional organ culture to allow us to assay cell differentiation and morphogenesis. When the pancreatic epithelium buds off the foregut endoderm, the pancreatic mesenchyme diffusely expresses laminin-1. This laminin subsequently organizes to the interface between the epithelium and the mesenchyme by E12.5. As gestation progresses, epithelial cells in direct contact with laminin-1 seem to differentiate into ducts and acini, whereas those spared intimate contact with laminin-1 appeared to organize into islets. Although basement membrane gel could induce pancreatic ductal morphogenesis of embryonic pancreatic epithelium, this induction was blocked when we added neutralizing antibodies against any of the following: 1) laminin (specifically laminin-1), 2) the "cross-region" of laminin-1, and 3) the alpha6 moiety of the integrin receptor, which is known to bind laminins. Immunohistochemistry, however, showed that pancreatic duct cell-specific differentiation (carbonic anhydrase II) without ductal morphogenesis was still present, despite the blockage of duct morphogenesis by the anti-laminin-1 neutralizing antibodies. Interestingly, there appeared to be a decrease in carbonic anhydrase II expression over time when the epithelia were grown in a collagen gel, rather than in a basement membrane gel. The pattern of laminin-1 expression in the embryonic pancreas supports the conclusion that laminin-1 is important in the induction of exocrine (ducts and acini) differentiation in the pancreas. Furthermore, our data demonstrate that 1) pancreatic ductal morphogenesis appears to require basement membrane laminin-1 and an alpha6-containing integrin receptor; 2) the cross-region of basement membrane laminin is a biologically active locus of the laminin molecule necessary for pancreatic ductal morphogenesis; 3) duct-specific cytodifferentiation, in the form of carbonic anhydrase II expression, is not necessarily coupled to duct morphogenesis; and 4) the basement membrane gel may contain components (e.g., growth factors) other than laminin-1 that can sustain both carbonic anhydrase II expression and, possibly, the capacity to form ducts, despite the absence of duct structures.
High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes.
Authors: Nelson B A , Robinson K A , Buse M G
Published: March 2007

Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes.
Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use.
Authors: Kurtzhals P , Schäffer L , Sørensen A , Kristensen C , Jonassen I , Schmid C , Trüb T
Published: March 2007

In recent years, analogs of human insulin have been engineered with the aim of improving therapy for people with diabetes. To ensure that the safety profile of the human hormone is not compromised by the molecular modifications, the toxico-pharmacological properties of insulin analogs should be carefully monitored. In this study, we compared the insulin and IGF-I receptor binding properties and metabolic and mitogenic potencies of insulin aspart (B28Asp human insulin), insulin lispro (B28Lys,B29Pro human insulin), insulin glargine (A21Gly,B31Arg,B32Arg human insulin), insulin detemir (NN304) [B29Lys(epsilon-tetradecanoyl), desB30 human insulin], and reference insulin analogs. Receptor affinities were measured using purified human receptors, insulin receptor dissociation rates were determined using Chinese hamster ovary cells overexpressing the human insulin receptor, metabolic potencies were evaluated using primary mouse adipocytes, and mitogenic potencies were determined in human osteosarcoma cells. Metabolic potencies correlated well with insulin receptor affinities. Mitogenic potencies in general correlated better with IGF-I receptor affinities than with insulin receptor off-rates. The 2 rapid-acting insulin analogs aspart and lispro resembled human insulin on all parameters, except for a slightly elevated IGF-I receptor affinity of lispro. In contrast, the 2 long-acting insulin analogs, glargine and detemir, differed significantly from human insulin. The combination of the B31B32diArg and A21Gly substitutions provided insulin glargine with a 6- to 8-fold increased IGF-I receptor affinity and mitogenic potency compared with human insulin. The attachment of a fatty acid chain to LysB29 provided insulin detemir with reduced receptor affinities and metabolic and mitogenic potencies but did not change the balance between mitogenic and metabolic potencies. The safety implications of the increased growth-stimulating potential of insulin glargine are unclear. The reduced in vitro potency of insulin detemir might explain why this analog is not as effective on a molar basis as human insulin in humans.
K(ATP) channel openers protect rat islets against the toxic effect of streptozotocin.
Authors: Kullin M , Li Z , Hansen J B , Björk E , Sandler S , Karlsson F A
Published: March 2007

We examined the influence of two K(ATP) channel openers, diazoxide and an analog (NNC 55-0118), on experimental beta-cell damage induced by streptozotocin (STZ; 0.5 mmol/l). Rat pancreatic islets were exposed to diazoxide or NNC 55-0118 for 30 min and were further incubated for 30 min after the addition of STZ. The islets were then washed and cultured for 24 h. Islets exposed to STZ alone showed extensive morphological damage, reduced glucose oxidation, low insulin content, and severely impaired glucose-stimulated insulin secretion and proinsulin biosynthesis. Islets treated with STZ in the presence of the channel openers (0.03-0.30 mmol/l) showed dose-dependent preservation of the morphology and improved glucose oxidation rates, insulin content, and secretion. NNC 55-0118 was capable of fully counteracting the STZ impairment, whereas diazoxide had a less protective effect. NNC 55-0118 did not counteract STZ-induced depression of islet NAD levels when examined 2 h after STZ exposure, which suggests that the mechanism of action by NNC 55-0118 is not through an inhibition of poly(ADP-ribose) polymerase. The results illustrate that K(ATP) channel openers can protect insulin-producing cells against toxic damage, an effect that may be of use in subjects with ongoing insulitis.
Induction of PEPCK gene expression in insulinopenia in rat small intestine.
Authors: Rajas F , Croset M , Zitoun C , Montano S , Mithieux G
Published: March 2007

PEPCK is a key enzyme of gluconeogenesis in liver and kidney. Recently, we have shown that small intestine also contributes to the endogenous glucose production in insulinopenia in rats and that glutamine is the main precursor of glucose synthesized in this tissue. The expression of the PEPCK gene in rat and human small intestine and the effect of streptozotocin-induced diabetes and fasting have been studied using reverse transcriptase-polymerase chain reaction, Northern blot analysis, and determination of enzyme activity. The PEPCK gene is expressed along the whole small intestine in adult rat and human. The abundance of PEPCK mRNA was increased approximately 30 times in the duodenum, 15 times in the jejunum, and only 3 times in the ileum from diabetic rats. PEPCK mRNA was downregulated in all parts of the tissue upon insulin treatment for 10 h. In 48-h fasted rats, the PEPCK mRNA abundance was increased 17 times in the duodenum and the jejunum and 3 times in the ileum, and it was normalized upon refeeding for 7 h. PEPCK activity was also increased 2-5 times in diabetic and fasted rats in the duodenum and jejunum but not in the ileum. We conclude that PEPCK is a crucial enzyme contributing to the induction of gluconeogenesis in small intestine, just as it is well known to be in the liver and kidney.
Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation.
Authors: Park J Y , Takahara N , Gabriele A , Chou E , Naruse K , Suzuma K , Yamauchi T , Ha S W , Meier M , Rhodes C J , King G L
Published: March 2007

Enhanced actions or levels of endothelin-1 (ET-1), a potent vasoconstrictor, have been associated with decreased blood flow in the retina and peripheral nerves of diabetic animals and may be related to the development of pathologies in these tissues. Hyperglycemia has been postulated to increase ET-1 secretion in endothelial cells. We have characterized the mechanism by which elevation of glucose is increasing ET-1 mRNA expression in capillary bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRPC). Elevation of glucose, but not mannitol, from 5.5 to 25 mmol/l for 3 days increased membranous protein kinase C (PKC) activities and ET-1 mRNA in parallel levels by 2-fold in BREC and BRPC. These effects were reversed by decreasing glucose levels to 5.5 mmol/l for an additional 2 days. Glucose-induced ET-1 overexpression was inhibited by a general PKC inhibitor, GF109203X, and a mitogen-activated protein kinase kinase inhibitor, PD98059, but not by wortmannin, a phosphatidylinositol 3-kinase inhibitor. By immunoblot analysis, PKC-beta2 and -delta isoforms in BREC were significantly increased relative to other isoforms in the membranous fractions when glucose level was increased. Overexpression of PKC-beta1 and -delta isoforms but not PKC-zeta isoform by adenovirus vectors containing the respective cDNA enhanced in parallel PKC activities, proteins, and basal and glucose-induced ET-1 mRNA expression by at least 2-fold. These results showed that enhanced ET-1 expression induced by hyperglycemia in diabetes is partly due to activation of PKC-beta and -delta isoforms, suggesting that inhibition of these PKC isoforms may prevent early changes in diabetic retinopathy and neuropathy.
A new mitochondrial DNA mutation at 14577 T/C is probably a major pathogenic mutation for maternally inherited type 2 diabetes.
Authors: Tawata M , Hayashi J I , Isobe K , Ohkubo E , Ohtaka M , Chen J , Aida K , Onaya T
Published: March 2007

From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted (rho0) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family.
Beta-cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy.
Authors: Kassem S A , Ariel I , Thornton P S , Scheimberg I , Glaser B
Published: March 2007

Hyperinsulinism of infancy (HI), also known as persistent hyperinsulinemic hypoglycemia of infancy, is a rare genetic disorder that occurs in approximately 1 of 50,000 live births. Histologically, pancreases from HI patients can be divided into 2 major groups. In the first, diffuse HI, beta-cell distribution is similar to that seen in normal neonatal pancreas, whereas in the second, focal HI, there is a discrete region of beta-cell adenomatous hyperplasia. In most patients, the clinical course of the disease suggests a slow progressive loss of beta-cell function. Using double immunostaining, we examined the proportion of beta-cells undergoing proliferation and apoptosis during the development of the normal human pancreas and in pancreases from diffuse and focal HI patients. In the control samples, our findings show a progressive decrease in beta-cell proliferation from 3.2 +/- 0.5% between 17 and 32 weeks of gestation to 0.13 +/- 0.08% after 6 months of age. In contrast, frequency of apoptosis is low (0.6 +/- 0.2%) in weeks 17-32 of gestation, elevated (1.3 +/- 0.3% ) during the perinatal period, and again low (0.08 +/- 0.3%) after 6 months of age. HI beta-cells showed an increased frequency of proliferation, with focal lesions showing particularly high levels. Similarly, the proportion of apoptotic cells was increased in HI, although this reached statistical significance only after 3 months of age. In conclusion, we demonstrated that islet remodeling normally seen in the neonatal period may be primarily due to a wave of beta-cell apoptosis that occurs at that time. In HI, our findings of persistently increased beta-cell proliferation and apoptosis provide a possible mechanism to explain the histologic picture seen in diffuse disease. The slow progressive decrease in insulin secretion seen clinically in these patients suggests that the net effect of these phenomena may be loss of beta-cell mass.
ATP4- mediates closure of pancreatic beta-cell ATP-sensitive potassium channels by interaction with 1 of 4 identical sites.
Authors: Markworth E , Schwanstecher C , Schwanstecher M
Published: March 2007

In pancreatic beta-cells, cytosolic [ATP(4-)] critically controls insulin secretion via inhibition of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel subunit (Kir6.2) plus a regulatory sulfonylurea receptor. To elucidate stoichiometry of ATP(4-) action, we analyzed ATP(4-) sensitivity of channels coassembled from wild-type Kir6.2 and a loss of ATP(4-) sensitivity mutant (G334D). Concentration-inhibition curves for cDNA ratios of 1:1 or 1:10 resembled those for channel block resulting from interaction with 1 of 4 sites, whereas models for inhibition requiring occupation of 2, 3, or 4 sites were incongruous. Random assembly of wild-type Kir6.2 with the G334D mutant was confirmed by controls, which assessed the effect of an additional mutation that induced strong rectification (N160D). We conclude 4 identical noncooperative ATP(4-) sites to be grouped within 1 KATP channel complex, with occupation of 1 site being sufficient to induce channel closure. This architecture might facilitate coupling of [ATP(4-)] to insulin secretion and may protect against diabetic dysregulation resulting from heterozygous mutations in Kir6.2.
Prevention of encephalomyocarditis virus-induced diabetes by live recombinant Mycobacterium bovis bacillus Calmette-Guérin in susceptible mice.
Authors: Choi B K , Cho S H , Bai G H , Kim S J , Hyun B H , Choe Y K , Bae Y S
Published: March 2007

The D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct cytolysis of pancreatic beta-cells. cDNA covering the major outer capsid protein (VP1) of the EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J mice immunized with live recombinant BCG-VP1 (rBCG-VP1) became diabetic when challenged with the highly diabetogenic EMC-D virus, but the control mice inoculated with normal BCG developed diabetes during the same challenge. VP1-specific antibodies (including neutralizing antibodies) were markedly increased over time and reached the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than 4 weeks. Mice and guinea pigs immunized with live rBCG-VP1 showed strong delayed-type hypersensitivity to the VP1 of the EMC-D virus. The preventive immunity still worked effectively 10 months after the primary immunization. At that time, the VP1-specific antibody was almost undetectable in the bloodstream, but a large number of VP1-specific lymphocytes was found in the spleen of the immunized mice. Our results show that live rBCG-VP1 elicits effective humoral and long-lasting cellular immune responses against EMC-D virus infection that results in the prevention of virus-induced diabetes in susceptible mice.
Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.
Authors: Barg S , Galvanovskis J , Göpel S O , Rorsman P , Eliasson L
Published: March 2007

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.
Obesity and Body Fat Distribution Induce Endothelial Dysfunction by Oxidative Stress
Authors: Perticone Francesco , Ceravolo Roberto , Candigliota Mafalda , Ventura Giorgio , Iacopino Saverio , Sinopoli Flora , Mattioli Pier L.
Published: March 2007

Endothelial dysfunction has been reported in obese subjects, but its mechanism has not been elucidated. We have therefore investigated 1) the possible relationship among BMI, waist-to-hip ratio (WHR), and endothelium-dependent vasodilation and 2) whether oxidative stress participates in endothelial dysfunction. We recruited 76 healthy subjects (50 men and 26 women aged 21-45 years) and measured their BMI (kg/m2), WHR, and insulin resistance (IR) estimated by the homeostasis model assessment (HOMA). Endothelium-dependent and -independent vasodilation were assessed by increasing doses of acetylcholine (ACh) (7.5, 15, and 30 μg · ml-1 · min-1) and sodium nitroprusside (SNP) (0.8, 1.6, and 3.2 μg · ml-1 · min-1) during saline and vitamin C coinfusion (24 mg/min). The effects of cyclooxygenase activity were evaluated by a dose-response curve to intrabrachial coinfusion of ACh and indomethacin (500 μg/min). Three different groups have been identified according to their BMI: group A (BMI <25), consisting of 10 men and 5 women; group B (BMI between 25 and 29), consisting of 16 men and 8 women; and group C (BMI ≥30), consisting of 24 men and 13 women. Obese subjects had significantly lower forearm blood flow (FBF) during ACh infusions (means ± SD): 19.8 ± 2.8, 10.8 ± 2.7, and 6.5 ± 1.8 ml · 100 ml-1 tissue · min-1 (P < 0.0001) for groups A, B, and C, respectively. SNP caused comparable increments in FBF in all groups. Regression analysis revealed a significant negative correlation between BMI (r = -0.676, P < 0.0001), WHR (r = -0.631, P < 0.0001), fasting insulin (r = -0.695, P < 0.0001), HOMA-IR (r = -0.633, P < 0.0001), and percent peak increase in FBF during ACh infusion. In obese subjects, both vitamin C and indomethacin increased the impaired vasodilating response to ACh, whereas the SNP effect was unchanged. In conclusion, in obese subjects, ACh-stimulated vasodilation is blunted, and the increase in FBF is inversely related to BMI, WHR, fasting insulin, and HOMA-IR. The effects of both vitamin C and indomethacin on impaired ACh-stimulated vasodilation support the hypothesis that oxidative stress contributes to endothelial dysfunction in human obesity.
A Genetic Defect in β-Cell Gene Expression Segregates Independently From the fa Locus in the ZDF Rat
Authors: Griffen Steven C. , Wang Juehu , German Michael S.
Published: March 2007

Type 2 diabetes is a strongly genetic disorder resulting from inadequate compensatory insulin secretion in the face of insulin resistance. The Zucker diabetic fatty (ZDF) rat is a model of type 2 diabetes and, like the human disease, has both insulin resistance (from a mutant leptin receptor causing obesity) and inadequate β-cell compensation. To test for an independently inherited β-cell defect, we examined β-cell function in fetuses of ZDF-lean rats, which have wild-type leptin receptors. β-Cell number and insulin content do not differ among wild-type, heterozygous, and homozygous ZDF-lean fetuses. However, insulin promoter activity is reduced 30-50% in homozygous ZDF-lean fetal islets, and insulin mRNA levels are similarly reduced by 45%. This is not a generalized defect in gene expression nor an altered transfection efficiency, because the islet amyloid polypeptide promoter and viral promoters are unaffected. Insulin promoter mapping studies suggest that the defect involves the critical A2-C1-E1 region. This study demonstrates that the ZDF rat carries a genetic defect in β-cell transcription that is inherited independently from the leptin receptor mutation and insulin resistance. The genetic reduction in β-cell gene transcription in homozygous animals likely contributes to the development of diabetes in the setting of insulin resistance.
Cell-Permeable Peptide Inhibitors of JNK
Authors: Bonny Christophe , Oberson Anne , Negri Stéphanie , Sauser Christelle , Schorderet Daniel F.
Published: March 2007

Stress conditions and proinflammatory cytokines activate the c-Jun NH2-terminal kinase (JNK), a member of the stress-activated group of mitogen-activated protein kinases (MAPKs). We recently demonstrated that inhibition of JNK signaling with the use of the islet-brain (IB) 1 and 2 proteins prevented interleukin (IL)-1β—induced pancreatic β-cell death. Bioactive cell-permeable peptide inhibitors of JNK were engineered by linking the minimal 20-amino acid inhibitory domains of the IB proteins to the 10-amino acid HIV-TAT sequence that rapidly translocates inside cells. Kinase assays indicate that the inhibitors block activation of the transcription factor c-Jun by JNK. Addition of the peptides to the insulin-secreting βTC-3 cell line results in a marked inhibition of IL-1β—induced c-jun and c-fos expression. The peptides protect βTC-3 cells against apoptosis induced by IL-1β. All-D retro-inverso peptides penetrate cells as efficiently as the L-enantiomers, decrease c-Jun activation by JNK, and remain highly stable inside cells. These latter peptides confer full protection against IL-1β—induced apoptosis for up to 2 weeks of continual treatment with IL-1β. These data establish these bioactive cell-permeable peptides as potent pharmacological compounds that decrease intracellular JNK signaling and confer long-term protection to pancreatic β-cells from IL-1β—induced apoptosis.
Resistin / Fizz3 Expression in Relation to Obesity and Peroxisome Proliferator–Activated Receptor-γ Action in Humans
Authors: Savage David B. , Sewter Ciaran P. , Klenk Ellen S. , Segal David G. , Vidal-Puig Antonio , Considine Robert V. , O’Rahilly Stephen
Published: March 2007

Recent studies in murine models suggest that resistin (also called Fizz3 [1]), a novel cysteine-rich protein secreted by adipocytes, may represent the long-sought link between obesity and insulin resistance (2). Furthermore, peroxisome proliferator–activated receptor-γ (PPAR-γ) agonists appear to inhibit resistin expression in murine adipocytes, providing a possible explanation for the mode of action of this class of insulin sensitizers (2). Using a fluorescent real-time reverse transcriptase–polymerase chain reaction–based assay, we found that resistin mRNA levels in whole adipose tissue samples were increased in morbidly obese humans compared with lean control subjects. However, in freshly isolated human adipocytes, resistin mRNA levels were very low and showed no correlation with BMI. Resistin mRNA was undetectable in preadipocytes, endothelial cells, and vascular smooth muscle cells, but it was readily detectable in circulating mononuclear cells. Although exposure of human mononuclear cells to PPAR-γ agonists markedly upregulated fatty acid–binding protein-4 expression, these agents had no effect on mononuclear cell resistin expression. Finally, resistin mRNA was undetectable in adipocytes from a severely insulin-resistant subject with a dominant-negative mutation in PPAR-γ (3). We conclude that the recently described relationships of murine resistin/Fizz3 expression with obesity, insulin resistance, and PPAR-γ action may not readily translate to humans. Further studies of this novel class of proteins are needed to clarify their roles in human metabolism.
Intrauterine Growth Retardation Leads to the Development of Type 2 Diabetes in the Rat
Authors: Simmons Rebecca A. , Templeton Lori J. , Gertz Shira J.
Published: March 2007

Intrauterine growth retardation has been linked to the development of type 2 diabetes in later life. The mechanisms underlying this phenomenon are unknown. We have developed a model of uteroplacental insufficiency, a common cause of intrauterine growth retardation, in the rat. Bilateral uterine artery ligation was performed on day 19 of gestation (term is 22 days) in the pregnant rat; sham-operated pregnant rats served as controls. Birth weights of intrauterine growth–retarded (IUGR) animals were significantly lower than those of controls until ∼7 weeks of age, when IUGR rats caught up to controls. Between 7 and 10 weeks of age, the growth of IUGR rats accelerated and surpassed that of controls, and by 26 weeks of age, IUGR rats were obese (P < 0.05 vs. controls). No significant differences were observed in blood glucose and plasma insulin levels at 1 week of age. However, between 7 and 10 weeks of age, IUGR rats developed mild fasting hyperglycemia and hyperinsulinemia (P < 0.05 vs. controls). At age 26 weeks, IUGR animals had markedly elevated levels of glucose (P < 0.05 vs. controls). IUGR animals were glucose-intolerant and insulin-resistant at an early age. First-phase insulin secretion in response to glucose was also impaired early in life in IUGR rats, before the onset of hyperglycemia. There were no significant differences in β-cell mass, islet size, or pancreatic weight between IUGR and control animals at 1 and 7 weeks of age. However, in 15-week-old IUGR rats, the relative β-cell mass was 50% that of controls, and by 26 weeks of age, β-cell mass was less than one-third that of controls (P < 0.05). The data presented here support the hypothesis that an abnormal intrauterine milieu can induce permanent changes in glucose homeostasis after birth and lead to type 2 diabetes in adulthood.
Changes in Regional Brain 18F-Fluorodeoxyglucose Uptake at Hypoglycemia in Type 1 Diabetic Men Associated With Hypoglycemia Unawareness and Counter-Regulatory Failure
Authors: Cranston Iain , Reed Laurence J. , Marsden Paul K. , Amiel Stephanie A.
Published: March 2007

We examined the effects of acute moderate hypoglycemia and the condition of hypoglycemia unawareness on regional brain uptake of the labeled glucose analog [18F]fluorodeoxyglucose (FDG) using positron emission tomography (PET). FDG-PET was performed in diabetic patients with (n = 6) and without (n = 7) hypoglycemia awareness. Each patient was studied at plasma glucose levels of 5 and 2.6 mmol/l, applied by glucose clamp techniques, in random order. Hypoglycemia-unaware patients were asymptomatic during hypoglycemia, with marked attenuation of their epinephrine responses (mean [± SD] peak of 0.77 ± 0.39 vs. 7.52 ± 2.9 nmol/l; P < 0.0003) and a reduced global brain FDG uptake ([mean ± SE] 2.592 ± 0.188 vs. 2.018 ± 0.174 at euglycemia; P = 0.027). Using statistical parametric mapping (SPM) to analyze images of FDG uptake, we identified a subthalamic brain region that exhibited significantly different behavior between the aware and unaware groups. In the aware group, there was little change in the normalized FDG uptake in this region in response to hypoglycemia ([mean ± SE] 0.654 ± 0.016 to 0.636 ± 0.013; NS); however, in the unaware group, the uptake in this region fell from 0.715 ± 0.015 to 0.623 ± 0.012 (P = 0.001). Our data were consistent with the human hypoglycemia sensor being anatomically located in this brain region, and demonstrated for the first time a change in its metabolic function associated with the failure to trigger a counter-regulatory response.
A Novel Variant of Glutamine
Authors: DeHaven John E. , Robinson Katherine A. , Nelson Bryce A. , Buse Maria G.
Published: March 2007

Glutamine:fructose-6-phosphate  amidotransferase(GFAT) is the rate-limiting enzyme of the hexosamine synthesis pathway. Products of this pathway have been implicated in insulin resistance and glucose toxicity. GFAT1 is ubiquitous, whereas GFAT2 is expressed mainly in the central nervous system. In the course of developing a competitive reverse transcriptase–polymerase chain reaction assay, we noted that GFAT1 cDNA from muscle but not from other tissues migrated as a doublet. Subsequent cloning and sequencing revealed two GFAT1 mRNAs in both mouse and human skeletal muscles. The novel GFAT1 mRNA (GFAT1Alt [muscle selective variant of GFAT1]) is likely a splice variant. It is identical to GFAT1 except for a 48 or 54 bp insert in the mouse and human, respectively, at nucleotide position 686 of the coding sequence, resulting in a 16 or 18 amino acid insert at position 229 of the protein. GFAT1Alt is the predominant GFAT1 mRNA in mouse hindlimb muscle, is weakly expressed in the heart, and is undetectable in the brain, liver, kidney, lung, intestine, spleen, and 3T3-L1 adipocytes. In humans, it is strongly expressed in skeletal muscle but not in the brain. GFAT1 and GFAT1Alt expressed by recombinant adenovirus infection in COS-7 cells displayed robust enzyme activity and kinetic differences. The apparent Km of GFAT1Alt for fructose-6-phosphate was approximately twofold higher than that of GFAT1, whereas Ki for UDP-N-acetylglucosamine was approximately fivefold lower. Muscle insulin resistance is a hallmark and predictor of type 2 diabetes. Variations in the expression of GFAT isoforms in muscle may contribute to predisposition to insulin resistance.
Protein Kinase C and Calcium Regulation of Adenylyl Cyclase in Isolated Rat Pancreatic Islets
Authors: Tian Yingrao , Laychock Suzanne G.
Published: March 2007

Rat islets express several isoforms of adenylyl cyclase (AC), and the regulation of AC activity in isolated islets by Ca2+ and protein kinase C (PKC) was investigated. At basal 2.8 mmol/l glucose, the muscarinic receptor agonist carbamylcholine chloride (CCh) evoked a concentration-dependent increase in cAMP generation with a maximum increase at least 4.5-fold above control. In contrast, forskolin and glucagon-like peptide 1 fragment 7-36 amide increased cAMP accumulation 23-fold and almost 10-fold, respectively. Cholecystokinin 26-33 sulfated amide (CCK) also stimulated cAMP production by up to eightfold, as did the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu and CCh or CCK responses were not additive. The effects of phorbol ester, CCh, and CCK were inhibited by as much as 75% by the PKC inhibitors GF 109203X and Ro-32-0432 and after PKC downregulation. In the absence of extracellular Ca2+, PDBu-, CCh-, and CCK-induced cAMP production was inhibited by ∼50% in each case. Chelation of intracellular Ca2+ with 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM) inhibited CCh- and CCK-stimulated cAMP generation by ∼50% but did not inhibit the stimulatory effect of PDBu. Stringent Ca2+ depletion by removal of extracellular Ca2+ and inclusion of BAPTA/AM allowed for increased cAMP production in response to CCh and CCK; PKC inhibitors and PKC downregulation prevented this stimulation. Glucose stimulation also increased islet cAMP production, but PDBu did not potentiate the glucose response. The results suggest that Ca2+ influx, Ca2+ mobilization, and PKC activation play important roles in the modulation of AC activity in pancreatic islets.
Islet Amyloid Develops Diffusely Throughout the Pancreas Before Becoming Severe and Replacing Endocrine Cells
Authors: Wang Feng , Hull Rebecca L. , Vidal Josep , Cnop Miriam , Kahn Steven E.
Published: March 2007

Islet amyloid occurs in >90% of type 2 diabetic patients and may play a role in the pathogenesis of this disease. To determine whether islet amyloid occurs diffusely throughout the pancreas, whether it affects islets equally, and whether it decreases islet endocrine cells, we characterized islet amyloidosis by computerized fluorescence microscopy in transgenic mice that develop typical islet amyloid. These mice produce the unique amyloidogenic component of human islet amyloid, human islet amyloid polypeptide (hIAPP). The prevalence of amyloid (number of islets containing amyloid/total number of islets × 100) and the severity of amyloid (Σamyloid area/Σislet area × 100) were found to be uniform throughout the pancreas. Furthermore, a high prevalence of amyloid was observed in islets when the severity of amyloid was only 1.5% of the islet area, suggesting a diffuse distribution of amyloid from the very early stages of islet amyloidosis. In 12 hIAPP transgenic mice with an amyloid severity of 9.6 ± 3.4%, the proportion of islets composed of β- and δ-cells was reduced in the transgenic mice compared with 6 nontransgenic mice that do not develop amyloid (β-cells: 62.9 ± 3.1% vs. 75.5 ± 0.9%, P = 0.02; δ-cells: 2.8 ± 0.5% vs. 4.4 ± 0.4%, P = 0.05), whereas the proportion of islets composed of α-cells did not significantly differ between the two groups of mice. In the individual islets in these transgenic mice, amyloid severity was inversely correlated with β-cell, (r = −0.59, P < 0.0001), α-cell (r = −0.32, P < 0.0001), and δ-cell (r = −0.25, P < 0.0001) areas. In conclusion, islet amyloidosis occurs uniformly throughout the pancreas, affecting all islets before becoming severe. A reduction in islet endocrine mass starts at this early stage of islet amyloid development and progresses as amyloid mass increases.
In Muscle-Specific Lipoprotein Lipase−Overexpressing Mice, Muscle Triglyceride Content Is Increased Without Inhibition of Insulin-Stimulated Whole-Body and Muscle-Specific Glucose Uptake
Authors: Voshol Peter J. , Jong Miek C. , Dahlmans Vivian E.H. , Kratky Dagmar , Levak-Frank Sanja , Zechner Rudolf , Romijn Johannes A. , Havekes Louis M.
Published: March 2007

In patients with type 2 diabetes, a strong correlation between accumulation of intramuscular triclycerides (TGs) and insulin resistance has been found. The aim of the present study was to determine whether there is a causal relation between intramuscular TG accumulation and insulin sensitivity. Therefore, in mice with muscle-specific overexpression of human lipoprotein lipase (LPL) and control mice, muscle TG content was measured in combination with glucose uptake in vivo, under hyperinsulinemic-euglycemic conditions. Overexpression of LPL in muscle resulted in accumulation of TGs in skeletal muscle (85.5 ± 33.3 vs. 25.7 ± 23.1 μmol/g tissue in LPL and control mice, respectively; P < 0.05). During the hyperinsulinemic clamp study, there were no differences in plasma glucose, insulin, and FFA concentrations between the two groups. Moreover, whole-body, as well as skeletal muscle, insulin-mediated glucose uptake did not differ between LPL-overexpressing and wild-type mice. Surprisingly, whole-body glucose oxidation was decreased by ∼60% (P < 0.05), whereas nonoxidative glucose disposal was increased by ∼50% (P < 0.05) in LPL-overexpressing versus control mice. In conclusion, overexpression of human LPL in muscle increases intramuscular TG accumulation, but does not affect whole-body or muscle-specific insulin-mediated uptake, findings that argue against a simple causal relation between intramuscular TG content and insulin resistance.
Rosiglitazone (BRL 49653) Enhances Insulin Secretory Response via Phosphatidylinositol 3-Kinase Pathway
Authors: Yang Chi , Chang Tien-Jyun , Chang Jen-Chieh , Liu Min-Wei , Tai Tong-Yuan , Hsu Walter H. , Chuang Lee-Ming
Published: March 2007

To elucidate the direct effect of rosiglitazone (RSG), a new thiazolidinedione antihyperglycemic agent, on pancreatic insulin secretion, an in situ investigation by rat pancreatic perfusion was performed. At a basal glucose concentration of 6 mmol/l, RSG (0.045–4.5 μmol/l) stimulated insulin release in a dose-dependent manner. In addition, 4.5 μmol/l RSG potentiated the glucose (10 mmol/l)-induced insulin secretion. Both the first and second phases of glucose-induced insulin secretion were significantly enhanced by RSG, by 80.7 and 52.4%, respectively. The effects of RSG on insulin secretion were inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. In contrast, the glucose-stimulated insulin secretion was not affected by LY294002. The potentiation effect of RSG on glucose-stimulated insulin secretion, in both the first and second phases, was significantly blocked by LY294002. These results suggest that RSG has a direct potentiation effect on insulin secretion in the presence of 10 mmol/l glucose, mediated through PI3K activity. The inability of LY294002 to inhibit glucose-induced insulin secretion suggests that different pathways are responsible for glucose and RSG signaling.
Effect of Diabetes on Nitric Oxide Metabolism During Cardiac Surgery
Authors: Matata Bashir M. , Galiñanes Manuel
Published: March 2007

The metabolism of nitric oxide (NO) during cardiac surgery is unclear. We studied the effect of diabetes on NO metabolism during cardiac surgery in 40 subjects (20 with diabetes and 20 without diabetes). The patients were randomized to receive an infusion of physiological saline or nitroglycerin (GTN) at 1 μg · kg–1 · min–1 starting 10 min before the initiation of cardiopulmonary bypass and then continuing for a period of 4 h. Blood and urine samples were collected at several time points for up to 8 h. NO metabolites were determined by the measurement of nitrate/nitrite (NOx, μmol/mmol creatinine) and cyclic guanosine monophosphate (cGMP, nmol/mmol creatinine) in plasma and urine. Plasma insulin levels were also determined at selected time points. Plasma NOx levels before surgery were significantly elevated in the group with diabetes compared with the group without diabetes (P < 0.001), and values were further increased during surgery in the former (P = 0.005) but not in the latter (P = 0.8). The greater plasma NOx values in patients with diabetes were matched by commensurate elevations in plasma cGMP levels (P = 0.01). Interestingly, infusion of GTN, an NO donor, significantly reduced plasma NOx (P < 0.001) and its urine elimination (P < 0.001) in patients with diabetes without reducing plasma cGMP levels (P = 0.89). Cardiac surgery increased plasma insulin in patients with and without diabetes; this increase was delayed by the infusion of GTN, but it was not related to the changes in NO production. In conclusion, NO production during cardiac surgery is increased in patients with diabetes, and this elevation can be blunted by the infusion of GTN in a rapid and reversible manner.
Dynamic Pattern of Disease-Associated Autoantibodies in Siblings of Children With Type 1 Diabetes
Authors: Savola Kaisa , Läärä Esa , Vähäsalo Paula , Kulmala Petri , Åkerblom Hans K. , Knip Mikael ,   Childhood Diabetes in Finland Study Group
Published: March 2007

To study the dynamics of disease-associated humoral immune responses, we analyzed autoantibodies to the IA-2 protein (IA-2A), glutamic acid decarboxylase (GADA), and insulin (IAA) and also islet cell antibodies (ICA) in a population-based, prospective, representative series of 710 siblings (<20 years of age) of children with type 1 diabetes. Positivity for single autoantibodies was observed in 8–13% of these siblings during an average follow-up of 4 years. The overall incidence rates per 1,000 years (number of cases/person-years in parentheses) for positive seroconversion of IA-2A were nine (19/2,123), followed by six (12/2,049) for GADA, 19 (40/2,111) for IAA, and 16 (31/1965) for ICA. Positive seroconversions seemed to be associated with a young age of the sibling, HLA DR3/DR4 heterozygosity, HLA identity, and a high initial number of detectable autoantibodies. The overall incidence rates per 1,000 years (number of cases/person-years in parentheses) for inverse seroconversion of IA-2A were 76 (12/157), followed by 42 (10/237) for GADA, 460 (32/70) for IAA, and 27 (9/331) for ICA. No consistent risk factor for inverse seroconversions was present, although seroconversions were most frequent in siblings with older age, male sex, HLA phenotypes other than DR3/DR4, a small family size, and no other autoantibodies detectable at seroconversion. Altogether, these observations indicate that β-cell autoimmunity may be induced at any age in childhood and adolescence. HLA-conferred genetic disease susceptibility is a strong determinant of persistent β-cell autoimmunity, but environmental factors may also contribute to such autoimmunity.
Loss of the Antiangiogenic Pigment Epithelium-Derived Factor in Patients With Angiogenic Eye Disease
Authors: Spranger Joachim , Osterhoff Martin , Reimann Manja , Möhlig Matthias , Ristow Michael , Francis Mary Kay , Cristofalo Vincent , Hammes Hans-Peter , Smith Gillian , Boulton Michael , Pfeiffer Andreas F.H.
Published: March 2007

Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P < 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P < 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.