bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Glycogen Synthase by Glucose and Glycogen
Authors: Halse Reza , Fryer Lee G.D. , McCormack James G. , Carling David , Yeaman Stephen J.
Published: March 2007

We report here use of human myoblasts in culture to study the relationships between cellular glycogen concentrations and the activities of glycogen synthase (GS) and AMP-activated protein kinase (AMPK). Incubation of cells for 2 h in the absence of glucose led to a 25% decrease in glycogen content and a significant decrease in the fractional activity of GS. This was accompanied by stimulation of both the α1 and α2 isoforms of AMPK, without significant alterations in the ratios of adenine nucleotides. When glucose was added to glycogen-depleted cells, a rapid and substantial increase in GS activity was accompanied by inactivation of AMPK back to basal values. Inclusion of the glycogen phosphorylase inhibitor, CP-91149, prevented the loss of glycogen during glucose deprivation but not the activation of AMPK. However, in the absence of prior glycogen breakdown, glucose treatment failed to activate GS above control values, indicating the crucial role of glycogen content. Activation of AMPK by either 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) or hydrogen peroxide was also associated with a decrease in the activity ratio of GS. AICAR treatment had no effect on total cellular glycogen content but led to a modest increase in glucose uptake. These data support a role for AMPK in both stimulating glucose uptake and inhibiting GS in intact cells, thus promoting glucose flux through glycolysis.
Measurement of Fractional Whole-Body Gluconeogenesis in Humans From Blood Samples Using 2H Nuclear Magnetic Resonance Spectroscopy
Authors: Kunert O. , Stingl H. , Rosian E. , Krššák M. , Bernroider E. , Seebacher W. , Zangger K. , Staehr P. , Chandramouli V. , Landau B.R. , Nowotny P. , Waldhäusl W. , Haslinger E. , Roden M.
Published: March 2007

Several problems limit quantification of gluconeogenesis. We applied in vitro 2H-nuclear magnetic resonance (NMR) spectroscopy to simultaneously measure 2H in all glucose carbons for direct assessment of gluconeogenesis. This method was compared with 2H measurement in carbons 5 and 2 using gas chromatography–mass spectrometry (hexamethylenetetramine [HMT]) and with in vivo 13C magnetic resonance spectroscopy (MRS). After 14 h of fasting, and following 2H2O ingestion, blood was obtained from nine healthy and seven type 2 diabetic subjects. Glucose was purified, acetylated, and analyzed for 2H in carbons 1–6 with 2H-NMR. Using 5:2 ratios, gluconeogenesis increased (P < 0.05) over time and mean gluconeogenesis was lower in control subjects than in type 2 diabetic patients (63 ± 3 vs. 75 ± 2%, P < 0.01). 13C-MRS revealed higher hepatic glycogenolysis in control subjects (3.9 ± 0.4 vs. 2.3 ± 0.2 μmol · kg−1 · min−1) yielding mean contribution of gluconeogenesis of 65 ± 3 and 77 ± 2% (P < 0.005). Measurement of gluconeogenesis by 2H-NMR correlated linearly with 13C-MRS (r = 0.758, P = 0.0007) and HMT (r = 0.759, P = 0.0007). In an additional protocol, 2H enrichments demonstrated a fast decline of gluconeogenesis from ∼100 to ∼68% (P < 0.02) within 4 h of galactose infusion after 40–44 h of fasting. Thus, in vitro 2H-NMR offers an alternative approach to determine fractional gluconeogenesis in good agreement with standard methods and allows monitoring of rapid metabolic alterations.
Long-Term Improvement in Insulin Sensitivity by Changing Lifestyles of People with Impaired Glucose Tolerance
Authors: Uusitupa Matti , Lindi Virpi , Louheranta Anne , Salopuro Titta , Lindström Jaana , Tuomilehto Jaakko ,   for the Finnish Diabetes Prevention Study Group
Published: March 2007

Lifestyle interventions reduce the incidence of type 2 diabetes among individuals with impaired glucose tolerance (IGT). However, it is unknown whether this is due to improved insulin sensitivity or insulin secretion. We investigated at baseline insulin sensitivity and insulin secretion applying frequently sampled intravenous glucose tolerance test (FSIGT) in 87 of 101 obese middle-aged subjects with IGT randomized into an intervention or a control group in the Finnish Diabetes Prevention Study. FSIGT was repeated after 4 years in 52 people. There were no significant differences in any of the baseline anthropometric or metabolic characteristics between the groups. The 4-year weight and waist circumference decreases were greater in the intervention than in the control group (P = 0.043 and P = 0.025, respectively). At 4-year examination, insulin sensitivity (Si) tended to be higher in the intervention group (the difference between the mean values 36%; P = 0.067, and P = 0.136 after adjustment for age, sex, BMI, and baseline Si value). There was strong correlation between the 4-year changes in Si and weight (r = −0.628 and r = −0.710, for intervention and control groups; P < 0.001 for both). In the entire group, Si improved by 64% in the highest tertile of weight loss but deteriorated by 24% in those who gained weight (lowest tertile). Acute insulin response declined significantly in the control group. In conclusion, Si markedly improved by weight reduction during the 4-year follow-up of individuals with IGT. Insulin secretion remained constant for years in individuals with IGT who were able to lose weight.
Cellular Aspects of Intestinal Lipoprotein Assembly in Psammomys Obesus
Authors: Zoltowska Monika , Ziv Ehud , Delvin Edgard , Sinnett Daniel , Kalman Ronny , Garofalo Carole , Seidman Ernest , Levy Emile
Published: March 2007

Although postprandial hypertriglyceridemia is a major contributing factor in the development of atherosclerosis, little information is available on the effect of insulin resistance and diabetes on intestinal fat transport. The aim of the present study was to examine intracellular events that govern lipid transport and apolipoprotein (apo) B-48-containing lipoprotein assembly in the small intestine of Psammomys obesus, a model of nutritionally induced insulin resistance and type 2 diabetes. Animals with normoglycemia/hyperinsulinemia and hyperglycemia/hyperinsulinemia exhibited high levels of triglycerides (TGs) in the plasma and intestine and postprandial plasma chylomicrons and apo B-48 compared with normoglycemic/normoinsulinemic animals. In vitro studies, using cultured jejunal explants incubated with either [14C]oleic acid or [35S]methionine, revealed their higher efficiency in de novo TG synthesis, apo B-48 biogenesis, and TG-rich lipoprotein assembly. Accordingly, enhanced monoacylglycerol and diacylglycerol acyltransferase activity was also discernible and concomitant with an increased content of L-fatty acid binding protein and in vivo chylomicron production rates. However, both the I-fatty acid binding protein amount and the apo B-48 proteasomal degradative pathway were decreased. Overall, our findings show that the development of an insulin-resistant/diabetic state in Psammomys obesus triggers the whole intra-enterocyte machinery, leading to lipoprotein assembly and favoring the intestinal oversecretion of apo B-48-lipoproteins, which may contribute to characteristic hypertriglyceridemia.
Reactive Oxygen Species from Mitochondria Induce Cyclooxygenase-2 Gene Expression in Human Mesangial Cells
Authors: Kiritoshi Shinsuke , Nishikawa Takeshi , Sonoda Kazuhiro , Kukidome Daisuke , Senokuchi Takahumi , Matsuo Tomoko , Matsumura Takeshi , Tokunaga Hiroshi , Brownlee Michael , Araki Eiichi
Published: March 2007

Hyperglycemia increases the production of reactive oxygen species (ROS) from the mitochondrial electron transport chain in bovine endothelial cells. Because several studies have postulated a role for prostaglandins (PGs) in the glomerular hyperfiltration seen in early diabetes, we evaluated the effect of mitochondrial ROS on expression of the inducible isoform of cyclooxygenase (COX-2) in cultured human mesangial cells (HMCs). We first confirmed that incubation of HMC with 30 mmol/l glucose significantly increased COX-2 mRNA but not COX-1 mRNA, compared with 5.6 mmol/l glucose. Similarly, incubation of HMCs with 30 mmol/l glucose significantly increased mitochondrial membrane potential, intracellular ROS production, COX-2 protein expression, and PGE2 synthesis, and these events were completely suppressed by thenoyltrifluoroacetone or carbonyl cyanide m-chlorophenylhydrazone, inhibitors of mitochondrial metabolism, or by overexpression of uncoupling protein-1 or manganese superoxide dismutase. Furthermore, increased expression of COX-2 mRNA and protein was confirmed in glomeruli of streptozotocin-induced diabetic mice. In addition, hyperglycemia induced activation of the COX-2 gene promoter, which was completely abrogated by mutation of two nuclear factor κB (NF-κB) binding sites in the promoter region. Our results suggest that hyperglycemia increases mitochondrial ROS production, resulting in NF-κB activation, COX-2 mRNA induction, COX-2 protein production, and PGE2 synthesis. This chain of events might contribute to the pathogenesis of diabetic nephropathy.
Translocation of Glomerular p47phox and p67phox by Protein Kinase C-β Activation Is Required for Oxidative Stress in Diabetic Nephropathy
Authors: Kitada Munehiro , Koya Daisuke , Sugimoto Toshiro , Isono Motohide , Araki Shin-ichi , Kashiwagi Atsunori , Haneda Masakazu
Published: March 2007

Oxidative stress is implicated to play an important role in the development of diabetic vascular complications, including diabetic nephropathy. It is unclear whether oxidative stress is primarily enhanced in the diabetic glomeruli or whether it is merely a consequence of diabetes-induced glomerular injury. To address this issue, we examined diabetic glomeruli to determine whether oxidative stress is enhanced, as well as examined the role of protein kinase C (PKC)-β activation in modulating NADPH oxidase activity. Urinary 8-hydroxydeoxyguanosine excretion and its intense immune-reactive staining in the glomeruli were markedly higher in diabetic than in control rats, and these alterations were ameliorated by a treatment with a selective PKC-β inhibitor, ruboxistaurin (RBX; LY333531) mesylate, without affecting glycemia. NADPH oxidase activity, which was significantly enhanced in diabetic glomeruli and the source of reactive oxygen species (ROS) generation, was also improved by RBX treatment by preventing the membranous translocation of p47phox and p67phox from cytoplasmic fraction without affecting their protein levels. Adenoviral-mediated PKC-β2 overexpression enhanced ROS generation by modulating the membranous translocation of p47phox and p67phox in cultured mesangial cells. We now demonstrate that oxidative stress is primarily enhanced in the diabetic glomeruli due to a PKC-β-dependent activation of NADPH oxidase resulting in ROS generation.
Kir6.2 Polymorphisms Sensitize β-Cell ATP-Sensitive Potassium Channels to Activation by Acyl CoAs
Authors: Riedel Michael J. , Boora Parveen , Steckley Diana , de Vries Gerda , Light Peter E.
Published: March 2007

The commonly occurring E23K and I337V Kir6.2 polymorphisms in the ATP-sensitive potassium (KATP) channel are more frequent in Caucasian type 2 diabetic populations. However, the underlying cellular mechanisms contributing to the pathogenesis of type 2 diabetes remain uncharacterized. Chronic elevation of plasma free fatty acids observed in obese and type 2 diabetic subjects leads to cytosolic accumulation of long-chain acyl CoAs (LC-CoAs) in pancreatic β-cells. We postulated that the documented stimulatory effects of LC-CoAs on KATP channels might be enhanced in polymorphic KATP channels. Patch-clamp experiments were performed on inside-out patches containing recombinant KATP channels (Kir6.2/SUR1) to record macroscopic currents. KATP channels containing Kir6.2 (E23K/I337V) showed significantly increased activity in response to physiological palmitoyl-CoA concentrations (100–1,000 nmol/l) compared with wild-type KATP channels. At physiological intracellular ATP concentrations (mmol/l), E23K/I337V polymorphic KATP channels demonstrated significantly enhanced activity in response to palmitoyl-CoA. The observed increase in KATP channel activity may result in multiple defects in glucose homeostasis, including impaired insulin and glucagon-like peptide-1 secretion and increased glucagon release. In summary, these results suggest that the E23K/I337V polymorphism may have a diabetogenic effect via increased KATP channel activity in response to endogenous levels of LC-CoAs in tissues involved in the maintenance of glucose homeostasis.
Hypothesis: Shifting the Equilibrium From Activity to Food Leads to Autonomic Unbalance and the Metabolic Syndrome
Authors: Kreier Felix , Yilmaz Ajda , Kalsbeek Andries , Romijn Johannes A. , Sauerwein Hans P. , Fliers Eric , Buijs Ruud M.
Published: March 2007

“The stability of the internal environment is the condition that life should be free and independent… So, far from the higher animal being indifferent to the external world, it is on the contrary in a precise and informed relation with it, in such a way that its equilibrium results from a continuous and delicate compensation, established as by the most sensitive of balances.”

Claude Bernard (1865)
Coordinated Upregulation of Oxidative Pathways and Downregulation of Lipid Biosynthesis Underlie Obesity Resistance in Perilipin Knockout Mice
Authors: Castro-Chavez Fernando , Yechoor Vijay K. , Saha Pradip K. , Martinez-Botas Javier , Wooten Eric C. , Sharma Saumya , O’Connell Peter , Taegtmeyer Heinrich , Chan Lawrence
Published: March 2007

Obesity is a major risk factor for diabetes and heart disease. We previously reported that the inactivation of the gene for perilipin (plin), an adipocyte lipid droplet surface protein, produced lean and obesity-resistant mice. To dissect the underlying mechanisms involved, we used oligonucleotide microarrays to analyze the gene-expression profile of white adipose tissue (WAT), liver, heart, skeletal muscle, and kidney of plin−/− and plin+/+ mice. As compared with wild-type littermates, the WAT of plin−/− mice had 270 and 543 transcripts that were significantly up- or downregulated. There was a coordinated upregulation of genes involved in β-oxidation, the Krebs cycle, and the electron transport chain concomitant with a downregulation of genes involved in lipid biosynthesis. There was also a significant downregulation of the stearoyl CoA desaturase-1 gene, which has been associated with obesity resistance. Thus, in response to the constitutive activation of lipolysis associated with absence of perilipin, WAT activated pathways to rid itself of the products of lipolysis and activated pathways of energy expenditure that contribute to the observed obesity resistance. The biochemical pathways involved in obesity resistance in plin−/− mice identified in this study may represent potential targets for the treatment of obesity.
Transgenic Neuronal Expression of Proopiomelanocortin Attenuates Hyperphagic Response to Fasting and Reverses Metabolic Impairments in Leptin-Deficient Obese Mice
Authors: Mizuno Tooru M. , Kelley Kevin A. , Pasinetti Giulio M. , Roberts James L. , Mobbs Charles V.
Published: March 2007

Hypothalamic proopiomelanocortin (POMC) gene expression is reduced in many forms of obesity and diabetes, particularly in those attributable to deficiencies in leptin or its receptor. To assess the functional significance of POMC in mediating metabolic phenotypes associated with leptin deficiency, leptin-deficient mice bearing a transgene expressing the POMC gene under control of the neuron-specific enolase promoter were produced. The POMC transgene attenuated fasting-induced hyperphagia in wild-type mice. Furthermore, the POMC transgene partially reversed obesity, hyperphagia, and hypothermia and effectively normalized hyperglycemia, glucosuria, glucose intolerance, and insulin resistance in leptin-deficient mice. Effects of the POMC transgene on glucose homeostasis were independent of the partial correction of hyperphagia and obesity. Furthermore, the POMC transgene normalized the profile of hepatic and adipose gene expression associated with gluconeogenesis, glucose output, and insulin sensitivity. These results indicate that central POMC is a key modulator of glucose homeostasis and that agonists of POMC products may provide effective therapy in treating impairments in glucose homeostasis when hypothalamic POMC expression is reduced, as occurs with leptin deficiency, hypothalamic damage, and aging.
Chronic Exposure to Interleukin-6 Causes Hepatic Insulin Resistance in Mice
Authors: Klover Peter J. , Zimmers Teresa A. , Koniaris Leonidas G. , Mooney Robert A.
Published: March 2007

Interleukin (IL)-6 is one of several proinflammatory cytokines associated with the insulin resistance of obesity and type 2 diabetes. There is, however, little direct evidence in vivo for a causative role of IL-6 in insulin resistance. Here, a 5-day constant subcutaneous infusion of hIL-6 before portal vein insulin challenge resulted in impairment of early insulin receptor signaling in the liver of mice. Importantly, the sixfold elevation of IL-6 attained with constant infusion was similar to levels reached in obesity. Consistent with an hepatic response to IL-6, STAT3 phosphorylation was increased in livers of IL-6-treated mice at 5 days. Chronic infusion of IL-6 also reduced hepatic insulin receptor autophosphorylation by 60% and tyrosine phosphorylation of insulin receptor substrates-1 and -2 by 60 and 40%, respectively. IL-6 had no effect on the mass of these proteins. IL-6 also decreased refeeding-dependent glucokinase mRNA induction by ∼40%. Insulin tolerance tests revealed reduced insulin sensitivity. In contrast to hepatic insulin receptor signal transduction, 5-day IL-6 exposure failed to suppress skeletal muscle insulin receptor signal transduction. These data suggest that chronic IL-6 treatment selectively impairs hepatic insulin signaling in vivo, further supporting a role for IL-6 in hepatic insulin resistance of obesity.
Adverse Effects of Dietary Glycotoxins on Wound Healing in Genetically Diabetic Mice
Authors: Peppa Melpomeni , Brem Harold , Ehrlich Paul , Zhang Jian-Gang , Cai Weijing , Li Zhu , Croitoru Anca , Thung Swan , Vlassara Helen
Published: March 2007

Advanced glycoxidation end products (AGEs) are implicated in delayed diabetic wound healing. To test the role of diet-derived AGE on the rate of wound healing, we placed female db/db (+/+) (n = 55, 12 weeks old) and age-matched control db/db (+/−) mice (n = 45) on two diets that differed only in AGE content (high [H-AGE] versus low [L-AGE] ratio, 5:1) for 3 months. Full-thickness skin wounds (1 cm) were examined histologically and for wound closure. Serum 24-h urine and skin samples were monitored for Nε-carboxymethyl-lysine and methylglyoxal derivatives by enzyme-linked immunosorbent assays. L-AGE-fed mice displayed more rapid wound closure at days 7 and 14 (P < 0.005) and were closed completely by day 21 compared with H-AGE nonhealed wounds. Serum AGE levels increased by 53% in H-AGE mice and decreased by 7.8% in L-AGE mice (P < 0.04) from baseline. L-AGE mice wounds exhibited lower skin AGE deposits, increased epithelialization, angiogenesis, inflammation, granulation tissue deposition, and enhanced collagen organization up to day 21, compared with H-AGE mice. Reepithelialization was the dominant mode of wound closure in H-AGE mice compared with wound contraction that prevailed in L-AGE mice. Thus, increased diet-derived AGE intake may be a significant retardant of wound closure in diabetic mice; dietary AGE restriction may improve impaired diabetic wound healing.
Linkage Analysis of a Composite Factor for the Multiple Metabolic Syndrome
Authors: Tang Weihong , Miller Michael B. , Rich Stephen S. , North Kari E. , Pankow James S. , Borecki Ingrid B. , Myers Richard H. , Hopkins Paul N. , Leppert Mark , Arnett Donna K.
Published: March 2007

Recent studies have demonstrated significant genetic and phenotypic correlation underlying the clustering of traits involved in the multiple metabolic syndrome (MMS). The aim of this study was to identify chromosomal regions contributing to MMS-related traits represented by composite factors derived from factor analysis. Data from the National Heart, Lung, and Blood Institute (NHLBI) Family Heart Study were subjected to a maximum likelihood–based factor analysis. These analyses generated an MMS factor that was loaded by BMI, waist-to-hip ratio, subscapular skinfold, triglycerides, HDL, homeostasis model assessment index, plasminogen activator inhibitor-1 antigen, and serum uric acid. Genetic data were obtained for 2,467 subjects from 387 three-generation families (402 markers, the NHLBI Mammalian Genotyping Service) and 1,082 subjects from 256 sibships (243 markers, the Utah Molecular Genetics Laboratory). Multipoint variance components linkage analysis (GENEHUNTER version 2.1) of the MMS factor was conducted in the combined marker set sample. The greatest evidence for linkage was found on chromosome 2, with a peak LOD of 3.34 at 240 cM. Suggestive linkage was also observed for regions on chromosomes 7, 12, 14, and 15. In summary, a genomic region on chromosome 2 may contain a pleiotropic locus contributing to the clustering of MMS-related phenotypes.
A Mammalian Protein Homologous to Fructosamine-3-Kinase Is a Ketosamine-3-Kinase Acting on Psicosamines and Ribulosamines but not on Fructosamines
Authors: Collard François , Delpierre Ghislain , Stroobant Vincent , Matthijs Gert , Van Schaftingen Emile
Published: March 2007

Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a d-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose—the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate fructosamines.
Agouti Expression in Human Adipose Tissue
Authors: Smith Steven R. , Gawronska-Kozak Barbara , Janderová Lenka , Nguyen Taylor , Murrell Angela , Stephens Jacqueline M. , Mynatt Randall L.
Published: March 2007

It is well recognized that the agouti/melanocortin system is an important regulator of body weight homeostasis. Given that agouti is expressed in human adipose tissue and that the ectopic expression of agouti in adipose tissue results in moderately obese mice, the link between agouti expression in human adipose tissue and obesity/type 2 diabetes was investigated. Although there was no apparent relationship between agouti mRNA levels and BMI, agouti mRNA levels were significantly elevated in subjects with type 2 diabetes. The regulation of agouti in cultured human adipocytes revealed that insulin did not regulate agouti mRNA, whereas dexamethasone treatment potently increased the levels of agouti mRNA. Experiments with cultured human preadipocytes and with cells obtained from transgenic mice that overexpress agouti demonstrated that melanocortin receptor (MCR) signaling in adipose tissue can regulate both preadipocyte proliferation and differentiation. Taken together, these results reveal that agouti can regulate adipogenesis at several levels and suggest that there are functional consequences of elevated agouti levels in human adipose tissue. The influence of MCR signaling on adipogenesis combined with the well-established role of MCR signaling in the hypothalamus suggest that adipogenesis is coordinately regulated with food intake and energy expenditure.
Insulin is Required for Prandial Ghrelin Suppression in Humans
Authors: Murdolo Giuseppe , Lucidi Paola , Di Loreto Chiara , Parlanti Natascia , De Cicco Arianna , Fatone Cristina , Fanelli Carmine G. , Bolli Geremia B. , Santeusanio Fausto , De Feo Pierpaolo
Published: March 2007

Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis. In normal subjects, circulating ghrelin concentrations decrease after meal ingestion and increase progressively before meals. At present, it is not clear whether nutrients suppress the plasma ghrelin concentration directly or indirectly by stimulating insulin secretion. To test the hypothesis that insulin regulates postprandial plasma ghrelin concentrations in humans, we compared the effects of meal ingestion on plasma ghrelin levels in six C-peptide-negative subjects with type 1 diabetes and in six healthy subjects matched for age, sex, and BMI. Diabetic subjects were studied during absence of insulin (insulin withdrawal study), with intravenous infusion of basal insulin (basal insulin study) and subcutaneous administration of a prandial insulin dose (prandial insulin study). Meal intake suppressed plasma ghrelin concentrations (nadir at 105 min) by 32 ± 4% in normal control subjects, 57 ± 3% in diabetic patients during the prandial insulin study (P < 0.002 vs. control subjects), and 38 ± 8% during basal insulin study (P = 0.0016 vs. hyperinsulinemia; P = NS vs. control subjects) but did not have any effect in the insulin withdrawal study (P < 0.001 vs. other studies). In conclusion, 1) insulin is essential for meal-induced plasma ghrelin suppression, 2) basal insulin availability is sufficient for postprandial ghrelin suppression in type 1 diabetic subjects, and 3) lack of meal-induced ghrelin suppression caused by severe insulin deficiency may explain hyperphagia of uncontrolled type 1 diabetic subjects.
On-Line Monitoring of Apoptosis in Insulin-Secreting Cells
Authors: Köhler Martin , Zaitsev Sergei V. , Zaitseva Irina I. , Leibiger Barbara , Leibiger Ingo B. , Turunen Mikael , Kapelioukh Iouri L. , Bakkman Linda , Appelskog Ioulia B. , Boutet de Monvel Jacques , Imreh Gabriela , Berggren Per-Olof
Published: March 2007

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3–like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate β-cell apoptosis was demonstrated by inhibiting caspase-3–like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose–and cytokine-induced apoptosis in the β-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 ± 23 min (n = 9) and 257 ± 59 min (n = 4; mean ± SE) after activation of apoptosis with staurosporine (6 μmol/l), showing that this method worked in insulin-producing cells.
Regulation of Apolipoprotein M Gene Expression by MODY3 Gene Hepatocyte Nuclear Factor-1α
Authors: Richter Symi , Shih David Q. , Pearson Ewan R. , Wolfrum Christian , Fajans Stefan S. , Hattersley Andrew T. , Stoffel Markus
Published: March 2007

Hepatocyte nuclear factor-1a (HNF-1α) is a transcription factor that plays an important role in regulation of gene expression in pancreatic β-cells, intestine, kidney, and liver. Heterozygous mutations in the HNF-1α gene are responsible for maturity-onset diabetes of the young (MODY3), which is characterized by pancreatic β-cell–deficient insulin secretion. HNF-1α is a major transcriptional regulator of many genes expressed in the liver. However, no liver defect has been identified in individuals with HNF-1α mutations. In this study, we show that Hnf-1α is a potent transcriptional activator of the gene encoding apolipoprotein M (apoM), a lipoprotein that is associated with the HDL particle. Mutant Hnf-1α−/− mice completely lack expression of apoM in the liver and the kidney. Serum apoM levels in Hnf-1α+/− mice are reduced ∼50% compared with wild-type animals and are absent in the HDL and HDLc fractions of Hnf-1α−/−. We analyzed the apoM promoter and identified a conserved HNF-1 binding site. We show that Hnf-1α is a potent activator of the apoM promoter, that a specific mutation in the HNF-1 binding site abolished transcriptional activation of the apoM gene, and that Hnf-1α protein can bind to the Hnf-1 binding site of the apoM promoter in vitro. To investigate whether patients with mutations in HNF-1α mutations (MODY3) have reduced serum apoM levels, we measured apoM levels in the serum of nine HNF-1α/MODY3 patients, nine normal matched control subjects (HNF-1α+/+), and nine HNF-4α/MODY1 subjects. Serum levels of apoM were decreased in HNF-1α/MODY3 subjects when compared with control subjects (P < 0.02) as well as with HNF-4α/MODY1 subjects, indicating that HNF-1α haploinsufficiency rather than hyperglycemia is the primary cause of decreased serum apoM protein concentrations. This study demonstrates that HNF-1α is required for apoM expression in vivo and that heterozygous HNF-1α mutations lead to an HNF-1α–dependent impairment of apoM expression. ApoM levels may be a useful serum marker for the identification of MODY3 patients.
Insulin Activation of Phosphatidylinositol 3-Kinase in the Hypothalamic Arcuate Nucleus
Authors: Niswender Kevin D. , Morrison Christopher D. , Clegg Deborah J. , Olson Ryan , Baskin Denis G. , Myers Martin G. , Seeley Randy J. , Schwartz Michael W.
Published: March 2007

In peripheral tissues, insulin signaling involves activation of the insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI3K) enzyme system. In the hypothalamus, insulin functions with leptin as an afferent adiposity signal important for the regulation of body fat stores and hepatic glucose metabolism. To test the hypothesis that hypothalamic insulin action involves intracellular PI3K signaling, we used histochemical and biochemical methods to determine the effect of insulin on hypothalamic IRS-PI3K activity. Here, we report that insulin induces tyrosine phosphorylation of the insulin receptor and IRS-1 and -2, increases binding of activated IRS-1 and -2 to the regulatory subunit of PI3K, and activates protein kinase B/Akt, a downstream target of PI3K. Using an immunohistochemical technique to detect PI 3,4,5-triphosphate, the main product of PI3K activity, we further demonstrate that in the arcuate nucleus, insulin-induced PI3K activity occurs preferentially within cells that contain IRS-2. Finally, we show that the food intake- lowering effects of insulin are reversed by intracerebroventricular infusion of either of two PI3K inhibitors at doses that have no independent feeding effects. These findings support the hypothesis that the IRS-PI3K pathway is a mediator of insulin action in the arcuate nucleus and, combined with recent evidence that leptin activates PI3K signaling in the hypothalamus, provide a plausible mechanism for neuronal cross-talk between insulin and leptin signaling.
Is the Energy Homeostasis System Inherently Biased Toward Weight Gain?
Authors: Schwartz Michael W. , Woods Stephen C. , Seeley Randy J. , Barsh Gregory S. , Baskin Denis G. , Leibel Rudolph L.
Published: March 2007

We describe a model of energy homeostasis to better understand neuronal pathways that control energy balance and their regulation by hormonal signals such as insulin and leptin. Catabolic neuronal pathways are those that both reduce food intake and increase energy expenditure (e.g., melanocortin neurons in the hypothalamic arcuate nucleus) and are stimulated by input from insulin and leptin. We propose that in the basal state, catabolic effectors are activated in response to physiological concentrations of leptin and insulin, and that this activation is essential to prevent excessive weight gain. In contrast, anabolic pathways (e.g., neurons containing neuropeptide Y) are those that stimulate food intake and decrease energy expenditure and are strongly inhibited by these same basal concentrations of insulin and leptin. In the basal state, therefore, catabolic effector pathways are activated while anabolic effector pathways are largely inhibited. The response to weight loss includes both activation of anabolic and inhibition of catabolic pathways and is, thus, inherently more vigorous than the response to weight gain (stimulation of already-activated catabolic pathways and inhibition of already-suppressed anabolic pathways). Teleological, molecular, physiological, and clinical aspects of this hypothesis are presented, along with a discussion of currently available supporting evidence.
Role of Leptin in the Regulation of Glucagon-Like Peptide-1 Secretion
Authors: Anini Younes , Brubaker Patricia L.
Published: March 2007

Glucagon-like peptide-1 (GLP-1), released from intestinal endocrine L cells, is a potent insulinotropic hormone. GLP-1 secretion is diminished in obese patients. Because obesity is linked to abnormal leptin signaling, we hypothesized that leptin may modulate GLP-1 secretion. Leptin significantly stimulated GLP-1 secretion (by up to 250% of control) from fetal rat intestinal cells, a mouse L cell line (GLUTag), and a human L cell line (NCI-H716) in a dose-dependent manner (P < 0.05–0.001). The long form of the leptin receptor was shown to be expressed, and leptin induced the phosphorylation of STAT3 in the three cell types. The leptin receptor was also expressed by rodent and human intestinal L cells, and leptin (1 mg/kg i.p.) significantly stimulated GLP-1 secretion in rats and ob/ob mice. To determine the effect of leptin resistance on GLP-1 secretion, C57BL/6 mice were fed a high-fat (45%) or low-fat (10%) diet for 8 weeks. Mice on the high-fat diet became obese; developed glucose intolerance, hyperinsulinemia, and hyperleptinemia; and were leptin resistant. Mice on the high-fat diet also had twofold lower basal plasma GLP-1 and a diminished GLP-1 response to oral glucose, by 28.5 ± 5.0% (P < 0.05). These results show for the first time that leptin stimulates GLP-1 secretion from rodent and human intestinal L cells, and they suggest that leptin resistance may account for the decreased levels of GLP-1 found in obese humans.
Effects of Free Fatty Acids Per Se on Glucose Production, Gluconeogenesis, and Glycogenolysis
Authors: Staehr Peter , Hother-Nielsen Ole , Landau Bernard R. , Chandramouli Visvanathan , Holst Jens Juul , Beck-Nielsen Henning
Published: March 2007

Insulin-independent effects of a physiological increase in free fatty acid (FFA) levels on fasting glucose production, gluconeogenesis, and glycogenolysis were assessed by administering [6,6-2H2]-glucose and deuteriated water (2H2O) in 12 type 1 diabetic patients, during 6-h infusions of either saline or a lipid emulsion. Insulin was either fully replaced (euglycemic group, n = 6), or underreplaced (hyperglycemic group, n = 6). During saline infusions, plasma FFA levels remained unchanged. Glucose concentrations decreased from 6.7 ± 0.4 to 5.3 ± 0.4 mmol/l and 11.9 ± 1.0 to 10.5 ± 1.0 mmol/l in the euglycemic and hyperglycemic group, respectively. Accordingly, glucose production declined from 84 ± 5 to 63 ± 5 mg · m−2 · min−1 and from 84 ± 5 to 68 ± 4 mg · m−2 · min−1, due to declining rates of glycogenolysis but unaltered rates of gluconeogenesis. During lipid infusions, plasma FFA levels increased twofold. In the euglycemic group, plasma glucose increased from 6.8 ± 0.3 to 7.8 ± 0.8 mmol/l. Glucose production declined less in the lipid study than in the saline study due to a stimulation of gluconeogenesis by 6 ± 1 mg · m−2 · min−1 and a decline in glycogenolysis that was 6 ± 2 mg · m−2 · min−1 less in the lipid study than in the saline study. In contrast, in the hyperglycemic group, there were no significant effects of elevated FFA on glucose production, gluconeogenesis, or glycogenolysis. In conclusion, a physiological elevation of plasma FFA levels stimulates glycogenolysis as well as gluconeogenesis and causes mild fasting hyperglycemia. These effects of FFA appear attenuated in the presence of hyperglycemia.
Sexual Differentiation, Pregnancy, Calorie Restriction, and Aging Affect the Adipocyte-Specific Secretory Protein Adiponectin
Authors: Combs Terry P. , Berg Anders H. , Rajala Michael W. , Klebanov Simon , Iyengar Puneeth , Jimenez-Chillaron José C. , Patti Mary Elizabeth , Klein Sabra L. , Weinstein Robert S. , Scherer Philipp E.
Published: March 2007

Adiponectin or adipocyte complement-related protein of 30 kDa (Acrp30) is a circulating protein produced exclusively in adipocytes. Circulating Acrp30 levels have been associated with insulin sensitivity in adult mice and humans, yet the Acrp30 profile over the lifespan and its hormonal regulation in vivo have not been previously described. Hence, we set forth to determine whether hormonal and metabolic changes associated with sexual maturation, reproduction, aging, and calorie restriction affect Acrp30. In mice, Acrp30 levels increase during sexual maturation by 4-fold in males and 10-fold in females. Neonatal castration (CX) allows Acrp30 of adults to reach female levels. CX in adults does not lead to female Acrp30 levels unless glucocorticoid exposure is elevated simultaneously by implant. Ovariectomy of infant mice does not interfere with the pubertal rise of Acrp30. However, ovariectomy in adults increases Acrp30. Estrogen suppressed Acrp30 in mice and 3T3-L1 adipocytes. In parallel to changes in estrogen action, Acrp30 decreased in late gestation but increased in both calorie-restricted and old (anovulatory) mice. The reduction of Acrp30 in lactating dams is consistent with a suppressive effect of prolactin and a stimulating effect of bromocriptine. In summary, Acrp30 levels in serum are under complex hormonal control and may play a key role in determining systemic insulin sensitivity under the respective conditions.
Gene Transfer of Manganese Superoxide Dismutase Extends Islet Graft Function in a Mouse Model of Autoimmune Diabetes
Authors: Bertera Suzanne , Crawford Megan L. , Alexander Angela M. , Papworth Glenn D. , Watkins Simon C. , Robbins Paul D. , Trucco Massimo
Published: March 2007

Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and β-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned ∼50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.
Age-Dependent Changes in Metabolism, Contractile Function, and Ischemic Sensitivity in Hearts From db/db Mice
Authors: Aasum Ellen , Hafstad Anne D. , Severson David L. , Larsen Terje S.
Published: March 2007

Glucose and palmitate metabolism and contractile function were measured with ex vivo perfused working hearts from control (db/+) and diabetic (db/db) female mice at 6, 10–12, and 16–18 weeks of age. Palmitate oxidation was increased by 2.2-fold in 6-week-old db/db hearts and remained elevated in 10- to 12- and 16- to 18-week-old hearts. Carbohydrate oxidation was normal at 6 weeks but was reduced to 27 and 23% of control at 10–12 and 16–18 weeks, respectively. At 6 weeks, db/db hearts exhibited a slight reduction in mechanical function, whereas marked signs of dysfunction were evident at 10–12 and 16–18 weeks. Mechanical function after ischemia-reperfusion was examined in hearts from male mice; at 6 weeks, db/db hearts showed normal recovery, whereas at 12 weeks it was markedly reduced. Fatty acid oxidation was the predominant substrate used after reperfusion. Thus, diabetic db/db hearts exhibit signs of a progressive cardiomyopathy; increased fatty acid oxidation preceded reductions in carbohydrate oxidation. Postischemic recovery of function was reduced in db/db hearts, in parallel with age-dependent changes in normoxic contractile performance. Finally, peroxisome proliferator-activated receptor-α treatment (3 weeks) did not affect sensitivity to ischemia-reperfusion, even though carbohydrate oxidation was increased and palmitate oxidation was decreased.
Preferential Stiffening of Central Over Peripheral Arteries in Type 2 Diabetes
Authors: Kimoto Eiji , Shoji Tetsuo , Shinohara Kayo , Inaba Masaaki , Okuno Yasuhisa , Miki Takami , Koyama Hidenori , Emoto Masanori , Nishizawa Yoshiki
Published: March 2007

Arterial stiffness affects cardiac functions, peripheral circulation, and cardiovascular mortality. We examined whether arterial stiffness in different regions is equally affected by diabetes and other factors. The subjects were 161 patients with type 2 diabetes and 129 healthy subjects comparable in age and sex. Arterial stiffness was evaluated by measuring pulse wave velocity (PWV) in the heart-carotid, heart-brachial, heart-femoral, and femoral-ankle segments using an automatic device. The diabetic patients had greater PWV than the healthy subjects in the four arterial regions, and the effect of diabetes on PWV was greater in the heart-carotid and heart-femoral segments (central) than in the heart-brachial and femoral-ankle regions (peripheral). PWV increased with age in the four arterial regions, and the effect of age on PWV was greater in the central than in peripheral arteries. In multiple regression analysis, age and systolic blood pressure had significant impacts on PWV of the four regions, whereas diabetes was significantly associated only with PWV of the central arteries. In contrast, sex was associated with PWV of the peripheral arteries. Thus, type 2 diabetes had greater impact on PWV of the central arteries, and different factors were involved in PWV among different arterial regions.
Prediction of Type 2 Diabetes Using Simple Measures of Insulin Resistance
Authors: Hanley Anthony J.G. , Williams Ken , Gonzalez Clicerio , D’Agostino Ralph B. , Wagenknecht Lynne E. , Stern Michael P. , Haffner Steven M.
Published: March 2007

To determine and formally compare the ability of simple indexes of insulin resistance (IR) to predict type 2 diabetes, we used combined prospective data from the San Antonio Heart Study, the Mexico City Diabetes Study, and the Insulin Resistance Atherosclerosis Study, which include well-characterized cohorts of non-Hispanic white, African-American, Hispanic American, and Mexican subjects with 5–8 years of follow-up. Poisson regression was used to assess the ability of each candidate index to predict incident diabetes at the follow-up examination (343 of 3,574 subjects developed diabetes). The areas under the receiver operator characteristic (AROC) curves for each index were calculated and statistically compared. In pooled analysis, Gutt et al.’s insulin sensitivity index at 0 and 120 min (ISI0,120) displayed the largest AROC (78.5%). This index was significantly more predictive (P < 0.0001) than a large group of indexes (including those by Belfiore, Avignon, Katz, and Stumvoll) that had AROC curves between 66 and 74%. These findings were essentially similar both after adjustment for covariates and when analyses were conducted separately by glucose tolerance status and ethnicity/study subgroups. In conclusion, we found substantial differences between published IR indexes in the prediction of diabetes, with ISI0,120 consistently showing the strongest prediction. This index may reflect other aspects of diabetes pathogenesis in addition to IR, which might explain its strong predictive abilities despite its moderate correlation with direct measures of IR.
Altered Homeostatic Adaptation of First- and Second-Phase β-Cell Secretion in the Offspring of Patients With Type 2 Diabetes
Authors: Bonadonna Riccardo C. , Stumvoll Michael , Fritsche Andreas , Muggeo Michele , Häring Hans , Bonora Enzo , van Haeften Timon W.
Published: March 2007

We adapted a minimal model to assess β-cell function during a hyperglycemic glucose clamp and to uncover peculiar aspects of the relationship among β-cell function, plasma glucose, and insulin sensitivity (IS) in offspring of Caucasian patients with type 2 diabetes (OfT2D). We pooled two data sets of OfT2D (n = 69) and control subjects (n = 45) with normal glucose regulation. Plasma C-peptide was measured during a hyperglycemic clamp (∼10 mmol/l) to quantify model-based first-phase secretion and glucose sensitivity of second-phase secretion (β). IS was quantified during the hyperglycemic clamp. In the pooled data, first-phase secretion was linearly and negatively related to fasting plasma glucose, but not IS; OfT2D lay on a distinct line shifted to the left of the control subjects. In contrast, β was negatively related to IS, and OfT2D lay on a distinct line shifted more and more to the left of the control subjects, as IS was worse. Thus, in OfT2D lower β-cell adaptive responses exist between ambient glycemia and first-phase insulin secretion and between IS and second-phase secretion. Under conditions leading to decreased insulin sensitivity, these disturbed relationships may lead to progression to diabetes in OfT2D.