bluuebunny/crossref_metadata_2025 huggingface dataset:
Impairment of Coronary Microvascular Dilation in Response to Cold Pressor–Induced Sympathetic Stimulation in Type 2 Diabetic Patients With Abnormal Stress Thallium Imaging
Authors: Nitenberg Alain , Ledoux Séverine , Valensi Paul , Sachs Régis , Attali Jean-Raymond , Antony Isabelle
Published: March 2007

Coronary microcirculation dysfunction may be associated with myocardial perfusion defects on thallium imaging in diabetic patients without coronary artery stenosis. Microvascular coronary adaptation to increased myocardial oxygen demand in response to sympathetic stimulation evoked by the cold pressor test was examined in 22 type 2 diabetic patients with thallium imaging defects and in 15 control subjects. Both the diabetic patients and control subjects had angiographically normal coronary arteries and no other risk factors. Despite a similar increase in the rate-pressure product in the two groups (22.6 ± 12.4% in diabetic patients and 31.8 ± 8.2% in control subjects, NS), coronary blood flow increase in the left anterior descending artery (mean flow velocity measured by intracoronary Doppler multiplied by the cross-sectional area measured by digital angiography) was significantly lower in diabetic patients than in control subjects (14.7 ± 19.8 vs. 75.5 ± 13.5%, respectively; P = 0.0001). In addition, when there was a positive correlation between the two parameters in control subjects (r = 0.651, P < 0.01), there was no relationship in diabetic patients (r = 0.054). In conclusion, vasodilation of the coronary microcirculation in response to sympathetic stimulation evoked by the cold pressor test is impaired in type 2 diabetic patients without epicardial artery lesions. This microvascular impairment during sympathetic stimulation may explain exercise-induced myocardial perfusion abnormalities observed in these patients and may impair microcirculatory coronary vasodilation during current life stress episodes such as exercise, mental stress, or cold exposition.
Hexokinase Isozyme Distribution in Human Skeletal Muscle
Authors: Ritov Vladimir B. , Kelley David E.
Published: March 2007

Two isoforms of hexokinase (type I and type II) are expressed in skeletal muscle; however, the intracellular distribution of these hexokinase isoforms in human skeletal muscle is unclear. The current study was undertaken to assess this issue because binding of hexokinase to subcellular structures is considered to be an important mechanism in the regulation of glucose phosphorylation. Vastus lateralis muscle was obtained from healthy lean individuals. Muscle homogenate was separated at 45,000g into particulate and cytosolic fractions. The activity and subcellular distribution of hexokinase isozymes in human skeletal muscle was determined using ion-exchange chromatography and a highly sensitive high-performance liquid chromatography–based hexokinase assay. This criterion method was used to validate a modified thermal inactivation method for distinguishing type I and type II isoforms. Mean hexokinase activity was 3.88 ± 0.65 U/g wet wt or 0.64 ± 0.11 U/mU creatine kinase (CrK) in the particulate fraction and 0.45 ± 0.22 U/g wet wt or 0.07 ± 0.03 U/mU CrK in the cytosolic fraction. Hexokinase I and II accounted for 70–75 and 25–30% of total hexokinase activity, respectively. Nearly all (95%) of hexokinase I activity (0.52 ± 0.09 U/mU CrK) was found in the particulate fraction, consistent with the known high affinity of hexokinase I for mitochondria. Hexokinase II activity was also largely bound to the particulate fraction (72%), but 28% was found within the cytosolic fraction. Thus, within the particulate fraction, the relative contributions of hexokinase I and hexokinase II were 81 and 19%, whereas within the cytosolic fraction, the relative contributions for hexokinase I and hexokinase II were 37 and 63%.
Stimulating Effects of Low-Dose Fructose on Insulin-Stimulated Hepatic Glycogen Synthesis in Humans
Authors: Petersen Kitt Falk , Laurent Didier , Yu Chunli , Cline Gary W. , Shulman Gerald I.
Published: March 2007

Fructose has been shown to have a catalytic effect on glucokinase activity in vitro; however, its effects on hepatic glycogen metabolism in humans is unknown. To address this question, we used 13C nuclear magnetic resonance (NMR) spectroscopy to noninvasively assess rates of hepatic glycogen synthesis and glycogenolysis under euglycemic (∼5 mmol/l) hyperinsulinemic conditions (∼400 pmol/l) with and without a low-dose infusion of fructose (∼3.5 μmol · kg–1 · min–1). Six healthy overnight-fasted subjects were infused for 4 h with somatostatin (0.1 μg · kg–1 · min–1) and insulin (240 pmol · m–2 · min–1). During the initial 120 min, [1-13C]glucose was infused to assess glycogen synthase flux followed by an ∼120-min infusion of unlabeled glucose to assess rates of glycogen phosphorylase flux. Acetaminophen was given to assess the percent contribution of the direct and indirect (gluconeogenic) pathways of glycogen synthesis by the 13C enrichment of plasma UDP-glucuronide and C-1 of glucose. In the control studies, the flux through glycogen synthase and glycogen phosphorylase was 0.31 ± 0.06 and 0.17 ± 0.04 mmol/l per min, respectively, and the rate of net hepatic glycogen synthesis was 0.14 ± 0.05 mmol/l per min. In the fructose studies, the glycogen synthase flux increased 2.5-fold to 0.79 ± 0.16 mmol/l per min (P = 0.018 vs. control), whereas glycogen phosphorylase flux remained unchanged (0.24 ± 0.06; P = 0.16 vs. control). The infusion of fructose resulted in a threefold increase in rates of net hepatic glycogen synthesis (0.54 ± 0.12 mmol/l per min; P = 0.008 vs. control) without affecting the pathways of hepatic glycogen synthesis (direct pathway ∼60% in both groups). We conclude that during euglycemic hyperinsulinemia, a low-dose fructose infusion causes a threefold increase in net hepatic glycogen synthesis exclusively through stimulation of glycogen synthase flux. Because net hepatic glycogen synthesis has been shown to be diminished in patients with poorly controlled type 1 and type 2 diabetes, stimulation of hepatic glycogen synthesis by this mechanism may be of potential therapeutic value.
Increased Uncoupling Protein-2 Levels in β-cells Are Associated With Impaired Glucose-Stimulated Insulin Secretion
Authors: Chan Catherine B. , De Leo Domenica , Joseph Jamie W. , McQuaid Timothy S. , Ha Xiao Fang , Xu Fang , Tsushima Robert G. , Pennefather Peter S. , Salapatek Anne Marie F. , Wheeler Michael B.
Published: March 2007

In pancreatic β-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H+ leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K+ (KATP) channels with glybenclamide elicited similar insulin secretion in control and UCP-2–overexpressing islets. The glucose-stimulated mitochondrial membrane (Ψm) hyperpolarization was reduced in β-cells overexpressing UCP-2. ATP content of UCP-2–induced islets was reduced by 50%, and there was no change in the efflux of Rb+ at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of β-cell insulin secretion and demonstrate that reduced ΔΨm and increased activity of KATP channels are mechanisms by which UCP-2–mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.
Decreased Susceptibility to Fatty Acid–Induced Peripheral Tissue Insulin Resistance in Women
Authors: Frias Juan P. , Macaraeg Gina B. , Ofrecio Jachelle , Yu Joseph G. , Olefsky Jerrold M. , Kruszynska Yolanta T.
Published: March 2007

Elevation of plasma nonesterified fatty acid (NEFA) levels has been shown in various studies to induce peripheral tissue insulin resistance and impair the suppression of endogenous glucose production (EGP). These studies have been conducted predominantly in men. We compared the effects of elevated plasma NEFA levels on basal and insulin-stimulated glucose metabolism in 8 normal women (age 42 ± 8 years [mean ± SD], BMI 25 ± 3 kg/m2) and 10 normal men (35 ± 6 years, 24 ± 3 kg/m2). Each subject underwent two 5-h 80 mU · m–2 · min–1 hyperinsulinemic-euglycemic clamps with measurement of glucose kinetics (intravenous [3-3H]glucose) and substrate oxidation. Plasma NEFA levels were elevated in one study for 3 h before and during the clamp (∼1 mmol/l in both groups) by infusion of 20% Intralipid (60 ml/h) and heparin (900 U/h). In the control studies, the men and women had similar insulin-stimulated glucose disposal rates (Rd) and substrate oxidation rates. In the men, elevated NEFA levels decreased insulin-stimulated glucose Rd during the final 40 min of the clamp by 23% (P < 0.001). By contrast, no significant change in glucose Rd was found in the women (control 10.4 ± 1.1, lipid study 9.9 ± 1.3 mg · kg–1 · min–1). Glucose Rd was also unchanged in six women studied at a lower insulin dose (40 mU · m–2 · min–1). During the last 40 min of the high–insulin dose clamps with elevated NEFA, glucose oxidation was decreased by 33% in the men (P < 0.001) and by 23% in the women (P < 0.02). Nonoxidative glucose Rd at this time was decreased by 15% in the men (P = 0.02) but was not significantly affected in women. Basal EGP was unaffected by elevation of plasma NEFA levels in both groups. Suppression of EGP during the glucose clamps, however, was impaired. At the insulin infusion rate used, the magnitude of this defect was comparable in men and women. In summary, our findings suggest that although the effects on EGP appear comparable, the inhibitory effects of NEFA on peripheral tissue insulin sensitivity are observed in men but cannot be demonstrated in women.
IGF-1 Overexpression Inhibits the Development of Diabetic Cardiomyopathy and Angiotensin II–Mediated Oxidative Stress
Authors: Kajstura Jan , Fiordaliso Fabio , Andreoli Anna Maria , Li Baosheng , Chimenti Stefano , Medow Marvin S. , Limana Federica , Nadal-Ginard Bernardo , Leri Annarosa , Anversa Piero
Published: March 2007

Stimulation of the local renin-angiotensin system and apoptosis characterize the diabetic heart. Because IGF-1 reduces angiotensin (Ang) II and apoptosis, we tested whether streptozotocin-induced diabetic cardiomyopathy was attenuated in IGF-1 transgenic mice (TGM). Diabetes progressively depressed ventricular performance in wild-type mice (WTM) but had no hemodynamic effect on TGM. Myocyte apoptosis measured at 7 and 30 days after the onset of diabetes was twofold higher in WTM than in TGM. Myocyte necrosis was apparent only at 30 days and was more severe in WTM. Diabetic nontransgenic mice lost 24% of their ventricular myocytes and showed a 28% myocyte hypertrophy; both phenomena were prevented by IGF-1. In diabetic WTM, p53 was increased in myocytes, and this activation of p53 was characterized by upregulation of Bax, angiotensinogen, Ang type 1 (AT1) receptors, and Ang II. IGF-1 overexpression decreased these biochemical responses. In vivo accumulation of the reactive O2 product nitrotyrosine and the in vitro formation of H2O2-˙OH in myocytes were higher in diabetic WTM than TGM. Apoptosis in vitro was detected in myocytes exhibiting high H2O2-˙OH fluorescence, and apoptosis in vivo was linked to the presence of nitrotyrosine. H2O2-˙OH generation and myocyte apoptosis in vitro were inhibited by the AT1 blocker losartan and the O2 scavenger Tiron. In conclusion, IGF-1 interferes with the development of diabetic myopathy by attenuating p53 function and Ang II production and thus AT1 activation. This latter event might be responsible for the decrease in oxidative stress and myocyte death by IGF-1.
The HIV Protease Inhibitor Nelfinavir Induces Insulin Resistance and Increases Basal Lipolysis in 3T3-L1 Adipocytes
Authors: Rudich Assaf , Vanounou Sharon , Riesenberg Klaris , Porat Michal , Tirosh Amir , Harman-Boehm Ilana , Greenberg Andrew S. , Schlaeffer Francisc , Bashan Nava
Published: March 2007

HIV protease inhibitors (HPIs) are potent antiretroviral agents clinically used in the management of HIV infection. Recently, HPI therapy has been linked to the development of a metabolic syndrome in which adipocyte insulin resistance appears to play a major role. In this study, we assessed the effect of nelfinavir on glucose uptake and lipolysis in differentiated 3T3-L1 adipocytes. An 18-h exposure to nelfinavir resulted in an impaired insulin-stimulated glucose uptake and activation of basal lipolysis. Impaired insulin stimulation of glucose up take occurred at nelfinavir concentrations >10 μmol/l (EC50 = 20 μmol/l) and could be attributed to impaired GLUT4 translocation. Basal glycerol and free fatty acid (FFA) release were significantly enhanced with as low as 5 μmol/l nelfinavir, displaying fivefold stimulation of FFA release at 10 μmol/l. Yet, the antilipolytic action of insulin was preserved at this concentration. Potential underlying mechanisms for these metabolic effects included both impaired insulin stimulation of protein kinase B Ser 473 phosphorylation with preserved insulin receptor substrate tyrosine phosphorylation and decreased expression of the lipolysis regulator perilipin. Troglitazone pre- and cotreatment with nelfinavir partly protected the cells from the increase in basal lipoysis, but it had no effect on the impairment in insulin-stimulated glucose uptake induced by this HPI. This study demonstrates that nelfinavir induces insulin resistance and activates basal lipolysis in differentiated 3T3-L1 adipocytes, providing potential cellular mechanisms that may contribute to altered adipocyte metabolism in treated HIV patients.
Phosphorylation of p38 Mitogen-Activated Protein Kinase Downstream of Bax-Caspase-3 Pathway Leads to Cell Death Induced by High d-Glucose in Human Endothelial Cells
Authors: Nakagami Hironori , Morishita Ryuichi , Yamamoto Kei , Yoshimura Shin-ichi , Taniyama Yoshiaki , Aoki Motokuni , Matsubara Hiroaki , Kim Shokei , Kaneda Yasufumi , Ogihara Toshio
Published: March 2007

Because high d-glucose significantly stimulates endothelial cell death, we examined the molecular mechanisms of high d-glucose–induced endothelial apoptosis. Treatment of human aortic endothelial cells with high d-glucose (25 mmol/l), but not mannitol and l-glucose, resulted in a significant decrease in cell number and a significant increase in apoptotic cells as compared with a physiological concentration (5 mmol/l). Interestingly, high d-glucose treatment significantly increased bax protein, accompanied by translocation of bax protein from cytosol to mitochondria-enriched heavy membrane fraction. In contrast, the expression and distribution of bcl-2 protein were not altered by high d-glucose. In addition, the activity of caspase-3 proteases was increased after exposure to high glucose, whereas caspase inhibitors prevented endothelial cell death induced by high d-glucose. On the other hand, p38 mitogen-activated protein kinase (MAPK) was markedly phosphorylated and showed sustained phosphorylation after stimulation. A specific inhibitor of p38 MAPK, SB 203580, and the overexpression of kinase-inactive p38 MAPK significantly attenuated cell death induced by high d-glucose in human aortic endothelial cells, whereas at 6 h after high d-glucose treatment, SB 203580 and overexpression of kinase-inactive p38 MAPK did not attenuate caspase-3 activation induced by high d-glucose. Importantly, caspase inhibitors significantly attenuated the sustained phosphorylation of p38 MAPK induced by high d-glucose. Thus, we finally focused the MAPK kinase (MEK) kinase 1 (MEKK1) to further examine the cross-talk between p38 MAPK and the bax-caspase proteases pathway. High d-glucose treatment induced MEKK1 cleavage, whereas caspase inhibitors significantly attenuated the cleavage. Importantly, kinase-inactive MEKK1 also blocked the phosphorylation of p38 MAPK induced by high d-glucose. Here, we demonstrated that high d-glucose induced apoptosis in human endothelial cells through activation of the bax-caspase proteases pathway and through phosphorylation of p38 MAPK mediated by MEKK1. Phosphorylation of p38 MAPK downstream of the bax-caspase pathway may play a pivotal role in endothelial apoptosis mediated by high d-glucose.
Novel Arguments in Favor of the Substrate-Transport Model of Glucose-6-Phosphatase
Authors: Gerin Isabelle , Noël Gaëtane , Van Schaftingen Emile
Published: March 2007

The purpose of this work was to discriminate between two models for glucose-6-phosphatase: one in which the enzyme has its catalytic site oriented toward the lumen of the endoplasmic reticulum, requiring transporters for glucose-6-phosphate, inorganic phosphate (Pi), and glucose (substrate-transport model), and a second one in which the hydrolysis of glucose-6-phosphate occurs inside the membrane (conformational model). We show that microsomes preloaded with yeast phosphoglucose isomerase catalyzed the detritiation of [2-3H]glucose-6-phosphate and that this reaction was inhibited by up to 90% by S3483, a compound known to inhibit glucose-6-phosphate hydrolysis in intact but not in detergent-treated microsomes. These results indicate that glucose-6-phosphate is transported to the lumen of the microsomes in an S3483-sensitive manner. Detritiation by intramicrosomal phosphoglucose isomerase was stimulated twofold by 1 mmol/l vanadate, a phosphatase inhibitor, indicating that glucose-6-phosphatase and the isomerase compete for the same intravesicular pool of glucose-6-phosphate. To investigate the site of release of Pi from glucose-6-phosphate, we incubated microsomes with Pb2+, which forms an insoluble complex with Pi, preventing its rapid exit from the microsomes. Under these conditions, ∼80% of the Pi that was formed after 5 min was intramicrosomal, compared with <10% in the absence of Pb2+. We also show that, when incubated with glucose-6-phosphate and mannitol, glucose-6-phosphatase formed mannitol-1-phosphate and that this nonphysiological product was initially present within the microsomes before being released to the medium. These results indicate that the primary site of product release by glucose-6-phosphatase is the lumen of the endoplasmic reticulum.
Liver-Derived IGF-I is of Importance for Normal Carbohydrate and Lipid Metabolism
Authors: Sjögren Klara , Wallenius Kristina , Liu Jun-Li , Bohlooly-Y Mohammad , Pacini Giovanni , Svensson Lennart , Törnell Jan , Isaksson Olle G.P. , Ahrén Bo , Jansson John-Olov , Ohlsson Claes
Published: March 2007

IGF-I is important for postnatal body growth and exhibits insulin-like effects on carbohydrate metabolism. The function of liver-derived IGF-I is still not established, although we previously demonstrated that liver-derived IGF-I is not required for postnatal body growth. Mice whose IGF-I gene in the liver was inactivated at 24 days of age were used to investigate the long-term role of liver-derived IGF-I for carbohydrate and lipid metabolism. Serum levels of leptin in these mice were increased by >100% at 3 months of age, whereas the fat mass of the mice was decreased by 25% at 13 months of age. The mice became markedly hyperinsulinemic and yet normoglycemic, indicating an adequately compensated insulin resistance. Furthermore, they had increased serum levels of cholesterol. We conclude that liver-derived IGF-I is of importance for carbohydrate and lipid metabolism.
Glucagon-Like Peptide-1 and Exendin-4 Stimulate β-Cell Neogenesis in Streptozotocin-Treated Newborn Rats Resulting in Persistently Improved Glucose Homeostasis at Adult Age
Authors: Tourrel Cécile , Bailbé Danielle , Meile Marie-Jo , Kergoat Micheline , Portha Bernard
Published: March 2007

In neonatal Wistar rats injected with streptozotocin (STZ) at birth (n0-STZ model), a recognized model of β-cell regeneration, we investigated the capacity of early treatment with glucagon-like peptide 1 (GLP-1) or exendin-4 to promote β-cell regeneration and thereby improve islet function in the long term, when animals become adults. To this end, n0-STZ rats were submitted to GLP-1 or exendin-4 from postnatal day 2 to day 6 only, and their β-cell mass and pancreatic functions were tested on day 7 and at 2 months. On day 7, both treatments increased body weight, decreased basal plasma glucose, decreased insulinemia, and increased pancreatic insulin content in n0-STZ rats. At the same age, the β-cell mass, measured by immunocytochemistry and morphometry methods, was strongly increased in n0-STZ/GLP-1 and n0-STZ/Ex rats compared with n0-STZ rats, representing 51 and 71%, respectively, of the β-cell mass in Wistar rats, whereas n0-STZ β-cell mass represented only 21% of the Wistar control value. Despite such early improved β-cell mass, which is maintained at adult age, the basal and glucose-stimulated insulin secretion (in vivo after intravenous glucose load or in vitro using perfused pancreas) were not improved in the 2-month-old n0-STZ rats previously treated with GLP-1 or exendin-4 compared with untreated n0-STZ rats. However, both treated groups significantly exhibited a decreased basal plasma glucose level and an increased plasma glucose clearance rate compared with the 2-month-old untreated n0-STZ group at adult age. These findings in the n0-STZ model indicate for the first time that GLP-1 or exendin-4 applied during the neonatal diabetic period exert both short- and long-term beneficial effects on β-cell mass recovery and glucose homeostasis. However, the increase in β-cell mass, which is still present in the adult n0-STZ rats previously treated, contrasts with the poor β-cell responsiveness to glucose. Further studies are needed to understand the dissociation between β-cell regeneration and the lack of improvement in β-cell function.
The Effect of Acute Hypoglycemia on Brain Function and Activation
Authors: Rosenthal J. Miranda , Amiel Stephanie A. , Yágüez Lidia , Bullmore Edward , Hopkins David , Evans Mark , Pernet Andrew , Reid Helen , Giampietro Vincent , Andrew Chris M. , Suckling John , Simmons Andrew , Williams Stephen C.R.
Published: March 2007

The authors’ aim was to examine the regional anatomy of brain activation by cognitive tasks commonly used in hypoglycemia research and to assess the effect of acute hypoglycemia on these in healthy volunteers. Eight right-handed volunteers performed a set of cognitive tasks—finger tapping (FT), simple reaction time (SRT), and four-choice reaction time (4CRT)—twice during blood oxygen level–dependent (BOLD) functional magnetic resonance imaging of the brain on two occasions. In study 1 (n = 6), plasma glucose was maintained at euglycemia (5 mmol/l) throughout. In study 2 (n = 6), plasma glucose was reduced to 2.5 mmol/l for the second set. Performance of the tasks resulted in specific group brain activation maps. During hypoglycemia, FT slowed (P = 0.026), with decreased BOLD activation in right premotor cortex and supplementary motor area and left hippocampus and with increased BOLD activation in left cerebellum and right frontal pole. Although there was no significant change in SRT, BOLD activation was reduced in right cerebellum and visual cortex. The 4CRT deteriorated (P = 0.020), with reduction in BOLD activation in motor and visual systems but increased BOLD signal in a large area of the left parietal association cortex, a region involved in planning. Hypoglycemia impairs simple brain functions and is associated with task-specific localized reductions in brain activation. For a task with greater cognitive load, the increased BOLD signal in planning areas is compatible with recruitment of brain regions in an attempt to limit dysfunction. Further investigation of these mechanisms may help devise rational treatment strategies to limit cortical dysfunction during acute iatrogenic hypoglycemia.
Glucose Effects on Skin Keratinocytes
Authors: Spravchikov Natalia , Sizyakov Galina , Gartsbein Marina , Accili Domenico , Tennenbaum Tamar , Wertheimer Efrat
Published: March 2007

Altered skin wound healing is a common cause of morbidity and mortality among diabetic patients. However, the molecular mechanisms whereby diabetes alters skin physiology have not been elucidated. In this study, we investigated the relative roles of hyperglycemia, insulin, and IGF-I, all of which are abnormal in diabetes, in primary murine skin keratinocytes. These cells proliferate and differentiate in vitro in a manner similar to skin in vivo. It was found that in the presence of high glucose (20 mmol/l), the glucose transport rate of primary proliferating or differentiating keratinocytes was downregulated, whereas at 2 mmol/l glucose, the transport rate was increased. These changes were associated with changes in the GLUT1 expression and with changes in the affinity constant (Km) of the transport. Exposure to high glucose was associated with changes in cellular morphology, as well as with decreased proliferation and enhancement of Ca2+-induced differentiation of keratinocytes. Furthermore, in the presence of high glucose, ligand-induced IGF-I receptor but not insulin receptor (IR) autophosphorylation was decreased. Consequently, in high glucose, the effects of IGF-I on glucose uptake and keratinocyte proliferation were inhibited. Interestingly, lack of IR expression in IR-null keratinocytes abolished insulin-induced glucose uptake and partially decreased insulin- and IGF-I–induced proliferation, demonstrating the direct involvement of the IR in these processes. Our results demonstrate that hyperglycemia and impaired insulin signaling might be directly involved in the development of chronic complications of diabetes by impairing glucose utilization of skin keratinocytes as well as skin proliferation and differentiation.
Lipoprotein Lipase Mediates the Uptake of Glycated LDL in Fibroblasts, Endothelial Cells, and Macrophages
Authors: Zimmermann Robert , Panzenböck Ute , Wintersperger Andrea , Levak-Frank Sanja , Graier Wolfgang , Glatter Otto , Fritz Gerhard , Kostner Gerhard M. , Zechner Rudolf
Published: March 2007

The nonenzymatic glycation of LDL is a naturally occurring chemical modification of apolipoprotein (apo)-B lysine residues by glucose. Once glycated, LDL is only poorly recognized by lipoprotein receptors including the LDL receptor (LDL-R), the LDL-R–related protein (LRP), and scavenger receptors. Glycated LDL (gLDL) is a preferred target for oxidative modifications. Additionally, its presence initiates different processes that can be considered “proatherogenic.” Thus, LDL glycation might contribute to the increased atherosclerotic risk of patients with diabetes and familial hypercholesterolemia. Here we investigate whether lipoprotein lipase (LPL) can mediate the cellular uptake of gLDL. The addition of exogenous LPL to the culture medium of human skin fibroblasts, porcine aortic endothelial cells, and mouse peritoneal macrophages enhanced the binding, uptake, and degradation of gLDL markedly, and the relative effect of LPL on lipoprotein uptake increased with the degree of apoB glycation. The efficient uptake of gLDL by LDL-R–deficient fibroblasts and LRP-deficient Chinese hamster ovary cells in the presence of LPL suggested a mechanism that was independent of the LDL-R and LRP. In macrophages, the uptake of gLDL was also correlated with their ability to produce LPL endogenously. Mouse peritoneal macrophages from genetically modified mice, which lacked LPL, exhibited a 75% reduction of gLDL uptake compared with normal macrophages. The LPL-mediated effect required the association of the enzyme with cell surface glycosaminoglycans but was independent of its enzymatic activity. The uptake of gLDL in different cell types by an LPL-mediated process might have important implications for the cellular response after gLDL exposure as well as the removal of gLDL from the circulation.
Actin and Annexins I and II Are Among the Main Endothelial Plasmalemma-Associated Proteins Forming Early Glucose Adducts in Experimental Diabetes
Authors: Ghitescu Lucian D. , Gugliucci Alejandro , Dumas France
Published: March 2007

An immunochemical and biochemical study was performed to reveal which of the endothelial plasma membrane proteins become glycated during the early phases of diabetes. The blood front of the lung microvascular endothelial plasmalemma was purified by the cationic colloidal silica method from normal and diabetic (streptozotocin-induced) rats and comparatively analyzed by two-dimensional electrophoresis. No major qualitative differences in the general spectrum of endothelial plasmalemmal proteins were recorded between normoglycemic and hyperglycemic animals. By probing with anti-glucitollysine antibodies, we found that at 1 month after the onset of diabetes, several endothelial membrane polypeptides contained glucose covalently linked to their lysyl residues. Ten days of insulin treatment restored euglycemia in the diabetic animals and completely abolished the membrane nonenzymatic glycosylation. All the glycated polypeptides of the endothelial plasma membrane belong to the peripheral type and are associated with its cytoplasmic face (cell cortex). They were solubilized by buffers of high pH and were not detected in the lung cytosolic fraction (100,000 g). By microsequencing, the major proteins labeled by the anti-glucitollysine have been identified as being actin, annexin I, annexin II, the p34 subunit of the Arp2/3 complex, and the Ras suppressor protein-1. Conversely, the intrinsic endothelial membrane proteins do not seem to be affected by hyperglycemia. This defines the internal face of the endothelial plasma membrane, particularly the cortical cytoskeleton, as a preferential target for nonenzymatic glycosylation in diabetes, with possible consequences on the fluidity of the endothelial plasmalemma and impairment of the endothelial mechanotransducing ability.
Glucose Metabolism and Pulsatile Insulin Release From Isolated Islets
Authors: Westerlund Johanna , Bergsten Peter
Published: March 2007

The effects of metabolic inhibition on insulin release and the cytoplasmic Ca2+ concentration ([Ca2+]i) were studied in individually perifused pancreatic islets from ob/ob mice. The modest basal secretion in the presence of 3 mmol/l glucose was pulsatile with a frequency of ∼0.2/min, although [Ca2+]i was stable at ∼100 nmol/l. Introduction of 11 mmol/l glucose resulted in large amplitude oscillations of [Ca2+]i and almost 20-fold stimulation of average secretion manifested as increased amplitude of the insulin pulses without change in frequency. Inhibition of glycolysis with iodoacetamide or mitochondrial metabolism with dinitrophenol or antimycin A reduced glucose-stimulated secretion back to basal levels with maintained pulsatility. The [Ca2+]i responses to the metabolic inhibitors were more complex, but in general there was an initial peak and eventually sustained elevation without oscillations. When introduced in the presence of 3 mmol/l glucose, the metabolic inhibitors tended to increase the amplitude of the insulin pulses, although the simultaneous elevation in [Ca2+]i occurred without oscillations. The data indicate that pulsatile secretion is regulated by factors other than [Ca2+]i under basal conditions and after metabolic inhibition. Although pulsatile secretion can be driven by oscillations in metabolism when [Ca2+]i is stable, it was not possible from the present data to determine whether insulin pulses have a glycolytic or mitochondrial origin.
Nonalcoholic Fatty Liver Disease
Authors: Marchesini Giulio , Brizi Mara , Bianchi Giampaolo , Tomassetti Sara , Bugianesi Elisabetta , Lenzi Marco , McCullough Arthur J. , Natale Stefania , Forlani Gabriele , Melchionda Nazario
Published: March 2007

Insulin sensitivity (euglycemic clamp, insulin infusion rate: 40 mU · m−2 · min−1) was studied in 30 subjects with biopsy-proven nonalcoholic fatty liver disease (NAFLD), normal glucose tolerance, and a BMI <30 kg/m2. Of those 30 subjects, 9 had pure fatty liver and 21 had evidence of steatohepatitis. In addition, 10 patients with type 2 diabetes under good metabolic control and 10 healthy subjects were studied. Most NAFLD patients had central fat accumulation, increased triglycerides and uric acid, and low HDL cholesterol, irrespective of BMI. Glucose disposal during the clamp was reduced by nearly 50% in NAFLD patients, as well as in patients with normal body weight, to an extent similar to that of the type 2 diabetic patients. Basal free fatty acids were increased, whereas insulin-mediated suppression of lipolysis was less effective (−69% in NAFLD vs. −84% in control subjects; P = 0.003). Postabsorptive hepatic glucose production (HGP), measured by [6,6-2H2]glucose, was normal. In response to insulin infusion, HGP decreased by only 63% of basal in NAFLD vs. 84% in control subjects (P = 0.002). Compared with type 2 diabetic patients, NAFLD patients were characterized by lower basal HGP, but with similarly reduced insulin-mediated suppression of HGP. There was laboratory evidence of iron overload in many NAFLD patients, but clinical, histological, and biochemical data (including insulin sensitivity) were not correlated with iron status. Four subjects were heterozygous for mutation His63Asp of the HFE gene of familiar hemochromatosis. We concluded that NAFLD, in the presence of normoglycemia and normal or moderately increased body weight, is characterized by clinical and laboratory data similar to those found in diabetes and obesity. NAFLD may be considered an additional feature of the metabolic syndrome, with specific hepatic insulin resistance.
Menopause in Type 1 Diabetic Women
Authors: Dorman Janice S. , Steenkiste Ann R. , Foley Thomas P. , Strotmeyer Elsa S. , Burke James P. , Kuller Lewis H. , Kwoh C. Kent
Published: March 2007

Women with type 1 diabetes have a delayed menarche and a greater prevalence of menstrual disorders than women without diabetes. However, little is known about the menopause transition among type 1 diabetic women. The Familial Autoimmune and Diabetes (FAD) Study recruited both adult individuals who were identified from the Children’s Hospital of Pittsburgh Type 1 Diabetes Registry for the years 1950–1964 and their family members. Unrelated nondiabetic control probands and their relatives were also evaluated. Women with type 1 diabetes (n = 143) compared with nondiabetic sisters (n = 186) or unrelated control subjects (n = 160) were more likely to have an older age at menarche (13.5, 12.5, and 12.6 years, respectively, P < 0.001), more menstrual irregularities before 30 years of age (45.7, 33.3, and 33.1%, respectively, P = 0.04), and a younger age at menopause (41.6, 49.9, and 48.0 years, respectively, P = 0.05). This resulted in a 6-year reduction in the number of reproductive years (30.0, 37.0, and 35.2 years, respectively, P = 0.05) for women with type 1 diabetes. Risk factors univariately associated with earlier menopause included type 1 diabetes (hazard ratio [HR] 1.99, P = 0.04), menstrual irregularities before 30 years of age (HR 1.87, P = 0.04), nulliparity (HR 2.14, P = 0.01), and unilateral oophorectomy (HR 6.51, P < 0.0001). Multivariate analysis confirmed that type 1 diabetes (HR 1.98, P = 0.056), menstrual irregularities by 30 years of age (HR 2.36, P = 0.01), and unilateral oophorectomy (HR 9.76, P < 0.0001) were independent determinants of earlier menopause in our cohort. We hypothesize that an earlier menopause, which resulted in a 17% decrease in reproductive years, is a major unstudied complication of type 1 diabetes.
Defective Insulin-Induced GLUT4 Translocation in Skeletal Muscle of High Fat–Fed Rats Is Associated With Alterations in Both Akt/Protein Kinase B and Atypical Protein Kinase C (ζ/λ) Activities
Authors: Tremblay Frédéric , Lavigne Charles , Jacques Hélène , Marette André
Published: March 2007

The cellular mechanism by which high-fat feeding induces skeletal muscle insulin resistance was investigated in the present study. Insulin-stimulated glucose transport was impaired (∼40–60%) in muscles of high fat–fed rats. Muscle GLUT4 expression was significantly lower in these animals (∼40%, P < 0.05) but only in type IIa–enriched muscle. Insulin stimulated the translocation of GLUT4 to both the plasma membrane and the transverse (T)-tubules in chow-fed rats. In marked contrast, GLUT4 translocation was completely abrogated in the muscle of insulin-stimulated high fat–fed rats. High-fat feeding markedly decreased insulin receptor substrate (IRS)-1–associated phosphatidylinositol (PI) 3-kinase activity but not insulin-induced tyrosine phosphorylation of the insulin receptor and IRS proteins in muscle. Impairment of PI 3-kinase function was associated with defective Akt/protein kinase B kinase activity (−40%, P < 0.01) in insulin-stimulated muscle of high fat–fed rats, despite unaltered phosphorylation (Ser473/Thr308) of the enzyme. Interestingly, basal activity of atypical protein kinase C (aPKC) was elevated in muscle of high fat–fed rats compared with chow-fed controls. Whereas insulin induced a twofold increase in aPKC kinase activity in the muscle of chow-fed rats, the hormone failed to further increase the kinase activity in high fat–fed rat muscle. In conclusion, it was found that GLUT4 translocation to both the plasma membrane and the T-tubules is impaired in the muscle of high fat–fed rats. We identified PI 3-kinase as the first step of the insulin signaling pathway to be impaired by high-fat feeding, and this was associated with alterations in both Akt and aPKC kinase activities.
Overexpression of Metallothionein in Pancreatic β-Cells Reduces Streptozotocin-Induced DNA Damage and Diabetes
Authors: Chen Hainan , Carlson Edward C. , Pellet Lori , Moritz Jon T. , Epstein Paul N.
Published: March 2007

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced β-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect β-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in β-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced β-cell death.
The Generalized Aminoaciduria Seen in Patients With Hepatocyte Nuclear Factor-1α Mutations Is a Feature of All Patients With Diabetes and Is Associated With Glucosuria
Authors: Bingham Coralie , Ellard Sian , Nicholls Anthony J. , Pennock Charles A. , Allen John , James Alan J. , Satchell Simon C. , Salzmann Maurice B. , Hattersley Andrew T.
Published: March 2007

Hepatocyte nuclear factor-1α (HNF-1α) mutations are the most common cause of maturity-onset diabetes of the young. HNF-1α homozygous knockout mice exhibit a renal Fanconi syndrome with glucosuria and generalized aminoaciduria in addition to diabetes. We investigated glucosuria and aminoaciduria in patients with HNF-1α mutations. Sixteen amino acids were measured in urine samples from patients with HNF-1α mutations, age-matched nondiabetic control subjects, and age-matched type 1 diabetic patients, type 2 diabetic patients, and patients with diabetes and chronic renal failure. The HNF-1α patients had glucosuria at lower glycemic control (as shown by HbA1c) than type 1 and type 2 diabetic patients, consistent with a lower renal glucose threshold. The HNF-1α patients had a generalized aminoaciduria with elevated levels of 14 of 16 amino acids and an increased mean Z score for all amino acids compared with control subjects (0.66 vs. 0.00; P < 0.0005). Generalized aminoaciduria was also present in type 1 diabetic (Z score, 0.80; P < 0.0001), type 2 diabetic (Z score, 0.71; P < 0.0002), and chronic renal failure (Z score, 0.65; P < 0.01) patients. Aminoaciduria was not associated with microalbuminuria or proteinuria but was associated with glucosuria (1.00 glucosuria vs. 0.19 no glucosuria; P = 0.002). In type 1 diabetic patients, urine samples taken on the same day showed significantly more aminoaciduria when glucosuria was present compared with when it was absent (P < 0.01). In conclusion, HNF-1α mutation carriers have a mutation-specific defect of proximal tubular glucose transport, resulting in increased glucosuria. In contrast, the generalized aminoaciduria seen in patients with HNF-1α mutations is a general feature of patients with diabetes and glucosuria. Glucose may depolarize and dissipate the electrical gradient of the sodium-dependent amino acid transporters in the proximal renal tubule, causing a reduction in amino acid resorption.
Use of a cDNA Array for the Identification of Genes Induced in Islets of Suckling Rats by a High-Carbohydrate Nutritional Intervention
Authors: Song Fei , Srinivasan Malathi , Aalinkeel Ravikumar , Patel Mulchand S.
Published: March 2007

Four-day-old rat pups that are raised artificially on a high-carbohydrate (HC) milk formula immediately develop hyperinsulinemia, which persists into adulthood without any further nutritional stimulus. cDNA array analysis was used to identify large-scale changes in gene expression patterns in islets from 12- and 100-day-old HC rats in response to the HC dietary modification during the suckling period. It was observed that the expression of several genes that belong to clusters involved in β-cell development and/or β-cell function was significantly upregulated in islets from 12- and 100-day-old HC rats. It is inferred that in addition to predicted changes in gene expression, for example preproinsulin gene, global changes in gene expression contribute to the hyperinsulinemic state in the HC rat.
Skeletal Muscle of Stroke-Prone Spontaneously Hypertensive Rats Exhibits Reduced Insulin-Stimulated Glucose Transport and Elevated Levels of Caveolin and Flotillin
Authors: James Declan J. , Cairns Fiona , Salt Ian P. , Murphy Gregory J. , Dominiczak Anna F. , Connell John M.C. , Gould Gwyn W.
Published: March 2007

Insulin resistance is of major pathogenic importance in several common human disorders, but the underlying mechanisms are unknown. The stroke-prone spontaneously hypertensive (SHRSP) rat is a model of human insulin resistance and is characterized by reduced insulin-mediated glucose disposal and defective fatty acid metabolism in isolated adipocytes (Collison et al. [Diabetes 49:2222–2226, 2000]). In this study, we have examined skeletal muscle and cultured skeletal muscle myoblasts for defects in insulin action in the male SHRSP rat model compared with the normotensive, insulin-sensitive control strain, Wistar-Kyoto (WKY). We show that skeletal muscle from SHRSP animals exhibits a marked decrease in insulin-stimulated glucose transport compared with WKY animals (fold increase in response to insulin: 1.4 ± 0.15 in SHRSP, 2.29 ± 0.22 in WKY; n = 4, P = 0.02), but the stimulation of glucose transport in response to activation of AMP-activated protein kinase was similar between the two strains. Similar reductions in insulin-stimulated glucose transport were also evident in myoblast cultures from SHRSP compared with WKY cultures. These differences were not accounted for by a reduction in cellular GLUT4 content. Moreover, analysis of the levels and subcellular distribution of insulin receptor substrates 1 and 2, the p85α subunit of phosphatidylinositol 3′-kinase, and protein kinase B (PKB)/cAKT in skeletal muscle did not identify any differences between the two strains; the insulin-dependent activation of PKB/cAKT was not different between the two strains. However, the total cellular levels of caveolin and flotillin, proteins implicated in insulin signal transduction/compartmentalization, were markedly elevated in skeletal muscles from SHRSP compared with WKY animals. Increased cellular levels of the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins syntaxin 4 and vesicle-associated membrane protein (VAMP)-2 were also observed in the insulin-resistant SHRSP strain. Taken together, these data suggest that the insulin resistance observed in the SHRSP is manifest at the level of skeletal muscle, that muscle cell glucose transport exhibits a blunted response to insulin but unchanged responses to activation of AMP-activated protein kinase, that alterations in key molecules in both GLUT4 trafficking and insulin signal compartmentalization may underlie these defects in insulin action, and that the insulin resistance of these muscles appears to be of genetic origin rather than a paracrine or autocrine effect, since the insulin resistance is also observed in cultured myoblasts over several passages.
Mutational Screening of the CART Gene in Obese Children
Authors: del Giudice Emanuele Miraglia , Santoro Nicola , Cirillo Grazia , D’Urso Luigi , Di Toro Rosario , Perrone Laura
Published: March 2007

Cocaine- and amphetamine-regulated transcript (CART) inhibits feeding and induces the expression of c-Fos in hypothalamic areas implicated in appetite regulation. Furthermore, the CART peptide is found in neurons regulating sympathetic outflow, which in turn play an integral role in regulating body temperature and energy expenditure. The CART gene was screened by single-strand conformation polymorphism and automatic sequencing in 130 (72 girls) unrelated obese Italian children and adolescents. Their Z-scores (mean ± SD) of relative to BMI percentiles was 3.9 ± 1.8, and the average age at obesity onset was 4.7 ± 2.6 years. Two previously described silent polymorphisms were found in the 3′ untranslated region: an adenine deletion at position 1457 in 9 patients (allele frequency 0.035) and an A/G substitution at position 1475 in 11 patients (allele frequency 0.042). We found no difference between the obese patients heterozygous for one of these polymorphisms and those patients homozygous for the wild allele with respect to their age of obesity onset, BMI Z-scores, and leptin levels. A missense mutation of G729C resulting in the substitution of Leu with Phe at codon 34, within the NH2-terminal CART region, has been detected in the heterozygous state in a 10-year-old obese boy who has been obese since the age of 2 years. The patient belongs to a large family of obese subjects. The mutation cosegregated with the severe obesity phenotype over three generations and was not found in the control population. Resting metabolic rates were lower than expected in the propositus (−14%) and his mother (−16%), who carried the mutation. Leucine at codon 34, conserved in this position in the human and in the rat sequences, immediately precedes a couple of lysine residues that may well represent a dibasic processing site. The Leu34Phe mutation might alter the susceptibility to proteolysis of this potential processing site, likely altering the CART effect on thermogenesis and energy expenditure.