bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin and Leptin Acutely Regulate Cholesterol Ester Metabolism in Macrophages by Novel Signaling Pathways
Authors: O’Rourke Lisa , Yeaman Steven J. , Shepherd Peter R.
Published: March 2007

Leptin is produced in adipose tissue and acts in the hypothalamus to regulate food intake. However, recent evidence also indicates a potential for direct roles for leptin in peripheral tissues, including those of the immune system. In this study, we provide direct evidence that macrophages are a target tissue for leptin. We found that J774.2 macrophages express the functional long form of the leptin receptor (ObRb) and that this becomes tyrosine-phosphorylated after stimulation with low doses of leptin. Leptin also stimulates both phosphoinositide 3-kinase (PI 3-kinase) activity and tyrosine phosphorylation of JAK2 and STAT3 in these cells. We investigated the effects of leptin on hormone-sensitive lipase (HSL), which acts as a neutral cholesterol esterase in macrophages and is a rate-limiting step in cholesterol ester breakdown. Leptin significantly increased HSL activity in J774.2 macrophages, and these effects were additive with the effects of cAMP and were blocked by PI 3-kinase inhibitors. Conversely, insulin inhibited HSL in macrophages, but unlike adipocytes, this effect did not require PI 3-kinase. These results indicate that leptin and insulin regulate cholesterol-ester homeostasis in macrophages and, therefore, defects in this process caused by leptin and/or insulin resistance could contribute to the increased incidence of atherosclerosis found associated with obesity and type 2 diabetes.
Chloride Channels Regulate HIT Cell Volume but Cannot Fully Account for Swelling-Induced Insulin Secretion
Authors: Kinard Tracie A. , Goforth Paulette B. , Tao Qing , Abood Mary E. , Teague Jeanette , Satin Leslie S.
Published: March 2007

Insulin-secreting pancreatic islet β-cells possess anion-permeable Cl− channels (ICl,islet) that are swelling-activated, but the role of these channels in the cells is unclear. The Cl− channel blockers 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and niflumic acid were evaluated for their ability to inhibit ICl,islet in clonal β-cells (HIT cells). Both drugs blocked the channel, but the blockade due to niflumic acid was less voltage-dependent than the blockade due to DIDS. HIT cell volume initially increased in hypotonic solution and was followed by a regulatory volume decrease (RVD). The addition of niflumic acid and, to a lesser extent, DIDS to the hypotonic solution potentiated swelling and blocked the RVD. In isotonic solution, niflumic acid produced swelling, suggesting that islet Cl− channels are activated under basal conditions. The channel blockers glyburide, gadolinium, or tetraethylammonium-Cl did not alter hypotonic-induced swelling or volume regulation. The Na/K/2Cl transport blocker furosemide produced cell shrinkage in isotonic solution and blocked cell swelling normally induced by hypotonic solution. Perifused HIT cells secreted insulin when challenged with hypotonic solutions. However, this could not be completely attributed to ICl,islet-mediated depolarization, because secretion persisted even when Cl− channels were fully blocked. To test whether blocker-resistant secretion occurred via a distal pathway, distal secretion was isolated using 50 mmol/l potassium and diazoxide. Under these conditions, glucose-dependent secretion was blunted, but hypotonically induced secretion persisted, even with Cl− channel blockers present. These results suggest that β-cell swelling stimulates insulin secretion primarily via a distal ICl,islet-independent mechanism, as has been proposed for KATP-independent glucose- and sulfonylurea-stimulated insulin secretion. Reverse transcriptase–polymerase chain reaction of HIT cell mRNA identified a CLC-3 transcript in HIT cells. In other systems, CLC-3 is believed to mediate swelling-induced outwardly rectifying Cl− channels. This suggests that the proximal effects of swelling to regulate cell volume may be mediated by CLC-3 or a closely related Cl− channel.
Pancreatic Biopsy as a Procedure for Detecting In Situ Autoimmune Phenomena in Type 1 Diabetes
Authors: Imagawa Akihisa , Hanafusa Toshiaki , Tamura Shinji , Moriwaki Makoto , Itoh Naoto , Yamamoto Koji , Iwahashi Hiromi , Yamagata Kazuya , Waguri Masako , Nanmo Takao , Uno Sae , Nakajima Hiromu , Namba Mitsuyoshi , Kawata Sumio , Miyagawa Jun-ichiro , Matsuzawa Yuji
Published: March 2007

To better understand the pathogenesis of type 1 diabetes, we have developed pancreatic biopsy under laparoscope for recent-onset type 1 diabetic patients. The patients included 29 acute-onset type 1 diabetic patients, 5 latent-onset type 1 diabetic patients, and 1 type 2 diabetic patient. Their median age was 28 years, and the duration of diabetes at the time of biopsy was ∼3 months. In 31 of 35 patients, we could obtain the pancreas tissue by punching. No serious complications, such as heavy bleeding, peritonitis, or pancreatitis, have been experienced. Pneumoderma was observed in two patients, and abdominal dull pain had continued for 2 days in two patients. However, special treatment was not necessary for these complications. T-cell–predominant infiltration to islets (insulitis) and hyperexpression of major histocompatibility complex class I antigens on islet cells were the two major findings and were observed in 17 of 29 recent-onset type 1 diabetic patients. These findings could be regarded as evidence of immune attack against β-cells, and their presence was closely correlated with the presence of either anti-GAD or anti–IA-2 antibodies (P = 0.02). In conclusion, pancreatic biopsy under laparoscope is a safe procedure without serious complications, according to our findings, for detecting in situ autoimmune phenomenon in recent-onset type 1 diabetic patients.
High Glucose Causes Apoptosis in Cultured Human Pancreatic Islets of Langerhans
Authors: Federici Massimo , Hribal Marta , Perego Lucia , Ranalli Marco , Caradonna Zaira , Perego Carla , Usellini Luciana , Nano Rita , Bonini Paolo , Bertuzzi Federico , Marlier Lionel N.J.L. , Davalli Alberto M. , Carandente Orazio , Pontiroli Antonio E. , Melino Gerry , Marchetti Piero , Lauro Renato , Sesti Giorgio , Folli Franco
Published: March 2007

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, β-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased β-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of β-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days’ culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase–polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in β-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring β-cell death.
The HIV Protease Inhibitor Indinavir Impairs Sterol Regulatory Element-Binding Protein-1 Intranuclear Localization, Inhibits Preadipocyte Differentiation, and Induces Insulin Resistance
Authors: Caron Martine , Auclair Martine , Vigouroux Corinne , Glorian Martine , Forest Claude , Capeau Jacqueline
Published: March 2007

Protease inhibitors used in the treatment of HIV infection have been causally associated with lipodystrophy and insulin resistance and were shown to alter adipocyte differentiation in cultured cells. We aimed to delineate the mechanism by which indinavir impaired adipocyte function. We report that indinavir altered neither the growth nor insulin sensitivity of 3T3-F442A preadipocytes, nor did it alter the initial step of their differentiation, i.e., clonal proliferation. However, adipose conversion was inhibited by indinavir (by 50–60%), as shown by 1) the decrease in the number of newly formed adipocytes; 2) the lower level of the adipogenic protein markers, sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator–activated receptor-γ (PPAR-γ), and the insulin receptor (IR); and 3) the lack of SREBP-1 and PPAR-γ immunoreactivity in the nucleus of most indinavir-treated cells. Partial adipose conversion also correlated with an accumulation of SREBP-1 at the nuclear periphery and an alteration in its electrophoretic mobility. Defective expression and nuclear localization of PPAR-γ probably resulted from the decreased level of nuclear SREBP-1. Indinavir also rendered 3T3-F442A adipocytes resistant to insulin for mitogen-activated protein kinase activation at a step distal to IR substrate-1 tyrosine phosphorylation. Hence, indinavir impairs differentiation at an early step of adipose conversion probably involving the process controlling SREBP-1 intranuclear localization.
The HIV Protease Inhibitor Indinavir Decreases Insulin- and Contraction-Stimulated Glucose Transport in Skeletal Muscle
Authors: Nolte Lorraine A. , Yarasheski Kevin E. , Kawanaka Kentaro , Fisher Jonathan , Le Ngan , Holloszy John O.
Published: March 2007

In many patients with human immunodeficiency virus (HIV) treated with HIV protease inhibitors, a complication develops that resembles abdominal obesity syndrome, with insulin resistance and glucose intolerance that, in some cases, progresses to diabetes. In this study, we tested the hypothesis that indinavir, an HIV-protease inhibitor, directly induces insulin resistance of glucose transport in skeletal muscle. Rat epitrochlearis muscles were incubated with a maximally effective insulin concentration (12 nmol/l) and 0, 1, 5, 20, or 40 μmol/l indinavir for 4 h. In control muscles, insulin increased 3-O-[3H]methyl-d-glucose (3MG) transport from 0.15 ± 0.03 to 1.10 ± 0.05 μmol · ml−1 · 10 min−1. Incubation of muscles with 5 μmol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 40%, whereas 20 μmol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 58%. Indinavir induced a similar reduction in maximally insulin-stimulated 3MG transport in the soleus muscle. The increase in glucose transport activity induced by stimulating epitrochlearis muscles to contract was also markedly reduced by indinavir. The insulin-stimulated increase in cell-surface GLUT4, assessed using the 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-[2-3H] (d-mannose-4-yloxy)-2-propylamine exofacial photolabeling technique, was reduced by ∼70% in the presence of 20 μmol/l indinavir. Insulin stimulation of phosphatidylinositol 3-kinase activity and phosphorylation of protein kinase B were not decreased by indinavir. These results provide evidence that indinavir inhibits the translocation or intrinsic activity of GLUT4 rather than insulin signaling.
Increased Prevalence of Microthromboses in Retinal Capillaries of Diabetic Individuals
Authors: Boeri Daria , Maiello Michele , Lorenzi Mara
Published: March 2007

In diabetic retinopathy, capillary nonperfusion and eventual obliteration can lead to retinal ischemia and sight-threatening neovascularization. The occurrence of retinal microthrombosis in human diabetes has long been suspected and occasionally observed but never systematically demonstrated. We used trypsin digestion to isolate the intact vascular network from retinas obtained postmortem from nine diabetic donors (age 64 ± 11 years, duration of diabetes 6 ± 4 years; mean ± SD) and eight age-matched nondiabetic donors. Topographically matched sectors (each one-sixth of retina) of diabetic and nondiabetic retinas were tested sequentially with antibodies to fibrin cross-linking factor XIII and platelet glycoprotein (GP)-IIIa to identify fibrin-platelet thrombi. In some trypsin digests, we also examined vascular cell apoptosis. The retina from a nondiabetic donor, 24 years of age, who had died of trauma, was used to exclude confounding influences caused by the postmortem period. When compared with those of nondiabetic donors, the retinas of diabetic donors showed double the number of capillary segments with colocalized immunostaining for factor XIII and GPIIIa (P = 0.02). The total area of the positive segments was fourfold greater in the diabetic than in the nondiabetic donors (P = 0.02) and correlated with the duration of diabetes (r = 0.71, P < 0.05). Large thrombi were six times more frequent in the diabetic donors (P = 0.03), and there was a significant topographical association of microthrombosis with apoptotic cells in both diabetic and nondiabetic vessels (P = 0.0001). Hence, diabetes of short duration was found to be associated with a greater than normal number and size of platelet-fibrin thrombi in the retinal capillaries. These thrombi can contribute to capillary obliteration and retinal ischemia and may be a practical target for early drug intervention.
Hypoxia Stimulates Osteopontin Expression and Proliferation of Cultured Vascular Smooth Muscle Cells
Authors: Sodhi Chhinder P. , Phadke Sarojini A. , Batlle Daniel , Sahai Atul
Published: March 2007

We examined the effect of hypoxia on proliferation and osteopontin (OPN) expression in cultured rat aortic vascular smooth muscle (VSM) cells. In addition, we determined whether hypoxia-induced increases in OPN and cell proliferation are altered under hyperglycemic conditions. Quiescent cultures of VSM cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and cell proliferation as well as the expression of OPN was assessed. Cells exposed to hypoxia for 24 h exhibited a significant increase in [3H]thymidine incorporation followed by a significant increase in cell number at 48 h in comparison with respective normoxic controls. Exposure to hypoxia produced significant increases in OPN protein and mRNA expression at 2 h followed by a gradual decline at 6 and 12 h, with subsequent significant increases at 24 h. Neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin prevented the hypoxia-induced increase in [3H]thymidine incorporation. Inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein (MAP) kinase also reduced the hypoxia-induced stimulation of proliferation and OPN synthesis. Exposure to high-glucose (HG) (25 mmol/l) medium under normoxic conditions also resulted in significant increases in OPN protein and mRNA levels as well as the proliferation of VSM cells. Under hypoxic conditions, HG further stimulated OPN synthesis and cell proliferation in an additive fashion. In conclusion, hypoxia-induced proliferation of cultured VSM cells is mediated by the stimulation of OPN synthesis involving PKC and p38 MAP kinase. In addition, hypoxia also enhances the effect of HG conditions on both OPN and proliferation of cultured VSM cells, which may have important implications in the development of diabetic atherosclerosis associated with arterial wall hypoxia.
Hyperglycemia Potentiates Collagen-Induced Platelet Activation Through Mitochondrial Superoxide Overproduction
Authors: Yamagishi Sho-ichi , Edelstein Diane , Du Xue-liang , Brownlee Michael
Published: March 2007

Alteration of platelet function contributes to microthrombus formation and may play an important role in the pathogenesis of diabetic micro- and macroangiopathies. However, the molecular mechanism for platelet dysfunction observed in patients with diabetes has not been fully elucidated. In this study, the direct effects of hyperglycemia on platelet function in vitro were investigated. Hyperglycemia increased reactive oxygen species generation in human platelets, and this effect was additive with that of collagen. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial electron transport chain complex II, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely prevented the effects of hyperglycemia, suggesting that reactive oxygen species arise from the mitochondrial electron transport chain. Hyperglycemia potentiated both platelet aggregation and the subsequent release of platelet-derived growth factor AB induced by a nonaggregating subthreshold concentration of collagen, which were also completely inhibited by TTFA or CCCP. Furthermore, hyperglycemia was found to inhibit protein tyrosine phosphatase (PTP) activity and increase phosphorylation of the tyrosine kinase Syk in platelets exposed to collagen. Hyperglycemia-induced PTP inhibition and Syk phosphorylation were found to be completely prevented by TTFA, CCCP, or Mn(III)tetrakis (4-benzoic acid) porphyrin, a stable cell-permeable superoxide dismutase mimetic. These results suggest that hyperglycemia-induced mitochondrial superoxide generation may play an important role in platelet dysfunction observed in patients with diabetes.
Dipeptidyl Peptidase IV Inhibition Reduces the Degradation and Clearance of GIP and Potentiates Its Insulinotropic and Antihyperglycemic Effects in Anesthetized Pigs
Authors: Deacon Carolyn F. , Danielsen Patricia , Klarskov Letty , Olesen Mette , Holst Jens J.
Published: March 2007

Glucose-dependent insulinotropic peptide (GIP) is known to be degraded by dipeptidyl peptidase IV (DPP IV), forming an inactive metabolite, but the extent of the enzyme’s role in regulating the biological activity of GIP in vivo is still largely unknown. In nonfasted anesthetized pigs given an intravenous infusion of GIP, the intact peptide (determined by a novel NH2-terminally directed radioimmunoassay) accounts for only 14.5 ± 2.5% of total immunoreactivity. This is increased (to 40.9 ± 0.9%, P < 0.0001) by coadministration of valine-pyrrolidide (a specific DPP IV inhibitor) at a dose that completely inhibits plasma DPP IV activity. The plasma t1/2 of intact GIP is prolonged by the inhibitor (from 3.3 ± 0.3 to 8.1 ± 0.6 min; P < 0.001), whereas the t1/2 for COOH-terminal immunoreactivity is unaffected (13.2 ± 0.5 and 11.5 ± 0.8 min, pre- and postinhibitor). Measurement of arteriovenous concentration differences revealed that the liver, kidney, and extremities are the main sites of removal of exogenous intact GIP (organ extractions, 28.0 ± 2.2, 26.3 ± 5.7, and 21.8 ± 3.0%, respectively). These organ extractions are reduced (P < 0.02) but not eliminated (kidney and extremities) by valine-pyrrolidide (to 6.5 ± 4.6, 14.1 ± 3.1, and 13.9 ± 2.4%, respectively). Valine-pyrrolidide potentiates the insulinotropic effect of GIP (P < 0.02), resulting in an enhanced glucose disappearance rate (k, from 8.0 ± 0.5 to 15.5 ± 2.2%/min; P < 0.01) and a reduction in the glucose excursion after an intravenous glucose load (area under the curve, from 133 ± 23 to 75 ± 9 min · mmol/l; P < 0.05). These results suggest that DPP IV plays an important role in GIP metabolism but is not the sole enzyme responsible for its NH2-terminal degradation. Nevertheless, DPP IV inhibition increases the proportion of intact peptide sufficiently to enhance its insulinotropic and antihyperglycemic effects.
Proteins Linked to a Protein Transduction Domain Efficiently Transduce Pancreatic Islets
Authors: Embury Jennifer , Klein Dagmar , Pileggi Antonello , Ribeiro Melina , Jayaraman Sundararajan , Molano R. Damaris , Fraker Christopher , Kenyon Norma , Ricordi Camillo , Inverardi Luca , Pastori Ricardo L.
Published: March 2007

The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused β-galactosidase to the 11–amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-XL and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-α in a pancreatic β-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient mice.
Expression of GAD65 and Islet Cell Antibody (ICA512) Autoantibodies Among Cytoplasmic ICA+ Relatives Is Associated With Eligibility for the Diabetes Prevention Trial−Type 1
Authors: Yu Liping , Cuthbertson David D. , Maclaren Noel , Jackson Richard , Palmer Jerry P. , Orban Tihamer , Eisenbarth George S. , Krischer Jeffrey P. ,   and the DPT-1 Participating Investigators
Published: March 2007

More than 71,000 relatives of type 1 diabetic patients have been screened for cytoplasmic islet cell antibodies (ICAs), GAD65 autoantibodies (GAAs), and ICA512 autoantibodies (ICA512AAs). Among those 71,148 relatives, 2,448 were cytoplasmic ICA+, and the remainder were ICA−. Of the ICA+ group, 1,229 (50.2%) were positive for GAAs and/or ICA512AAs. Among ICA− relatives, 1,897 (2.76%) were positive for GAAs and/or ICA512AAs. Given the large number of relatives positive for cytoplasmic ICA and negative for “biochemically” determined autoantibodies, and the converse, we analyzed the proportion of ICA+ relatives found eligible to participate in the intervention phase of Diabetes Prevention Trial−Type 1 (DPT-1). To be eligible for the parenteral insulin DPT-1 trial, a relative had to have first-phase insulin secretion below the 1st percentile of cut-points (for parents) or below the 10th percentile (for siblings and offspring). To be eligible for the oral insulin trial, a relative had to have first-phase insulin secretion above cut-points (>1st percentile for parents, >10th percentile for siblings/offspring) and be positive for anti-insulin autoantibodies. For both trials, DQB1*0602 was an exclusion criteria, cytoplasmic ICA positivity had to be confirmed, and an oral glucose tolerance test had to result in nondiabetic levels. Of 572 relatives found to be eligible for trial entry, 442 (77.3%) were positive for GAAs and/or ICA512AAs, although overall only 50.2% of ICA+ relatives were positive for GAAs and/or ICA512AAs. The positive predictive value for trial eligibility for ICA+ relatives with GAAs or ICA512AAs who completed staging was 51.0%. In contrast, only 11.9% of ICA+ but GAA− and ICA512AA− relatives were found to be eligible by DPT criteria for trial entry. Positivity for biochemically determined autoantibodies among cytoplasmic antibody–positive relatives is associated with eligibility for the DPT-1 study.
Characterization of Preparations of GAD65, Proinsulin, and the Islet Tyrosine Phosphatase IA-2 for Use in Detection of Autoreactive T-Cells in Type 1 Diabetes
Authors: Peakman Mark , Tree Timothy I. , Endl Josef , van Endert Peter , Atkinson Mark A. , Roep Bart O.
Published: March 2007

The identification, quantification, and characterization of T-cells reactive with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphatase–like molecules IA-2 and phogrin are major research goals in type 1 diabetes. In the Immunology of Diabetes Society First Workshop on Autoreactive T-Cells, the quality of recombinant preparations of these autoantigens was identified as a significant weakness, a finding that may account for much of the inconsistency in published studies of peripheral blood T-cell reactivity to islet autoantigens. Poor antigen quality has also hampered the development of novel technologies for the detection of islet-reactive T-cells. For these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to stimulate a panel of relevant T-cell clones, and inhibitory effects on proliferation to unrelated third-party antigens. Through this process, we have been able to identify preparations of GAD65 and IA-2, generated in insect cells using the baculovirus expression system, that stimulate relevant clones and display low inhibitory effects on third-party antigens. In addition, we characterized a PI preparation generated in bacteria as being free of effects on proliferation to third-party antigens and low in endotoxin content. These preparations are important to promote the development of robust and sensitive assays of islet-reactive T-cells in patients with type 1 diabetes or patients at high risk for developing the disease.
Nerve Growth Factor Increases Insulin Secretion and Barium Current in Pancreatic β-Cells
Authors: Rosenbaum T. , Sánchez-Soto M.C. , Hiriart M.
Published: March 2007

We analyzed the effect of a brief exposure to nerve growth factor (NGF) on insulin secretion and macroscopic barium currents of single adult rat pancreatic β-cells. After a 1-h exposure to NGF (50 ng/ml), single β-cells show a 2.5-fold increase in the insulin secretion index in 5.6 mmol/l glucose and a nearly twofold increase in 15.6 mmol/l glucose compared with control cells. We have recently demonstrated that pancreatic β-cells synthesize and secrete NGF. We analyzed the effect of endogenous NGF on insulin secretion by incubating islet cells in the presence of an anti-NGF monoclonal antibody for 1 h in different glucose concentrations. Although the basal insulin secretion index (5.6 mmol/l glucose) is not affected, glucose-stimulated insulin secretion (15.6 mmol/l glucose) is decreased by 41% in the presence of the antibody. This effect is mediated by the activation of the NGF receptor TrkA because the specific inhibitor of Trk phosphorylation K252a also blocks NGF-induced increase in insulin secretion, both in the presence and absence of exogenous NGF. Using the whole-cell variation of the patch-clamp technique, we found that cells exposed to NGF for 5 min exhibit a 32% increase in the average barium current density. These results suggest that the effects of NGF on insulin secretion are partially mediated by an increase in calcium current through Ca channels. These results further suggest that NGF plays an important autoregulatory role in pancreatic β-cell function. Two targets of short-term NGF-modulation are insulin secretion and calcium-channel activity.
Effect of Physiological Hyperinsulinemia on Gluconeogenesis in Nondiabetic Subjects and in Type 2 Diabetic Patients
Authors: Gastaldelli Amalia , Toschi Elena , Pettiti Maura , Frascerra Silvia , Quiñones-Galvan Alfredo , Sironi Anna Maria , Natali Andrea , Ferrannini Ele
Published: March 2007

Gluconeogenesis (GNG) is enhanced in type 2 diabetes. In experimental animals, insulin at high doses decreases the incorporation of labeled GNG precursors into plasma glucose. Whether physiological hyperinsulinemia has any effect on total GNG in humans has not been determined. We combined the insulin clamp with the 2H2O technique to measure total GNG in 33 subjects with type 2 diabetes (BMI 29.0 ± 0.6 kg/m2, fasting plasma glucose 8.1 ± 0.3 mmol/l) and in 9 nondiabetic BMI-matched subjects after 16 h of fasting and after euglycemic hyperinsulinemia. A primed-constant infusion of 6,6-2H-glucose was used to monitor endogenous glucose output (EGO); insulin (40 mU · min−1 · m−2) was then infused while clamping plasma glucose for 2 h (at 5.8 ± 0.1 and 4.9 ± 0.2 mmol/l for diabetic and control subjects, respectively). In the fasting state, EGO averaged 15.2 ± 0.4 μmol · min−1 · kgffm−1 (62% from GNG) in diabetic subjects and 12.2 ± 0.7 μmol · min−1 · kgffm−1 (55% from GNG) in control subjects (P < 0.05 or less for both fluxes). Glycogenolysis (EGO – GNG) was similar in the two groups (P = NS). During the last 40 min of the clamp, both EGO and GNG were significantly (P < 0.01 or less, compared with fasting) inhibited (EGO 7.1 ± 0.9 and 3.6 ± 0.5 and GNG 7.9 ± 0.5 and 4.5 ± 1.0 μmol · min−1 · kgffm−1 in diabetic and control subjects, respectively) but remained significantly (P < 0.05) higher in diabetic subjects, whereas glycogenolysis was suppressed completely and equally in both groups. During hyperinsulinemia, GNG was reciprocally related to plasma glucose clearance. In conclusion, physiological hyperinsulinemia suppresses GNG by ∼20%, while completely blocking glycogenolysis. Resistance of GNG (to insulin suppression) and resistance of glucose uptake (to insulin stimulation) are coupled phenomena. In type 2 diabetes, the excess GNG of the fasting state is carried over to the insulinized state, thereby contributing to glucose overproduction under both conditions.
Low Acute Insulin Secretory Responses in Adult Offspring of People With Early Onset Type 2 Diabetes
Authors: Gautier Jean-François , Wilson Charlton , Weyer Christian , Mott Dave , Knowler William C. , Cavaghan Melissa , Polonsky Kenneth S. , Bogardus Clifton , Pratley Richard E.
Published: March 2007

The offspring of Pima Indians with early onset type 2 diabetes are at high risk for developing diabetes at an early age. This risk is greater among those whose mothers were diabetic during pregnancy. To define the metabolic abnormalities predisposing individuals in these high-risk groups to diabetes, we conducted a series of studies to measure insulin secretion and insulin action in healthy adult Pima Indians. In 104 normal glucose-tolerant subjects, acute insulin secretory response (AIR) to a 25-g intravenous glucose challenge correlated with the age at onset of diabetes in the mother (r = 0.23, P = 0.03) and, in multiple regression analyses, the age at onset of diabetes in the father (P = 0.02), after adjusting for maternal age at onset and after allowing for an interaction between these terms. In contrast, insulin action (hyperinsulinemic glucose clamp) did not correlate with the age at onset of diabetes in the parents. To determine whether early onset diabetes in the parents affected insulin secretion in the offspring across a range of glucose concentrations, responses to a stepped glucose infusion were measured in 23 subjects. Insulin secretion rates were lower in individuals whose mothers had developed diabetes before 35 years of age (n = 8) compared with those whose parents remained nondiabetic until at least 49 years of age (n = 15) (average insulin secretory rates: geometric mean [95% CI] 369 [209–652] vs. 571 [418–780] pmol/min, P = 0.007). Finally, the AIR was lower in individuals whose mothers were diabetic during pregnancy (n = 8) than in those whose mothers developed diabetes at an early age but after the birth of the subject (n = 41) (740 [510–1,310] vs. 1,255 [1,045–1,505] pmol/l, P < 0.02). Thus, insulin secretion is lower in normal glucose tolerant offspring of people with early onset type 2 diabetes. This impairment may be worsened by exposure to a diabetic environment in utero.
Glucose Regulates the Transcription of Human Genes Relevant to HDL Metabolism
Authors: Tu An-Yue , Albers John J.
Published: March 2007

Phospholipid transfer protein (PLTP) plays an important role in human plasma HDL metabolism. Clinical data have recently indicated that plasma PLTP activity and mass were both higher in diabetic patients concomitant with hyperglycemia. The present study shows that high glucose increases both PLTP mRNA and functional activity in HepG2 cells, due to a significant increase in the promoter activity of human PLTP gene. The glucose-responsive elements are located between −759 and −230 of the PLTP 5′-flanking region, within which two binding motifs (−537 to –524 and −339 to –327) for either peroxisome proliferator–activated receptor or farnesoid X-activated receptor are involved in this glucose-mediated transcriptional regulation. This finding suggests that high glucose upregulates the transcription of human PLTP gene via nuclear hormone receptors. In addition, high glucose increases mRNA levels for several genes that are functionally important in HDL metabolism, including human ATP-binding cassette transporter A1, apolipoprotein A-I, scavenger receptor BI, and hepatic lipase. The functional promoter activities of these genes are enhanced by high glucose in three cell lines tested, indicating that glucose may also regulate these genes at the transcriptional level. Our findings provide a molecular basis for a role of hyperglycemia in altered HDL metabolism.
The Neuropeptide Pituitary Adenylate Cyclase–Activating Polypeptide and Islet Function
Authors: Filipsson Karin , Kvist-Reimer Martina , Ahrén Bo
Published: March 2007

The neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) is ubiquitously distributed in both the central and peripheral nervous systems and exerts a variety of effects. PACAP is a neuropeptide in pancreatic islets, where it has been suggested as a parasympathetic and sensory neurotransmitter. PACAP stimulates insulin secretion in a glucose-dependent manner, by an effect executed mainly through augmenting the formation of cAMP and stimulating the uptake of calcium. Accumulating evidence in animal studies points to a physiological importance of PACAP in the regulation of the insulin response to feeding. This review summarizes the current knowledge of islet actions and mechanisms and the function of PACAP.
A Role for Hormone-Sensitive Lipase in Glucose-Stimulated Insulin Secretion
Authors: Roduit Raphaël , Masiello Pellegrino , Wang Shu Pei , Li Hong , Mitchell Grant A. , Prentki Marc
Published: March 2007

Endogenous lipid stores are thought to be involved in the mechanism whereby the β-cell adapts its secretory capacity in obesity and diabetes. In addition, hormone-sensitive lipase (HSL) is expressed in β-cells and may provide fatty acids necessary for the generation of coupling factors linking glucose metabolism to insulin release. We have recently created HSL-deficient mice that were used to directly assess the role of HSL in insulin secretion and action. HSL−/− mice were normoglycemic and normoinsulinemic under basal conditions, but showed an ∼30% reduction of circulating free fatty acids (FFAs) with respect to control and heterozygous animals after an overnight fast. An intraperitoneal glucose tolerance test revealed that HSL-null mice were glucose-intolerant and displayed a lack of a rise in plasma insulin after a glucose challenge. Examination of plasma glucose during an insulin tolerance test suggested that HSL-null mice were insulin-resistant, because plasma glucose was barely lowered after the injection of insulin. Freshly isolated islets from HSL-deficient mice displayed elevated secretion at low (3 mmol/l) glucose, failed to release insulin in response to high (20 mmol/l) glucose, but had a normal secretion when challenged with elevated KCl. The phenotype of heterozygous mice with respect to the measured parameters in vitro was similar to that of wild type. Finally, the islet triglyceride content of HSL−/− mice was 2–2.5 fold that in HSL−/+ and HSL+/+ animals. The results demonstrate an important role of HSL and endogenous β-cell lipolysis in the coupling mechanism of glucose-stimulated insulin secretion. The data also provide direct support for the concept that some lipid molecule(s), such as FFAs, fatty acyl-CoA or their derivatives, are implicated in β-cell glucose signaling.
Expression of the Receptor Tyrosine Kinase KIT in Mature β-Cells and in the Pancreas in Development
Authors: Rachdi Latif , El Ghazi Lynda , Bernex Florence , Panthier Jean-Jacques , Czernichow Paul , Scharfmann Raphael
Published: March 2007

In the pancreas, ligands of receptor tyrosine kinases (RTKs) are thought to be implicated in the development and function of the islets of Langerhans, which represent the endocrine part of the pancreas. In a previous study, we randomly screened by reverse transcriptase−polymerase chain reaction for RTKs expressed in the embryonic pancreas. One cDNA fragment that was cloned during this screen corresponded to the KIT receptor. The objective of the present study was to analyze the pattern of Kit expression in the pancreas. We demonstrated that Kit is expressed and functional in terms of signal transduction in the insulin-producing cell line INS-1. Indeed, upon treatment with the KIT ligand (KITL), the extracellular signal-regulated protein kinase was phosphorylated, and the expression of early responsive genes was induced. We also demonstrated that Kit mRNAs are present in fetal and adult rat islets. We next used mice that had integrated the lacZ reporter gene into the Kit locus. In these mice, β-galactosidase (β-gal) served as a convenient marker for expression of the endogenous Kit gene. Kit was found to be specifically transcribed in β-cells (insulin-expressing cells), whereas no expression was found in other endocrine cell types or in the exocrine tissue. Interestingly, not all mature β-cells expressed Kit, indicating that Kit is a marker of a subpopulation of β-cells. Finally, by following β-gal expression in the pancreas during fetal life, we found that at E14.5, Kit is expressed in both insulin- and glucagon-expressing cells present at that stage, and also in a specific cell population present in the epithelium that stained negative for endocrine markers. These data suggest that these Kit-positive/endocrine-negative cells could represent a subpopulation of endocrine cell precursors.
Reduced Oral Wound Healing in the NOD Mouse Model for Type 1 Autoimmune Diabetes and Its Reversal by Epidermal Growth Factor Supplementation
Authors: Nagy Akos , Nagashima Hiroyuki , Cha Seunghee , Oxford Gregory E. , Zelles Tivadar , Peck Ammon B. , Humphreys-Beher Michael G.
Published: March 2007

Using the NOD mouse, a model for type 1 diabetes, we examined how reduced concentrations of epidermal growth factor (EGF) in the saliva, after onset of type 1 diabetes, affect oral wound healing. Diabetic NOD/LtJ mice on insulin therapy, prediabetic NOD/LtJ, and age- and sex-matched BALB/cJ mice were given a cutaneous tongue punch and allowed to undergo normal healing. With diabetes onset and a reduction in saliva-derived growth factor levels, the rate of tongue wound healing was reduced compared with nondiabetic NOD/LtJ and healthy BALB/cJ mice. Addition of exogenous EGF to the drinking water did not accelerate the rate of healing in BALB/cJ or prediabetic NOD/LtJ; however, diabetic NOD/LtJ mice exhibited accelerated wound healing similar to healthy mice. These results demonstrate that loss of growth factors from saliva is associated with profoundly reduced oral wound healing, suggesting that therapeutic treatment with topical delivery may be beneficial to patients with type 1 diabetes and oral wound complications.
Depressed Levels of Ca2+-Cycling Proteins May Underlie Sarcoplasmic Reticulum Dysfunction in the Diabetic Heart
Authors: Netticadan Thomas , Temsah Rana M. , Kent Ardeep , Elimban Vijayan , Dhalla Naranjan S.
Published: March 2007

In view of the depressed sarcoplasmic reticulum (SR) Ca2+-pump and Ca2+-release activities in the diabetic heart and the critical role of phosphorylation in regulating the SR function, we examined the status of Ca2+-calmodulin–dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA)-mediated phosphorylations in the diabetic heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (65 mg/kg i.v.), and the animals were killed 6 weeks later for assessment of the ventricular SR function. Depressed cardiac performance and SR Ca2+-uptake and -release activities in diabetic animals were accompanied by a significant decrease in the level of SR Ca2+-cycling proteins, such as ryanodine receptor, Ca2+-pump ATPase, and phospholamban. On the other hand, the CaMK- and PKA-mediated phosphorylations of these Ca2+-cycling proteins, the endogenous SR CaMK and PKA activities, and the endogenous SR and cytosolic phosphatase activities were increased in the diabetic heart. Treatment of 3-week diabetic animals with insulin partially or fully prevented the diabetes-induced changes in cardiac performance, SR Ca2+-uptake and -release activites, and SR protein content, whereas the diabetes-induced changes in SR CaMK- and PKA-mediated phosphorylations and activities, as well as phosphatase activities, were not significantly affected. These results suggest that the reduced content of the Ca2+-cycling proteins, unlike alterations in PKA and phosphatase activities, appear to be the major defect underlying SR dysfunction in the diabetic heart.
Intrahepatic Mechanisms Underlying the Effect of Metformin in Decreasing Basal Glucose Production in Rats Fed a High-Fat Diet
Authors: Mithieux Gilles , Guignot Ludovic , Bordet Jean-Claude , Wiernsperger Nicolas
Published: March 2007

The aim of this study was to understand by which intrahepatic mechanism metformin (Met) may inhibit basal hepatic glucose production (HGP) in type 2 diabetes. We studied rats that were fed for 6 weeks a high-fat (HF) diet, supplemented (HF-Met) or not (HF) with Met (50 mg · kg−1 · day−1). Basal HGP, assessed by 3-[3H]glucose tracer dilution, was lower by 20% in HF-Met rats compared with HF-rats: 41.6 ± 0.7 vs. 52 ± 1.5 μmol · kg−1 · min−1 (means ± SE, n = 5; P < 0.01). Glucose-6 phosphatase (Glc6Pase) activity, assayed in a liver lobe freeze-clamped in situ, was lower by 25% in HF-Met rats compared with HF-rats (7.9 ± 0.4 vs. 10.3 ± 0.9 μmol · min−1 · g−1 wet liver; P < 0.05). Glucose-6 phosphate and glycogen contents, e.g., 42 ± 5 nmol/g and 3.9 ± 2.4 mg/g, respectively, in HF-rats were dramatically increased by three to five times in HF-Met rats, e.g., 118 ± 12 nmol/g and 19.6 ± 4.6 mg/g (P < 0.05 and P < 0.01, respectively). Glucose-6 phosphate dehydrogenase activity was increased in HF-Met compared with HF rats (1.51 ± 0.1 vs. 1.06 ± 0.08 μmol · min−1 · g−1; P < 0.01). Intrahepatic lactate concentration tended to be lower in the Met-group (−30%; NS), whereas plasma lactate concentration was higher in HF-Met rats (1.59 ± 0.15 mmol/l) than in HF rats (1.06 ± 0.06 mmol/l; P < 0.05). We concluded that Met decreases HGP in insulin-resistant HF-fed rats mainly by an inhibition of hepatic Glc6Pase activity, promoting glycogen sparing. Additional mechanisms might involve the diversion of glucose-6 phosphate into the pentose phosphate pathway and an inhibition of hepatic lactate uptake.
Insulin Resistance in Morbid Obesity
Authors: Greco Aldo V. , Mingrone Gertrude , Giancaterini Annalisa , Manco Melania , Morroni Manrico , Cinti Saverio , Granzotto Marnie , Vettor Roberto , Camastra Stefania , Ferrannini Ele
Published: March 2007

Obesity is a frequent cause of insulin resistance and poses a major risk for diabetes. Abnormal fat deposition within skeletal muscle has been identified as a mechanism of obesity-associated insulin resistance. We tested the hypothesis that dietary lipid deprivation may selectively deplete intramyocellular lipids, thereby reversing insulin resistance. Whole-body insulin sensitivity (by the insulin clamp technique), intramyocellular lipids (by quantitative histochemistry on quadriceps muscle biopsies), muscle insulin action (as the expression of Glut4 glucose transporters), and postprandial lipemia were measured in 20 morbidly obese patients (BMI = 49 ± 8 [mean ± SD] kg · m−2) and 7 nonobese control subjects. Patients were restudied 6 months later after biliopancreatic diversion (BPD; n = 8), an operation that induces predominant lipid malabsorption, or hypocaloric diet (n = 9). At 6 months, BPD had caused the loss of 33 ± 10 kg through lipid malabsorption (documented by a flat postprandial triglyceride profile). Despite an attained BMI still in the obese range (39 ± 8 kg · m−2), insulin resistance (23 ± 3 μmol/min per kg of fat-free mass; P < 0.001 vs. 53 ± 13 of control subjects) was fully reversed (52 ± 11 μmol/min per kg of fat-free mass; NS versus control subjects). In parallel with this change, intramyocellular—but not perivascular or interfibrillar—lipid accumulation decreased (1.63 ± 1.06 to 0.22 ± 0.44 score units; P < 0.01; NS vs. 0.07 ± 0.19 of control subjects), Glut4 expression was restored, and circulating leptin concentrations were normalized. In the diet group, a weight loss of 14 ± 12 kg was accompanied by very modest changes in insulin sensitivity and intramyocellular lipid contents. We conclude that lipid deprivation selectively depletes intramyocellular lipid stores and induces a normal metabolic state (in terms of insulin-mediated whole-body glucose disposal, intracellular insulin signaling, and circulating leptin levels) despite a persistent excess of total body fat mass.
Leptin Effect on Endothelial Nitric Oxide Is Mediated Through Akt–Endothelial Nitric Oxide Synthase Phosphorylation Pathway
Authors: Vecchione Carmine , Maffei Angelo , Colella Salvatore , Aretini Alessandra , Poulet Roberta , Frati Giacomo , Gentile Maria Teresa , Fratta Luigi , Trimarco Valentina , Trimarco Bruno , Lembo Giuseppe
Published: March 2007

Recent evidence suggests that besides its action on the central nervous system, leptin can modulate vascular tone through local mechanisms involving nitric oxide (NO) release. In this study, using a fluorescent probe for direct determination of NO, we demonstrated both in endothelial cells and in vessels that leptin is able to stimulate NO release. The effect of leptin on NO is abolished by erbstatin A, a Ca2+-independent tyrosine kinase inhibitor, whereas it is not influenced by calcium removal or by other protein phosphorylation inhibitors, such as genistein (an ATP-dependent tyrosine-kinase inhibitor) or wortmannin and LY294002 (two different phosphatidylinositol [PI] 3-kinase inhibitors). Accordingly, leptin-induced vasorelaxation in aortic rings was abolished only by erbstatin A. Furthermore, immunoblotting studies revealed that leptin evokes Akt phosphorylation, with a comparable time course in both endothelial cells and vessels. Also in this experimental system, the effect of leptin was abolished by erbstatin A and not by other inhibitors. Finally, a considerable increase in endothelial NO synthase (eNOS) phosphorylation in Ser1177 was found when vessels were treated with leptin. In conclusion, leptin induces NO production by activating a PI 3-kinase–independent Akt-eNOS phosphorylation pathway.