bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Trait Linkage Analysis of Lipid-Related Traits in Familial Type 2 Diabetes
Authors: Elbein S.C. , Hasstedt S.J.
Published: March 2007

Macrovascular disease is a major complication of type 2 diabetes. Epidemiological data suggest that the risk of macrovascular complications may predate the onset of hyperglycemia. Hypertriglyceridemia, low levels of HDL cholesterol, and an atherogenic profile characterize the insulin resistance/metabolic syndrome that is also prevalent among nondiabetic members of familial type 2 diabetic kindreds. To identify the genes for lipid-related traits, we first performed a 10-cM genome scan using 440 markers in 379 members of 19 multiplex families ascertained for two diabetic siblings (screening study). We then extended findings for three regions with initial logarithm of odds (LOD) scores >1.5 to an additional 23 families, for a total of 576 genotyped individuals (extended study). We found heritabilities for all lipid measures in the range of 0.31 to 0.52, similar to those reported by others in unselected families. However, we found the strongest evidence for linkage of triglyceride levels to chromosome 19q13.2, very close to the ApoC2/ApoE/ApoC1/ApoC4 gene cluster (LOD 2.56) in the screening study; the LOD increased to 3.16 in the extended study. Triglyceride-to-HDL cholesterol ratios showed slightly lower LOD scores (2.73, extended family) in this same location. Other regions with LOD scores >2.0 included HDL linkage to chromosome 1q21-q23, where susceptibility loci for both familial type 2 diabetes and familial combined hyperlipidemia have been mapped, and to chromosome 2q in the region of the NIDDM1 locus. Neither region showed stronger evidence for linkage in the extended studies, however. Our results suggest that genes in or near the ApoE/ApoC2/ApoC1/ApoC4 cluster on 19q13.2 may contribute to the commonly observed hypertriglyceridemia and low HDL seen in diabetic family members and their offspring, and thus may be a candidate locus for the insulin resistance syndrome.
Metabolic Oscillations in β-Cells
Authors: Kennedy Robert T. , Kauri Lisa M. , Dahlgren Gabriella M. , Jung Sung-Kwon
Published: March 2007

Whereas the mechanisms underlying oscillatory insulin secretion remain unknown, several models have been advanced to explain if they involve generation of metabolic oscillations in β-cells. Evidence, including measurements of oxygen consumption, glucose consumption, NADH, and ATP/ADP ratio, has accumulated to support the hypothesis that energy metabolism in β-cells can oscillate. Where simultaneous measurements have been made, these oscillations are well correlated with oscillations in intracellular [Ca2+] and insulin secretion. Considerable evidence has been accumulated to suggest that entry of Ca2+ into cells can modulate metabolism both positively and negatively. The main positive effect of Ca2+ is an increase in oxygen consumption, believed to involve activation of mitochondrial dehydrogenases. Negative feedback by Ca2+ includes decreases in glucose consumption and decreases in the mitochondrial membrane potential. Ca2+ also provides negative feedback by increasing consumption of ATP. The negative feedback provided by Ca2+ provides a mechanism for generating oscillations based on a model in which glucose stimulates a rise in ATP/ADP ratio that closes ATP-sensitive K+ (KATP) channels, thus depolarizing the cell membrane and allowing Ca2+ entry through voltage-sensitive channels. Ca2+ entry reduces the ATP/ADP ratio and allows reopening of the KATP channel.
Dynamic Imaging of Endoplasmic Reticulum Ca2+ Concentration in Insulin-Secreting MIN6 Cells Using Recombinant Targeted Cameleons
Authors: Varadi Aniko , Rutter Guy A.
Published: March 2007

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca2+ concentrations ([Ca2+]cyt) and hence in insulin secretion from pancreatic β-cells. However, the molecular mechanisms involved in both the uptake and release of Ca2+ from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca2+ fluxes across the ER membrane in single live MIN6 β-cells, we have imaged changes in the ER intralumenal free Ca2+ concentration ([Ca2+]ER) using ER-targeted cameleons. Resting [Ca2+]ER (∼250 μmol/l) was markedly reduced after suppression (by ∼40%) of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca2+-ATPase in this cell type. Nutrient secretagogues that elevated [Ca2+]cyt also increased [Ca2+]ER, an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca2+]ER. Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca2+ and marked increases in [Ca2+]cyt. Furthermore, photolysis of caged cyclic ADP ribose increased [Ca2+]cyt, and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse β-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca2+ changes in the β-cell and thus in the regulation of insulin secretion.
Relationships Among Age, Proinsulin Conversion, and β-Cell Function in Nondiabetic Humans
Authors: Fritsche Andreas , Madaus Alexander , Stefan Norbert , Tschritter Otto , Maerker Elke , Teigeler Anna , Häring Hans , Stumvoll Michael
Published: March 2007

The aim of the present study was to examine the relationships among β-cell function, proinsulin conversion to insulin, and age. We studied insulin and proinsulin secretion in nondiabetic subjects during an oral glucose tolerance test (OGTT) using published indexes of β-cell function (n = 379, age 16–68 years) and a modified hyperglycemic clamp (10 mmol/l, additional glucagon-like peptide [GLP-1] infusion, final arginine bolus; n = 50, age 19–68 years). Proinsulin conversion to insulin was assessed using proinsulin/insulin (PI/I) ratios immediately after an acute stimulus (OGTT, 30 min; hyperglycemic clamp, 2.5–5.0 min after glucose and arginine). There was a negative correlation between age and β-cell function (adjusted for insulin sensitivity, BMI, and fasting glucose) in the OGTT (r = −0.21, P < 0.001) and first phase of the hyperglycemic clamp (r = −0.30, P = 0.03), but not second phase (r = −0.08, P = 0.6) or arginine-induced insulin secretion (r = 0.06, P = 0.7). There was a positive correlation between age and the PI/I ratio in the OGTT (r = 0.24, P < 0.001). Analogously, there was also a positive correlation between age and the PI/I ratio during first phase (r = 0.37, P = 0.009) and arginine stimulation (r = 0.33, P = 0.01) of the hyperglycemic clamp. First-phase insulin secretion of the hyperglycemic clamp was inversely correlated with the PI/I ratio (r = −0.60, P < 0.001). Interestingly, adjusting first-phase secretion rate for the PI/I ratio abolished the linear relationship with age (r = −0.06, P = 0.7). In conclusion, aging is associated with deteriorating β-cell function and deteriorating proinsulin conversion to insulin. The age effect on insulin secretion appears to be attributable at least in part to an impairment of proinsulin conversion to insulin.
Ultradian Oscillations of Insulin Secretion in Humans
Authors: Simon Chantal , Brandenberger Gabrielle
Published: March 2007

Ultradian rhythmicity appears to be characteristic of several endocrine systems. As described for other hormones, insulin release is a multioscillatory process with rapid pulses of about 10 min and slower ultradian oscillations (50–120 min). The mechanisms underlying the ultradian circhoral oscillations of insulin secretion rate (ISR), which arise in part from a rhythmic amplification of the rapid pulses, are not fully understood. In humans, included in the same period range is the alternation of rapid eye movement (REM) and non-REM (NREM) sleep cycles and the associated opposite oscillations in sympathovagal balance. During sleep, the glucose and ISR oscillations were amplified by about 150%, but the REM-NREM sleep cycles did not entrain the glucose and ISR ultradian oscillations. Also, the latter were not related to either the ultradian oscillations in sympathoagal balance, as inferred from spectral analysis of cardiac R-R intervals, or the plasma fluctuations of glucagon-like peptide-1 (GLP-1), an incretin hormone known to potentiate glucose-stimulated insulin. Other rhythmic physiological processes are currently being examined in relation to ultradian insulin release.
Reduced Early Insulin Secretion in the Etiology of Type 2 Diabetes Mellitus in Pima Indians
Authors: Bogardus Clifton , Tataranni P. Antonio
Published: March 2007

We report the results of cross-sectional, prospective, and longitudinal studies identifying etiologic metabolic factors in the susceptibility to type 2 diabetes mellitus of the Pima Indians of Arizona, whose prevalence and incidence rates of the disease are the highest in the world. Diabetic Pima Indians are metabolically prototypic, with obesity, insulin resistance, a reduced acute insulin response to glucose, and increased endogenous glucose production. Cross-sectional studies show that the acute insulin response is absent in diabetic subjects and lower in impaired than in normal glucose-tolerant subjects. Prospective studies using proportional hazards analyses indicate that insulin resistance and a relatively low acute insulin response predict diabetes independently of age, gender, and each other, with obesity increasing susceptibility by worsening one or both predictors. Longitudinal studies show that glucose tolerance deteriorates as the degree of obesity increases due to worsening insulin resistance and decreases in early insulin secretion. Furthermore, since the children of diabetic pregnancies are at much greater risk of developing diabetes at a young age than those of nondiabetic pregnancies, the diabetic uterine environment may induce insulin resistance and/or reduced insulin secretion: early evidence confirms that adult normal glucose-tolerant offspring show a substantially decreased acute insulin response—the clearest demonstration yet of an environmental condition increasing susceptibility to type 2 diabetes mellitus. However, the genetic determinants require elucidation: correlation of the acute insulin response with the age of parental diabetes onset in fathers as well as mothers indicates a mechanism independent of the diabetic uterine environment.
Increased Insulin Resistance in Obese Children Who Have Both 972 IRS-1 and 1057 IRS-2 Polymorphisms
Authors: Le Fur Sophie , Le Stunff Catherine , Bougnères Pierre
Published: March 2007

In two cohorts of 174 and 165 obese Caucasian children, we measured insulin sensitivity and genotyped insulin receptor substrate IRS-1 and IRS-2 genes for the Arg972Gly and the Asp1057Gly variants, respectively. Because IRS-1 and IRS-2 have complementary roles in insulin signaling, we classified the genotypes in three categories: those with none of the variants in IRS-1 or IRS-2, those with one variant in IRS-1 or IRS-2, and those with variants in both IRS-1 and 2 proteins. The obese children with either the IRS-1 or IRS-2 variant had a mean insulin sensitivity index (2.9 ± 0.2 in cohort 1, 2.7 ± 0.1 in cohort 2) only slightly lower than the children having no variant in either gene (3.1 ± 0.2 and 3.5 ± 0.3, respectively). However, patients having variant alleles in both IRS-1 and IRS-2 genes showed a 25–35% decrease in sensitivity (2.3 ± 0.2 and 2.0 ± 0.2, respectively) when compared with nonvariant homozygotes (P < 0.001). These observations are reminiscent of the insulin sensitivity phenotypes in double IRS-1+/− IRS-2+/− heterozygous knockout mice. Our results stress the need for combined genotype analysis when candidate genes are functionally involved in the same pathway.
Decreased Glibenclamide Uptake in Hepatocytes of Hepatocyte Nuclear Factor-1α-Deficient Mice
Authors: Boileau Pascal , Wolfrum Christian , Shih David Q. , Yang Tien-An , Wolkoff Allan W. , Stoffel Markus
Published: March 2007

Diabetes in subjects with hepatocyte nuclear factor (HNF)-1α gene mutations (maturity-onset diabetes of the young [MODY]-3) is characterized by impaired insulin secretion. Surprisingly, MODY3 patients exhibit hypersensitivity to the hypoglycemic actions of sulfonylurea therapy. To study the pharmacogenetic mechanism(s), we have investigated glibenclamide-induced insulin secretion, glibenclamide clearance from the blood, and glibenclamide metabolism in wild-type and Hnf-1α-deficient mice. We show that despite a profound defect in glucose-stimulated insulin secretion, diabetic Hnf-1α−/− mice have a robust glibenclamide-induced insulin secretory response. We demonstrate that the half-life (t1/2) of glibenclamide in the blood is increased in Hnf-1α−/− mice compared with wild-type littermates (3.9 ± 1.3 vs. 1.5 ± 1.8 min, P ≤ 0.05). The clearance of glibenclamide from the blood during the first hours after intravenous administration was reduced approximately fourfold in Hnf-1α−/− mice compared with Hnf-1α+/+ littermates. Glibenclamide uptake into hepatocytes was dramatically decreased in vivo and in vitro. To study the metabolism of glibenclamide in Hnf-1α−/− animals, we analyzed liver extracts from [3H]glibenclamide-injected animals by reverse-phase chromatography. We found that the ratio of the concentrations of glibenclamide and its metabolites was moderately increased in livers of Hnf-1α−/− mice, suggesting that hepatic glibenclamide metabolism was not impaired in animals with Hnf-1α deficiency. Our data demonstrate that high serum glibenclamide concentrations and an increased t1/2 of glibenclamide in the blood of Hnf-1α−/− mice are caused by a defect in hepatic uptake of glibenclamide. This suggests that hypersensitivity to sulfonylureas in MODY3 patients may be due to impaired hepatic clearance and elevated plasma concentrations of the drug.
The Common Single Nucleotide Polymorphism E23K in KIR6.2 Sensitizes Pancreatic β-Cell ATP-Sensitive Potassium Channels Toward Activation Through Nucleoside Diphosphates
Authors: Schwanstecher Christina , Neugebauer Bettina , Schulz Miriam , Schwanstecher Mathias
Published: March 2007

E23K, a common polymorphism in the pore-forming subunit KIR6.2 of pancreatic β-cell ATP-sensitive K+ (KATP) channels, is functionally relevant and thus might play a major role in the pathophysiology of common type 2 diabetes. In this study, we show that in the simultaneous presence of activatory and inhibitory nucleotides, the polymorphism exerts opposite effects on the potencies of these modulators: channel opening through nucleoside diphosphates is facilitated, whereas sensitivity toward inhibition through ATP is slightly decreased. The results support the conclusion that E23K predisposes to type 2 diabetes by changing the channel’s response to physiological variation of cytosolic nucleotides, resulting in KATP overactivity and discrete inhibition of insulin release.
Interaction of the Cytosolic Domains of the Kir6.2 Subunit of the KATP Channel Is Modulated by Sulfonylureas
Authors: Lippiat Jonathan D. , Albinson Sophie L. , Ashcroft Frances M.
Published: March 2007

The NH2- and COOH-termini of the ATP-sensitive potassium (KATP) channel pore-forming subunit, Kir6.2, both lie intracellularly and interact with one another. To study this interaction, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were fused to the NH2- and COOH-termini of Kir6.2, respectively (CFP-Kir6.2-YFP). These fluorescent proteins have sufficient spectral overlap to allow distance-dependent fluorescence resonance energy transfer (FRET). When CFP-Kir6.2-YFP was expressed in human embryonic kidney cells and illuminated at 440 nm to excite CFP, significant fluorescence was recorded at 535 nm, the peak of the YFP emission spectrum. This indicated that FRET was occurring and thus that the NH2- and COOH-termini of Kir6.2 lie in close proximity to one another. The emission ratio, F535/F480, was increased by co-expression of SUR2A, but not SUR1, suggesting that SUR2A but not SUR1 influences the Kir6.2 NH2- and COOH-terminal interaction. This interaction was reduced by the sulfonylureas tolbutamide and gliclazide, but not by the pore blocker barium. The properties of the tolbutamide response indicate that the drug disrupts the interaction between the NH2- and COOH-termini of Kir6.2 by binding to a low-affinity site on Kir6.2.
Genetic Manipulations of Fatty Acid Metabolism in β-Cells Are Associated With Dysregulated Insulin Secretion
Authors: Eto Kazuhiro , Yamashita Tokuyuki , Matsui Junji , Terauchi Yasuo , Noda Mitsuhiko , Kadowaki Takashi
Published: March 2007

Triacylglyceride (TG) accumulation in pancreatic β-cells is associated with impaired insulin secretion, which is called lipotoxicity. To gain a better understanding of the pathophysiology of lipotoxicity, we generated three models of dysregulated fatty acid metabolism in β-cells. The overexpression of sterol regulatory element binding protein-1c induced lipogenic genes and TG accumulation. Under these conditions, we observed a decrease in glucose oxidation and upregulation of uncoupling protein-2, which might be causally related to the decreased glucose-stimulated insulin secretion. The overexpression of AMP-activated protein kinase was accompanied by decreased lipogenesis, increased fatty acid oxidation, and decreased glucose oxidation; insulin secretions to glucose and depolarization stimuli were decreased, probably because of the decrease in glucose oxidation and cellular insulin content. It was notable that the secretory response to palmitate was blunted, which would suggest a role of the fatty acid synthesis pathway, but not its oxidative pathway in palmitate-stimulated insulin secretion. Finally, we studied islets of PPAR-γ+/− mice that had increased insulin sensitivity and low TG content in white adipose tissue, skeletal muscle, and liver. On a high-fat diet, glucose-stimulated insulin secretion was decreased in association with increased TG content in the islets, which might be mediated through the elevated serum free fatty acid levels and their passive transport into β-cells. These results revealed some aspects about the mechanisms by which alterations of fatty acid metabolism affect β-cell functions.
Glutaminolysis and Insulin Secretion
Authors: Kelly Andrea , Li Changhong , Gao Zhiyong , Stanley Charles A. , Matschinsky Franz M.
Published: March 2007

Identification of regulatory mutations of glutamate dehydrogenase (GDH) in a form of congenital hyperinsulinism (GDH-HI) is providing a model for basal insulin secretion (IS) and amino acid (AA)-stimulated insulin secretion (AASIS) in which glutaminolysis plays a key role. Leucine and ADP are activators and GTP is an inhibitor of GDH. GDH-HI mutations impair GDH sensitivity to GTP inhibition, leading to fasting hypoglycemia, leucine hypersensitivity, and protein-induced hypoglycemia, indicating the importance of GDH in basal secretion and AASIS. The proposed model for glutaminolysis in IS is based on GDH providing NADH and α-ketoglutarate (α-KG) to the Krebs cycle, hence increasing the β-cell ATP-to-ADP ratio to effect insulin release. The process operates with 1) sufficient lowering of β-cell phosphate potential (i.e., fasting) and when 2) AAs provide leucine for allosteric activation and glutamate from transaminations. To test this hypothesis, IS studies were performed in rat and GDH-HI mouse models. In the rat study, rat islets were isolated, cultured, and then perifused in Krebs-Ringer bicarbonate buffer with 2 mmol/l glutamine using 10 mmol/l 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) or a BCH ramp after 50 or 120 min of glucose deprivation. In the GDH-HI mouse study, the H454Y GDH-HI mutation driven by the rat insulin promoter was created for H454Y β-cell-specific expression. Cultured, isolated islets were perifused in leucine 0–10 mmol/l with 2 mmol/l glutamine 0–25 mmol/l, AA 0–10 mmol/l, or glucose 0–25 mmol/l. Rat islets displayed enhanced BCH-stimulated IS after 120 min of glucose deprivation, but not when energized by fuel. H454Y and control islets had similar glucose-stimulated IS, but H454Y mice had lower random blood glucose. Leucine-stimulated IS and AASIS occurred at lower thresholds and were greater in H454Y versus control islets. Glutamine stimulated IS in H454Y but not control islets. The clinical manifestations of GDH-HI and related animal studies suggest that GDH regulates basal IS and AASIS. Energy deprivation enhanced GDH-mediated IS, and H454Y mice were hypoglycemic, substantiating roles for GDH and its regulation by the phosphate potential in basal IS. Excessive IS from H454Y islets upon exposure to GDH substrates or stimuli indicate that regulation of GDH by the β-cell phosphate potential plays a critical role in AASIS. These findings provide a foundation for defining pathways of basal secretion and AASIS, augmenting our understanding of β-cell function.
Fructose Improves the Ability of Hyperglycemia Per Se to Regulate Glucose Production in Type 2 Diabetes
Authors: Hawkins Meredith , Gabriely Ilan , Wozniak Robert , Vilcu Cristian , Shamoon Harry , Rossetti Luciano
Published: March 2007

The ability of hyperglycemia per se to suppress endogenous glucose production (GP) is blunted in type 2 diabetes. This could be due in part to decreased glucose-induced flux through glucokinase (GK). Because fructose activates hepatic GK, we examined whether catalytic amounts of fructose could restore inhibition of GP by hyperglycemia in humans with type 2 diabetes. Glucose fluxes ([3-3H]glucose) were measured during euglycemia (5 mmol/l) and after abrupt onset of hyperglycemia (10 mmol/l; variable dextrose infusion) under fixed hormonal conditions (somatostatin infusion for 6 h with basal insulin/glucagon/growth hormone replacement). A total of 10 subjects with moderately controlled type 2 diabetes and 7 age- and BMI-matched nondiabetic subjects were studied on up to three separate occasions under the following conditions: without fructose (F−) or with infusion of fructose at two dosages: 0.6 mg/kg · min (low F) and 1.8 mg/kg · min (high F). Although GP failed to decrease in response to hyperglycemia in type 2 diabetes, the coinfusion of both doses of fructose was associated with comparable decreases in GP in response to hyperglycemia (low F = −27%, high F = −33%; P < 0.01 vs. F− at both dosages), which approached the 44% decline in GP observed without fructose in the nondiabetic subjects. GP responses to hyperglycemia were not altered by the addition of fructose in the nondiabetic group (low F = −47%, high F = −42%; P > 0.05 vs. F−). Thus, the administration of small amounts of fructose to type 2 diabetic subjects partially corrected the regulation of GP by hyperglycemia per se, yet did not affect this regulation in the nondiabetic subjects. This suggests that the liver’s inability to respond to hyperglycemia in type 2 diabetes, likely caused by impaired GK activity, contributes substantially to the increased GP in these individuals.
Combination Therapy With Sirolimus and Interleukin-2 Prevents Spontaneous and Recurrent Autoimmune Diabetes in NOD Mice
Authors: Rabinovitch Alex , Suarez-Pinzon Wilma L. , Shapiro A.M. James , Rajotte Ray V. , Power Robert
Published: March 2007

Sirolimus is an immunosuppressant that inhibits interleukin (IL)-2 signaling of T-cell proliferation but not IL-2-induced T-cell apoptosis. Therefore, we hypothesized that administration of IL-2, together with sirolimus, might shift T-cell proliferation to apoptosis and prevent autoimmune destruction of islet β-cells. We found that sirolimus and IL-2 therapy of female NOD mice, beginning at age 10 weeks, was synergistic in preventing diabetes development, and disease prevention continued for 13 weeks after stopping sirolimus and IL-2 therapy. Similarly, sirolimus and IL-2 were synergistic in protecting syngeneic islet grafts from recurrent autoimmune destruction after transplantation in diabetic NOD mice, and diabetes did not recur after stopping sirolimus and IL-2 combination therapy. Immunocytochemical examination of islet grafts revealed significantly decreased numbers of leukocytes together with increased apoptosis of these cells in mice treated with sirolimus and IL-2, whereas β-cells were more numerous, and significantly fewer were apoptotic. In addition, Th1-type cells (γ-interferon-positive and IL-2+) were decreased the most, and Th2-type cells (IL-4+ and IL-10+) and Th3-type cells (transforming growth factor-β1+) were increased the most in islet grafts of sirolimus and IL-2-treated mice. We conclude that 1) combination therapy with sirolimus and IL-2 is synergistic in protecting islet β-cells from autoimmune destruction; 2) diabetes prevention continues after withdrawal of therapy; and 3) the mechanism of protection involves a shift from Th1- to Th2- and Th3-type cytokine-producing cells, possibly due to deletion of autoreactive Th1 cells.
Hypoglycemia-Associated Autonomic Failure in Advanced Type 2 Diabetes
Authors: Segel Scott A. , Paramore Deanna S. , Cryer Philip E.
Published: March 2007

We tested the hypotheses that the glucagon response to hypoglycemia is reduced in patients who are approaching the insulin-deficient end of the spectrum of type 2 diabetes and that recent antecedent hypoglycemia shifts the glycemic thresholds for autonomic (including adrenomedullary epinephrine) and symptomatic responses to hypoglycemia to lower plasma glucose concentrations in type 2 diabetes. Hyperinsulinemic stepped hypoglycemic clamps (85, 75, 65, 55, and 45 mg/dl steps) were performed on two consecutive days, with an additional 2 h of hypoglycemia (50 mg/dl) in the afternoon of the first day, in 13 patients with type 2 diabetes—7 treated with oral hypoglycemic agents (OHA RX; mean [±SD] HbA1c 8.6 ± 1.1%) and 6 requiring therapy with insulin for an average of 5 years and with reduced C-peptide levels (insulin RX, HbA1c 7.5 ± 0.7%)—and 15 nondiabetic control subjects. The glucagon response to hypoglycemia was virtually absent (P = 0.0252) in the insulin-deficient type 2 diabetic patients (insulin RX mean [±SE] final values of 52 ± 16 vs. 93 ± 15 pg/ml in control subjects and 98 ± 16 pg/ml in type 2 diabetic patients, OHA RX on day 1). Glucagon (P = 0.0015), epinephrine (P = 0.0002), and norepinephrine (P = 0.0138) responses and neurogenic (P = 0.0149) and neuroglycopenic (P = 0.0015) symptom responses to hypoglycemia were reduced on day 2 after hypoglycemia on day 1 in type 2 diabetic patients; these responses were not eliminated, but their glycemic thresholds were shifted to lower plasma glucose concentrations. In addition, the glycemic thresholds for these responses were at higher-than-normal plasma glucose concentrations (P = 0.0082, 0.0028, 0.0023, and 0.0182, respectively) at baseline (day 1) in OHA RX type 2 diabetic patients, with relatively poorly controlled diabetes. Because the glucagon response to falling plasma glucose levels is virtually absent and the glycemic thresholds for autonomic and symptomatic responses to hypoglycemia are shifted to lower glucose concentrations by recent antecedent hypoglycemia, patients with advanced type 2 diabetes, like those with type 1 diabetes, are at risk for hypoglycemia-associated autonomic failure and the resultant vicious cycle of recurrent iatrogenic hypoglycemia.
Attenuation of Diabetic Hyperphagia in Neuropeptide Y–Deficient Mice
Authors: Sindelar Dana K. , Mystkowski Paul , Marsh Donald J. , Palmiter Richard D. , Schwartz Michael W.
Published: March 2007

The combined effects of increased hypothalamic signaling by neuropeptide Y (NPY) and decreased signaling by melanocortins are hypothesized to stimulate food intake when body fat stores are depleted. To investigate NPY’s role in the hyperphagic response to uncontrolled diabetes, streptozotocin (STZ) (200 mg/kg intraperitoneally) or saline vehicle was given to NPY-deficient (Npy–/–) and wild-type (Npy+/+) mice. In Npy+/+ mice, STZ-induced diabetes increased mean daily food intake to plateau values 50% above baseline intake (+2.0 ± 0.6 g/day; P ≤ 0.05), an effect that was not seen in STZ-treated Npy–/– mice (+0.8 ± 0.1 g/day; NS), despite comparably elevated levels of plasma glucose and comparably decreased levels of body weight, fat content, and plasma leptin. Unlike the impaired feeding response to uncontrolled diabetes, Npy–/– mice exhibit intact hyperphagic responses to fasting (Erickson et al. [1], Nature 381:415–418, 1996). To investigate whether differences in hypothalamic melanocortin signaling can explain this discrepancy, we used in situ hybridization to compare the effects of STZ-diabetes and fasting on pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) mRNA levels in the hypothalamic arcuate nucleus (ARC) of Npy–/– and Npy+/+ mice. AgRP mRNA levels were increased by both fasting and STZ-diabetes, but the increase in STZ-diabetes was small (50–80%) compared with the effect of fasting (∼20-fold increase of AgRP mRNA). STZ-diabetes also lowered POMC mRNA levels by 65% in the ARC of Npy+/+ mice (P ≤ 0.05) but by only 11% in Npy–/– mice (NS); fasting significantly lowered POMC mRNA levels in both genotypes. We conclude that NPY is required for both the increase of food intake and the decrease of hypothalamic POMC gene expression induced by uncontrolled diabetes. In contrast, NPY is not required for either of these responses when the stimulus is food deprivation. Moreover, fasting is a more potent stimulus to hypothalamic AgRP gene expression than is STZ-diabetes. Therefore, central nervous system melanocortin signaling appears to be suppressed more effectively by fasting than by uncontrolled diabetes, which provides a plausible explanation for differences in the feeding response to these two stimuli in mice lacking NPY.
The Fate of [U-13C]Palmitate Extracted by Skeletal Muscle in Subjects With Type 2 Diabetes and Control Subjects
Authors: Blaak Ellen E. , Wagenmakers Anton J.M.
Published: March 2007

The current study investigated the fate of a [U-13C]palmitate tracer extracted by forearm muscle in type 2 diabetic and control subjects. We studied seven healthy lean male subjects and seven obese male subjects with type 2 diabetes using the forearm muscle balance technique with continuous intravenous infusion of the stable isotope tracer [U-13C]palmitate under baseline conditions and during intravenous infusion of the nonselective β-agonist isoprenaline (ISO; 20 ng · kg−1 lean body mass · min−1). In skeletal muscle of control subjects, there was a significant release of 13C-labeled oxidation products in the form of 13CO2 (15% of 13C uptake from labeled palmitate) and a significant release of 13C-labeled glutamine (release of 13C-labeled atoms from glutamine was 6% of 13C uptake from labeled palmitate), whereas in type 2 diabetic subjects there was no detectable release of 13CO2 and 13C-glutamine, despite a significant uptake of [U-13C]palmitate (60% of control value). There was net uptake of arterial 13C-labeled glutamate by forearm muscle in both groups. Also, the ISO-induced increase in arterial glutamine enrichment and arterial concentration of 13C-glutamine was more pronounced in the diabetic group relative to control subjects. In view of the diminished ISO-induced release of 13C-glutamine from type 2 diabetic muscle, the latter data indicate that more [U-13C]palmitate entered the liver in the diabetic group and was incorporated into newly synthesized glutamine and glutamate molecules. Thus, the lack of release of 13C-labeled oxidation products by type 2 diabetic muscle during β-adrenergic stimulation, despite significant [U-13C]palmitate uptake, indicates differences in the handling of fatty acids between type 2 diabetic subjects and healthy control subjects.
Effects of Treatment With Sulfonylurea Drugs or Insulin on Ischemia-Induced Myocardial Dysfunction in Type 2 Diabetes
Authors: Scognamiglio Roldano , Avogaro Angelo , Vigili de Kreutzenberg Saula , Negut Christian , Palisi Monica , Bagolin Eros , Tiengo Antonio
Published: March 2007

In patients with diabetes and coronary artery disease, the potential negative role of sulfonylurea drugs is under intensive investigation. We assessed the effects of treatment with glibenclamide or insulin on the extension of left ventricular myocardial dysfunction induced by acute ischemia. Nineteen consecutive patients with type 2 diabetes and coronary artery disease entered the study. Each patient was randomly assigned to either insulin or glibenclamide therapy. Treatment was crossed over after 12 weeks and maintained for another 12 weeks. At the end of each treatment, left ventricular myocardial function at rest and during dipyridamole infusion was studied by two-dimensional echocardiography under the same conditions of metabolic control. Glibenclamide or insulin treatment did not influence the rest values of left ventricular dimensions, left ventricular ejection fraction (LVEF), or wall motion score index (WMSI). Dipyridamole infusion, in patients receiving glibenclamide treatment, decreased LVEF (43 ± 7 vs. 37 ± 12%, P < 0.005) and increased WMSI (1.4 ± 0.28 vs. 1.98 ± 0.24, P < 0.001) compared with baseline values; during insulin treatment, LVEF (46 ± 8 vs. 45 ± 11%, NS) and WMSI (1.4 ± 0.29 vs. 1.6 ± 0.4, NS) did not change significantly. Peak stress LVEF was higher (45 ± 11 vs. 37 ± 12%, P < 0.001) and WMSI lower (1.6 ± 0.4 vs. 1.98 ± 0.24, P < 0.001) in patients receiving insulin. The results indicate that in patients with type 2 diabetes and coronary artery disease, ischemic myocardial dysfunction induced by dipyridamole infusion is less severe during treatment with insulin than with glibenclamide. Restitution of a preconditioning mechanism in insulin-treated patients may be the potential beneficial mechanism.
A Genome-Wide Scan for Loci Linked to Plasma Levels of Glucose and HbA1c in a Community-Based Sample of Caucasian Pedigrees
Authors: Meigs James B. , Panhuysen Carolien I. M. , Myers Richard H. , Wilson Peter W.F. , Cupples L. Adrienne
Published: March 2007

Elevated blood glucose levels are the hallmark of type 2 diabetes as well as a powerful risk factor for development of the disease. We conducted a genome-wide search for diabetes-related genes, using measures of glycemia as quantitative traits in 330 pedigrees from the Framingham Heart Study. Of 3,799 attendees at the 5th Offspring Study exam cycle (1991–1995), 1,461, 1,251, and 771 men (49%) and women provided information on levels of 20-year mean fasting glucose, current fasting glucose, and HbA1c, respectively, and 1,308 contributed genotype data (using 401 microsatellite markers with an average spacing of 10 cM). Levels of glycemic traits were adjusted for age, cigarette smoking, alcohol and estrogen use, physical activity, and BMI. We ranked standardized residuals from these models, created normalized deviates from the ranks, and used the variance component model implemented in SOLAR (Sequential Oligogenic Linkage Analysis Routines) to evaluate linkage to normalized deviates as quantitative traits. We found peak evidence for linkage to 20-year mean fasting glucose levels on chromosome 1 at ∼247 cM from p-telomere (pter) (multipoint logarithm of odds [LOD] 2.33) and on chromosome 10 at ∼86 cM from pter (multipoint LOD 2.07); to current fasting glucose levels on chromosome 1 at ∼218 cM from pter (multipoint LOD 1.80) and on chromosome 10 at ∼96 cM from pter (multipoint LOD 2.15); and to HbA1c levels on chromosome 1 at ∼187 cM (multipoint LOD 2.81). This analysis of unselected European Caucasian pedigrees suggests localization of quantitative trait loci influencing glucose homeostasis on chromosomes 1q and 10q. Findings at ∼187–218 cM on chromosome 1 appear to replicate linkage reported in previous studies of other populations, pointing to this large chromosomal region as worthy of more detailed scrutiny in the search for type 2 diabetes susceptibility genes.
Relationship Between TaqIB Cholesteryl Ester Transfer Protein Gene Polymorphism and Macrovascular Complications in Japanese Patients With Type 2 Diabetes
Authors: Kawasaki Isao , Tahara Hideki , Emoto Masanori , Shoji Tetsuo , Nishizawa Yoshiki
Published: March 2007

Cholesteryl ester transfer protein (CETP) is a key regulating factor of lipid metabolism, and the polymorphism of its gene may therefore be a candidate for modulating the lipid parameters, altering the susceptibility to atherosclerosis in type 2 diabetic subjects. In a group of 443 unrelated Japanese patients with type 2 diabetes, we studied the B1B2 polymorphism at the CETP locus, which is detectable with the restriction enzyme TaqI. Patients were separated into three groups according to genotype and compared based on their clinical characteristics, lipid parameters, and macrovascular complications. The B2 allele was associated in a dose-dependent fashion with higher HDL cholesterol and apolipoprotein AI levels, together with lower CETP concentrations. Furthermore, the prevalence of macrovascular complications, such as coronary heart disease, arteriosclerosis obliterans, and cerebral vascular disease, was significantly higher in subjects with the B1B1 genotype. Multiple logistic regression analysis also showed that the B1 allele of CETP genotype was associated with the incidence of these three complications independently of other risk factors. Thus, in type 2 diabetic patients, the B1B2 polymorphism of CETP gene is likely to be a strong genetic predictor of macrovascular complications.
Blood Letting in High-Ferritin Type 2 Diabetes
Authors: Fernández-Real José Manuel , Peñarroja Georgina , Castro Antoni , García-Bragado Fernando , Hernández-Aguado Ildefonso , Ricart Wifredo
Published: March 2007

Iron-related insulin-resistance is improved by iron depletion or treatment with iron chelators. The aim of this study was to evaluate insulin sensitivity and insulin secretion after blood letting in patients who had high-ferritin type 2 diabetes and were randomized to blood letting (three phlebotomies [500 ml of blood] at 2-week intervals, group 1) or to observation (group 2). Insulin secretion and sensitivity were tested at baseline and 4 and 12 months thereafter. The two groups were matched for age, BMI, pharmacologic treatment, and chronic diabetic complications. All patients were negative for C282Y mutation of hereditary hemochromatosis. Baseline glycated hemoglobin (6.27 ± 0.9% vs. 6.39 ± 1.2%), insulin sensitivity (2.75 ± 1.8 vs. 3.2 ± 2.1 mg · dl−1 · min−1), and area under the curve for C-peptide (AUCC-peptide; 38.7 ± 11.6 vs. 37.6 ± 14.1 ng · ml−1 · min−1) were not significantly different between the two groups of patients. Body weight, blood pressure, blood hematocrit levels, and drug treatment remained essentially unchanged during the study period. As expected, serum ferritin, transferrin saturation index, and blood hemoglobin decreased significantly at 4 months only in patients who received blood letting. In parallel to this changes, blood HbA1c decreased significantly only in group 1 subjects (mean differences, −0.61; 95% CI, −0.17 to −1.048; P = 0.01). AUCC-peptide decreased by −10.2 ± 6.3% after blood letting. In contrast, a 10.4 ± 6.4% increase in AUCC-peptide was noted in group 2 subjects at 4 months (P = 0.032). At 12 months, AUCC-peptide returned to values not significantly different from baseline in the two groups of subjects. At 4 months, the change in insulin sensitivity from baseline was significantly different between the two groups (80.6 ± 43.2% vs. −8.6 ± 9.9% in groups 1 and 2, respectively, P = 0.049). At 12 months, the differences between the two groups were even more marked (55.5 ± 24.8% vs. −26.8 ± 9.9%; P = 0.005). When the analysis was restricted to those subjects who completed the follow-up until 12 months, results did not show differences compared with the changes observed at 4 months, except for insulin sensitivity. A statistically significant increase in insulin sensitivity was observed in the blood-letting group (from 2.30 ± 1.81 to 3.08 ± 2.55 mg · dl−1 · min−1 at 4 months, to 3.16 ± 1.85 mg · dl−1 · min−1 at 12 months; P = 0,045) in contrast with group 2 subjects (from 3.24 ± 1.9 to 3.26 ± 2.05 mg · dl−1 · min−1 at 4 months, to 2.31 ± 1.35 mg · dl−1 · min−1 at 12 months). In summary, blood letting led simultaneously to decreased blood HbA1c levels and to changes in insulin secretion and insulin resistance that were significantly different from those observed in a matched observational group of subjects with high-ferritin type 2 diabetes. The mechanisms for improvement in peripheral insulin sensitivity after blood letting should be investigated further.
Specific Inhibition of PTEN Expression Reverses Hyperglycemia in Diabetic Mice
Authors: Butler Madeline , McKay Robert A. , Popoff Ian J. , Gaarde William A. , Witchell Donna , Murray Susan F. , Dean Nicholas M. , Bhanot Sanjay , Monia Brett P.
Published: March 2007

Signaling through the phosphatidylinositol 3′-kinase (PI3K) pathway is crucial for metabolic responses to insulin, and defects in PI3K signaling have been demonstrated in type 2 diabetes. PTEN (MMAC1) is a lipid/protein phosphatase that can negatively regulate the PI3K pathway by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, but it is unclear whether PTEN is physiologically relevant to insulin signaling in vivo. We employed an antisense oligonucleotide (ASO) strategy in an effort to specifically inhibit the expression of PTEN. Transfection of cells in culture with ASO targeting PTEN reduced PTEN mRNA and protein levels and increased insulin-stimulated Akt phosphorylation in α-mouse liver-12 (AML12) cells. Systemic administration of PTEN ASO once a week in mice suppressed PTEN mRNA and protein expression in liver and fat by up to 90 and 75%, respectively, and normalized blood glucose concentrations in db/db and ob/ob mice. Inhibition of PTEN expression also dramatically reduced insulin concentrations in ob/ob mice, improved the performance of db/db mice during insulin tolerance tests, and increased Akt phosphorylation in liver in response to insulin. These results suggest that PTEN plays a significant role in regulating glucose metabolism in vivo by negatively regulating insulin signaling.
Acute Hyperglycemia Induces Nitrotyrosine Formation and Apoptosis in Perfused Heart From Rat
Authors: Ceriello Antonio , Quagliaro Lisa , D’Amico Michele , Di Filippo Clara , Marfella Raffaele , Nappo Francesco , Berrino Liberato , Rossi Francesco , Giugliano Dario
Published: March 2007

This study investigated coronary perfusion pressure, nitric oxide (NO) and superoxide production, nitrotyrosine (NT) formation, and cardiac cell apoptosis in isolated hearts perfused with high glucose concentration. Coronary perfusion pressure; NO and superoxide anion generation; immunostaining for NT, inducible NO synthase (iNOS), and the constitutive type of NO synthase (NOS) eNOS; iNOS and eNOS mRNA expression by Western blot and RT-PCR; and apoptosis of cardiac cells were studied in hearts perfused for 2 h with solutions containing d-glucose at a concentration of 11.1 mmol/l (control), d-glucose at the concentration of 33.3 mmol/l (high glucose), or d-glucose (33.3 mmol/l) plus glutathione (0.3 mmol/l). Perfusion of isolated hearts in conditions of high glucose concentration caused a significant increase of coronary perfusion pressure (P < 0.001) and an increase of both NO and superoxide generation. However, superoxide production was 300% higher than baseline, whereas NO production was 40% higher (P < 0.001 for both). This effect was accompanied by the formation of NT, and an increase of iNOS expression. eNOS remained unchanged. At the end of the experiments, cardiac cell apoptosis was evident in hearts perfused with high glucose. The effects of high glucose were significantly prevented by glutathione. This study demonstrates that high glucose for 2 h is enough to increase iNOS gene expression and NO release in working rat hearts. Upregulation of iNOS and raised NO generation are accompanied by a marked concomitant increase of superoxide production, a condition favoring the production of peroxynitrite, a powerful pro-oxidant that can mediate the toxic effects of high glucose on heart by itself and/or via the formation of nitrotyrosine, as suggested by the detection of cell apoptosis.
Decreased Expression of Heat Shock Protein 72 In Skeletal Muscle of Patients With Type 2 Diabetes Correlates With Insulin Resistance
Authors: Kurucz Istvan , Morva Ágota , Vaag Allan , Eriksson Karl-Fredrik , Huang Xudong , Groop Leif , Koranyi Laszlo
Published: March 2007

Oxidative stress has been ascribed a role in the pathogenesis of diabetes and its complications, and stress proteins have been shown to protect organisms in vitro and in vivo against oxidative stress. To study the putative role of one of the most abundant cytoprotective stress proteins, inducible cytoplasmic 72-kDa-mass heat shock protein (Hsp-72), in the pathogenesis of diabetes, we measured its mRNA concentration in muscle biopsies from six type 2 diabetic patients and six healthy control subjects (protocol 1) as well as in 12 twin pairs discordant for type 2 diabetes and 12 control subjects undergoing a euglycemic-hyperinsulinemic clamp in combination with indirect calorimetry (protocol 2). The amount of Hsp-72 mRNA in muscle was significantly lower in type 2 diabetic patients than in healthy control subjects (in protocol 1: 5.2 ± 2.2 vs. 53 ± 32 million copies of Hsp-72 mRNA/μg total RNA, n = 6, P = 0.0039; in protocol 2: 3.2 ± 3.3 vs. 43 ± 31 million copies of Hsp-72 mRNA/μg total RNA, n = 12, P = 0.0001). Hsp-72 mRNA levels were also markedly reduced in the nondiabetic co-twins compared with healthy control subjects (5.8 ± 5.0 vs. 43 ± 31, n = 12, P = 0.0001), but they were also statistically significantly different from their diabetic co-twins when the difference between the pairs was compared (P = 0.0280). Heat shock protein mRNA content in muscle of examined patients correlated with the rate of glucose uptake and other measures of insulin-stimulated carbohydrate and lipid metabolism. In conclusion, the finding of decreased levels of Hsp-72 mRNA in skeletal muscle of patients with type 2 diabetes and its relationship with insulin resistance raises the question of whether heat shock proteins are involved in the pathogenesis of skeletal muscle insulin resistance in type 2 diabetes.
Dose-Dependent Vasodilating Effects of Insulin on Adenosine-Stimulated Myocardial Blood Flow
Authors: Sundell Jan , Nuutila Pirjo , Laine Hanna , Luotolahti Matti , Kalliokoski Kari , Raitakari Olli , Knuuti Juhani
Published: March 2007

In the peripheral vasculature, insulin induces time- and dose-dependent vasodilation. We have recently demonstrated that insulin potentiates adenosine-stimulated myocardial blood flow. However, it is unknown whether insulin’s effects on the coronary vasculature are dose dependent. In this study, we quantitated myocardial blood flow and adenosine-stimulated coronary flow (140 μg · kg−1 · min−1 for 5 min) in 10 healthy men (age, 32 ± 6 years; BMI, 24.1 ± 1.8 kg/m2) using positron emission tomography and 15O-labeled water. Hyperemic myocardial blood flow was measured in the basal state, during euglycemic physiological hyperinsulinemia (serum insulin ∼65 mU/l) and during supraphysiological hyperinsulinemia (serum insulin ∼460 mU/l). Basal myocardial blood flow was 0.84 ± 0.17 ml · g−1 · min−1. Physiological hyperinsulinemia increased the adenosine-stimulated flow by 20% (from 3.92 ± 1.17 to 4.72 ± 0.96 ml · g− 1 · min−1; P < 0.05). Supraphysiological hyperinsulinemia further enhanced the adenosine-stimulated flow by 19% (to 5.61 ± 1.03 ml · g−1 · min−1; P < 0.05). These effects were not explained by changes in systemic hemodynamics, since coronary resistance decreased during each insulin infusion (P < 0.05). In addition, hyperemic myocardial blood flow responses during insulin stimulation were positively correlated with whole-body glucose uptake. The results demonstrate that insulin is able to enhance hyperemic myocardial blood flow in a dose-dependent manner in healthy subjects. These effects might contribute to the known beneficial dose-dependent effects of insulin on myocardial ischemia.