bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Hyperglycemia on Nerve Conduction and Structure Is Age Dependent
Authors: Malone John I , Lowitt Saul , Korthals Jan K , Salem Angela , Miranda Candy
Published: September 2013

The nerve conduction velocity (NCV) of nondiabetic male Wistar rats continues to increase until ∼ 26 weeks of age. Rats made hyperglycemic at 6 weeks of age manifest reduced NCV by 10 weeks of age and show morphological differences in the sciatic tibial nerve after 5 months of hyperglycemia when compared with age-matched controls. Fiber diameter, myelin width, and the number of large myelinated fibers were decreased in the tibial nerves of the hyperglycemic animals. Rats made hyperglycemic at 26 weeks of age had elevated glycosylated hemoglobin and sciatic nerve sorbitol levels but maintained normal NCVs and had little change in morphology after 7 months of hyperglycemia. Thus, animals with maturing peripheral nerve structure and function exposed to chronic hyperglycemia manifest greater pathological alterations than those that occur when more matured nerves are exposed to similarly elevated glucose concentrations for an even greater duration. We suggest that immature animal models commonly used to study diabetic peripheral neuropathy may not be appropriate for understanding a process that commonly develops in humans who become hyperglycemic after maturation of the peripheral nerves.
Mitochondrial Glycerol-3-Phosphate Dehydrogenase: Cloning of an Alternatively Spliced Human Islet-Cell cDNA, Tissue Distribution, Physical Mapping, and Identification of a Polymorphic Genetic Marker
Authors: Ferrer Jorge , Aoki Minoru , Behn Philip , Nestorowicz Ann , Riggs Andrew , Permutt M Alan
Published: September 2013

Pancreatic β-cell mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) plays a major role in glucose-induced insulin secretion. Decreased activity of this enzyme has thus been proposed to play a role in the pathogenesis of NIDDM. Cloning of human insulinoma mGPDH cDNAs disclosed the existence of two variant transcripts with different 5′ ends. Reverse transcription polymerase chain reaction (PCR) confirmed the presence of both mGPDH mRNAs in purified native human pancreatic islets and other tissues. A major 6.5-Kb mGPDH transcript was detected by Northern blot analysis in RNA from human and rat pancreatic islets, with distinctly lower levels in other human tissues, indicating that previously reported high mGPDH enzymatic activity in β-cells is determined by high transcript levels. The mGPDH gene was mapped to chromosome 2 by PCR analysis of genomic DNA from human/rodent somatic cell hybrids, and five independent overlapping yeast artificial chromosome (YAC) clones containing the mGPDH sequence were identified from the Centre d'Etude du Polymorphisme Humain YAC library. Analysis of these YAC clones identified a highly polymorphic chromosome 2q21-q33 dinucleotide repeat genetic marker (D2S141) physically linked to the mGPDH gene. These studies provide the means to investigate the role of the human mGPDH gene in the pathogenesis of NIDDM and illustrate the value of a novel strategy to identify genetic markers for diabetes candidate genes.
Regulation of Glycogen Synthase and Protein Phosphatase-1 by Hexosamines
Authors: Crook Errol D , McClain Donald A
Published: September 2013

The hexosamine biosynthesis pathway has been hypothesized to be involved in mediating some of the adverse effects of high glucose. We have previously shown that glucose downregulates basal glycogen synthase (GS) activity in Rat-1 cells and that overexpressing the rate-limiting enzyme in the hexosamine biosynthesis pathway (glutamine:fructose-6-phosphate amidotransferase [GFA]) makes the cells more sensitive to these effects of glucose. GFA overexpression also leads to a reduction in insulin sensitivity of GS. Here we examine the effects of glucose and glucosamine on insulin-stimulated GS activity and on protein phosphatase-1 (PP1) activity. These activities were assayed in cytoplasmic extracts from Rat-1 fibroblasts overexpressing human GFA and cultured in varying glucose concentrations. Both maximal insulin-stimulated GS activity and insulin sensitivity decreased with increasing glucose. Overexpression of GFA leads to a further reduction in insulin sensitivity but not in maximal insulin-stimulated GS activity. Because there were no differences in total (glucose-6-phosphate-dependent) GS activity between cell lines or as a function of glucose concentration, these results most likely reflect a change in the phosphorylation state of the synthase. Activity of PP1, a potential mediator of these effects, was responsive to glucose and hexosamines. Control cells showed a 9.3 ± 4.3% decrease in PP1 activity with increasing glucose. GFA cells showed a greater response to glucose, with PP1 activity decreasing 34.2 ± 5.5% with increasing glucose. Glucosamine was more potent than glucose in decreasing PP1 activity in control cells. Cells overexpressing the normal human insulin receptor (HIRc-B) were used to facilitate analysis of insulin-stimulated PP1 activity. Stimulation with 1.7 mmol/l insulin led to a 37.6 ± 9.9% increase in PP1 activity in HIRc-B cells cultured in 1 mmol/l glucose, while cells cultured in 5 mmol/l glucosamine or 20 mmol/l glucose demonstrated only 3.79 ± 0.60 or 1.6 ± 0.75% increases, respectively. We conclude that both basal and insulin-stimulable GS and PP1 activity are downregulated by high glucose in fibroblasts and this regulation is mediated by products of the hexosamine biosynthesis pathway.
Long-Term Normoglycemic Remission in Black Newly Diagnosed NIDDM Subjects
Authors: Banerji Mary Ann , Chaiken Rochelle L , Lebovitz Harold E
Published: September 2013

We have defined and characterized the natural history of spontaneous near-normoglycemic remission off of antidiabetic medication in 79 black NIDDM subjects. They had initially presented with plasma glucose levels of 37.8 ± 19.3 mmol/l. Baseline clinical metabolic and 8-year prospective data were obtained (51 men and 28 women, mean age 45 ± 10 years, islet-cell or GAD antibody negative). After hospitalization and intensive outpatient treatment, near-normoglycemic remission (fasting plasma glucose 6.1 ± 0.83 mmol/l and HbA1c 0.95 ± 0.10 of upper limit of normal) occurred within 8 ± 10 months of insulin or sulfonylurea therapy. This was unrelated to the resolution of stress or significant weight loss (1.9 ± 4.97 kg). Metabolic studies performed during remission showed 17% normal, 33% impaired, and 50% diabetic glucose tolerance. Glucose disposal (1 mU · kg−1 · min−1) euglycemic insulin clamp with D-[3-3)H]glucose) was higher in the normal glucose tolerance group compared with the impaired and diabetic groups (37.8 ± 10.2 vs. 26.1 ± 10.7 and 26.7 ± 12.0 μmol · kg−1 · min−1; P < 0.05) despite similar BMIs in all three groups (28.8 ± 3.7 kg/m2). Insulin secretion was below the normal range. Of 79 patients, 27 relapsed. A Kaplan-Meier survival analysis gives a median time of 40 months to relapse. Higher presenting plasma glucose and male sex predicted earlier relapse. Near-normoglycemic remission may occur in up to 30% of black new-onset NIDDM patients. It appears to be associated with intensive initial glycemic regulation and may be a method of decreasing the development of microvascular complications in NIDDM.
Prostacyclin-Stimulating Factor, Novel Protein, and Diabetic Angiopathy
Authors: Umeda Fumio , Ono Yasuhiro , Masakado Mitsunori , Sekiguchi Naotaka , Yamauchi Teruaki , Hashimoto Toshihiko , Nawata Hajime
Published: September 2013

We recently purified and cloned a new protein that stimulates the synthesis of prostacyclin (PGI2) by the vascular endothelial cells (ECs). We have termed this protein “PGI2-stimulating factor” (PSF). The present study evaluated the expression of PSF mRNA in tissues of Wistar rats, including the kidneys of rats with streptozotocin-induced diabetes, and in cultured cells. Furthermore, we evaluated the presence of PSF in human sera and the immunohistochemical localization of PSF in tissues of patients obtained at autopsy. The latter included a coronary atherosclerotic lesion of a patient who died of acute myocardial infarction. PSF was observed by Northern blot analysis to be expressed in all rat tissues examined (brain, lung, liver, kidney, skeletal muscle, and fat tissue) and was expressed in cultured vascular ECs, smooth muscle cells (SMCs), and flbroblast cells (FCs). A decreased expression of PSF was observed in the kidneys of diabetic rats versus those of normal rats. The presence of PSF in human serum was confirmed by Western blot analysis. In humans, PSF was mainly localized in vascular ECs and SMCs of arterial media and in SMCs of bronchi. Reduced staining for PSF was found in an atherosclerotic versus a normal coronary artery of humans. PSF may be involved in the production of PGI2 in the vessel wall and may participate in the maintenance of vascular homeostasis. PSF abnormalities may be involved in the development of such vascular lesions as atherosclerosis and diabetic angiopathy.
Atherogenic Modified LDL in Diabetes
Authors: Sobenin Igor A , Tertov Vladimir V , Orekhov Alexander N
Published: September 2013

This paper summarizes the recent findings on LDL atherogenic modifications in diabetic patients. LDL from diabetic patients, unlike LDL from healthy subjects, caused a significant increase in cholesterol content of cells cultured from unaffected human aortic intima, i.e., produced a direct atherogenic effect. LDL was divided into two fractions (nonbound and bound) by affinity chromatography on Ricinus communis agglutinin-agar-ose. The amount of bound LDL was significantly higher in diabetic patients compared with healthy subjects. Bound LDL was characterized by significantly lowered sialic acid content and a significantly increased fructosyl lysine level compared with unbound LDL, i.e., was desialylated and nonenzymatically glycosylated lipoprotein. The bound (desialylated), but not unbound (sialylated), LDL subfraction induced cholesterol accumulation in cultured cells. Bound LDL was also characterized by a significantly lowered content of neutral lipids and demonstrated increased electrophoretic mobility on agarose gel electro-phoresis. Bound and nonbound LDL differed significantly in hydrated density and particle size, as was determined by density gradient ultracentrifugation and native poly-acrylamide gradient gel electrophoresis. The results of this study have shown that the in vivo modified atherogenic LDL subfraction in the blood of diabetic patients is represented by small, dense, more electronegative, desialylated, and glycated LDL.
Effects of Insulin and IGF-I on Vascular Smooth Muscle Glucose and Cation Metabolism
Authors: Sowers James R
Published: September 2013

Data is accumulating indicating that impaired insulin action predisposes to increased vascular smooth muscle (VSM) tone, the hallmark of hypertension associated with diabetes. During the last several years, it has been established that VSM is an insulin-sensitive tissue like skeletal muscle and adipocytes. Investigators have shown that insulin regulates VSM intracellular cation metabolism through attenuating effects on inward calcium (Ca2+) currents and by direct effects on VSM cells (VSMCs) Na+, K+-ATPase pump expression and activity and that insulin and IGF-I stimulate glucose uptake in VSMCs. Furthermore, recent data suggest that IGF-I, like insulin, attenuates cytosolic calcium [Ca2+]i transients and vasoconstrictive responses. IGF-I, like insulin, also stimulates the production of nitric oxide from both the endothelium and VSMCs. IGF-I and insulin are structurally related, share receptors, and have similar postreceptor actions. Unlike insulin, which must transverse the endothelium before acting on VSMCs in vivo, IGF-I is synthesized by VSMCs. Thus, it is likely that IGF-I has more relevance than insulin in regulating physiological parameters in VSMCs.
Mechanism and Clinical Implication of Insulin Resistance Syndrome
Authors: Suzuki Masaaki , Ikebuchi Motoyoshi , Shinozaki Kazuya , Hara Yasushi , Tsushima Motoo , Matsuyama Tatsuo , Harano Yutaka
Published: September 2013

The insulin resistance syndrome has been noted as an interesting and important new risk factor for coronary artery disease. The syndrome consists of hypertension, glucose intolerance, and dyslipidemia, all of which are likely to be derived from insulin insensitivity. In subjects with nonobese and nondiabetic essential hypertension, steady-state plasma glucose (SSPG) was higher than in normotensive subjects during an insulin sensitivity test, indicating reduced insulin sensitivity to glucose metabolism in the hypertensive group. SSPG correlated with the percentage decrease of branched chain amino acids, free fatty acids, and serum potassium during the insulin sensitivity test. With a 2-h insulin infusion, serum norepinephrine, epinephrine, plasminogen activator inhibitor 1, and intraplatelet Ca2+ decreased significantly, but 6-ke-to-prostaglandin (PG) F1α and PGE2 did not change. Insulin resistance decreased by using antihypertensive treatments with bunazosin, cilazapril, amlodipine, and benidipine in hypertensive subjects. Diagnostic criteria for the insulin resistance syndrome, including clinical values for each risk factor, were developed. Lowered insulin sensitivity and hyperinsulinemia were demonstrated in subjects with both vasospastic and coronary artery stenotic angina. The insulin resistance syndrome together with hyperinsulinemia is likely to induce atherosclerotic changes, possibly through reduced rather than excessive action of insulin.
Diabetes and Coronary Heart Disease in American Indians: The Strong Heart Study
Authors: Howard Barbara V , Lee Elisa T , Fabsitz Richard R , Robbins David C , Yeh Jeunliang L , Cowan Linda D , Welty Thomas K
Published: September 2013

Coronary heart disease (CHD) is the leading cause of death among American Indians. However, information on the prevalence of CHD and its association with known risk factors is limited. The purpose of the Strong Heart Study is to quantify CHD and its risk factors among three geographically diverse groups of American Indians. The population consists of 4,549 adults between 45 and 74 years of age in 13 Indian communities in Arizona, Oklahoma, and South and North Dakota. The phase I examination (1989–1991) revealed very high prevalence rates of diabetes that ranged from 33 to 72% in men and women in the three centers. Prevalence rates of definite myocardial infarction (MI) and definite CHD were higher in men than in women in all three centers (P < 0.0001) and in those with diabetes (P = 0.002 and P = 0.0003 in women and men, respectively). Diabetes was associated with a relatively greater increase in prevalence of MI (prevalence rate = 3.8 vs. 1.9) and CHD (prevalence rate = 4.6 vs. 1.8) in women than in men. Logistic regression analysis indicated that the prevalence of CHD among American Indians was significantly related to age, diabetes, hypertension, albuminuria, percentage of body fat, smoking, high concentrations of plasma insulin, and low concentrations of HDL cholesterol. Lower prevalence rates of CHD were found in Arizona despite higher rates of diabetes, obesity, hypertension, and albuminuria; these lower rates may be in part related to lower smoking frequency and lower concentrations of total and LDL cholesterol. These findings from the baseline Strong Heart Study examination emphasize the relative importance of diabetes and its associated variables as risk factors for CHD among American Indian populations.
Glycation, Oxidative Stress, and Scavenger Activity: Glucose Metabolism and Radical Scavenger Dysfunction in Endothelial Cells
Authors: Kashiwagi Atunori , Asahina Takayuki , Nishio Yoshihiko , Ikebuchi Motoyoshi , Tanaka Yasushi , Kikkawa Ryuichi , Shigeta Yukio
Published: September 2013

It has been reported that oxidative stress is increased in vivo in the diabetic state. Increased oxidative stress is caused not only by accelerated production of oxygen-free radicals but also by decreased scavenging of those molecules. Endothelial cells are extremely sensitive to oxidative stress, resulting in impairments of various endothelial cell function. In this report, we studied the association of intracellular glucose metabolism and oxygen radical scavenging function via the glutathione redox (GR) cycle in cells exposed to high-glucose conditions using cultured human umbilical vein endothelial cells. Glutathione-dependent H2O2 degradation in cells exposed to 33 mmol/1 glucose (HG) for 5–7 days was reduced by 48% vs. 5.5 mmol/1 glucose (NG). This impairment under the oxidative stress was D-glucose-specific and concentration-dependent and was also associated with a 42% decrease in intracellular NADPH content. Exposure of cells to 200 μmol/1 H2O2 stimulated the GR cycle and the pentose phosphate pathway (PPP) at the same time. In the HG condition, activation of PPP was reduced by 50%, which was consistent with a decrease in NADPH content. Inhibition of glycolysis by H2O2 was less marked in HG cells versus NG cells. Activation of polyol pathway in HG cells is not responsible for the decrease in intracellular NADPH content. These results indicate that activation of the PPP and NADPH supply to the GR cycle is impaired in HG cells exposed to H2O2, which may result in increased oxidative stress to endothelial cells.
Differential Responsiveness of Protein Synthesis and Degradation to Amino Acid Availability in Humans
Authors: Giordano Mauro , Castellino Pietro , DeFronzo Ralph A
Published: September 2013

We investigated the effects of graded hyperaminoacidemia on protein metabolism in eight healthy, young (25 ± 2 years), normal weight (BMI = 25 ± 1 kg/m2), overnight-fasted human subjects. A balanced amino acid solution was infused for 180 min at five different rates: 0.5 (study I), 1.0 (study II), 2.0 (study III), 4.0 (study IV), and 6.0 (study V) mg · kg−1 · min−1 on separate days in random order. Studies were performed with [1-14C]leucine infusion and indirect calorimetry to calculate leucine oxidation (LOX), nonoxidative leucine disposal (NOLD) (an index of protein synthesis), and endogenous leucine flux (ELF) (an index of proteolysis). Basal total plasma amino acid concentrations averaged 1.85 ± 0.1 nunol/1 and increased to 2.27 ± 0.1, 2.70 ± 0.2, 3.84 ± 0.2, 5.87 ± 0.4, and 7.52 ± 0.3 mmol/1 in studies I–V, respectively. ELF decreased from a basal value of 2.27 ± 0.2 to 2.12 ± 0.2, 1.97 ± 0.1, 1.73 ± 0.2, 1.67 ± 0.3, and 1.65 ± 0.1 μmol · kg−1 · min−1 in studies I–V, respectively (P < 0.05 for study I vs. basal, P < 0.01 for studies II–V vs. basal, and NS for studies IV and V vs. study III). LOX increased from a basal value of 0.31 ± 0.04 to 0.38 ± 0.05, 0.41 ± 0.02, 0.64 ± 0.04, 1.11 ± 0.07, and 1.56 ± 0.05 μmol kg−1 · min−1 in studies I–V (all P < 0.01 vs. basal; P < 0.05–0.01 for each study vs. preceding study). Basal NOLD averaged 1.96 ± 0.2 and did not change significantly in studies I and II (2.03 ± 0.2 and 2.10 ± 0.1 μmol · kg−1 · min−1). In contrast, a significant increase in NOLD was observed in studies III, IV, and V (to 2.3 ± 0.15, 2.74 ± 0.2, and 3.25 ± 0.7 (μmol · kg−1 · min−1 , respectively; all P < 0.01 vs. basal; P < 0.05–0.01 for each study vs. preceding study). The net leucine balance (difference between ELF and NOLD) (−0.31 ± 0.06 (μmol · kg−1 · min−1) became less negative in study I (P < 0.01 vs. basal) and positive during studies II–V when the rise in plasma total amino acid levels was ≥50% above basal level (P < 0.01 vs. each preceding study). In conclusion, NOLD, ELF, and LOX exhibit a differential responsiveness to acute changes in substrate availability: 1) small increments (25–50%) in plasma amino acid levels inhibit ELF and stimulate LOX but have no effect on NOLD; 2) stimulation of NOLD is observed only with increments in plasma amino acid levels ≥100% above basal values; and 3) increments in plasma amino acid concentrations >100% above basal values cause a progressive dose-related increase in LOX and NOLD but do not induce any further inhibition of ELF.
Polymorphism at the rad Gene Is Not Associated With NIDDM in Finns
Authors: Orho Marju , Carlsson Martin , Kanninen Timo , Groop Leif C
Published: September 2013

Recently, a trinucleotide repeat polymorphism at the rad (ras associated with diabetes) locus (RAD1) on chromosome 16q was described in association with NIDDM in white Americans. In an attempt to replicate this finding, we screened RAD1 and another microsatellite marker at the D16S265 loci, which is located near the rad locus, with a radioactive polymerase chain reaction method in 290 unrelated Finnish NIDDM patients and 270 control subjects and related the findings to measures of insulin sensitivity. Both groups were randomly selected from the western (189 NIDDM patients and 184 control subjects) and southern (101 NIDDM and 86 control subjects) parts of Finland. The allele frequency distributions of RAD1 and D16S265 did not differ between NIDDM patients and control subjects in the studied population groups. The genotype distribution was also analyzed by structural classes of the RAD1 polymorphism, and no difference was detected between the NIDDM and control groups. In addition, carriers of allele classes I, II, and IV (reported to be preferentially associated with NIDDM in white Americans) did not differ from the class III homozygotes with respect to age at onset of NIDDM, BMI, or rates of insulin-stimulated glucose disposal. In conclusion, we found no association between the rad locus and NIDDM or insulin resistance in Finnish NIDDM patients.
Hyperglucagonemia Stimulates Phenylalanine Oxidation in Humans
Authors: Tessari Paolo , Inchiostro Sandro , Barazzoni Rocco , Zanetti Michela , Vettore Monica , Biolo Gianni , Iori Elisabetta , Kiwanuka Edward , Tiengo Antonio
Published: September 2013

Glucagon stimulates in vitro liver phenylalanine (Phe) degradation, thus inducing net protein catabolism. Whether these effects occur also in vivo in humans is not known. Therefore, we studied the effects of physiological hyperglucagonemia on Phe rate of appearance (Rα), hydroxylation, and oxidation in seven normal volunteers during infusions of somatostatin with replacement doses of insulin and growth hormone. Steady-state Phe kinetics were evaluated using the l-[1-14C]Phe tracer both at the end of a 3-h basal glucagon replacement period (glucagon concentration: 212 ± 115 ng/l) and after a 3-h hormone infusion at the rate of ∼ 3 ng · kg−1 · min−1 (→654 ± 280 ng/l). Hyperglucagonemia did not change plasma Phe concentration and Ra but increased Phe oxidation by ∼ 30% (P < 0.01). Oxidation was also increased by ∼ 24% (P < 0.01) using plasma [14C]tyrosine (Tyr) specific activity as a precursor pool. Phe hydroxylation to Tyr estimated by assuming a fixed ratio of Tyr to Phe Rα (0.73) did not change. Nonhydroxylated Phe disposal decreased by ∼ 6% (P = 0.08). These data show that in humans in the postabsorptive state, hyperglucagonemia, with near maintenance of basal insulin and growth hormone concentrations, stimulates Phe oxidation but not Phe hydroxylation, suggesting a different regulation of these two Phe catabolic steps. Glucagon may also reduce Phe availability for protein synthesis.
Phenotypic Modulation of the Mesangium Reflected by Contractile Proteins in Diabetes
Authors: Makino Hirofumi , Kashihara Naoki , Sugiyama Hitoshi , Kanao Koichiro , Sekikawa Takashi , Okamoto Kazunori , Maeshima Yohei , Ota Zensuke , Nagai Ryozo
Published: September 2013

The phenotypic change of the mesangial cell is considered to play a pivotal role in the accumulation of extracellular matrix in diabetic nephropathy. This investigation was undertaken to evaluate the expression of the various isoforms of contractile proteins in the streptozocin (STZ)-induced diabetic rat kidney and in renal biopsy specimens from patients with diabetic nephropathy. Specific antibodies to myosin heavy chain isoforms (SM1, SM2, SMemb), caldesmon, and α-smooth muscle actin and cDNAs for SMemb were used. Increased expression of SMemb at the mRNA and protein levels was demonstrated at 1 week after STZ administration in the rat. Both levels were increased at 4 weeks. Mesangial staining of caldesmon was observed at 4 weeks and that of α-smooth muscle actin at 24 weeks. Immunohistochemical mesangial staining of the contractile proteins was pronounced in patients with diabetic nephropathy in contrast to the trace mesangial staining in normal control subjects. These results indicate that the phenotypic change in mesangial cells occurs in the early stages of diabetes and that several stages in phenotypic changes may exist. Expression of the contractile protein isoforms, especially SMemb, should serve as a new marker for the subsequent glomerular hypertrophy and sclerosis.
Ontogeny and Tissue Distribution of Human GAD Expression
Authors: Mally Martin I , Cirulli Vincenzo , Otonkoski Timo , Soto Gabriel , Hayek Alberto
Published: September 2013

One of the major β-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with α- and β-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.
Evidence of a Glycemic Threshold for the Formation of Pentosidine in Diabetic Dog Lens but Not in Collagen
Authors: Nagaraj Ramanakoppa H , Kern Timothy S , Sell David R , Fogarty John , Engerman Ronald L , Monnier Vincent M
Published: September 2013

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 ± 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 ± 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.
Indication for Linkage of the Human OB Gene Region With Extreme Obesity
Authors: Clement Karine , Garner Chad , Hager Jorg , Philippi Anne , LeDuc Carrie , Carey Alisoun , Harris Timothy JR , Jury Corinne , Cardon Lon R , Basdevant Arnaud , Demenais Florence , Guy-Grand Bernard , North Michael , Froguel Philippe
Published: September 2013

Obesity is one of the most significant risk factors for hypertension, coronary heart disease, and NIDDM (Frayn KN, Coppack SW: Insulin resistance, adipose tissue and coronary heart disease. Clin Sci 82:1–8, 1992; Kaplan NM: The deadly quartet: upper-body obesity, glucose intolerance, hypertriglyceridemia, and hypertension. Arch Intern Med 149:1514–1520, 1989). While family segregation, adoption, and twin studies have indicated that degree of adiposity has a significant genetic component (Stunkard AJ, Harris JR, Pedersen NL, McClearn GE: The body-mass index of twins who have been reared apart. N Engl J Med 322:1483–1487, 1990; Bouchard C, Despres J-P, Mauriege P: Genetic and nongenetic determinants of regional fat distribution. Endocr Rev 14:72–93, 1993), the genes and predisposing mutations remain poorly understood. This is in contrast to several well-defined genetic models for obesity in rodents, particularly the mouse obese (ob) gene, in which loss-of-function mutations cause severe obesity. Recent studies have demonstrated a substantial reduction in body fat when recombinant ob protein (leptin) is administered to mice. To test the relevance of these observations to human obesity, the location of the human homologue (OB) was established by radiation hybrid mapping and eight microsatellite markers spanning the OB gene region (7q3l.3) were genotyped in 101 obese French families. Affected–sib-pair analyses for extreme obesity, defined by BMI >35 kg/m2, revealed suggestive evidence for linkage to three markers located within 2 cM of the OB gene (D7S514, D7S680, and D7S530). The OB gene is therefore a candidate for genetic predisposition to extreme obesity in a subset of these families.
Expression of a Specific Subset of CD44 Variant Transcripts in NOD Pancreatic Islets
Authors: Milde Kerstin F , Alonso Miriam , Shen Kong Shen , Alejandro Rodolfo , Mintz Daniel H , Pastori Ricardo L
Published: September 2013

Adhesion of lymphocytes to the endothelial venules inside the islets of Langerhans seems to initiate the infiltration of islets in NOD mice. An overexpression of the lymphocyte surface molecule CD44 in infiltrated NOD islets compared with peripheral blood lymphocytes was recently reported. The CD44 protein family includes a variety of molecules generated by alternative RNA splicing from 10 variant exons (v1–v10). By using reverse transcriptase–polymerase chain reaction followed by Southern blotting and hybridization to exon-specific cDNA probes, we investigated the expression of CD44 isoforms in highly purified islets of Langerhans from 4- and 10-week-old NOD mice. At least six CD44 isoforms were strongly overexpressed in NOD islets at 4 and 10 weeks when compared with age-matched BALB/c islets. Controls in different tissues indicate that these variants are specifically increased in the islets from the NOD strain. Islets from the NOD-scid/scid strain also expressed these variant exons. Splenocytes from BALB/c did not express CD44 isoforms, whereas splenocytes from 4-week-old NOD mice did express CD44 variants. Treatment with inflammatory mediators induced new isoforms; however, these transcripts have a different variant exon composition from that found in NOD mice islets. These results suggest that some isoforms are expressed very early in the development of insulitis by a component of the NOD islet itself and underscore a possible role of CD44 in islet infiltration.
The Gly40Ser Mutation in the Human Glucagon Receptor Gene Associated With NIDDM Results in a Receptor With Reduced Sensitivity to Glucagon
Authors: Hansen Lars H , Abrahamsen Niels , Hager Jörg , Jelinek Laura , Kindsvogel Wayne , Froguel Philippe , Nishimura Erica
Published: September 2013

The pancreatic islet hormone, glucagon, stimulates hepatic glucose production and has also been shown to potentiate glucose-induced insulin secretion. Because glucagon is a key regulator of glucose homeostasis, its receptor, which mediates the actions of glucagon, was considered a candidate gene involved in the pathogenesis of NIDDM. We have previously reported that a single heterozygous missense mutation in exon 2 of the glucagon receptor gene, which changes a glycine to a serine (Gly40Ser), is associated with NIDDM in a French population. In the present study, the signaling properties of this mutant receptor were examined in baby hamster kidney cells and rat insulinoma cells (REV-5AH) stably transfected with either the wild type or Gly40Ser mutant human glucagon receptor cDNAs. Competition assays using 125 I-labeled glucagon were performed, and in both cell types, the Gly40Ser mutant receptor was found to bind glucagon with an approximately threefold lower affinity compared with the wild type receptor. In both cell types, the production of cAMP in response to glucagon was decreased in cells expressing the mutant receptor compared with those expressing the wild type. Finally, glucagon-stimulated insulin secretion by RIN cells expressing the mutant receptor was decreased such that the dose-response curve was shifted to the right in comparison to that obtained with cells expressing the wild type receptor. These results indicate that this single-point mutation located in the extracellular region of the glucagon receptor decreases the sensitivity of target tissues to glucagon.
Microalbuminuria in NIDDM Is Caused by Increased Excretion of Unmodified Albumin
Authors: Ishibashi Fukashi
Published: September 2013

After an intravenous infusion of L-arginine to inhibit tubular reabsorption of albumin, glomerular clearance, renal clearance, and tubular reabsorption of unmodified albumin (UMA) and glycated albumin (GA) were determined in 72 patients with NIDDM without (NIDDM-I; n = 47) or with microalbuminuria (NIDDM-II; n = 25) and in 24 healthy control subjects. Samples of serum albumin and dialyzed urine obtained 60 min before and during L-arginine infusion were applied to an affinity column to separate GA from UMA, and their albumin contents were assayed. The serum level of GA in NIDDM patients was higher than that in control subjects (P < 0.0001). Both UMA and GA were excreted in excess in NIDDM-II as compared with the other two groups (P < 0.0001), and UMA comprised 80% of total albumin excretion. In NIDDM-II, the glomerular clearance of UMA (2.5 ± 0.16 > NIDDM-I [1.8 ± 0.1] > control subjects [1.3 ± 0.1 μl/min], P < 0.001) and of GA (1.7 ± 0.13 > NIDDM-I = control subjects [1.1 ± 0.1 μl/min], P < 0.001) were enhanced, as compared with the other two groups. Renal clearance of UMA (1.3 ± 0.13 μl/min) and GA (0.89 ± 0.09 μl/min) in NIDDM-II was greater than that in control subjects (0.27 ± 0.03, 0.19 ± 0.02 μl/min) or in NIDDM-I (0.30 ± 0.03, 0.11 ± 0.01 μl/min). Tubular reabsorption of UMA, as assessed by the difference between glomerular and renal clearances of albumin, in NIDDM-II (1.1 ± 0.1 μl/min) was less than in NIDDM-I (1.50 ± 0.09 μl/min), and that of GA in NIDDM-II was lower than that in the other two groups, despite exaggerated glomerular clearance of GA and UMA in NIDDM-II. In summary, microalbuminuria in NIDDM is caused by increased excretion of UMA resulting from the decompensated ability of tubular reabsorption, which is exceeded by increased glomerular clearance of UMA.
LDL From Patients With Well-Controlled IDDM Is Not More Susceptible to In Vitro Oxidation
Authors: Jenkins Alicia J , Klein Richard L , Chassereau Charlyne N , Hermayer Kathie L , Lopes-Virella Maria F
Published: September 2013

Increased susceptibility of LDL to oxidation has been shown to be associated with the presence of coronary heart disease and may account for the accelerated vascular disease seen in diabetes. The response of LDL to in vitro oxidative stress has been proposed as a measure of the predisposition of LDL to the in vivo subendothelial oxidative stress. Increased susceptibility to oxidation has been demonstrated recently in diabetic patients with poorly controlled IDDM. Thus, we conducted studies to determine whether the increased susceptibility of LDL to oxidation was secondary to diabetes per se or to the level of glycemic control. Fifteen IDDM patients with good glycemic control and with no evidence of macrovascular disease or proteinuria were compared with healthy age-, sex-, race-, and BMI-matched nondiabetic subjects. Fasting blood glucose levels averaged 12.1 ± 1.1 (mean ± SE) vs. 4.9 ± 0.1 mmol/l in the diabetic versus the control groups, respectively. HbAlc levels averaged 7.7 ± 0.5 vs. 4.4 ± 0.2%, reflecting well-controlled diabetes (P < 0.0001). Total, LDL, VLDL, and HDL cholesterol, triglyceride, and lipoprotein(a) levels did not differ between the groups. The particle size, lipid composition, fatty acid content, antioxidant content, and glycation were similar for LDL isolated from both groups. A rapid LDL preparation technique was used to compare LDL susceptibility to oxidation under the following conditions: final LDL cholesterol concentration of 100 μg/ml, 5 μmol/l of CuCl2 at 25°C. There was no difference in the susceptibility to in vitro oxidation of LDL isolated from IDDM patients compared with control subjects. There was no correlation of glycemic control with any of the parameters of the in vitro oxidation of LDL. LDL from patients with well-controlled IDDM does not differ in composition or in susceptibility to in vitro oxidative stress compared with LDL from nondiabetic subjects.
Strain-Dependent Differences in Sensitivity of Rat β-Cells to Interleukin 1β In Vitro and In Vivo
Authors: Reimers Jesper I , Anderson Herik U , Mauricio Dídac , Pociot Flemming , Karlsen Allan , Peterson Jacob S , Mandrup-Poulsen Thomas , Nerup Jørn
Published: September 2013

The aim of this study was to investigate whether straindependent differences in β-cell sensitivity to interleukin (IL) 1β exist in vitro and in vivo and if so, whether these differences correlate to variations in IL-1β-induced islet inducible nitric oxide synthase (iNOS) mRNA expression and nitrite production in vitro and islet iNOS protein content in vivo. Isolated islets of Langerhans in vitro from Wistar-Kyoto/Møllegården (WK/Mol) rats were sensitive to the inhibitory effect of IL-1β on accumulated and acute insulin secretion, whereas islets from Brown Norway/Charles River (BN/CR) rats were resistant. Furthermore, IL-1β induced higher islet iNOS mRNA expression and nitric oxide production from WK/Mol islets compared with BN/CR islets. WK/Mol, WK/CR, BN/Mol, BN/CR, and Lewis-Scripps/Mol (LS/Mol) rats received one daily injection of recombinant human IL-1β (4.0 μg/kg) or vehicle for 5 days. All the strains investigated were susceptible to IL-1β–induced changes in body weight, food intake, temperature, and plasma glucagon and corticosterone. However, IL-1β induced hyperglycemia and impairment of β-cell glucose responsiveness in WK/Mol and LS/Mol rats, but not in BN rats. Furthermore, IL-1β–induced islet iNOS expression in vivo determined by immunostaining was greater in WK/Mol rats compared with WK/CR and BN/CR rats. No restriction fragment length polymorphisms, using 20 restriction enzymes, were identified in the iNOS gene in six rat strains including BioBreeding rats. In conclusion, the relative resistance of BN rat islets to IL-1β–induced inhibition of β-cell function in vitro was associated with lower islet iNOS mRNA expression and nitrite production in this strain. Further, the resistance of BN rats to IL-1β–induced hyperglycemia was associated with a lower islet iNOS expression in vivo.
Cellular Immune Response to Diverse Islet Cell Antigens in IDDM
Authors: Durinovic-Bellò Ivana , Hummel Michael , Ziegler Anette-G
Published: September 2013

In IDDM, T-cells are postulated to mediate the destruction of pancreatic β-cells. We analyzed peripheral blood mononuclear cell (PBMC) responses to human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase ICA512, glucagon, membrane preparations of RIN cells and human pancreas, and three control antigens (La = nuclear cell antigen, tetanus toxoid, and phytohemagglutinin). A total of 28 patients with newly diagnosed IDDM, 9 antibody-positive (Ab+) first-degree relatives, and 16 healthy control subjects were included. Increased proliferative responses to pancreatic islet cell antigens were observed in diabetic patients and in Ab+ relatives compared with control subjects, whereas T-cell reactivity to nonpancreatic control antigens was similar between the study groups. The highest differences in the magnitude of proliferative responses were seen for ICA512, followed by membrane preparations of RIN cells, GAD65, and human pancreas. Few subjects reacted with insulin or glucagon. Interestingly, Ab+ relatives showed higher T-cell reactivity with respect to stimulation indexes and prevalences than newly diagnosed diabetic patients, and as many as 89% of Ab+ relatives showed proliferation to more than one islet cell antigen preparation in comparison to 43% of newly diagnosed diabetic patients and none of the control subjects. Statistical analysis revealed significant positive correlation of insulin autoantibody levels with the levels of insulin-specific T-cells in Ab+ relatives, but no relation of PBMC responses to age, sex, or HLA-DR haplotypes. Our results demonstrate the simultaneous existence of various autoreactive T-cells specific for islet cell antigens in the prediabetic period. These T-cells may play a significant role in the pathogenesis of the disease.
Sequence Variants in the Sulfonylurea Receptor (SUR) Gene Are Associated With NIDDM in Caucasians
Authors: Inoue Hiroshi , Ferrer Jorge , Welling Cris M , Elbein Steven C , Hoffman Michael , Mayorga Rachel , Warren-Perry Margaret , Zhang Yun , Millns Helen , Turner Robert , Province Mike , Bryan Joseph , Permutt M Alan , Aguilar-Bryan Lydia
Published: September 2013

NIDDM is a common heterogeneous disorder, the genetic basis of which has yet to be determined. The sulfonylurea receptor (SUR) gene, now known to encode an integral component of the pancreatic β-cell ATP-sensitive potassium channel, IKATP, was investigated as a logical candidate for this disorder. The two nucleotide-binding fold (NBF) regions of SUR are known to be critical for normal glucose regulation of insulin secretion. Thus, singlestrand conformational polymorphism analysis was used to find sequence changes in the two NBF regions of the SUR gene in 35 NIDDM patients. Eight variants were found; and three were evaluated in two Northern European white populations (Utah and the U.K.): 1) a missense mutation in exon 7 (S1370A) was found with equal frequency in patients (n = 223) and control subjects (n = 322); 2) an ACC→ACṮ silent variant in exon 22 (T761T) was more common in patients than in control subjects (allele frequencies 0.07 vs. 0.02, P = 0.0008, odds ratio (OR) 3.01, 95% CI 1.54–5.87); and 3) an intronic t→c change located at position –3 of the exon 24 splice acceptor site was also more common in patients than in control subjects (0.62 vs. 0.46, P < 0.0001, OR 1.91, 95% Cl 1.50–2.44). The combined genotypes of exon 22 C/T or T/T and intron 24 –3c/–3c occurred in 8.9% of patients and 0.5% of control subjects (P < 0.0001, OR 21.5,95% CI 2.91–159.6). These results suggest that defects at the SUR locus may be a major contributor to the inherited basis of NIDDM in Northern European Caucasians.
Glutamine and Alanine Metabolism in NIDDM
Authors: Stumvoll Michael , Perriello Gabriele , Nurjhan Nurjahan , Welle Stephen , Gerich John , Bucci Arthur , Jansson Per-Anders , Dailey George , Bier Dennis , Jenssen Trond , Gerich John
Published: September 2013

Gluconeogenesis is increased in NIDDM. We therefore examined the metabolism of glutamine and alanine, the most important gluconeogenic amino acids, in 14 postabsorptive NIDDM subjects and 18 nondiabetic volunteers using a combination of isotopic ([6-3H] glucose (20 µCi, 0.2 µCi/min), [U-14C]glutamine (20 µCi, 0.2 µCi/min), [3-13C]alanine (99% 13C, 2 mmol, 20 µmol/min), [ring-2H5]phenylalanine (99% 2H, 2 µmol/kg, 0.03 µmol · kg-1 · min-1), and limb balance techniques. Alanine turnover (4.54 ± 0.24 vs. 5.64 ± 0.33 µmol · kg-1 · min-1), de novo, synthesis (3.00 ± 0.25 vs. 4.01 ± 0.33 µmol · kg-1 · min-1) were increased in NIDDM subjects (all P < 0.001), while its forearm release (0.45 ± 0.04 vs. 0.39 ± 0.04 µmol · kg-1 · min-1) was unaltered. Although glutamine turnover (4.81 ± 0.23 vs. 4.40 ± 0.31 µmol · kg-1 · min-1) was unaltered in NIDDM, its conversion to glucose (0.57 ± 0.04 vs. 1.08 ± 0.10 µmol · kg-1 · min-1) and to alanine (0.10 ± 0.01 vs. 0.34 ± 0.04 µmol · kg-1 · min-1) (both P = 0.001) was increased while its oxidation (2.84 ± 0.27 vs. 1.84 ± 0.15 µmol kg-1 · min-1 P = 0.03) and forearm release (0.77 ± 0.05 vs. 0.62 ± 0.09 µmol · kg-1 · min-1 P < 0.008) were both reduced. Our results thus demonstrate that there are substantial alterations of glutamine and alanine metabolism in NIDDM. Conversion of both amino acids to glucose and the proportion of their turnover used for gluconeogenesis are increased; release of both amino acids from tissues other than skeletal muscle seems to be increased. Finally, the reduction in glutamine oxidation, possibly the result of competition with glucose and free fatty acids as fuels, makes more glutamine available for gluconeogenesis without a change in its turnover.
Effect of IGF-I on Phosphatidylinositol 3-Kinase in Soleus Muscle of Lean and Insulin-Resistant Obese Mice
Authors: Jullien Dominique , Heydrick Stanley J , Gautier Nadine , Obberghen Emmanuel Van , Marchand-Brustel Yannick Le
Published: September 2013

Insulin and IGF-I induced a similar stimulation of glucose transport in isolated soleus muscle. These actions require phosphatidylinositol (PI) 3-kinase activation since the PI 3-kinase inhibitor, wortmannin, blocked the stimulation by both peptides. We compared IGF-I with insulin in the ability to activate PI 3-kinase in the isolated soleus muscle from lean and gold thioglucose-induced obese insulin-resistant mice. In muscles from lean mice, IGF-I and insulin were able to activate PI 3-kinase with a similar time course, the effects being maximal within 3–5 min of stimulation. However, the IGF-I concentrations required to obtain similar effects on PI 3-kinase were about 10 times higher than the corresponding insulin doses. To determine through which receptor IGF-I was activating PI 3-kinase, the ability of IGF-I to activate both its own receptor and insulin receptor was simultaneously measured. Whatever the dose used (100 or 500 nmol/L), IGF-I activated to a nearly similar extent both the tyrosine kinase activity of its own receptor and that of the insulin receptor, suggesting that IGF-I was not only activating its receptor but was also able to stimulate the insulin receptor kinase. In muscles of obese insulinresistant mice, although the defect of PI 3-kinase activation in response to IGF-I was relatively less pronounced (45%) than in response to insulin (70%) when compared with lean mice, PI 3-kinase stimulation was still markedly altered in response to IGF-I.