bluuebunny/crossref_metadata_2025 huggingface dataset:
Connective Tissue Growth Factor and IGF-I Are Produced by Human Renal Fibroblasts and Cooperate in the Induction of Collagen Production by High Glucose
Authors: Lam Suzanne , van der Geest Reinier N. , Verhagen Nicole A.M. , van Nieuwenhoven Frans A. , Blom Ingrid E. , Aten Jan , Goldschmeding Roel , Daha Mohamed R. , van Kooten Cees
Published: March 2007

Tubulointerstitial fibrosis is an important component in the development of diabetic nephropathy. Various renal cell types, including fibroblasts, contribute to the excessive matrix deposition in the kidney. Although transforming growth factor-β (TGF-β) has been thought to play a major role during fibrosis, other growth factors are also involved. Here we examined the effects of connective tissue growth factor (CTGF) and IGF-I on collagen type I and III production by human renal fibroblasts and their involvement in glucose-induced matrix accumulation. We have demonstrated that both CTGF and IGF-I expressions were increased in renal fibroblasts under hyperglycemic conditions, also in the absence of TGF-β signaling. Although CTGF alone had no effect on collagen secretion, combined stimulation with IGF-I enhanced collagen accumulation. Furthermore, IGF-I also had a synergistic effect with glucose on the induction of collagens. Moreover, we observed a partial inhibition in glucose-induced collagen secretion with neutralizing anti-CTGF antibodies, thereby demonstrating for the first time the involvement of endogenous CTGF in glucose-induced effects in human renal fibroblasts. Therefore, the cooperation between CTGF and IGF-I might be involved in glucose-induced matrix accumulation in tubulointerstitial fibrosis and might contribute to the pathogenesis of diabetic nephropathy.
A Genome-Wide Search for Genes Involved in Type 2 Diabetes in a Recently Genetically Isolated Population From the Netherlands
Authors: Aulchenko Yurii S. , Vaessen Norbert , Heutink Peter , Pullen Jan , Snijders Pieter J.L.M. , Hofman Albert , Sandkuijl Lodewijk A. , Houwing-Duistermaat Jeanine J. , Edwards Mark , Bennett Simon , Oostra Ben A. , van Duijn Cornelia M.
Published: March 2007

Multiple genes, interacting with the environment, contribute to the susceptibility to type 2 diabetes. We performed a genome-wide search to localize type 2 diabetes susceptibility genes in a recently genetically isolated population in the Netherlands. We identified 79 nuclear families with type 2 diabetes who were related within 13 generations and performed a 770-marker genome-wide scan search for shared founder alleles. Twenty-six markers yielded a logarithm of odds (LOD) score >0.59 (nominal P < 0.05), of which 7 reached LOD scores >1.17 (nominal P < 0.01). The strongest evidence for a type 2 diabetes locus was at marker D18S63 on chromosome 18p (LOD 2.3, P = 0.0006). This region was investigated further using additional markers. For one of these markers (D18S1105), we found a significant association with type 2 diabetes (odds ratio 6.7 [95% CI 1.5–30.7], P = 0.005 for the 97-bp allele, assuming a dominant model), which increased when limiting the analysis to patients with high BMI (12.25 [2.1–71], P = 0.003). A locus on chromosome 18p in patients with high BMI was suggested earlier by Parker et al. Our study is the first to confirm this locus.
Differential Effects of Rosiglitazone and Metformin on Adipose Tissue Distribution and Glucose Uptake in Type 2 Diabetic Subjects
Authors: Virtanen Kirsi A. , Hällsten Kirsti , Parkkola Riitta , Janatuinen Tuula , Lönnqvist Fredrik , Viljanen Tapio , Rönnemaa Tapani , Knuuti Juhani , Huupponen Risto , Lönnroth Peter , Nuutila Pirjo
Published: March 2007

We evaluated the effects of rosiglitazone (4 mg b.i.d.) and metformin (1 g b.i.d.) monotherapy for 26 weeks on adipose tissue insulin-stimulated glucose uptake in patients (n = 41) with type 2 diabetes. Before and after the treatment, glucose uptake was measured using 2-[18F]fluoro-2-deoxyglucose and positron emission tomography and adipose tissue masses were quantified using magnetic resonance imaging. Rosiglitazone improved insulin-stimulated whole-body glucose uptake by 44% (P < 0.01 vs. placebo). Mean body weight was unchanged in the rosiglitazone group, while it decreased by 2.0 kg in the metformin group (P < 0.05 vs. placebo). In visceral adipose tissue, glucose uptake increased by 29% (from 17.8 ± 2.0 to 23.0 ± 2.6 μmol · kg−1 · min−1, P < 0.05 vs. placebo) in the rosiglitazone group but to a lesser extent (17%) in the metformin group (from 16.2 ± 1.5 to 18.9 ± 1.7 μmol · kg−1 · min−1, P < 0.05 vs. baseline). Because the visceral adipose tissue mass simultaneously decreased with both treatments (P < 0.05), no change was observed in total visceral glucose uptake per depot. Rosiglitazone significantly enhanced glucose uptake in the femoral subcutaneous area, either when expressed per tissue mass (from 10.8 ± 1.2 to 17.1 ± 1.7 μmol · kg−1 · min−1, P < 0.01 vs. placebo) or per whole-fat depot (P < 0.05 vs. placebo). In conclusion, metformin treatment resulted in improvement of glycemic control without enhancement of peripheral insulin sensitivity. The improved insulin sensitivity of the nonabdominal subcutaneous adipose tissue during treatment with rosiglitazone partly explains the enhanced whole-body insulin sensitivity and underlies the central role of adipose tissue for action of peroxisome proliferator-activated receptor γ agonist in vivo.
STAT5A Promotes Adipogenesis in Nonprecursor Cells and Associates With the Glucocorticoid Receptor During Adipocyte Differentiation
Authors: Floyd Z. Elizabeth , Stephens Jacqueline M.
Published: March 2007

The differentiation of adipocytes is regulated by the activity of a variety of transcription factors, including peroxidase proliferator-activated receptor (PPAR)-γ and C/EBPα. Our current study demonstrates that ectopic expression of STAT5A, such as that of PPAR-γ and C/EBPα, promotes adipogenesis in two nonprecursor fibroblast cell lines. Using morphologic and biochemical criteria, we have demonstrated that STAT5A and the combination of STAT5A and STAT5B are sufficient to induce the expression of early and late adipogenic markers in BALB/c and NIH-3T3 cells. Yet, the ectopic expression of STAT5B alone does not induce the expression of adipocyte genes, but enhances the induction of these genes in cells also expressing STAT5A. This finding suggests that STAT5A and STAT5B do not function identically in adipocytes. In addition, these studies demonstrate that the phosphorylation of STAT5 proteins may play a role in adipogenesis. Moreover, we have shown that STAT5A is associated with the glucocorticoid receptor during adipogenesis in a highly regulated manner.
Hyperactivity and Reduced Energy Cost of Physical Activity in Serotonin 5-HT2C Receptor Mutant Mice
Authors: Nonogaki Katsunori , Abdallah Luna , Goulding Evan H. , Bonasera Stephen J. , Tecott Laurence H.
Published: March 2007

We have observed late-onset obesity in mutant mice lacking the serotonin 5-HT2C receptor. Despite chronically elevated food intake, young adult mutants exhibit neither elevated adiposity nor altered glucose or fat homeostasis. However, obesity subsequently develops after 6 months of age without increases in their level of hyperphagia. In this study, we investigated determinants of energy expenditure in 5-HT2C receptor mutant mice. Young adult mutants displayed patterns of elevated activity levels that were enhanced by fasting and tightly associated with repeated visits to a food source. Surprisingly, subsequent obesity development occurred despite persisting locomotor hyperactivity and without age-related declines in resting metabolic rate. Rather, substantial reductions in the energy cost of locomotor activity (LA) were observed in 5-HT2C receptor mutant mice. Moreover, both mutant and wild-type mice displayed age-related declines in the energy cost of LA, indicating that this process may be regulated by both aging and serotonergic signaling. These results indicate that a mutation of the 5-HT2C receptor gene (htr2c) increases LA, which contributes to the maintenance of normal body composition in young adult mutants despite their hyperphagia. Moreover, age-dependent reductions in the energy cost of physical activity could contribute to the subsequent development of late-onset obesity in 5-HT2C receptor mutant mice.
BETA2 Activates Transcription From the Upstream Glucokinase Gene Promoter in Islet β-Cells and Gut Endocrine Cells
Authors: Moates J. Michael , Nanda Sarmistha , Cissell Michelle A. , Tsai Ming-Jer , Stein Roland
Published: March 2007

Glucokinase (GK) gene transcription initiates in the islet (β-cell), gut, and brain from promoter sequences residing ∼35 kbp upstream from those used in liver. Expression of βGK is controlled in β-cells by cell-enriched (i.e. pancreatic duodenal homeobox 1 [PDX-1]) and ubiquitously (i.e., Pal) distributed factors that bind to and activate from conserved sequence motifs within the upstream promoter region (termed βGK). Here, we show that a conserved E-box element also contributes to control in the islet and gut. βGK promoter-driven reporter gene activity was diminished by mutating the specific sequences involved in E-box-mediated basic helix-loop-helix factor activator binding in islet β-cells and enteroendocrine cells. Gel shift assays demonstrated that the βGK and insulin gene E-box elements formed the same cell-enriched (BETA2:E47) and generally distributed (upstream stimulatory factor [USF]) protein-DNA complexes. βGK E-box-driven activity was stimulated in cotransfection assays performed in baby hamster kidney (BHK) cells with BETA2 and E47, but not USF. Chromatin immunoprecipitation assays performed with BETA2 antisera showed that BETA2 occupies the upstream promoter region of the endogenous βGK gene in β-cells. We propose that BETA2 (also termed NeuroD1) regulates βGK promoter activity.
Diabetes Activates Cell Death Pathway After Transient Focal Cerebral Ischemia
Authors: Muranyi Marianna , Fujioka Masayuki , He QingPing , Han Angela , Yong Gregory , Csiszar Katalin , Li Ping-An
Published: March 2007

It is well known that diabetes aggravates brain damage in experimental and clinical stroke subjects. Diabetes accelerates maturation of neuronal damage, increases infarct volume, and induces postischemic seizures. The mechanism by which diabetes increases ischemic brain damage is still elusive. Our previous experiments indicate that mitochondria dysfunction may play a role in neuronal death. The objective of this study is to determine whether streptozotocin-induced diabetes activates cell death pathway after a brief period of focal cerebral ischemia. Both diabetic and nondiabetic rats were subjected to 30 min of transient middle cerebral artery occlusion, followed by 0, 0.5, 3, and 6 h of reperfusion. We first determined the pathological outcomes after 7 days of recovery by histopathology, and then detected key components of programmed cell death pathway using immunocytochemistry coupled with confocal laser-scanning microscopy and Western blot analysis. The results show that the cytosolic cytochrome c increased mildly after reperfusion in nondiabetic samples. This increase was markedly enhanced in diabetic rats in both ischemic focus and penumbra. Subsequently, caspase-3 was activated and poly-ADP ribose polymerase (PARP) was cleaved. Our results suggest that activation of apoptotic cell death pathway may play a pivotal role in exaggerating brain damage in diabetic subjects.
Glucose-Induced Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase-Dependent Upregulation of the Platelet-Derived Growth Factor-β Receptor Potentiates Vascular Smooth Muscle Cell Chemotaxis
Authors: Campbell Malcolm , Allen William E. , Silversides Jonathan A. , Trimble Elisabeth R.
Published: March 2007

The aim of this study was to investigate the effects of elevated d-glucose concentrations on vascular smooth muscle cell (VSMC) expression of the platelet-derived growth factor (PDGF)β receptor and VSMC migratory behavior. Immunoprecipitation, immunofluorescent staining, and RT-PCR of human VSMCs showed that elevated d-glucose induced an increase in the PDGFβ receptor that was inhibited by phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors. Exposure to 25 mmol/l d-glucose (HG) induced increased phosphorylation of protein kinase B (PKB) and extracellular-regulated kinase (ERK). All HG chemotaxis assays (with either 10 days’ preincubation in HG or no preincubation) in a FCS or PDGF-BB gradient showed positive chemotaxis, whereas those in 5 mmol/l d-glucose did not. Assays were also run with concentrations ranging from 5 to 25 mmol/l d-glucose. Chemotaxis was induced at concentrations ≥9 mmol/l d-glucose. An anti-PDGFβ receptor antibody inhibited glucose-potentiated VSMC chemotaxis, as did the inhibitors for the PI3K and MAPK pathways. This study has shown that small increases in d-glucose concentration, for a short period, increase VSMC expression of the PDGFβ receptor and VSMC sensitivity to chemotactic factors in serum, leading to altered migratory behavior in vitro. It is probable that similar processes occur in vivo with glucose-enhanced chemotaxis of VSMCs, operating through PDGFβ receptor-operated pathways, contributing to the accelerated formation of atheroma in diabetes.
The C-174G Promoter Polymorphism of the IL-6 Gene Affects Energy Expenditure and Insulin Sensitivity
Authors: Kubaszek Agata , Pihlajamäki Jussi , Punnonen Kari , Karhapää Pauli , Vauhkonen Ilkka , Laakso Markku
Published: March 2007

Interleukin-6 (IL-6) is a pleiotropic cytokine expressed in many tissues. IL-6 null mice show low energy expenditure, but the effect of the variants of the IL-6 gene on energy expenditure has not been previously studied in humans. Therefore, we investigated the effect of the C-174G promoter polymorphism of the IL-6 gene on energy expenditure, measured by indirect calorimetry in healthy Finnish subjects (n = 124). We also measured insulin sensitivity by the hyperinsulinemic-euglycemic clamp. Subjects with the C-174C genotype of the IL-6 gene had significantly lower energy expenditure than subjects with the G-174C or G-174G genotypes both in fasting (CC 13.68 ± 1.98, CG 14.73 ± 1.57, GG 14.81 ± 2.01 kcal · kg−1 · min−1; P = 0.012) and during the euglycemic-hyperinsulinemic clamp (CC 15.24 ± 2.05, CG 16.62 ± 2.06, GG 16.66 ± 2.50 kcal · kg−1 · min−1; P = 0.007). Moreover, subjects homozygous for the C allele had lower rates of whole-body glucose uptake than carriers of the G allele (CC 50.95 ± 13.91, CG 59.40 ± 14.17, GG 59.21 ± 15.93 μmol · kg−1 · min−1; P = 0.016). The rates of both oxidative (P = 0.013) and nonoxidative (P = 0.016) glucose disposal were significantly affected by the IL-6 promoter polymorphism. In conclusion, the C-174C promoter polymorphism of the IL-6 gene influences energy expenditure and insulin sensitivity in healthy normoglycemic subjects. Whether this polymorphism is a risk factor for obesity or type 2 diabetes can be estimated only in prospective population-based studies.
Systematic Search for Single Nucleotide Polymorphisms in the FOXC2 Gene
Authors: Osawa Haruhiko , Onuma Hiroshi , Murakami Akiko , Ochi Masaaki , Nishimiya Tatsuya , Kato Kenichi , Shimizu Ikki , Fujii Yasuhisa , Ohashi Jun , Makino Hideichi
Published: March 2007

FOXC2, a forkhead/winged helix transcription factor, represents a promising candidate gene for type 2 diabetes since transgenic mice that specifically overexpress this gene in adipocytes are lean and insulin sensitive. To determine whether there are single nucleotide polymorphisms (SNPs) in this gene that are associated with type 2 diabetes, sequences of the coding and ∼1 kb of 5′ flanking regions in 24 Japanese type 2 diabetic subjects were initially analyzed using PCR direct sequencing, and the regions containing the identified polymorphisms were then examined. In 200 control subjects, three frequent SNPs were found (g. −512C>T [32.3%] and −350G>T [13.0%] in the 5′ flanking region and +1548C>T [10.0%] in the 3′ flanking region). Linkage disequilibria were found between all three pairs of these SNPs. Of the eight possible haplotypes defined by these SNPs, only four were found. When the frequencies of these SNPs and the four common haplotypes between 195 type 2 diabetic and 200 control subjects were compared, no association was evident. The +898C>T (Pro300Ser), +907C>A (Leu303Met), 1167_1169delCCA (389delHis), and +1251C>A (Ala417Ala) identified in the coding region were rare, although +907C>A could be higher in type 2 diabetic subjects (1.5%) than in control subjects (0.3%). Thus, the SNPs identified in the FOXC2 gene are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.
Large-Scale Association Studies of Variants in Genes Encoding the Pancreatic β-Cell KATP Channel Subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) Confirm That the KCNJ11 E23K Variant Is Associated With Type 2 Diabetes
Authors: Gloyn Anna L. , Weedon Michael N. , Owen Katharine R. , Turner Martina J. , Knight Bridget A. , Hitman Graham , Walker Mark , Levy Jonathan C. , Sampson Mike , Halford Stephanie , McCarthy Mark I. , Hattersley Andrew T. , Frayling Timothy M.
Published: March 2007

The genes ABCC8 and KCNJ11, which encode the subunits sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel (Kir6.2) of the β-cell ATP-sensitive potassium (KATP) channel, control insulin secretion. Common polymorphisms in these genes (ABCC8 exon 16–3t/c, exon 18 T/C, KCNJ11 E23K) have been variably associated with type 2 diabetes, but no large (∼2,000 subjects) case-control studies have been performed. We evaluated the role of these three variants by studying 2,486 U.K. subjects: 854 with type 2 diabetes, 1,182 population control subjects, and 150 parent-offspring type 2 diabetic trios. The E23K allele was associated with diabetes in the case-control study (odds ratio [OR] 1.18 [95% CI 1.04–1.34], P = 0.01) but did not show familial association with diabetes. Neither the exon 16 nor the exon 18 ABCC8 variants were associated with diabetes (1.04 [0.91–1.18], P = 0.57; 0.93 [0.71–1.23], P = 0.63, respectively). Meta-analysis of all case-control data showed that the E23K allele was associated with type 2 diabetes (K allele OR 1.23 [1.12–1.36], P = 0.000015; KK genotype 1.65 [1.34–2.02], P = 0.000002); but the ABCC8 variants were not associated. Our results confirm that E23K increases risk of type 2 diabetes and show that large-scale association studies are important for the identification of diabetes susceptibility alleles.
Acute Hyperglycemia Causes Intracellular Formation of CML and Activation of ras, p42/44 MAPK, and Nuclear Factor κB in PBMCs
Authors: Schiekofer Stephan , Andrassy Martin , Chen Jiang , Rudofsky Gottfried , Schneider Jochen , Wendt Thoralf , Stefan Norbert , Humpert Per , Fritsche Andreas , Stumvoll Michael , Schleicher Erwin , Häring Hans-Ulrich , Nawroth Peter P. , Bierhaus Angelika
Published: March 2007

Twenty-three nondiabetic volunteers were divided into three groups. In group A (n = 9), the glucose infusion was adjusted to maintain blood glucose at 5 mmol/l (euglycemic clamp). In group B (n = 9), the glucose infusion was adjusted to maintain blood glucose at 10 mmol/l (hyperglycemic clamp) over 2 h. Group C consisted of five volunteers who were studied as the control group. Peripheral blood mononuclear cells (PBMCs) were isolated before and at the end of a 2-h clamp. In group C, PBMCs were isolated before and after 2 h without performing a clamp. The euglycemic clamp as well as “no clamp” had no effects on all parameters studied. In contrast, a significant increase in carboxymethyllysine (CML) content and p21ras and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation was observed at the end of a 2-h hyperglycemic clamp. The nuclear factor (NF)-κB (but not Oct-1) binding activity increased significantly in the hyperglycemic clamp. Western blots confirmed NF-κB-p65-antigen translocation into the nucleus. IκBα did not change significantly in both groups. Hyperglycemia-mediated NF-κB activation and increase of CML content, p21ras, and p42/44 MAPK phosphorylation was also seen in ex vivo–isolated PBMCs stimulated with 5 or 10 mmol/l glucose. Addition of insulin did not influence the results. Inhibition of activation of ras, MAPK, or protein kinase C blocked hyperglycemia-mediated NF-κB activation in ex vivo–isolated PBMCs stimulated with 10 mmol/l glucose. Similar data were obtained using an NF-κB-luciferase reporter plasmid. Therefore, we can conclude that an acute hyperglycemia-mediated mononuclear cell activation is dependent on activation of ras, p42/p44 MAPK phosphorylation, and subsequent NF-κB activation and results in transcriptional activity in PBMCs.
Gene Expression Profiles of Nondiabetic and Diabetic Obese Mice Suggest a Role of Hepatic Lipogenic Capacity in Diabetes Susceptibility
Authors: Lan Hong , Rabaglia Mary E. , Stoehr Jonathan P. , Nadler Samuel T. , Schueler Kathryn L. , Zou Fei , Yandell Brian S. , Attie Alan D.
Published: March 2007

Obesity is a strong risk factor for the development of type 2 diabetes. We have previously reported that in adipose tissue of obese (ob/ob) mice, the expression of adipogenic genes is decreased. When made genetically obese, the BTBR mouse strain is diabetes susceptible and the C57BL/6J (B6) strain is diabetes resistant. We used DNA microarrays and RT-PCR to compare the gene expression in BTBR-ob/ob versus B6-ob/ob mice in adipose tissue, liver, skeletal muscle, and pancreatic islets. Our results show: 1) there is an increased expression of genes involved in inflammation in adipose tissue of diabetic mice; 2) lipogenic gene expression was lower in adipose tissue of diabetes-susceptible mice, and it continued to decrease with the development of diabetes, compared with diabetes-resistant obese mice; 3) hepatic expression of lipogenic enzymes was increased and the hepatic triglyceride content was greatly elevated in diabetes-resistant obese mice; 4) hepatic expression of gluconeogenic genes was suppressed at the prediabetic stage but not at the onset of diabetes; and 5) genes normally not expressed in skeletal muscle and pancreatic islets were expressed in these tissues in the diabetic mice. We propose that increased hepatic lipogenic capacity protects the B6-ob/ob mice from the development of type 2 diabetes.
Monounsaturated Fatty Acids Prevent the Deleterious Effects of Palmitate and High Glucose on Human Pancreatic β-Cell Turnover and Function
Authors: Maedler Kathrin , Oberholzer José , Bucher Pascal , Spinas Giatgen A. , Donath Marc Y.
Published: March 2007

Glucotoxicity and lipotoxicity contribute to the impaired β-cell function observed in type 2 diabetes. Here we examine the effect of saturated and monounsaturated fatty acids at different glucose concentrations on human β-cell turnover and secretory function. Exposure of cultured human islets to saturated fatty acid and/or to an elevated glucose concentration for 4 days increased β-cell DNA fragmentation and decreased β-cell proliferation. In contrast, the monounsaturated palmitoleic acid or oleic acid did not affect DNA fragmentation and induced β-cell proliferation. Moreover, each monounsaturated fatty acid prevented the deleterious effects of both palmitic acid and high glucose concentration. The cell-permeable ceramide analogue C2-ceramide mimicked both the palmitic acid-induced β-cell apoptosis and decrease in proliferation. Furthermore, the ceramide synthetase inhibitor fumonisin B1 blocked the deleterious effects of palmitic acid on β-cell turnover. In addition, palmitic acid decreased Bcl-2 expression and induced release of cytochrome c from the mitochondria into the cytosol, which was prevented by fumonisin B1 and by oleic acid. Finally, each monounsaturated fatty acid improved β-cell secretory function that was reduced by palmitic acid and by high glucose. Thus, in human islets, the saturated palmitic acid and elevated glucose concentration induce β-cell apoptosis, decrease β-cell proliferation, and impair β-cell function, which can be prevented by monounsaturated fatty acids. The deleterious effect of palmitic acid is mediated via formation of ceramide and activation of the apoptotic mitochondrial pathway, whereas Bcl-2 may contribute to the protective effect of monounsaturated fatty acids.
The Insulin Secretory Granule Is the Major Site of KATP Channels of the Endocrine Pancreas
Authors: Geng Xuehui , Li Lehong , Watkins Simon , Robbins Paul D. , Drain Peter
Published: March 2007

With ATP sites on Kir6.2 that inhibit activity and ADP sites on SUR1 that antagonize the inhibition, ATP-sensitive potassium channels (KATP channels) are designed as exquisite sensors of adenine nucleotide levels that signal changes in glucose metabolism. If pancreatic KATP channels localize to the insulin secretory granule, they would be well positioned to transduce changes in glucose metabolism into changes in granule transport and exocytosis. Tests for pancreatic KATP channels localized to insulin secretory granules led to the following observations: fluorescent sulfonylureas that bind the pancreatic KATP channel specifically label intracellular punctate structures in cells of the endocrine pancreas. The fluorescent glibenclamides colocalize with Ins-C-GFP, a live-cell fluorescent reporter of insulin granules. Expression of either SUR1-GFP or Kir6.2-GFP fusion proteins, but not expression of GFP alone, directs GFP fluorescence to insulin secretory granules. An SUR1 antibody specifically labels insulin granules identified by anti-insulin. Two different Kir6.2 antibodies specifically label insulin secretory granules identified by anti-insulin. Immunoelectron microscopy showed Kir6.2 antibodies specifically label perimeter membrane regions of the secretory granule. Relatively little or no labeling of other structures, including the plasma membrane, was found. Our results demonstrate that the insulin secretory granule is the major site of KATP channels of the endocrine pancreas.
The Focal Form of Persistent Hyperinsulinemic Hypoglycemia of Infancy
Authors: Sempoux Christine , Guiot Yves , Dahan Karin , Moulin Pierre , Stevens Martine , Lambot Virginie , Lonlay Pascale de , Fournet Jean-Christophe , Junien Claudine , Jaubert Francis , Nihoul-Fekete Claire , Saudubray Jean-Marie , Rahier Jacques
Published: March 2007

Paternal mutation of ATP-sensitive K+ (KATP) channel genes and loss of heterozygosity (LOH) of the 11p15 region including the maternal alleles of ABCC8, IGF2, and CDKN1C characterize the focal form of persistent hyperinsulinemic hypoglycemia of infancy (FoPHHI). We aimed to understand the actual nature of FoPHHI in comparison with insulinoma. In FoPHHI, the lesion consists in clusters of β-cells surrounded by non–β-cells. Compared with adjacent islets, proinsulin mRNA is similar and proinsulin production higher (P ≤ 0.02), indicating regulation at a translational level, with slightly lower insulin stock and lower ABCC8 peptide labeling (P<0.05). Insulinomas, composed of β-cell nests or cords, have similar proinsulin mRNA compared with adjacent islets, highly variable proinsulin production, lower insulin stock (P ≤ 0.02), and higher ABCC8 peptide labeling (P<0.05). Proinsulin mRNA is lower than in FoPHHI (P<0.001). Islets adjacent to FoPHHI appear to be resting, in contrast to those adjacent to insulinomas, evidencing intrapancreatic regulation of islet β-cell activity. IGF2 peptide is present inside and outside both lesions, but IGF2 mRNA is restricted to the lesions. The 11p15 LOH and absence of CDKN1C peptide staining are demonstrated in all FoPHHI but also in three of eight insulinomas. Despite some molecular similarities, FoPHHI is thus fundamentally different from insulinoma.
The Gly972→Arg IRS-1 Variant Is Associated With Type 1 Diabetes in Continental Italy
Authors: Federici Massimo , Petrone Antonio , Porzio Ottavia , Bizzarri Carla , Lauro Davide , D’Alfonso Rossella , Patera Ippolita , Cappa Marco , Nisticò Lorenza , Baroni Marco , Sesti Giorgio , di Mario Umberto , Lauro Renato , Buzzetti Raffaella
Published: March 2007

The Arg972 insulin receptor substrate-1 (IRS-1) variant has been hypothesized to play a role in pancreatic β-cell stimulus-coupled insulin secretion and survival. We analyzed the relations between type 1 diabetes and the Arg972 IRS-1 variant. The frequency of the IRS-1 Arg972 variant was investigated in two independent sets of unrelated patients: a case-control study and a collection of type 1 diabetes simplex families. In the former group, frequency of the IRS-1 Arg972 variant was significantly increased in the patients (P = 0.0008), conferring an OR of 2.5. Transmission disequilibrium analysis of data obtained from the family set revealed that the Arg972 IRS-1 variant was transmitted from heterozygous parents to affected probands at a frequency of 70.2% (P < 0.02). Arg972 IRS-1 frequency showed no significant correlation with HLA genotypic risk for type 1 diabetes. Arg972 IRS-1 type 1 diabetic patients also had lower fasting plasma concentrations of C-peptide at the time of diagnosis with respect to patients carrying the wild-type IRS-1 (0.49 ± 0.058, n = 34, and 0.76 ± 0.066, n = 134, respectively [means ± SE]; P = 0.051). Our findings suggest a role for Arg972 IRS-1 in conferring risk for the development of type 1 diabetes.
A Gly482Ser Missense Mutation in the Peroxisome Proliferator-Activated Receptor γ Coactivator-1 Is Associated With Altered Lipid Oxidation and Early Insulin Secretion in Pima Indians
Authors: Muller Yunhua Li , Bogardus Clifton , Pedersen Oluf , Baier Leslie
Published: March 2007

Peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) is a transcriptional coactivator of peroxisome proliferator-activated receptor γ and α, which play important roles in adipogenesis and lipid metabolism. A single nucleotide polymorphism within the coding region of the PGC-1 gene predicts a glycine to serine substitution at amino acid 482 and has been associated with type 2 diabetes in a Danish population. In this study, we examined whether this Gly482Ser polymorphism is associated with type 2 diabetes or obesity, or metabolic predictors of these diseases, in Pima Indians. There was no association of the Gly482Ser polymorphism with either type 2 diabetes or BMI (n = 984). However, among nondiabetic Pima Indians (n = 183–201), those with the Gly/Gly genotype had a lower mean insulin secretory response to intravenous and oral glucose and a lower mean rate of lipid oxidation (over 24 h in a respiratory chamber) despite a larger mean subcutaneous abdominal adipocyte size and a higher mean plasma free fatty acid concentration. These data indicate that the Gly482Ser missense polymorphism in PGC-1 has metabolic consequences on lipid metabolism that could influence insulin secretion.
Mechanisms for the Deterioration in Glucose Tolerance Associated With HIV Protease Inhibitor Regimens
Authors: Woerle Hans J. , Mariuz Peter R. , Meyer Christian , Reichman Richard C. , Popa Emilia M. , Dostou Jean M. , Welle Stephen L. , Gerich John E.
Published: March 2007

The mechanisms responsible for the deterioration in glucose tolerance associated with protease inhibitor–containing regimens in HIV infection are unclear. Insulin resistance has been implicated as a major factor, but the affected tissues have not been identified. Furthermore, β-cell function has not been evaluated in detail. The present study was therefore undertaken to assess the effects of protease inhibitor–containing regimens on hepatic, muscle, and adipose tissue insulin sensitivity as well as pancreatic β-cell function. We evaluated β-cell function in addition to glucose production, glucose disposal, and free fatty acid (FFA) turnover using the hyperglycemic clamp technique in combination with isotopic measurements in 13 HIV-infected patients before and after 12 weeks of treatment and in 14 normal healthy volunteers. β-Cell function and insulin sensitivity were also assessed by homeostasis model assessment (HOMA). Treatment increased fasting plasma glucose concentrations in all subjects (P < 0.001). Insulin sensitivity as assessed by HOMA and clamp experiments decreased by ∼50% (P < 0.003). Postabsorptive glucose production was appropriately suppressed for the prevailing hyperinsulinemia, whereas glucose clearance was reduced (P < 0.001). β-Cell function decreased by ∼50% (P = 0.002), as assessed by HOMA, and first-phase insulin release decreased by ∼25%, as assessed by clamp data (P = 0.002). Plasma FFA turnover and clearance both increased significantly (P < 0.001). No differences at baseline or in responses after treatment were observed between drug naïve patients who were started on a nucleoside reverse transcriptase inhibitor (NRTI) plus a protease inhibitor and patients who had been on long-term NRTI treatment and had a protease inhibitor added. The present study indicates that protease inhibitor–containing regimens impair glucose tolerance in HIV-infected patients by two mechanisms: 1) inducement of peripheral insulin resistance in skeletal muscle and adipose tissue and 2) impairment of the ability of the β-cell to compensate.
Elevation in Tanis Expression Alters Glucose Metabolism and Insulin Sensitivity in H4IIE Cells
Authors: Gao Yuan , Walder Ken , Sunderland Terry , Kantham Lakshmi , Feng Helen C. , Quick Melissa , Bishara Natalie , de Silva Andrea , Augert Guy , Tenne-Brown Janette , Collier Gregory R.
Published: March 2007

Increased hepatic glucose output and decreased glucose utilization are implicated in the development of type 2 diabetes. We previously reported that the expression of a novel gene, Tanis, was upregulated in the liver during fasting in the obese/diabetic animal model Psammomys obesus. Here, we have further studied the protein and its function. Cell fractionation indicated that Tanis was localized in the plasma membrane and microsomes but not in the nucleus, mitochondria, or soluble protein fraction. Consistent with previous gene expression data, hepatic Tanis protein levels increased more significantly in diabetic P. obesus than in nondiabetic controls after fasting. We used a recombinant adenovirus to increase Tanis expression in hepatoma H4IIE cells and investigated its role in metabolism. Tanis overexpression reduced glucose uptake, basal and insulin-stimulated glycogen synthesis, and glycogen content and attenuated the suppression of PEPCK gene expression by insulin, but it did not affect insulin-stimulated insulin receptor phosphorylation or triglyceride synthesis. These results suggest that Tanis may be involved in the regulation of glucose metabolism, and increased expression of Tanis could contribute to insulin resistance in the liver.
Association Between Adiponectin and Mediators of Inflammation in Obese Women
Authors: Engeli Stefan , Feldpausch Mareike , Gorzelniak Kerstin , Hartwig Frauke , Heintze Ute , Janke Jürgen , Möhlig Matthias , Pfeiffer Andreas F.H. , Luft Friedrich C. , Sharma Arya M.
Published: March 2007

Low plasma levels of the anti-inflammatory factor adiponectin characterize obesity and insulin resistance. To elucidate the relationship between plasma levels of adiponectin, adiponectin gene expression in adipose tissue, and markers of inflammation, we obtained blood samples, anthropometric measures, and subcutaneous adipose tissue samples from 65 postmenopausal healthy women. Adiponectin plasma levels and adipose-tissue gene expression were significantly lower in obese subjects and inversely correlated with obesity-associated variables, including high-sensitive C-reactive protein (hs-CRP) and interleukin-6 (IL-6). Despite adjustment for obesity-associated variables, plasma levels of adiponectin were significantly correlated to adiponectin gene expression (partial r = 0.38, P < 0.05). Furthermore, the inverse correlation between plasma levels of hs-CRP and plasma adiponectin remained significant despite correction for obesity-associated variables (partial r = −0.32, P < 0.05), whereas the inverse correlation between adiponectin plasma levels or adiponectin gene expression in adipose tissue with plasma IL-6 were largely dependent on the clustering of obesity-associated variables. In conclusion, our data suggest a transcriptional mechanism leading to decreased adiponectin plasma levels in obese women and demonstrate that low levels of adiponectin are associated with higher levels of hs-CRP and IL-6, two inflammatory mediators and markers of increased cardiovascular risk.
Programming of Islet Functions in the Progeny of Hyperinsulinemic/Obese Rats
Authors: Srinivasan Malathi , Aalinkeel Ravikumar , Song Fei , Patel Mulchand S.
Published: March 2007

Neonatal female rat pups that were raised artificially on a high-carbohydrate (HC) milk formula during their suckling period developed hyperinsulinemia immediately, maintained chronic hyperinsulinemia in the postweaning period on laboratory diet, and developed obesity in adulthood. Pups (second-generation HC [2-HC]) born to such female rats (first-generation HC [1-HC]) spontaneously developed chronic hyperinsulinemia and adult-onset obesity (HC phenotype) without the requirement for any dietary intervention in their suckling period. Leftward shift in the insulin secretory response to a glucose stimulus, increase in hexokinase activity, and increased preproinsulin gene transcription were observed in islets from 28-day-old 2-HC rats, and these adaptations are similar to those reported for islets from 12-day-old and 100-day-old 1-HC rats. Unlike 1-HC islets, the ability to secrete moderate amounts of insulin in the absence of glucose and calcium and the incretin input for augmentation of insulin secretion were not observed in 2-HC islets. These results show that a dietary modification in the early postnatal life of the 1-HC female rat sets up a vicious cycle of spontaneous transfer of the HC phenotype to its progeny, implicating a new component to the growing list of factors that contribute to the fetal origins of adult-onset diseases.
Insulin Does Not Mediate Glucose Stimulation of Proinsulin Biosynthesis
Authors: Leibowitz Gil , Oprescu Andrei I. , Üçkaya Gökhan , Gross David J. , Cerasi Erol , Kaiser Nurit
Published: March 2007

It has recently been suggested that insulin augments its own production by a physiologically important feed-forward autocrine loop. We studied the kinetics of glucose-regulated proinsulin gene expression and proinsulin biosynthesis in normal rat islets with emphasis on the potential role of insulin as a mediator of the glucose effect. There was a time-dependent increase in steady-state proinsulin mRNA in islets cultured at 16.7 mmol/l compared with 3.3 mmol/l glucose; no early (1–3 h) increase in proinsulin gene expression was observed. In contrast, there was a threefold increase in proinsulin biosynthesis within 1 h of glucose stimulation that was not affected by inhibition of glucose-stimulated proinsulin gene transcription with actinomycin D. In addition, inhibition of glucose-stimulated insulin secretion with diazoxide had no effect on glucose-stimulated proinsulin mRNA or biosynthesis. Furthermore, addition of different concentrations of insulin to islets cultured in low glucose failed to affect proinsulin biosynthesis. Taken together, our data suggest that the early glucose-dependent increase in proinsulin biosynthesis is mainly regulated at the translational level, rather than by changes in proinsulin gene expression. Moreover, we could not demonstrate any effect of insulin on islet proinsulin mRNA level or rate of proinsulin biosynthesis. Thus, if insulin has any effect on the proinsulin biosynthetic apparatus, it is a minor one. We conclude that the secreted insulin is not an important mediator of insulin production in response to glucose.
Hepatic VLDL Production in ob/ob Mice Is Not Stimulated by Massive De Novo Lipogenesis but Is Less Sensitive to the Suppressive Effects of Insulin
Authors: Wiegman Coen H. , Bandsma Robert H.J. , Ouwens Margriet , van der Sluijs Fjodor H. , Havinga Rick , Boer Theo , Reijngoud Dirk-Jan , Romijn Johannes A. , Kuipers Folkert
Published: March 2007

Type 2 diabetes in humans is associated with increased de novo lipogenesis (DNL), increased fatty acid (FA) fluxes, decreased FA oxidation, and hepatic steatosis. In this condition, VLDL production is increased and resistant to suppressive effects of insulin. The relationships between hepatic FA metabolism, steatosis, and VLDL production are incompletely understood. We investigated VLDL-triglyceride and -apolipoprotein (apo)-B production in relation to DNL and insulin sensitivity in female ob/ob mice. Hepatic triglyceride (5-fold) and cholesteryl ester (15-fold) contents were increased in ob/ob mice compared with lean controls. Hepatic DNL was increased ∼10-fold in ob/ob mice, whereas hepatic cholesterol synthesis was not affected. Basal rates of hepatic VLDL-triglyceride and -apoB100 production were similar between the groups. Hyperinsulinemic clamping reduced VLDL-triglyceride and -apoB100 production rates by ∼60% and ∼75%, respectively, in lean mice but only by ∼20% and ∼20%, respectively, in ob/ob mice. No differences in hepatic expression of genes encoding apoB and microsomal triglyceride transfer protein were found. Hepatic expression and protein phosphorylation of insulin receptor and insulin receptor substrate isoforms were reduced in ob/ob mice. Thus, strongly induced hepatic DNL is not associated with increased VLDL production in ob/ob mice, possibly related to differential hepatic zonation of apoB synthesis (periportal) and lipid accumulation (perivenous) and/or relatively low rates of cholesterogenesis. Insulin is unable to effectively suppress VLDL-triglyceride production in ob/ob mice, presumably because of impaired insulin signaling.
Successful Human Islet Isolation Utilizing Recombinant Collagenase
Authors: Brandhorst Heide , Brandhorst Daniel , Hesse Friederike , Ambrosius Dorothee , Brendel Mathias , Kawakami Yoshiyuki , Bretzel Reinhard G.
Published: March 2007

The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 ± 0.8 vs. 85.6 ± 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.
Overexpression of PACAP in Transgenic Mouse Pancreatic β-Cells Enhances Insulin Secretion and Ameliorates Streptozotocin-induced Diabetes
Authors: Yamamoto Kyohei , Hashimoto Hitoshi , Tomimoto Shuhei , Shintani Norihito , Miyazaki Jun-ichi , Tashiro Fumi , Aihara Hiroyuki , Nammo Takao , Li Ming , Yamagata Kazuya , Miyagawa Jun-ichiro , Matsuzawa Yuji , Kawabata Yuki , Fukuyama Yuji , Koga Kazumi , Mori Wakaba , Tanaka Kazuhiro , Matsuda Toshio , Baba Akemichi
Published: March 2007

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal peptide/secretin/glucagon family, stimulates insulin secretion from islets in a glucose-dependent manner at femtomolar concentrations. To assess PACAP’s pancreatic function in vivo, we generated transgenic mice overexpressing PACAP in the pancreas under the control of human insulin promoter. Northern blot and immunohistochemical analyses showed that PACAP is overexpressed in pancreatic islets, specifically in transgenic mice. Plasma glucose and glucagon levels during a glucose tolerance test were not different between PACAP transgenic mice and nontransgenic littermates. However, plasma insulin levels in transgenic mice were higher after glucose loading. Also, increases of streptozotocin-induced plasma glucose were attenuated in transgenic compared with nontransgenic mice. Notably, an increase in 5-bromo-2-deoxyuridine-positive β-cells in the streptozotocin-treated transgenic mice was observed but without differences in the staining patterns by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Morphometric analysis revealed that total islet mass tends to increase in 12-month-old transgenic mice but showed no difference between 12-week-old transgenic and nontransgenic littermates. This is the first time that PACAP has been observed to play an important role in the proliferation of β-cells.
Sleep-Related Hypoglycemia-Associated Autonomic Failure in Type 1 Diabetes
Authors: Banarer Salomon , Cryer Philip E.
Published: March 2007

Given that iatrogenic hypoglycemia often occurs during the night in people with type 1 diabetes, we tested the hypothesis that physiological, and the resulting behavioral, defenses against developing hypoglycemia—already compromised by absent glucagon and attenuated epinephrine and neurogenic symptom responses—are further compromised during sleep in type 1 diabetes. To do so, we studied eight adult patients with uncomplicated type 1 diabetes and eight matched nondiabetic control subjects with hyperinsulinemic stepped hypoglycemic clamps (glucose steps of ∼85, 75, 65, 55, and 45 mg/dl) in the morning (0730–1230) while awake and at night (2100–0200) while awake throughout and while asleep from 0000 to 0200 in random sequence. Plasma epinephrine (P = 0.0010), perhaps norepinephrine (P = 0.0838), and pancreatic polypeptide (P = 0.0034) responses to hypoglycemia were reduced during sleep in diabetic subjects (the final awake versus asleep values were 240 ± 86 and 85 ± 47, 205 ± 24 and 148 ± 17, and 197 ± 45 and 118 ± 31 pg/ml, respectively), but not in the control subjects. The diabetic subjects exhibited markedly reduced awakening from sleep during hypoglycemia. Sleep efficiency (percent time asleep) was 77 ± 18% in the diabetic subjects, but only 26 ± 8% (P = 0.0109) in the control subjects late in the 45-mg/dl hypoglycemic steps. We conclude that autonomic responses to hypoglycemia are reduced during sleep in type 1 diabetes, and that, probably because of their reduced sympathoadrenal responses, patients with type 1 diabetes are substantially less likely to be awakened by hypoglycemia. Thus both physiological and behavioral defenses are further compromised during sleep. This sleep-related hypoglycemia-associated autonomic failure, in the context of imperfect insulin replacement, likely explains the high frequency of nocturnal hypoglycemia in type 1 diabetes.