bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel MODY3 Mutations in the Hepatocyte Nuclear Factor-1α Gene: Evidence for a Hyperexcitability of Pancreatic β-cells to Intravenous Secretagogues in a Glucose-Tolerant Carrier of a P447L Mutation
Authors: Hansen Torben , Eiberg Hans , Rouard Mathias , Vaxillaire Martine , Møller Ann M , Rasmussen Søren K , Fridberg Marianne , Urhammer Søren A , Holst Jens J , Almind Katrine , Echwald Søren M , Hansen Lars , Bell Graeme I , Pedersen Oluf
Published: September 2013

One form of maturity-onset diabetes of the young (MODY3) results from mutations in the hepatocyte nuclear factor (HNF)-1α gene, located on chromosome 12q24.2. The primary objective of the present study was to search for genetic variation in the HNF-1α gene in nine nonrelated Danish Caucasian subjects with MODY. Direct sequencing of the coding region and intron-exon boundaries of the HNF-1α gene revealed 2 novel and 1 previously reported missense mutations and 2 novel frameshift mutations in five of nine MODY subjects. These five mutations were found in neither 84 NIDDM patients nor 84 control subjects. One glucose-tolerant lean male with a P447L missense mutation, which in his relatives caused MODY, underwent an oral glucose tolerance test (OGTT), a tolbutamide modified frequently sampled intravenous glucose tolerance test, and a glucagon test to examine for a possible early β-cell abnormality. He had a low insulin secretion rate during an OGTT, but a twofold increase in pancreatic β-cell response after intravenous glucose and a 2.5- to 4-fold increase in β-cell response after either intravenous tolbutamide or intravenous glucagon loads. In conclusion, 1) mutations in the HNF-1α gene are common in Danish Caucasian MODY patients, and 2) early stages in the pathogenesis of MODY3 caused by the P447L mutation may be characterized by a hyperexcitability of β-cells to intravenous secretagogues.
Differential Activation of Mitogen-Activated Protein Kinase by Insulin and Epidermal Growth Factor in 3T3-L1 Adipocytes: A Possible Involvement of PI3-Kinase in the Activation of the MAP Kinase by Insulin
Authors: Suga Junko , Yoshimasa Yasunao , Yamada Kazunori , Yamamoto Yuji , Inoue Gen , Okamoto Motozumi , Hayashi Tatsuya , Shigemoto Michika , Kosaki Atsushi , Kuzuya Hideshi , Nakao Kazuwa
Published: September 2013

Mitogen-activated protein (MAP) kinase plays crucial roles in cell growth and differentiation. It has recently been shown that the MAP kinase cascade in growth factor signaling diverges and cross-talks with other signaling pathways. In the present study, we examined the effects of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), on the activation of Ras, Raf-1 kinase, and MAP kinase by insulin and epidermal growth factor (EGF). The effect of LY294002, a structurally distinct PI3-kinase inhibitor, on the activation of Raf-1 kinase by both ligands was also examined. In 3T3-L1 adipocytes, 25 nmol/l wortmannin inhibited the insulin-induced activation of Raf-1 kinase to the basal level, whereas the same dose of wortmannin had little effect on the EGF-induced activation of Raf-1 kinase. One hundred μmol/1 LY294002 blocked insulin-induced activation of Raf-1 kinase without affecting EGF-induced activation of this kinase. Twenty-five nmol/l wortmannin inhibited the insulin-induced activation of MAP kinase to the basal level with no effect on the EGF-induced activation of this kinase. But the same dose of wortmannin did not affect the formation of guanosine 5′-triphosphate (GTP)-bound Ras stimulated by either ligand. In KB cells, results similar to those in 3T3-L1 adipocytes were obtained. In contrast, in Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-HIR cells), neither wortmannin nor LY294002 inhibited the insulin-induced activation of Raf-1 kinase, and wort-mannin had little effect on the activation of MAP kinase by insulin. These results indicate that 1) PI3- kinase or wortmannin-sensitive molecules are involved in the interaction between activated Ras and Raf-1 kinase in the insulin signaling in 3T3-L1 adipocytes, 2) the involvement of PI3-kinase or wortmannin-sensitive molecules in the insulin-induced activation of MAP kinase appears to be cell-type specific, and 3) differential mechanisms to activate Raf-1 kinase and MAP kinase by insulin and EGF exist.
Insulin Stimulates Nitric Oxide Synthesis in Human Platelets and, Through Nitric Oxide, Increases Platelet Concentrations of Both Guanosine-3′, 5′-Cyclic Monophosphate and Adenosine-3′, 5′-Cyclic Monophosphate
Authors: Trovati Mariella , Anfossi Giovanni , Massucco Paola , Mattiello Luigi , Costamagna Costanzo , Piretto Valentina , Mularoni Elena , Cavalot Franco , Bosia Amalia , Ghigo Dario
Published: September 2013

The insulin-induced platelet anti-aggregating effect is attributed to a nitric oxide (NO)-mediated increase of cyclic guanosine monophosphate (cGMP). The aim of this work, carried out in human platelets, is to show whether insulin increases NO synthesis in platelets and whether it enhances not only cGMP but also cyclic adenosine monophosphate (cAMP) in these cells. We observed that 1) insulin dose-dependently increases NO production, evaluated as citrulline synthesis from Larginine (n = 4, P = 0.015); 2) insulin dose-dependently increases not only cGMP but also cAMP: for instance, after 8 min of insulin incubation at 1,920 pmol/l, cAMP increased from 39.8 ± 1.4 to 121.3 ± 12.6 pmol/l/109platelets (in = 16, P = 0.0001);3) when insulin is incubated for 120 min, the increase of cGMP and cAMP shows a plateau between 2 and 20 min, and while the effect on cGMP is significant until 120 min, the effect on cAMP is no more significant at 60 and 120 min; 4) insulin increases the effects on cAMP of the adenylate cyclase agonists Iloprost and forskolin (n = 5, P = 0.0001) and enhances their platelet anti-aggregating effects (n = 6 and 8, respectively; P = 0.0001); and 5) the inhibition of NO synthase by NG-monomethyl-L-arginine blunts both the insulin effects on basal cGMP and cAMP (n = 4) and those on the Iloprost- and forskolin-induced cAMP increase (n = 5). Thus, insulin increases NO synthesis in human platelets, and, through NO, enhances both cGMP and cAMP. The platelet antiaggregating effect exerted by insulin is, therefore, a NO-mediated phenomenon involving both cGMP and cAMP.
Insulinotropic Glucagon-Like Peptide I Receptor Expression in Glucagon-Producing α-Cells of the Rat Endocrine Pancreas
Authors: Heller R Scott , Kieffer Timothy J , Habener Joel F
Published: September 2013

Glucagon-like peptide I (GLP-I), an intestine-derived incretin hormone, is a potent stimulator of insulin and somatostatin secretion. In some studies, GLP-I is an inhibitor of glucagon secretion. It remains uncertain, however, whether the effect of GLP-I on the inhibition of glucagon secretion is direct, owing to interactions with GLP-I receptors on ケ-cells, or indirect, via paracrine suppression by insulin or somatostatin. The localization of the GLP-I receptor on insulin and somatostatin-producing cells in the islets is well established. Whether the GLP-I receptor also resides on the glucagon-producing α-cells remains controversial and is reported to be absent on rat α-cells. To investigate the distribution of the GLP-I receptor on islet cells, we examined the expression of GLP-I receptor mRNA in phenotypically distinct islet cell lines and islets, and the presence of immunoreactive GLP-I receptor in dispersed rat islet cells using a specific antiserum. GLP-I receptor mRNA was readily detected by reverse transcription–polymerase chain reaction (RT-PCR) in both rat islets and in established islet cell lines representing distinct α-, β, and δ-cell phenotypes. In addition, GLP I receptor expression was detected in single rat α-cells by single-cell RT-PCR. In dispersed rat islet cells analyzed by double immunofluorescent staining, 90% of the insulin, 76% of the somatostatin, and 20% of the glucagon positive cells colocalized with the GLP-I receptor immunoreactivity. Thus, a substantial population of glucagon immunoreactive α-cells express the GLP-I receptor. These findings imply that GLP-I may have a direct receptor-mediated action in the regulation of the physiological functions on a substantial subpopulation of α-cells. We suggest that a possible role for GLP-I receptors on α-cells may be to provide positive autocrine feedback control on glucagon secretion during fasting and/or to dampen the potent paracrine suppression of glucagon secretion by insulin during feeding.
Mitogen-Activated Protein Kinase Cascade Is Activated in Glomeruli of Diabetic Rats and Glomerular Mesangial Cells Cultured Under High Glucose Conditions
Authors: Haneda Masakazu , Araki Shin-ichi , Togawa Masaki , Sugimoto Toshiro , Motohide Isono , Kikkawa Ryuichi
Published: September 2013

The activation of protein kinase C (PKC) found in diabetic glomeruli and glomerular mesangial cells cultured under high glucose conditions has been proposed to contribute to the development of diabetic nephropathy. However, the abnormalities distal to PKC have not been fully elucidated yet. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) cascade, an important kinase cascade downstream to PKC and an activator of cytosolic phospholipase A2 (cPLA2) by direct phosphorylation, is activated in glomeruli isolated from streptozotocin-induced diabetic rats. MAPK cascade was also activated in glomerular mesangial cells cultured under high glucose (27.8 mmol/1) conditions for 5 days, and the activation of MAPK cascade was inhibited by treating the cells with calphostin C, an inhibitor of PKC. Furthermore, the activities of cPLA2 also increased in cells cultured under the same conditions and this activation was inhibited by both calphostin C and PD 098059, an inhibitor of MEK (MAPK or extracellular signal-regulated kinase [ERK] kinase). These results indicate that MAPK cascade is activated in glomeruli and mesangial cells under the diabetic state possibly through the activation of PKC. Activated MAPK, in turn, may induce various functional changes of mesangial cells at least through the activation of cPLA2 and contribute to the development of diabetic nephropathy.
Production of Plasminogen Activator Inhibitor 1 by Human Adipose Tissue: Possible Link Between Visceral Fat Accumulation and Vascular Disease
Authors: Alessi M C , Peiretti F , Morange P , Henry M , Nalbone G , Juhan-Vague I
Published: September 2013

Plasminogen activator inhibitor type 1 (PAI-1) contributes to the pathogenesis of atherothrombosis. Its plasma level is strongly correlated with parameters that define the insulin resistance syndrome, in particular with BMI and visceral accumulation of body fat, suggesting that PAI-1 may be an adipose tissue–derived circulating peptide. The present study was designed to investigate PAI-1 expression by human adipose tissue and its different cellular fractions. Special interest has been paid to the amount of PAI-1 antigen produced by omental versus subcutaneous fat. PAI-1 protein detected by immunolocalization was present at the stromal and adipocyte levels. PAI-1 mRNA was detected in stromal vascular cells freshly isolated and under culture conditions. It was also detected in whole adipose tissue and adipocyte fraction under culture conditions. The mRNA signal from the adipocyte fraction was detected as early as 2 h of incubation. The increase in PAI-1 mRNA was followed by an increase in PAI-1 antigen in the conditioned medium that was suppressed by treatment with cycloheximide. Transforming growth factor-β1 significantly increased PAI-1 antigen production by the adipocyte fraction, whereas tumor necrosis factor-α did not have any effect. Interestingly, after 5 h of incubation, omental tissue explants produced significantly more PAI-1 antigen than did subcutaneous tissue from the same individual, whereas similar production of leptin by the two territories was observed. These results strongly suggest that human adipose tissue, in particular visceral tissue, can be an important contributor to the elevated plasma PAI-1 levels observed in central obesity.
Cloning and Characterization of an Uncoupling Protein Homolog: A Potential Molecular Mediator of Human Thermogenesis
Authors: Gimeno Ruth E , Dembski Marlene , Weng Xun , Deng Nanhua , Shyjan Andrew W , Gimeno Carlos J , Iris Francois , Ellis Stephen J , Woolf Elizabeth A , Tartaglia Louis A
Published: September 2013

We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncoupling protein (UCP). Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tissues, with predominant expression in human white adipose tissue and skeletal muscle. In the white adipose tissue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean littermate controls. Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions. Furthermore, UCPH expression in yeast causes a decrease in the mitochondrial membrane potential, as judged by staining with the potential-sensitive dye DiOC6. These observations suggest that UCPH, like UCP, uncouples oxidative phosphorylation. The possibility that the UCPH protein is an important mediator of human thermogenesis is discussed.
Development of Autoimmune Diabetes in NOD Mice Is Associated With the Formation of Peroxynitrite in Pancreatic Islet β-Cells
Authors: Suarez-Pinzon Wilma L , Szabó Casaba , Rabinovitch Alex
Published: September 2013

Peroxynitrite (ONOO−) is a highly reactive oxidant species produced by the reaction of the free radicals superoxide (O2·–) and nitric oxide (NO·). Here we report a marked increase in nitrotyrosine (NT), a marker of peroxynitrite, in islet cells from NOD mice developing spontaneous autoimmune diabetes. By using specific antibodies and immunohistochemical methods, we found that NT-positive cells were significantly more frequent in islets from acutely diabetic NOD mice (22 ± 6%) than in islets from normoglycemic NOD mice (7 ± 1%) and control BALB/c mice (2 ± 1%). The NT+ cells in islets were identified to be macrophages and also β-cells. Most of the β-cells in islets from acutely diabetic NOD mice were NT+ (73 ± 8%), whereas significantly fewer β-cells were NT+ in islets from normoglycemic NOD mice (18 ± 4%) and BALB/c mice (5 ± 1%). Also, the percentage of β-cells in islets from NOD mice (normoglycemic and diabetic) correlated inversely with the frequency of NT+ β-cells. This study demonstrates for the first time that peroxynitrite, a reaction product of superoxide and nitric oxide, is formed in pancreatic islet β-cells of NOD mice developing autoimmune diabetes. This suggests that both oxygen and nitrogen free radicals contribute to β-cell destruction in IDDM via peroxynitrite formation in the islet β-cells.
Acute Postchallenge Hyperinsulinemia Predicts Weight Gain: A Prospective Study
Authors: Sigal Ronald J , El-Hashimy Mona , Martin Blaise C , Soeldner J Stuart , Krolewski Andrzej S , Warram James H
Published: September 2013

The relationships of insulin secretion and insulin action to body weight are incompletely understood. Obesity is associated with reduced sensitivity to insulin and high fasting and postprandial serum insulin levels. However, it is unknown whether insulin secretion rises to compensate for insulin resistance or high insulin secretion promotes body weight gain and the development of insulin resistance. To shed light on this question, we examined weight gain over an interval of 16.7 ± 3.9 years (mean ± SD) in 107 glucose-tolerant offspring (48 men, 59 women) of two parents with NIDDM. The offspring had a baseline intravenous glucose tolerance test, at which time they were aged 32.9 ± 9.7 years, and only those who did not develop diabetes during the follow-up period were included. We estimated insulin sensitivity with the insulin sensitivity index from Bergman's minimal model of glucose disposal and acute insulin secretion from the incremental area under the insulin curve in the first 10 min of the intravenous glucose tolerance test. Weight-gain rate (g/year) was defined as the regression slope of each subject's body weight over time. High acute insulin secretion, young age, and low baseline percent ideal body weight (IBW) were each associated with a high rate of weight gain. After adjustment for differences in age and IBW, statistically significant effects of insulin sensitivity (P = 0.05) as well as acute insulin secretion (P = 0.001) were obtained. To estimate the effects of acute insulin secretion and insulin sensitivity on the average rate of weight gain (adjusting for age and IBW), the study group was stratified into four subgroups by dividing it at the medians of these two variables. Among those with low acute insulin secretion, weight-gain rate was the same regardless of whether insulin sensitivity was low or high (176 and 152 g/year, respectively). Among those with high acute insulin secretion, mean weight-gain rate was still rather low in those with low insulin sensitivity (271 g/year), but it was quite high in those with high insulin sensitivity (672 g/year; significantly higher than in all other subgroups). Therefore a high first-phase insulin response to intravenous glucose is a risk factor for long-term weight gain, and this effect is particularly manifested in insulin-sensitive individuals.
Skeletal Muscle Triglyceride Levels Are Inversely Related to Insulin Action
Authors: Pan D A , Lillioja S , Kriketos A D , Milner M R , Baur L A , Bogardus C , Jenkins A B , Storlien L H
Published: September 2013

In animal studies, increased amounts of triglyceride associated with skeletal muscle (mTG) correlate with reduced skeletal muscle and whole body insulin action. The aim of this study was to test this relationship in humans. Subjects were 38 nondiabetic male Pima Indians (mean age 28 ± 1 years). Insulin sensitivity at physiological (M) and supraphysiological (MZ) insulin levels was assessed by the euglycemic clamp. Lipid and carbohydrate oxidation were determined by indirect calorimetry before and during insulin administration. mTG was determined in vastus lateralis muscles obtained by percutaneous biopsy. Percentage of body fat (mean 29 ± 1%, range 14–44%) was measured by underwater weighing. In simple regressions, negative relationships were found between mTG (mean 5.4 ± 0.3 μmol/g, range 1.3–1.9 μmol/g) and log10M (r = −0.53, P ≤ 0.001), MZ (r = −0.44, P = 0.006), and nonoxidative glucose disposal (r = −0.48 and −0.47 at physiological and supraphysiological insulin levels, respectively, both P = 0.005) but not glucose or lipid oxidation. mTG was not related to any measure of adiposity. In multiple regressions, measures of insulin resistance (log10M, MZ, log10[fasting insulin]) were significantly related to mTG independent of all measures of obesity (percentage of body fat, BMI, waist-to-thigh ratio). In turn, all measures of obesity were related to the insulin resistance measures independent of mTG. The obesity measures and mTG accounted for similar proportions of the variance in insulin resistance in these relationships. The results suggest that in this human population, as in animal models, skeletal muscle insulin sensitivity is strongly influenced by local supplies of triglycerides, as well as by remote depots and circulating lipids. The mechanism(s) underlying the relationship between mTG and insulin action on skeletal muscle glycogen synthesis may be central to an understanding of insulin resistance.
Inhibition of Diabetes by an Insulin-Reactive CD4 T-Cell Clone in the Nonobese Diabetic Mouse
Authors: Zekzer Dan , Wong F Susan , Wen Li , Altieri Martha , Gurlo Tatyana , von Grafenstein Hermann , Sherwin Robert S
Published: September 2013

A cloned Thl cell line was isolated from pancreatic lymph nodes of NOD mice that carries a T-cell receptor encoding Vβ14 and proliferates in response to NOD islets, islet supernatant, and crystalline bovine and rat insulin, specifically to a B-chain peptide bound to IAg7. The response to islet supernatant was reduced by 75% by anti-insulin antibody treatment. The insulin-reactive clone reduced insulitis and totally blocked the development of spontaneous diabetes in NOD mice (n = 8) as well as the adoptive transfer of diabetes into irradiated NOD mice following the injection of splenocytes from diabetic mice (n = 13). Trafficking of the adoptively transferred cells was assessed by labeling the clone or diabetic splenocytes with a fluorescent marker (Dil). The labeled clone was detected in the islet periphery, whereas labeled splenocytes alone invaded the islets by 3 days. In contrast, the protective clone dramatically delayed and reduced the number of labeled diabetic splenocytes infiltrating the islet, although their appearance in the spleen was unaffected. In vitro, the clone as well as supernatant derived from the clone blocked the proliferation of diabetic NOD splenocytes to islets. This inhibitory effect was diminished by anti–transforming growth factor-β. In conclusion, an insulin-specific Thl cell was isolated from NOD mice that traffics to the islet and prevents the spontaneous development and the adoptive transfer of diabetes. It appears to act locally by releasing transforming growth factor-β and/or other factors that inhibit homing to and/or proliferation of diabetic splenocytes within the islet. These findings may provide insights into and suggest mechanisms for the protective effects of insulin therapy against diabetes.
Glucose Stimulates Islet β-Cell Mitogenesis Through GTP-Binding Proteins and by Protein Kinase C–Dependent Mechanisms
Authors: Sjöholm Åke
Published: September 2013

Glucose is a cardinal secretory and mitogenic stimulus for the insulin-producing pancreatic ö-cell both in vitro and in vivo, but the mechanisms by which the sugar acts mitogenically remain largely elusive. In this study, the intracellular pathways that convey glucose-induced mitogenic and secretory signaling in ö-cells were investigated. For this purpose, fetal rat pancreatic islets enriched in ö-cells were cultured in 3.3 or 16.7 mmol/l glucose for 3 days. It was found that glucose stimulated ö-cell replication, insulin secretion, and cAMP content. These effects were mimicked by agonists of cAMP-dependent protein kinases but not by guanosine- 3′,5′-cyclic monophosphate (cGMP). Antagonists of cAMP-dependent protein kinases failed to block the glucose-induced increments in β-cell replication and insulin secretion. Glucose is known to activate protein kinase C, and a protein kinase C–activating phorbol ester was found to promote β-cell mitogenesis and insulin secretion. Conversely, when protein kinase C was inhibited, the mitogenic (but not secretory) response to glucose was attenuated. There were no additive or synergistic effects on β-cell replication when cAMP and phorbol ester were combined, whereas insulin secretion was potentiated by this combination. Artificially causing Ca2+ inlet by glibenclamide or ionomycin did not result in a stimulated mitogenic response, and preventing Ca2+ influx by blocking plasma membrane Ca2+ channels did not abolish the mitogenicity of glucose, although it reduced insulin secretion. Pretreatment of islets with pertussis toxin, known to regulate transduction of signals through heterotrimeric GTP-binding proteins, completely prevented the stimulatory effect of glucose on +-cell mitogenesis but not on insulin secretion. We conclude that specific activation of protein kinase C or cAMP synthesis is sufficient to increase β-cell mitogenesis and insulin secretion, whereas cGMP appears not to affect these processes. However, cAMP does not seem to mediate the mitogenicity or secretory action of glucose. The results instead suggest that signaling through GTP-binding proteins and protein kinase C activation is required for transduction of the mitogenic, but not secretory, message of the sugar in the β-cell.
Hexokinase Shift to Mitochondria Is Associated With an Increased Sensitivity to Glucose in Rat Pancreatic Islets
Authors: Rabuazzo Agata Maria , Patanè Giovanni , Anello Marcello , Piro Salvatore , Vigneri Riccardo , Purrello Francesco
Published: September 2013

When rat pancreatic islets are incubated in 5.5 or 16.7 mmol/1 glucose for 3 h, an increased sensitivity is observed in islets pre-exposed to high glucose, as indicated by a shift to the left of the glucose dose-response curve (EC50 7.1 ± 0.9 and 11.5 ± 1.2 in high- and low-glucose-exposed islets, respectively; n = 5, P < 0.05). To investigate the mechanism(s) responsible for this effect, we measured hexokinase and glucokinase activity both in the cytosolic fraction and in a mitochondrion-enriched fraction, since binding to the outer mitochondrial membrane has been reported to result in an increased enzyme activity. In islets cultured at 16.7 mmol/1 glucose, the cytosolic hexokinase activity was similar to control islets, but mitochondrial enzyme activity was significantly increased (124 ± 7 vs. 51 ± 9 nmol · µg−1 · 90 min−1, P < 0.01). As a consequence, the cytosolicrmitochondrial fraction ratio was altered in comparison with control islets. In contrast, glucokinase activity in the two groups of islets was similar in the cytosolic fraction and undetectable in the mitochodrial fraction. Hexokinase I quantitation by Western blot confirmed the enzyme translocation from the free cytosolic to the mitochondria-bound form in islets cultured at 16.7 mmol/l glucose. Glucose-induced alterations were reversible after 1 h exposure to 5.5 mmol/l glucose. Moreover, in islets exposed to 16.7 mmol/l glucose, inhibition of hexokinase binding to mitochondria by the addition of 20 nmol/1 dicyclohexylcarbodiimide resulted in no increase of glucose sensitivity (EC50 10.9 ± 0.4, n = 3, similar to that of control islets). These data indicate that after chronic exposure to high glucose, the β-cell becomes more sensitive to glucose before eventually getting desensitized. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity secondary to a subcellular shift of the enzyme from the free cytosolic to the mitochondriabound, more active form.
Long-Term Intensive Therapy of IDDM Patients With Clinically Overt Autonomic Neuropathy: Effects on Hypoglycemia Awareness and Counterregulation
Authors: Fanelli Carmine , Pampanelli Simone , Lalli Carlo , Sindaco Paola Del , Ciofetta Marco , Lepore Mauro , Porcellati Francesca , Bottini Paolo , Di Vincenzo Antonio , Brunetti Paolo , Bolli Geremia B
Published: September 2013

To test the hypothesis that hypoglycemia unawareness and impaired counterregulation are reversible after meticulous prevention of hypoglycemia in IDDM patients with diabetic autonomic neuropathy (DAN), 21 patients (8 without DAN [DAN−]; 13 with DAN [DAN+]; of the latter, 7 had orthostatic hypotension [DAN+PH+] and 6 did not [DAN+PH−]) and 15 nondiabetic subjects were studied during stepped hypoglycemia (plateau plasma glucose decrements from 5.0 to 2.2 mmol/l) before and 6 months after prevention of hypoglycemia (intensive therapy). After 6 months, frequency of mild hypoglycemia decreased from ∼20 to ∼2 episodes/patient-month while HbA1c increased from 6.2 ± 0.3 to 6.9 ± 0.2% (P < 0.05). Responses of adrenaline improved more in DAN− patients (from 1.17 ± 0.12 to 2.4 ± 0.22 nmol/l) than in DAN+PH− (from 0.75 ± 0.25 to 1.56 ± 0.23 nmol/l) and DAN+PH+ patients (from 0.80 ± 0.24 to 1.15 ± 0.27 nmol/l, P < 0.05) but remained lower than in nondiabetic subjects (4.9 ± 0.37 nmol/1, P < 0.05), whereas glycemic thresholds normalized only in DAN−, not DAN+. Autonomic symptoms of hypoglycemia improved but remained lower in DAN− (6.2 ± 0.6) than in nondiabetic subjects (8.1 ± 1.1) and lower in DAN+PH+ (4 ± 0.8) than in DAN+PH− subjects (5.1 ± 0.8, P < 0.05), whereas neuroglycopenic symptoms normalized (NS). Cognitive function deteriorated less before than after prevention of hypoglycemia (P < 0.05). Thus, intensive therapy with emphasis on preventing hypoglycemia reverses hypoglycemia unawareness in DAN+ patients despite marginal improvement of adrenaline responses, results in low frequency of hypoglycemia despite impaired counterregulation, and maintains HbA1c in the range of intensive therapy. We conclude that DAN, long IDDM duration per se, and antecedent recent hypoglycemia contribute to different extents to impaired adrenaline responses and hypoglycemia unawareness.
Optimization of Glycemic Control by Insulin Therapy Decreases the Proportion of Small Dense LDL Particles in Diabetic Patients
Authors: Caixàs Assumpta , Ordóñez-Llanos Jordi , de Leiva Alberto , Payés Amalia , Homs Rosa , Pérez Antonio
Published: September 2013

Small dense LDL particles (B phenotype) are considered to be more atherogenic than large buoyant LDL particles. The influence of glycemic control on LDL particle size and density is still under debate. The aim of this study was to determine LDL subfraction phenotype in both IDDM and NIDDM patients in poor glycemic control compared with that of respective matched control groups. In addition, we evaluated the effect of a 3-month period of optimized glycemic control on this parameter. Thirty-seven IDDM patients and 33 NIDDM patients, together with two respective age-, sex-, and BMI-matched control groups were studied. Non-A phenotype prevalence in IDDM patients before (19%) and after blood glucose optimization (11%) was similar to that of their control group (12%). However, NIDDM patients displayed a higher proportion of the non-A phenotype (51%) than did the control group (28%), but it became closer (30%, P < 0.05) after glycemic control improved. All subjects with non-A phenotype that changed to A phenotype showed triglyceride levels below 1.63 mmol/1 and a greater decrease in HbA1c than did subjects whose phenotype did not change (4.9 ± 1.5 vs. 3.1 ± 1.4%, P < 0.05). A higher proportion of small dense LDL was observed in NIDDM women than in nondiabetic women (LDL5 10.0 ± 4.8 vs. 6.3 ± 1.5%, LDL6 6.1 ± 2.2 vs. 4.2 ± 0.8%, P < 0.05) during both stages of glycemic control, but no differences were observed between NIDDM and nondiabetic men. In conclusion, these findings provide new evidence for the relevance of near-normal glycemic control in the prevention of macrovascular disease and could contribute to an explanation of the loss of protection for cardiovascular disease in diabetic women.
Scientific and Political Impediments to Successful Islet Transplantation
Authors: Weir Gordon C , Susan Bonner-Weir
Published: September 2013

Islet transplantation is a treatment for diabetes that has the potential to normalize glucose levels and prevent the development of complications. In spite of the simplicity of the concept and the urgent need to provide such a treatment to patients, there has been a frustrating lack of progress. This perspective delves into the scientific and political impediments to success. The scientific barriers are the need to find a satisfactory source of insulin-producing tissue and the requirement to prevent this tissue from being destroyed by immune rejection and autoimmunity. The problems and potential of allografts, xenografts, and the development of cell lines are discussed. Multiple approaches to the prevention of immune destruction are considered, including immunobarrier devices, immunosuppression, development of tolerance, and genetic manipulation. The political barriers discussed include the problems of high expectations, the controversy surrounding targeted research, the balance between basic and applied research, the roles of industry and academia, the concerns about xenotransplantation, and the difficulties in developing a planned approach to the problem.
Demonstration of Two Different Processes of β-Cell Regeneration in a New Diabetic Mouse Model Induced by Selective Perfusion of Alloxan
Authors: Waguri Masako , Yamamoto Koji , Miyagawa Jun-ichiro , Tochino Yoshihiro , Yamamori Katsumi , Kajimoto Yoshitaka , Nakajima Hiromu , Watada Hirotaka , Yoshiuchi Issei , Itoh Naoto , Imagawa Akihisa , Namba Mitsuyoshi , Kuwajima Masamichi , Yamasaki Yoshimitsu , Hanafusa Toshiaki , Matsuzawa Yuji
Published: September 2013

To clarify the regeneration process of pancreatic β-cells, we established a new mouse model of diabetes induced by selective perfusion of alloxan after clamping the superior mesenteric artery. In this model, diabetes could be induced by the destruction of β-cells in alloxan-perfused segments, while β-cells in nonperfused segments were spared. Intraperitoneal glucose tolerance tests showed glucose intolerance, which gradually ameliorated and was completely normalized in 1 year with a concomitant increase of insulin content in the pancreas. Histological examination showed neoislet formation in the alloxan-perfused segment and the proliferation of spared β-cells in the nonperfused segment. In the alloxan-perfused segment, despite a marked reduction of islets in size and number at an early stage, both the number of islets, including islet-like cell clusters (ICCs), and the relative islet area significantly increased at a later stage. Increased single β-cells and ICCs were located in close contact with duct cell lining, suggesting that they differentiated from duct cells and that such extra-islet precursor cells may be important for β-cell regeneration in β-cell–depleted segment. In addition to β-cells, some nonhormone cells in ICCs were positive for nuclear insulin promoter factor 1, which indicated that most, if not all, nonhormone cells positive for this factor were β-cell precursors. In the nonperfused segment, the islet area increased significantly, and the highest 5-bromo-2-deoxyuridine–labeling index in β-cells was observed at day 5, while the number of islets did not increase significantly. This indicated that the regeneration of islet endocrine cells occurs mostly through the proliferation of preexisting intra-islet β-cells in the nonperfused segment. In conclusion, the regeneration process of β-cells varied by circumstance. Our mouse model is useful for studying the mechanism of regeneration, since differentiation and proliferation could be analyzed separately in one pancreas.
Fasting Hyperinsulinemia Is a Predictor of Increased Body Weight Gain and Obesity in Pima Indian Children
Authors: Odeleye Olalekan E , de Courten Maximilian , Pettitt David J , Ravussin Eric
Published: September 2013

Hyperinsulinemia is commonly associated with obesity, but it has not been determined which defect comes first. Some have proposed that hyperinsulinemia may precede obesity in populations prone to NIDDM, such as Pima Indians or Pacific Islanders. In contrast, longitudinal studies in adults show that insulin sensitivity and low fasting insulin concentrations are associated with increased weight gain, whereas insulin resistance seems to protect against weight gain. The present study examined whether fasting plasma hyperinsulinemia is a risk factor for weight gain in prepubertal children in the Pima Indian population—a population that is prone to obesity. Fasting plasma insulin concentration was measured in 328 5- to 9-year-old Pima Indian children (147 boys and 181 girls) with normal glucose tolerance. Follow-up weight was obtained an average of 9.3 ± 1.9 years (means ± SD) later at age 15–19 years. Fasting plasma insulin concentration correlated with the rate of weight gain per year in both boys (r = 0.42; P < 0.0001) and girls (r = 0.20; P < 0.01) and was associated with the rate of weight gain, independent of known determinants of weight change, i.e., initial relative weight, change in height, age, and sex. Similar relationships were found between fasting plasma insulin concentration and the change in relative weight and in triceps skinfold thickness—two indicators of obesity. In conclusion, fasting hyperinsulinemia may be a risk factor for the development of obesity in young children.
4-Hydroxyisoleucine: A Novel Amino Acid Potentiator of Insulin Secretion
Authors: Sauvaire Yves , Petit Pierre , Broca Christophe , Manteghetti Michèle , Baissac Yves , Fernandez-Alvarez Josepha , Gross Renè , Roye Michèle , Leconte Agnès , Gomis Ramon , Ribes Gèrard
Published: September 2013

We report the characterization of a new insulinotropic compound, 4-hydroxyisoleucine. This amino acid has been extracted and purified from fenugreek seeds, which are known in traditional medicine for their antidiabetic properties. 4-Hydroxyisoleucine increases glucose-induced insulin release, in the concentration range of 100 μmol/l to 1 mmol/l, through a direct effect on isolated islets of Langerhans from both rats and humans. The stimulating effect of 4-hydroxyisoleucine was strictly glucose dependent; indeed, ineffective at low (3 mmol/l) or basal (5 mmol/l) glucose concentrations, the amino acid potentiated the insulin secretion induced by supranormal (6.6–16.7 mmol/l) concentrations of glucose. In addition, in the isolated perfused rat pancreas, we could show 1) that the pattern of insulin secretion induced by 4-hydroxyisoleucine was biphasic, 2) that this effect occurred in the absence of any change in pancreatic α-and δ-cell activity, and 3) that the more glucose concentration was increased, the more insulin response was amplified. Moreover, 4-hydroxyisoleucine did not interact with other agonists of insulin secretion (leucine, arginine, tolbutamide, glyceraldehyde). Therefore, we conclude that 4-hydroxyisoleucine insulinotropic activity might, at least in part, account for fenugreek seeds' antidiabetic properties. This secretagogue may be considered as a novel drug with potential interest for the treatment of NIDDM.
Plasma Leptin Concentrations Do Not Appear to Decrease Insulin-Mediated Glucose Disposal or Glucose-Stimulated Insulin Secretion in Women With Normal Glucose Tolerance
Authors: Carantoni Marcello , Abbasi Fahim , Azhar Salman , Chen Yii-Der I , Klebanov Mark , Wang Pei-Wen , Warmerdam Fabienne , Reaven Gerald M
Published: September 2013

The aim of this study was to test the hypothesis that plasma leptin concentrations contributed to the pathophysiology of NIDDM by decreasing both insulin-mediated glucose disposal and glucose-stimulated insulin secretion. The study was performed in 60 women with normal oral glucose tolerance. Differences in insulinmediated glucose disposal were determined by comparing the steady-state plasma glucose (SSPG) concentrations attained at the end of a 180-min constant infusion of somatostatin, glucose, and insulin, while comparisons of glucose-stimulated insulin secretion were based on the incremental increase in insulin concentration 30 min after an oral glucose challenge (ΔIns) as compared with the fasting value. The results showed that the higher the fasting plasma leptin concentration, the greater the degree of insulin resistance (r = 0.47, P < 0.01). Furthermore, multiple regression analysis indicated that the relationship between leptin and SSPG was independent of age and degree of obesity as estimated by BMI. However, since the total integrated plasma insulin response was highly correlated with both SSPG (r = 0.80, P < 0.001) and leptin (r = 0.55, P < 0.01), multiple regression analysis was repeated, adding total insulin response to the model. When this was done, the significant relationship between leptin and SSPG disappeared, whereas both BMI (P < 0.03) and insulin response (P < 0.001) were correlated with SSPG. A significant relationship between leptin and AIns was seen, but it was a positive one (r = 0.31, P < 0.02), not a negative one as would be expected if circulating levels of leptin inhibited glucose-stimulated insulin secretion. Furthermore, multiple regression analysis could only confirm an independent relationship between AIns and SSPG, but not between AIns and leptin. The results of these studies do not support the view that circulating leptin has a primary effect on either insulin action or secretion in normal female volunteers. It seems more likely that chronic hyperinsulinemia in insulin-resistant individuals acts to increase adipose tissue leptin synthesis and secretion, leading to higher ambient leptin concentrations.
Molecular Basis for HLA-DQ Associations With IDDM
Authors: Nepom Gerald T , Kwok William W
Published: September 2013

Autoimmune diabetes is the clinical end point for a sequential cascade of immunologic events that occur in a genetically susceptible individual. Structural and functional analysis of the HLA class II susceptibility genes in IDDM suggests likely molecular mechanisms for several of the key steps in this cascade of autoimmune events. We outline a pathway in which the HLA-DQ genes associated with IDDM bias the immunologic repertoire toward autoimmune specificities, creating an autoimmune-prone individual, followed by amplification and triggering events that promote subsequent immune activation. There are several direct links between genetics and autoimmune disease in this pathway: the developmental maturation of T-cells in a genetically susceptible individual occurs through molecular interactions between the T-cell receptor and the HLApeptide complex. Selection of T-cells with receptors likely to contribute to autoreactivity may preferentially occur in the context of specific HLA-DQ alleles that are diabetes prone, because of inefficiencies in the peptide-MHC structural interactions of these molecules. Subsequent activation of these T-cells in the context of recognizing islet-associated antigens can trigger a poorly regulated immune response that results in progressive islet destruction. These subsequent diabetes-specific events are also directed by specific HLA genes, most prominently by the binding of specific antigenic peptides by the disease-associated HLA molecules. In this sequential cascade, opportunities for environmental influences and modulation by non-HLA genes are identified that likely act in concert with the predominant genetic susceptibility contributed by the HLA molecules themselves. Clarification of the steps in this pathway extends our understanding of the prevailing role of HLA genes in IDDM pathogenesis and suggests opportunities to intervene at discrete initiating, disease-promoting, or regulatory steps in IDDM development.
Fibronectin Fragments Modulate Human Retinal Capillary Cell Proliferation and Migration
Authors: Grant Maria B , Caballero Sergio , Bush David M , Spoerri Polyxenie E
Published: September 2013

Capillary morphogenesis involves cell-cell and cellmatrix interactions. Proteases elaborated by capillary cells modify the extracellular matrix (ECM) to facilitate capillary tube formation. Previously, we detected the presence of fibronectin fragments (Fn-f) associated with the proform of matrix metalloprotease-2 (MMP-2) in conditioned medium of human retinal endothelial cells (HRECs). Association of this fragment to latent MMP-2 prevented autocatalytic activation of MMP-2, suggesting a modulatory role of Fn-f in MMP-2 activation. In this report, we examined the potential role of Fn-f on two processes involved in angiogenesis, proliferation and migration of vascular cells. The effects of Fn-f on proliferation were determined by DNA synthesis and cell counts. Their effects on migration were assessed using modified Boyden chambers. Seven Fn-f were tested on vascular cell migration and/or proliferation. Three Fn-f induced migration. Fn-f of 30-kDa and 120-kDa size positively affected proliferation of microvascular cells but not macrovascular cells. A 45-kDa gelatin binding fragment of Fn inhibited HREC proliferation but stimulated pericyte and smooth muscle cell proliferation. The potency of these fragments exceeded that of the known angiogenic growth factor, basic fibroblast growth factor (bFGF), on HREC migration. ECM components such as fibronectin may influence capillary morphogenesis by the generation of fragments that can modulate proliferation, migration, and protease activation. In the setting of diabetes, excess Fn is generated and is available for degradation. Thus, the production of Fn-f may be specifically relevant to the angiogenesis observed in proliferative diabetic retinopathy.
Human Preadipocytes Display a Depot-Specific Susceptibility to Apoptosis
Authors: Niesler Carola U , Siddle Kenneth , Prins Johannes B
Published: September 2013

Adipose tissue mass is determined by both the number and volume of adipose cells. Adipose cell number reflects the balance of cell acquisition and cell loss, whereas adipose cell volume represents the balance of lipolysis and lipogenesis. It is well recognized that insulin resistance, NIDDM, and other metabolic disorders are associated more strongly with increased omental adiposity than with subcutaneous adiposity. Depotrelated differences exist in adipocyte responses to lipolytic and lipogenic stimuli, in adipocyte apoptosis, and in the potential for preadipocyte replication and differentiation. In the present study, we address the question of whether there might also be a site-specific difference in the susceptibility of human preadipocytes to apoptosis. Paired samples of human omental and subcutaneous preadipocytes from 12 individuals were cultured, and apoptosis was induced by serum deprivation or treatment with tumor necrosis factor (TNF)-α for 4 h. Cells were then stained with acridine orange, and apoptotic indices were calculated as the fraction of cells showing nuclear condensation. Under both conditions, in 9 of 11 subjects, apoptotic indices were substantially greater in preadipocytes from the omental depot than in those from the subcutaneous depot, and mean apoptotic indices were more than twofold higher in omental cells (serum-free medium: P < 0.05; TNF-α: P < 0.02; paired t test). Omental preadipocytes are therefore more susceptible to two different apoptotic stimuli than subcutaneous preadipocytes, demonstrating another intrinsic site-specific difference between human adipose cells of the two depots. These results suggest that the regulation of adipose tissue distribution in humans could involve depot-specific differences in rates of preadipocyte apoptosis.
Diabetes is Associated with Autoimmunity in the New Zealand Obese (NZO) Mouse
Authors: Melez Kathleen A , Harrison Len C , Gilliam James N , Steinberg Alfred D
Published: September 2013

The New Zealand Obese (NZO) mouse was studied as a potential model for autoimmune diabetes. NZO mice develop obesity, glucose intolerance, and insulin resistance, and have low-titer IgM antibodies to the insulin receptor. It is shown that they have circulating antibodies to both native DNA and denatured, singlestranded DNA. The antibody levels are higher in females, and, up to 6 mo of age, are comparable to those found in the related NZB × NZW F1 (NZB/W) mouse, a model for systemic lupus erythematosus. After 6 mo of age the antibody levels in NZO mice fall toward normal, in contrast to the persistently elevated levels in NZB/W mice. NZB/W mice are known to succumb to immune complex-mediated proliferative glomerulonephritis before 1 yr of age, whereas NZO mice survive. NZO kidneys exhibit light microscopic features of both diabetic and lupus nephropathies: glomerular proliferation, mesangial deposits, mild basement membrane thickening, glomerulosclerosis, eosinophilic nodules in some glomeruli, occasional hyalinization of the glomerular arterioles, and healing arteriolar inflammation. These changes are associated with glomerular deposition of immunoglobulin, especially IgM, in a granular pattern on fluorescent staining. The NZO mouse, therefore, has evidence of a generalized immune disorder and provides a model for studying the relationship between autoimmunity, obesity, and diabetes.