bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Defects in Glucose Metabolism in Obese Patients With NIDDM
Authors: Kelley David E , Mokan Marian , Mandarino Lawrence J
Published: September 2013

Skeletal muscle insulin resistance in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) is characterized by decreased glucose uptake. Although reduced glycogen synthesis is thought to be the predominant cause for this deficit, studies supporting this notion often have been conducted at supraphysiological insulin concentrations in which glucose storage is the overwhelming pathway of glucose disposal. However, at lower, more physiological insulin concentrations, decreased muscle glucose oxidation could play a significant role. This study was undertaken to determine whether, under euglycemic conditions, insulin resistance for leg muscle glucose uptake in NIDDM patients is due primarily to decreased glucose storage or to oxidation. The leg balance technique and leg indirect calorimetry were used under steady-state euglycemic conditions to estimate muscle glucose uptake, storage, and oxidation in eight moderately obese NIDDM patients and eight matched-control subjects. Leg muscle biopsies also were performed to determine whether alterations in muscle pyruvate dehydrogenase or glycogen synthase activities could explain defects in glucose oxidation or storage. At insulin concentrations of ∼ 500–600 pM, leg glucose uptake, oxidation, and storage in the NIDDM group (2.03 ± 0.42, 1.00 ± 0.13, 0.66 ± 0.36 μmol · min−1 · 100 ml−1) were significantly lower (P < 0.05) than rates in control subjects (5.14 ± 0.64, 1.92 ± 0.17, 2.80 ± 0.54). Pyruvate dehydrogenase and glycogen synthase activities were also decreased, consistent with the in vivo metabolic defects. The average deficit in leg glucose uptake in NIDDM was 3.11 ± 0.42 μmol · min−1 μ 100 ml−1. Of this deficit, 66% (2.14 ± 0.36 μmol min−1 · 100 ml−1) was due to decreased leg glucose storage and 33% (0.92 ± 0.13 μmol min−1 · 100 ml−1) to decreased leg glucose oxidation. Our findings confirm that decreased muscle glucose storage during hyperinsulinemia is the largest defect in glucose metabolism but also reveal a major defect in glucose oxidation. These studies reinforce the notion that muscle insulin resistance in obese NIDDM patients is characterized by a panoply of intracellular defects in glucose metabolism and insulin action.
Variability of the Insulin Gene in American Blacks With NIDDM: Analysis by Single-Strand Conformational Polymorphisms
Authors: Olansky Leann , Janssen Rachel , Welling Cris , Permutt M Alan
Published: September 2013

Previous studies of the insulin gene–utilized restriction-fragment–length polymorphisms as markers for potential mutations at this locus. This indirect type of analysis could not define the number of variants that might exist within the structural portions and regulatory regions of the gene in non-insulin-dependent diabetes mellitus (NIDDM) patients. New technology has allowed us to examine insulin genes at the single nucleotide level from 100 American black NIDDM patients. Genomic DNA from patients was amplified by the polymerase chain reaction with primers flanking four regions of the gene: 1) the proximal promoter from positions −182 to 42 (including most of exon 1); 2) exon 1 from 14 to 259, which included the rest of exon 1 and all of the 1st intron; 3) exon 2 from 216 to 452; and 4) exon 3 from 1188 to 1433. One of the primers in each reaction was 32P–end labeled and the resulting products denatured into single strands and electrophoresed on nondenaturing sequencing gels such that mobility was a function of composition and size (single-strand conformational polymorphism or SSCP). Under these conditions, single-base changes in fragments up to 245 nucleotides were detected. Analysis of the proximal promoter region revealed several SSCP patterns in individuals. Direct genomic sequencing of DNA representative of these patterns showed the presence of a common C to G change at position −56 and a C deletion at position −90 in three patients. For exon 1, there were eight SSCP patterns, which on sequencing were found to represent combinations of three polymorphisms within the first intron, a 4-bp insertion (TTGC) at position 46 (close to the 5′-splice donor site of the 1st intron), a G-to-A change at 80, and a previously described T-to-A change at 216. Exon 2 analysis revealed a newly described G-to-A change at 274, resulting in a silent polymorphism within the prepeptide of the gene. For exon 3, two previously described allelic variants in the 3′-untranslated region(C to T at 1367 and A to C at 1380) were found to be polymorphic in this population. In summary, analysis of 200 insulin genes from NIDDM patients revealed the full extent of variability at this locus. Not a single mutation in the coding portions of the gene was found. Four previously described allelic variants were confirmed and four new variants observed, with only one in the coding region. Haplotypes were described for the first time, and seven new alleles identified. Two of four newly described polymorphisms might have biological consequences. The C deletion at −90 in the proximal promoter might alter transcription of the gene. The four-nucleotide insertion at 46 alters the consensus sequence of the 5′-splice donor site of the first intron, which might alter proinsulin mRNA processing. These hypotheses can be tested.
Promotion of Pancreatic Islet Allograft Survival By Intrathymic Transplantation of Bone Marrow
Authors: Posselt Andrew M , Odorico Jon S , Barker Clyde F , Naji Ali
Published: September 2013

An important goal in the treatment of insulin-dependent diabetes by pancreatic islet transplantation is the development of strategies that allow permanent survival of islet allografts without continuous host immunosuppression. In this study, we demonstrate that inoculation of allogeneic bone marrow into the thymus of adult rats treated with a single dose of anti-lymphocyte serum induces an unresponsive state that permits survival of subsequent pancreatic islet allografts transplanted to an extrathymic site. This effect is donor specific, cannot be reproduced by systemic administration of bone marrow, and is associated with persistence of chimeric cells in the thymus of the recipient. In addition, lymph node cells from long-term recipients of intrathymic bone marrow display markedly reduced proliferative responses to donor alloantigens in mixed lymphocyte culture. Interaction of maturing thymocytes with foreign alloantigens may produce the unresponsiveness. This model offers a potential approach for establishing donor-specific allograft acceptance in adult recipients.
Heterogeneity in Pancreatic β-cell Population
Authors: Pipeleers Daniel G
Published: September 2013

All pancreatic β-cells are identified by specific morphological characteristics. Similarity in microscopic features is not necessarily associated with identity in functional properties. In vitro studies on isolated rat β-cells have indicated intercellular differences in the threshold for glucose-induced shifts in metabolic redox state. The cellular heterogeneity in glucose sensitivity results in a dose-dependent recruitment of glucose-exposed β-cells into biosynthetic and secretory activities. The molecular basis of this diversity is not known. Indirect evidence supports the concept that the in situ pancreatic β-cell population is also composed of functionally diverse subpopulations. The heterogeneity in glucose responsiveness is expected to create subpopulations of β-cells with either constant, fluctuating, or occasional glucose-dependent functions; whether any subpopulation is preferentially responsive to other regulatory factors and/or committed to other activities is unknown. Morphological markers may help identify β-cell subpopulations in situ and quantify their size in conditions known to affect total β-cell mass or function. The concept of a functionally heterogeneous β-cell population influences views on the role of pancreatic β-cells in health and disease.
Human Islet Glucokinase Gene: Isolation and Sequence Analysis of Full-Length cDNA
Authors: Koranyi Laszlo I , Tanizawa Yukio , Welling Cris M , Rabin Daniel U , Permutt M Alan
Published: September 2013

Pancreatic islet glucokinase (ATP:d-hexose-6-phosphotransferase) cDNAs were isolated from a human islet cDNA library in λ-gt11. One clone (hlGLK2), 2723 bp plus additional poly(A) residues, appeared to be full length because its size was consistent with a single 2.9-kb glucokinase mRNA on Northern-blot analysis of islet RNA. This cDNA contained an open reading frame of 1395 bp from an ATG codon at position 459, encoding a predicted protein of 465 amino acids (52,000 Mr). Comparison of the nucleotide sequences of the human islet glucokinase cDNA with that of the recently isolated human liver glucokinase cDNA revealed that the two cDNAs differed completely on their 5′-ends, followed by an identical 2204-bp overlap extending to the 3′-ends. The 5′-ends of islet and liver glucokinase cDNAs predicted proteins that differ by 15 NH2-terminal residues. The overall sequence identity (70%) between the first exons of the human islet and rat islet cDNAs suggested that the islet promoter regions, like the liver promoter regions, have been conserved through evolution. Thus, NH2-terminal differences for human liver and islet enzymes might be explained by use of alternate promoters between the two tissues, analogous to the NH2-terminal differences of the rat liver and rat islet enzymes. If so, this relationship predicts important tissue-specific regulatory functions of these regions. Variations in the glucokinase gene are likely to occur in humans. Isolation of a human islet glucokinase cDNA has provided the sequence necessary to determine whether these variants are important determinants in the genetic predisposition for diabetes mellitus.
Impaired Glucose Sensitivity of ATP-Sensitive K+ Channels in Pancreatic β-cells in Streptozotocin-Induced NIDDM Rats
Authors: Tsuura Yoshiyuki , Ishida Hitoshi , Okamoto Yoshimasa , Tsuji Kozuo , Kurose Takeshi , Horie Minoru , Imura Hiroo , Okada Yasunobu , Seino Yutaka
Published: September 2013

ATP-sensitive K+ channels (KATP channels) are known to play a key role in the cellular mechanism of insulin secretion from pancreatic β cells. In order to examine the possible impairment of KATP channel function in non-insulin-dependent diabetes mellitus (NIDDM), we have studied the properties of the KATP channels in single β cells of neonatally streptozotocin-induced diabetic rats (NSZ rats) using the patch-clamp technique. The unitary conductance of the channel in diabetic β-cells was virtually identical to that in control beta cells and there was no difference in the sensitivity to ATP and glibenclamide of KATP channels between the NIDDM and control groups. In response to glucose, the activity of the KATP channels was diminished in a dose-dependent manner in both control and diabetic cells. However, the inhibition of the KATP channels in β-cells of NSZ rats was significantly < that in control cells. Even in the presence of 11.1 mM glucose, the openings of a few single KATP channels were consistently observed in cell-attached patch membranes of diabetic, but not control, β-cells. Thus, it appears that the impaired insulinotropic action of glucose in β-cells in NSZ rats is associated with a reduced sensitivity of the KATP channel to glucose, but not to ATP, presumably due to a deficiency in glucose metabolism.
Experimental Diabetic Neuropathy: Effect of Ganglioside Treatment on Axonal Transport of Cytoskeletal Proteins
Authors: Figliomeni Bruna , Bacci Barbara , Panozzo Cristina , Fogarolo Fabiano , Triban Chiara , Fiori Mario G
Published: September 2013

Abnormalities in axonal transport of proteins are thought to play an important role in the pathogenesis of diabetic neuropathy. Gangliosides exert a positive action on numerous alterations in biochemistry and physiology of diabetic nerves. This study was undertaken to assess the effects of exogenous gangliosides on the axonal transport of structural proteins such as actin and tubulin in the sensory fibers of short-term (9-wk) and long-term (6-mo) diabetic rats. Adult Sprague-Dawley rats were made diabetic with a single injection of 70 mg/kg streptozocin i.p. Subgroups were injected daily with either highly purified ganglioside mixture (10 mg/kg i.p.) or saline for 1 mo, beginning either 2 or 17 wk after streptozocin injection. Age-matched rats were used as controls. Axonal transport was studied by the pulse-labeling technique. Three weeks after labeling, sciatic nerves were dissected out and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In diabetic rats of both experimental designs, the transport rate of tubulin and actin was decreased by ∼ 30% compared with control rats. Ganglioside treatment counteracted such alterations in both 9-wk and 6-mo diabetic rats. These data suggest a pharmacological effect that could be correlated with molecular interactions between integral membrane glycolipids and cytoskeletal elements.
Islet Cell and Thyrogastric Antibodies in 633 Consecutive 15- to 34-Yr-Old Patients in the Diabetes Incidence Study in Sweden
Authors: Landin-Olsson Mona , Karlsson F Anders , Lernmark Åke , Sundkvist Göran ,   The Diabetes Incidence Study in Sweden Group
Published: September 2013

The effect of age on ICA and thyrogastric antibodies at diagnosis of IDDM was evaluated in 633 consecutively diagnosed Swedish diabetic patients aged 15–34 yr and in 282 volunteers of the same age. ICAs were present in 61% (383 of 633) of the patients and in 2% (5 of 282) of control subjects. When the initial classification was considered, ICAs were detected in 69% (327 of 473) of patients with IDDM, 23% (19 of 83) of those with NIDDM, 50% (36 of 72) of those with unclassifiable diabetes, and 20% (1 of 5) of those with secondary diabetes. The frequency of ICA fell significantly (P < 0.001) with age in IDDM patients from 77% (104/135) in those 15–19 yr old to 52% (50 of 96) in 30- to 34-yr-old IDDM patients. The low frequency of ICA in 30- to 34-yr-old IDDM patients was confined to men (42%, 28 of 66). The frequency of gastric (H+, K+-ATPase) antibodies was significantly (P < 0.05) higher in IDDM patients (10%, 47 of 449) than in patients with NIDDM (3%, 3 of 80) and unclassifiable diabetes (4%, 3 of 72). In conclusion, the frequency of ICA at the diagnosis of IDDM in young adult subjects decreases with increasing age, particularly in men. The frequent finding of ICA in patients considered to have NIDDM or unclassifiable diabetes indicates that misclassification of diabetes is frequent in young adult patients recently diagnosed with diabetes.
Exercise Training Increases Glucose Transporter Content in Skeletal Muscles More Efficiently From Aged Obese Rats Than Young Lean Rats
Authors: Ezaki Osamu , Higuchi Mitsuru , Nakatsuka Haruko , Kawanaka Kentaro , Itakura Hiroshige
Published: September 2013

Glucose uptake in rat skeletal muscles decreases with age and obesity, but increases with chronic exercise training. The purpose of our study was to determine whether the GLUT4 content in several skeletal muscles from 1-mo-old young, lean rats and 12-mo-old aged, obese rats alters with exercise training. For exercise, a treadmill run of ∼ 1 km/day was made for 4 wk by both groups of rats. The concentration of GLUT4 per protein in membrane fraction from several skeletal muscles was measured by immunoblotting. The amount of GLUT4 in the gastrocnemius and white quadriceps from aged rats slightly but significantly decreased to 73% and 78% of that from young rats, respectively. However, no significant difference in GLUT4 amount in the soleus, plantaris, and red quadriceps was observed between young and aged rats. The exercise training resulted in a larger increase in the amount of GLUT4 in each muscle from aged rats than in muscles from young rats. In aged rats, GLUT4 amount increased significantly with exercise training by 30, 33, 41, and 27% in the soleus, plantaris, gastrocnemius, and red quadriceps, respectively, compared with the sedentary controls. However, in young rats, exercise-induced increase of GLUT4 amount was significant only in the plantaris, and the increase was 17%. In exercised aged, obese rats, decreases of body weight, plasma triglyceride levels, and plasma free fatty acid were also observed. These findings suggest that the amount of GLUT4 in some skeletal muscles decreases slightly in aged, obese rats, exercise training increases the amount of GLUT4 more efficiently in aged rats than in young rats and ameliorates the GLUT4 decrease observed in aged rats, and lipid metabolism may be related to the regulation of GLUT4 protein content in muscles.
Linkage Analysis of Maturity-Onset Diabetes of the Young With Microsatellite Polymorphisms: No Linkage to ADA or GLUT2 Genes in Two Families
Authors: Patel Pushpa , Lo Y -M Dennis , Hattersley Andrew , Bell Graeme I , Tybjaerg-Hansen Anne , Nerup Jorn , Turner Robert C , Wainscoat James S
Published: September 2013

MODY is a form of NIDDM inherited as an autosomal dominant condition. We studied the linkage of MODY to two loci: ADA and GLUT2 in two large pedigrees with nonradioactive microsatellite polymorphic systems. A positive linkage of ADA to MODY was recently demonstrated in the large RW pedigree. Formal linkage analysis excluded a tight linkage between ADA and MODY with a LOD score of −5.82 and −2.24 at a recombination fraction of 0.01 in the two families. This result suggests genetic heterogeneity in the molecular basis of MODY. GLUT2 is a candidate gene that is expressed in the liver and β-cells of pancreatic islets. In the two families studied, the disease did not cosegregate with GLUT2 alleles. The LOD scores for GLUT2 were −7.79 and −1.9 at a recombination fraction of 0.001 in the two families, thus providing evidence against the involvement of GLUT2 in MODY.
In Vitro Effects of Amylin on Carbohydrate Metabolism in Liver Cells
Authors: Ciaraldi Theodore P , Goldberg Michele , Odom Rosanne , Stolpe Matthew
Published: September 2013

Amylin, a peptide found in pancreatic amyloid deposits, may be involved in NIDDM. The effects of biosynthetic human amylin on multiple aspects of carbohydrate metabolism were studied in freshly isolated and cultured liver cells (rat hepatocytes and HepG2 cells). Acute exposure of culture liver cells to amylin had no effect on glucose incorporation into glycogen. Amylin directly reduced glucose oxidation through the hexose monophosphate shunt. The glycolytic pathway was unaffected. Amylin stimulated both glycogenolysis and gluconeogenesis. These effects were largest at amylin concentrations of 1–10 pM. Insulin partially inhibited both of these responses. Glucagon stimulated glycogenolysis and gluconeogenesis to a similar extent as amylin but required concentrations 100- to 500-fold as high. Thus, amylin, at physiologic concentrations, can impair some aspects of glucose use in liver cells and is also capable of directly stimulating glucose production, suggesting a possible involvement of amylin in the impaired glucose disposal and elevated hepatic glucose output of NIDDM.
Comparison of Peripheral and Portal (Via the Umbilical Vein) Routes of Insulin Infusion in IDDM Patients
Authors: Shishko Peter I , Kovalev Pavel A , Goncharov Valentin G , Zajarny Igor U
Published: September 2013

Twelve subjects with insulin-dependent diabetes mellitus were treated using continuous subcutaneous insulin infusion (CSII) and intraportal insulin infusion (IPII) via the umbilical vein for 4 mo. Glucose control improved in both CSII and IPII groups, but a decrease in glucose and HbA1c was more rapid and more significant in the IPII group than in CSII, even though insulin requirement was lower during IPII than CSII (40 ± 2 vs. 50 ± 2 U/day, P < 0.05). The insulin plasma fasting levels were different (88 ± 10.7 in the IPII group vs. 263 ± 23 pM in CSII, P < 0.001). High plasma levels of lactate, pyruvate, alanine, cortisol, and growth hormone were decreased in both groups, with their full normalization only in the IPII group. Glucagon concentrations were low in both groups at the beginning of the study (30.0 ± 4.1 in the CSII group and 32.3 ± 1.8 ng/L in IPII); they were equalized to control values in the IPII group and were low in the CSII group at the study's end (46.0 ± 3.7 in IPII vs. 31.7 ± 3.1 ng/L in CSII, P < 0.05. We conclude that intraportal administration of insulin via the umbilical vein at rates of 0.01–0.05 U · kg−1 hr−1 reduces plasma levels of glucose, three carbon precursors, cortisol, and growth hormone by a direct action on the liver, and the hepatic action of peripherally administered insulin is manifested only when the infusion rate is increased to 0.1–0.3 U · kg−1 · hr−1. Thus the intraportal route via the umbilical vein may offer improved physiological insulin delivery over the subcutaneous route.
Effects of Oleate and Insulin on Glucose Uptake, Oxidation, and Glucose Transporter Proteins in Rat Adipocytes
Authors: Murer Erik , Boden Guenther , Gyda Michael , Deluca Francesco
Published: September 2013

We examined effects of Na oleate on glucose uptake, glucose transporter protein concentrations, and glucose oxidation in isolated adipocytes from fed rats. Na oleate increased basal 14C-glucose uptake in a dose-dependent manner (+42% with 1.0 mM, +79% with 2.8 mM Na oleate), but had no statistically significant effect on insulin-stimulated glucose uptake. Insulin (100 nM) resulted in a redistribution of GLUT4 protein concentration from the LDM fraction (−42%) to the PM fraction (+266%) but did not affect the distribution of GLUT1. Na oleate had no effect on basal or insulin-stimulated concentrations of GLUT1 or GLUT4 proteins in the PM or LOM fractions. Na oleate (2.8 mM) had no statistically significant effect on basal glucose oxidation, but inhibited insulin-stimulated glucose oxidation by 48% (P < 0.01). In summary, Na oleate inhibited insulin-stimulated glucose oxidation and stimulated basal glucose uptake in isolated adipocytes without affecting PM or LDM distribution of GLUT1 or GLUT4 proteins. We conclude that the stimulatory effect of Na oleate on basal glucose uptake in adipocytes may be mediated by changes in the intrinsic activity of the glucose transporters.
Glucose Transporter Number, Function, and Subcellular Distribution in Rat Skeletal Muscle After Exercise Training
Authors: Goodyear Laurie J , Hirshman Michael F , Valyou Patricia M , Horton Edward S
Published: September 2013

Endurance exercise training can result in increased rates f insulin-stimulated glucose uptake in skeletal muscle; however, this effect may be lost rapidly once training ceases. To examine a mechanism for these changes, the skeletal-muscle glucose transport system of female rats exercise-trained in wheelcages for 6 wk were studied against a group of untrained female rats. The trained rats were studied immediately following and 2 and 5 days after removal from wheelcages; both trained and untrained rats were studied 30 min after insulin (90 nmol/rat, intraperitoneal) or saline injection. The total number of skeletal-muscle plasma-membrane glucose transporters (Ro), total muscle-homogenate and plasma-membrane GLUT4 protein, and rates of plasma-membrane vesicle D-facilitated glucose transport were higher in the exercise-trained rats immediately after exercise training and did not decrease significantly during the 5 days after cessation of training. On the other hand, exercise training did not alter microsomal-membrane total glucose-transporter number or GLUT4 protein, nor did training alter GLUT1 protein in total muscle homogenates nor either membrane fraction. The carrier-turnover number, an estimate of average functional activity of glucose transporters in the plasma membrane, was elevated slightly, but not significantly, in the trained muscle. In both the trained and untrained muscle, insulin administration resulted in translocation of glucose transporters from the microsomal-membrane fraction to the plasma membrane and an increase in the carrier-turnovernumber. These data suggest that increased rates of glucose uptake in endurance-trained skeletal muscle results primarily from an increase in the number—and not an increase in the average functional activity—of glucose transporters present in the plasma membrane. Furthermore, these increases persist for several days after cessation of exercise training. The specific increase in the GLUT4, but not the GLUT1 glucose-transporter isoform, in response to training demonstrates that a common, chronic physiological stimulus can regulate the expression of the two glucose-transporter isoforms present in skeletal-muscle tissue differentially.
Diminished Glomerular Capillary Hydraulic Conductivity Precedes Morphologic Changes in Experimental Diabetes Mellitus in the Rat
Authors: Ellis Eileen N , Wiegmann Thomas B , Savin Virginia J
Published: September 2013

Giomerular ultrafiltration coefficient, Kf, is diminished in established diabetic nephropathy. To determine whether Kf is decreased because of a decrease in capillary area, A, and or in hydraulic conductivity, Lp, glomerular kf and morphometric parameters were measured, and Lp was calculated in glomeruli of young rats with STZ-induced DM and in control rats. STZ was administered to Fischer 344 rats that weighed 50–75 g; glomeruli were examined after 3 or 5 mo of DM, and their structure and function was compared with that of control rats. The effects of insulin or of an ACEI, enalapril, also were assessed after 3 or 5 mo. Growth of DM rats was markedly impaired, and their ratio of kidney weight to body weight was increased. Ccr was proportional to rat weight, and the ratio of Ccr to body weight was not different in DM and control rats. At 3 mo, average volume of glomeruli isolated from DM rats was less than that of glomeruli from control rats. In contrast, glomerular volume after 5 mo was equal in DM and control rats. No increase in GBM thickness or mesangial volume was observed, nor was any decrease seen in GBM area in DM rats at 5 mo. Kf was lower in DM rats than controls after 3 mo, but not after 5 mo. The Lp of DM and control glomeruli did not differ at 3 mo, but was lower in DM at 5 mo. Insulin therapy improved somatic growth and increased kidney and glomerular size in DM rats; the kidney weight/body weight ratio remained elevated. Insulin therapy was associated with an increase in capillary volume and GBM area and with a decrease in total slit pore length. The ACEI did not change somatic or renagrowth but diminished GBM area and total slit pore length. Neither insulin nor ACEI therapy affected the Lp of DM rats. We conclude that diminished Lp occurs in established DM in the rat; it precedes structural glomerular changes and is not corrected by insulin nor an ACEI in the dosage used.
Tissue-Specific Regulation of Insulin Receptor mRNA Levels in Rats With STZ-Induced Diabetes Mellitus
Authors: Sechi Leonardo A , Griffin Chandi A , Grady Eileen F , Grunfeld Carl , Kalinyak Judith E , Schambelan Morris
Published: September 2013

In rats with STZ-induced diabetes mellitus, a reduction in insulin secretion is associated with increased insulin binding in the liver, muscle, fat, and kidney, but not in the brain. To test the hypothesis that tissue-specific modulation of insulin receptors (IRs) in STZ-induced diabetes occurs at the level of mRNA, IR mRNA levels were measured in the liver, kidney, and brain of Sprague-Dawley rats 15 days after intravenous administration of STZ (60 mg/kg body weight) and compared with those of control rats. Diabetic rats were either left untreated or given differing insulin regimens that were designed to achieve varying degrees of metabolic control. IR mRNA levels were measured by slot blot hybridization with a 32P-labeled rlR probe and standardized by 28S ribosomal RNA determination. Hepatic IR mRNA levels were increased significantly in both untreated diabetic rats and in those that received low-dose (2 U/day) insulin therapy. In contrast, hepatic IR mRNA levels did not differ significantly from controls in those that received moderate doses of insulin (3–8 U/day) and were significantly less than controls in those that received the highest doses (6–10 U/day). Renal IR mRNA levels also were increased significantly in the untreated diabetic rats but not in those that received low- or moderate-dose insulin therapy, and were significantly less than controls in those that received the highest doses. A highly significant negative correlation was observed between the level of hepatic (r = −0.84, P <0.001) and renal (r = −0.64, P < 0.001) IR mRNA, and the plasma concentration of insulin obtained at the time of death. No significant difference was observed in brain IR mRNA levels between untreated diabetic and control rats. Thus, in rats with insulin deficiency, modulation of insulin binding in the liver and kidney can be attributed, at least in part, to a change in steady-state IR mRNA levels.
Incidence of NIDDM and Impaired Glucose Tolerance in Hypertensive Subjects: The San Antonio Heart Study
Authors: Morales Philip A , Mitchell Braxton D , Valdez Rodolfo A , Hazuda Helen P , Stern Michael P , Haffner Steven M
Published: September 2013

Hypertension often occurs in association with NIDDM and IGT. We examined the association of hypertension at baseline to the 8-yr incidence of NIDDM and IGT in 1471 subjects who participated in the San Antonio Heart Study. Subjects who were hypertensive at baseline had a higher incidence of NIDDM (8.9 vs. 4.9%, P = 0.041) and IGT (25.2 vs. 10.0%, P < 0.001) than subjects who were normotensive at baseline. After adjusting for age, sex, ethnicity, obesity, body fat distribution, fasting glucose, and insulin, this excess was eliminated for NIDDM, but not for IGT. Specifically, the adjusted OR for NIDDM in hypertensive versus normotensive patients was 0.73 (95% Cl 0.34–1.58), whereas the adjusted OR for IGT was 1.87 (95% Cl 1.08–3.22). The excess risk of NIDDM in hypertensive patients can be explained by their greater age, obesity, more unfavorable body fat distribution, and hyperinsulinemia, whereas the excess risk of IGT is independent of these factors.
Fibronectin-Induced Increase in Mesangial Cell Prostaglandin Release: Effect of Hyperglycemia and PKC Inhibition
Authors: Dunlop Marjorie , Keogh Rosemary , Larkins Richard G
Published: September 2013

Glomerular accumulation of extracellular matrix in diabetes is a potential regulator of mesangial cell-matrix interactions through transmembrane matrix receptors. We now provide evidence that PG production from rat glomerular mesangial cells is increased by Fn. An increase in PG (measured as PGE) was demonstrated in mesangial cell-enriched glomerular cores after 1-h exposure (149 ± 8% of timed control) and was sustained over a 24-h period (214 ±7%). Increased PG production followed exposure to a chymotryptic fragment (120,000 Mr) of Fn and occurred concomitant with an increase in participate PKC activity. A tetrapeptide (Arg-Gly-Asp-Ser) with the Arg-Gly-Asp sequence, contained in Fn and the chymotryptic fragment and recognized by specific membrane receptors (integrin matrix-binding proteins), also raised PG levels. As has been shown previously, exposure to high glucose concentration can increase mesangial cell PGE production (from 677 ± 61 pg mg protein−1 · 2 h−1 at 5.6 mM glucose to 1561 ± 132 pg · mg protein−1 · 2 h−1 at 50 mM glucose, P < 0.001). The response to the chymotryptic fragment of Fn also was enhanced by concurrent exposure to high glucose concentration (from 2560 ± 199 pg · mg protein−1 · 2 h−1 at 5.6 mM glucose to 4672 ± 358 pg · mg protein−1 · 2 h at 50 mM glucose, P < 0.001). Coincubation with H-7, an inhibitor of PKC, abolished the PG response to glucose and the chymotryptic fragment. Involvement of PKC was supported further by abrogation of the effect of chymotryptic fragment in mesangial cells cultured for a prior prolonged period with phorbol ester. Additionally, heparin significantly reduced the Fn- and chymotryptic fragment-induced response. Thus, the autocrine environment of the mesangial cell is altered in the presence of Fn. We propose that this mechanism contributes to the raised PG levels seen in diabetes and the attendant hemodynamic consequences.
Glucose Transport in Human Skeletal Muscle: The In Vivo Response to Insulin
Authors: Bonadonna Riccardo C , Saccomani Maria Pia , Seely Lynn , Zych Kathleen Starick , Ferrannini Eleuterio , Cobelli Claudio , DeFronzo Ralph A
Published: September 2013

Transmembrane glucose transport plays a key role in determining insulin sensitivity. We have measured in vivo WBGU, FGU, and KIn and Kout of 3-O-methyl-D-glucose in forearm skeletal muscle by combining the euglycemic clamp technique, the forearm-balance technique, and a novel dual-tracer (1-[3H]-L-glucose and 3-O-[4C]-methyl-D-glucose) technique for measuring in vivo transmembrane transport. Twenty-seven healthy, lean subjects were studied. During saline infusion, insulin concentration, FGU (n = 6), KIn, and Kout (n = 4) were similar to baseline. During SRIF-induced hypoinsulinemia (insulin <15 pM, n = 4) WBGU was close to 0, and FGU, KIn, and Kout were unchanged from basal (insulin = 48 pM) values. During insulin clamps at plasma insulin levels of ∼180 (n = 4), ∼420 (n = 5), ∼3000 (n = 4), and ∼9500 pM (n = 4), WBGU was 14.2 ± 1.3, 34.2 ± 4.1 (P < 0.05 vs. previous step), 55.8 ± 1.8 (P < 0.05 vs. previous step), and 56.1 ± 6.3 ixmol · min−1 · kg−1 of body weight (NS vs. previous step), respectively. Graded hyperinsulinemia concomitantly increased FGU from a basal value of 4.7 ± 0.5 μmol · min−1 · kg−1 up to 10.9 ± 2.3 (P < 0.05 vs. basal value), 26.6 ± 4.5 (P < 0.05 vs. previous step), 54.8 ± 4.3 (P < 0.05 vs. previous step), and 61.1 ± 10.8 μmol · min−1 · kg−1 of forearm tissues (NS vs. previous step), respectively. KIn of 3-O-methyl-D-glucose in forearm skeletal muscle was increased by hyperinsulinemia from a basal value of 6.6 · 10−2 ± 0.38 · 10−2 to 10.0 · 10−2 ± 1.4 · 10(p < 0.05 vs. baseline), 17.2 · 10−2 ± 2.2 · 10−2 (P < 0.05 vs. previous step), 26.3 · 10−2 ± 1.8 · 10−2 (P < 0.05 vs. previous step), and 29.8 · 10−2 ± 5.3 · 10−2 · min−1 (NS vs. previous step), respectively. FGU and Kln were positively correlated (r = 0.88, P < 0.01). Kout of 3-O-methyl-D-glucose did not change from the basal value at the lowest insulin dose (3.9 · 10−2 ± 1.1 · 10−2 vs. 3.8 ·10−2 ± 0.33 · 10−2 · 10−2 · min−1, NS), but rose significantly at the following insulin steps to 6.1 · 10−2 ± 0.8 · 10−2, 6.9 ·10−2 ± 0.5 · 10−2, and 11.9 · 10−2 ± 0.3 · 10−2 · min−1 (P < 0.05 for all three vs. baseline). Thus, in human skeletal muscle, in vivo, insulin stimulates K, n and uptake of glucose in a parallel fashion, whereas SRIF-induced acute hypoinsulinemia does not seem to affect transmembrane transport or uptake of glucose.
Effects of Ethanol on Carbohydrate Metabolism in the Elderly
Authors: Boden Guenther , Chen Xinhua , Desantis Richard , White John , Mozzoli Maria
Published: September 2013

We have previously reported that in young men, ethanol caused acute insulin resistance, but compensatory insulin secretion prevented deterioration of glucose tolerance (1). In this study, we tested the hypothesis that elderly men, because of their pre-existing insulin resistance and compromised insulin secretory capacity, may experience worsening of their glucose tolerance after ethanol. Nine elderly men (65.7 ± 0.8 yr, BMI 25.8 ± 1.4 kg/m2) received ethanol (13 mmol/kg for 30 min i.v.) or saline followed 30 min later by i.v. glucose (2.8 mmol/kg for 5 min). To determine the mechanism of the ethanol effect, six of the men underwent euglycemic-hyperinsulinemic (∼350 pM) clamping with simultaneous infusion of ethanol or saline. Muscle biopsies were obtained before and 1 and 4 h after insulin infusion. In all nine men, glucose concentrations after i.v. glucose were higher after ethanol than after saline, whereas insulin was the same and glucose tolerance decreased by 23% (Kg 2.41 ± 0.2 vs. 1.86 ± 0.1%/min, P < 0.01). Ethanol reduced insulin-stimulated glucose uptake from 40.6 ± 3.1 to 25.6 ±1.9 μmo; · kg−1 · min−1 (−37%, P < 0.05), glucose oxidation from 11.7 ± 1.1 to 7.0 ±0.7 μmol · kg−1 · min−1 (−33%, P < 0.01), and glucose storage from 28.7 ± 2.4 to 18.6 ±1.7 μmol · kg−1 · min−1(−35%, P <0.01). Ethanol increased muscle lactate concentration from 0.49 ± 0.14 to 1.99 ± 0.99 μmol/mg protein (P < 0.05), but had no effects on muscle concentration of freeglucose, G-6-P, and citrate concentrations, nor did it affect muscle GS activity. We concluded that modest amounts of ethanol in elderly men impaired glucose oxidation and caused insulin resistance, which because of lack of compensatory insulin secretion, resulted in deterioration of glucose tolerance.
Adoptive Transfer of Diabetes Into Immunodeficient NOD-scid/scid Mice: Relative Contributions of CD4+ and CD8+ T-Cells From Diabetic Versus Prediabetic NOD.NON-Thy-1a Donors
Authors: Christianson Sherri W , Shultz Leonard D , Leiter Edward H
Published: September 2013

Precise definition of the role of both CD4 and CD8 T-cell subsets from NOD mice in the adoptive transfer of diabetes has been complicated by the possibility that endogenous T-cells may be recruited. Two newly created NOD congenic stocks, NOD.NON-Thy-1aand NOD/LtSz-scid, have been used as T-cell donors and recipients, respectively, to eliminate contributions from endogenous T-cells and thus to define the requirement for transferred T-cell subsets as a function of underlying diabetes development in the NOD donor. Total T-cells and T-cell subsets prepared from either prediabetic or diabetic NOD.NON-Thy-1a donors were adoptively transferred into 6-wk-old NOD-scid/scid recipients that were monitored for diabetes development. Both flow cytometric and histological analysis of recipient spleen and pancreas after adoptive transfer showed lymphocytes of donor (Thy1.1+) origin exclusively. Total T-cell and enriched CD4+ T-cell preparations from both diabetic and young prediabetic donors transferred diabetes to NOD-scid/scid recipients. However, the mean time to diabetes onset was doubled when CD4+ lymphocytes were isolated from prediabetic versus diabetic donors, and t,+ cells over time. Enriched CD8+ populations alone were unable to transfer disease. More rigorous exclusion of CD8+ cells by means of anti-CD8 MoAb treatment in vivo of the recipients of enriched CD4+ cells demonstrated a significant difference in the diabetogenic potency of CD4+ lymphocytes from diabetic versus nondiabetic donors. Diabetes was adoptively transferred to 58% of the recipients of enriched CD4+ lymphocytes from diabetic donors. In contrast, none of the recipients of enriched CD4+ lymphocytes from young prediabetic donors developed diabetes after MoAb treatment in vivo. The ability of a T-cell population to produce severe insulitis and sialitis in NOD-scid/scid recipients of T-cells closely paralleled its ability to induce diabetes. In an effort to suppress insulitis by suppression of macrophage migration to the islets, NOD-scid/scid mice were treated with silica in conjunction with adoptive transfer of T-cells from diabetic donors. Chronic silica treatment failed to deplete tissue macrophages and did not prevent diabetes development after transfer of unfractionated T-cells. Evidence is discussed indicating that the age-associated differences in ability of CD4+ T-cells to adoptively transfer diabetes in the absence of the CD8+ T-cells subset is a function of prior, chronic exposure of the CD4+ lymphocytes to β-cell antigens in the donor. This study confirms that both CD4+ and CD8+ T-cells are required to initiate β-cell destruction in NOD mice.
Interaction of lnterleukin-1 With Islet β-Cells: Distinction Between Indirect, Aspecific Cytotoxicity and Direct, Specific Functional Suppression
Authors: Ling Zhidong , Veld Peter A In'T , Pipeleers Daniel G
Published: September 2013

A 5-day culture of adult rat islets with human recombinant IL-1β (3 U/ml) resulted in the death of most oc-cells and 50% of β-cells. The IL-1-exposed islet tissue contained—in addition to poorly granulated β-cells—patches of outgrowing monolayers and dispersed activated macrophages. In purified α- and β-cell preparations, no cytodestructive effects of IL-1 (as high as 30 U/ml) were noticed, indicating that the cytokine is in itself not a β-cell-selective killer. Pure β-cells were, on the other hand, more sensitive (from 0.3 U/ml on) than intact islets to an IL-1-induced suppression of hormone synthesis. This inhibitory action was reversible and affected predominantly the production of insulin, leading to degranulated cells with modified shape and attachment. Further studies with IL-1 should take into account that isolated islet preparations do not allow distinction between its irreversible, indirect, and aspecific β-cell toxicity and its reversible, direct, and specific suppression of β-cell functions. It is not yet known whether IL-1-suppressed β-cells exhibit an altered sensitivity to β-cell-toxic conditions.
Nuclear Protein—Binding Analysis of a GC-Rich Insulin-Receptor Promoter Regulatory Region
Authors: Levy James R , Hug Victoria
Published: September 2013

We previously demonstrated that activation of the insulin-receptor promoter occurs between Xho I and Hindlll restriction enzyme sites located 877 and 578 bp upstream, respectively, from the translational initiation site. Deletion mutants 5' of this promoter region were constructed with the reporter gene, CAT, and transiently transfected into HepG2 and MCF-7 cells. This study demonstrated that most of the promoter activity could be localized to a 40-bp region between –618 and –578. When attached to a heterologous SV40 early promoter, this 40-bp insulin-receptor regulatory region, in either orientation, stimulated the SV40 early promoter by approximately two- to threefold after transfection into HepG2 cells. EMSA demonstrated that the purified transcription factor, Sp1, binds to this transcription activator. DNA binding of protein obtained from crude HepG2 nuclear extracts demonstrated electrophoretically retarded bands that competed for the consensus Sp1 element; these bands were not observed in a similar analysis with crude MCF-7 nuclear extracts. However, in both HepG2 and MCF-7 cells, a protein was identified that specifically binds to this important insulin-receptor promoter region, but does not bind to the Sp1 consensus element. We conclude that activation of insulin-receptor gene transcription occurs in a 40 bp region 578 bp upstream from the translational initiation site, and that Sp1 and another nuclear factor other than Sp1 may be important in regulating transcription In HepG2 cells.
Differential Regulation of Glucose Transport and Transporters by Glucose in Vascular Endothelial and Smooth Muscle Cells
Authors: Kaiser Nurit , Sasson Shlomo , Feener Edward P , Boukobza-Vardi Nizza , Higashi Shinya , Moller David E , Davidheiser Sandra , Przybylski Ronald J , King George L
Published: September 2013

Hyperglycemia has been implicated in the pathogenesis of both micro- and macrovascular complications in diabetes. Little is known, however, about glucose transporters and their regulation in thevascular system. In this study, the regulation of glucose transporters by glucose was examined in cultured BAECs and BSMCs, and in human arterial smooth muscle cells. Both BAECs and BSMCs transported glucose via the facilitated diffusion transport system. Glucose-transport activity in vascular smooth muscle cells was inversely and reversibly regulated by glucose. Exposure of BSMCs and HSMCs to high glucose decreased Vmax for 2DG and 3-O-MG uptake, whereas Km remained unchanged. The hexose-transport system of BAECs exhibited lower 2DG and 3-O-MG uptake compared with BSMCs and showed little or no adaptation to changes in ambient glucose. Northern blot analysis. GLUT1 protein, quantified by Western blot analysis, was more abundant in BSMCs than in BAECs and was decreased by ∼50% when medium glucose was elevated from 1.2 to 22 mM for 24 h. The alterations in the level of GLUT1 protein correlated with the changes observed in transport activity. These observations suggest differential regulation of glucose transporter in response to glucose between smooth muscle and endothelial cells. The sites of autoregulation may involve translational control and/or the stability of the protein in the smooth muscle cells. The ability of vascular smooth muscle cells to down-regulate glucose transport in response to chronic hyperglycemia may serve as a protective mechanism against possible adverse effects of increased intracellular glucose.
Decreased Risk of Type I Diabetes in Offspring of Mothers Who Acquire Diabetes During Adrenarchy
Authors: Bleich David , Polak Michel , Eisenbarth George S , Jackson Richard A
Published: September 2013

Fathers with type I diabetes transmit diabetes to their offspring 2–3 times more frequently than mothers with type I diabetes. This phenomenon has provoked both genetic and nongenetic hypotheses, but the mechanism remains obscure. We find that mothers who develop diabetes before age 8 transmit diabetes at the same rate as diabetic fathers, and that the sex difference in diabetes transmission is explained by a decreased transmission rate in mothers who acquired diabetes after age 8. We constructed a data base containing 2156 nondiabetic and diabetic offspring of parents with type I diabetes. Families were selected from our main data base, which contains demographic information and diabetes autoantibody test results on >8000 first-degree relatives of patients with type I diabetes and diabetic probands. Identification of offspring was made through diabetic parents who had participated in our autoantibody screening program at the Joslin Diabetes Center between 1983 and 1990. Questionnaires were sent to all other family members to determine the number of diabetic and nondiabetic offspring in each family. The 20-yr life-table risk of diabetes in offspring of diabetic fathers and mothers is 8.9 ± 1.0 and 3.4 ± 0.6%, respectively. For mothers acquiring diabetes before or after age 8, the risk of diabetes in offspring is 13.9 ± 4.4 and 2.4 ± 0.6% at 20 yr of age, respectively. Furthermore, we find that duration of diabetes in mothers before pregnancy has no effect on the risk of diabetes in their offspring. The increased transmission rate of diabetes in diabetic fathers is explained by a decreased transmission rate of diabetes in mothers who acquire diabetes after age 8. Because women acquiring diabetes after age 8 transmit less diabetes to their offspring, we propose that adrenarchy may have an apparent protective effect on diabetes transmission. We speculate that mothers who acquire diabetes after age 8 are more susceptible to this disorder (similar to susceptibility to other organ-specific autoimmune diseases) and may possess and transmit fewer genetic susceptibility determinants to their offspring.