bluuebunny/crossref_metadata_2025 huggingface dataset:
Signals Derived From Glucose Metabolism Are Required for Glucose Regulation of Pancreatic Islet GLUT2 mRNA and Protein
Authors: Ferrer Jorge , Gomis Rramon , Fernández Alvarez Josefa , Casamitjana Roser , Vilardell Enric
Published: September 2013

Pancreatic islet GLUT2 mRNA is known to be regulated in vitro and in vivo by glucose. We have investigated several potential mechanisms mediating the response of islet GLUT2 to glucose. GLUT2 mRNA and protein were measured from isolated rat islets cultured for up to 24 h under selected conditions. Glucose at 11 mM stimulated GLUT2 mRNA 10-fold compared with 2 mM glucose, with no additional increase at 16.7 mM glucose, whereas maximal 4-fold induction of the protein was attained with 16 mM glucose. Time course studies showed a 2.5-fold induction of GLUT2 mRNA apparent after only 8 h of culture at 16.7 mM glucose. Glycolysis inhibitor mannoheptulose suppressed the stimulatory effect of 16.7 mM glucose on GLUT2 mRNA and protein. Metabolizable sugars mannose and glyceraldehyde enhanced transporter mRNA levels, in contrast with the lack of stimulation by nonmetabolizable 2-deoxy-D-glucose. Stimulation by different sugars and glycolysis inhibition led to analogous changes of proinsulin mRNA, suggesting that common signaling mechanisms are shared in glucose regulation of proinsulin and GLUT2 gene expression. Preexposure to mannoheptulose, however, failed to suppress glucose-stimulated insulin release. Tunicamycin, a glycoprotein synthesis inhibitor, did not block the effect of 16 mM glucose on GLUT2 mRNA levels. RNA and protein synthesis inhibitors actinomycin and cycloheximide abolished the enhancing effects of high glucose on GLUT2 mRNA. These findings indicate that glucose metabolism, but not glycoprotein synthesis or substrate interaction with the transporter protein, is instrumental in the stimulatory effects of glucose on β-cell GLUT2 mRNA accumulation. In addition, ongoing RNA and protein synthesis are required for this effect.
Regulation of Insulin-Stimulated Glycogen Synthase Activity by Overexpression of Glutamine: Fructose-6-Phosphate Amidotransferase in Rat-1 Fibroblasts
Authors: Crook Errol D , Daniels Marc C , Smith Thomas M , McClain Donald A
Published: September 2013

High glucose concentrations such as are seen in diabetes mellitus are known to have deleterious effects on cells, but the pathways by which glucose induces these effects are unknown. One hypothesis is that metabolism of glucose to glucosamine might be involved. For example, it has been shown that glucosamine is more potent than glucose in inducing insulin resistance in cultured adipocytes and in regulating the transcription of the growth factor transforming growth factor alpha in smooth muscle cells. The rate-limiting step in glucosamine synthesis is the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the enzyme glutamine:fructose-6-phosphate amidotransferase. To test the hypothesis that this hexosamine biosynthesis pathway is involved in the induction of insulin resistance, we have overexpressed the enzyme glutamine:fructose-6-phosphate amidotransferase in Rat-1 fibroblasts and investigated its effects on insulin action in those cells. We electroporated Rat-1 fibroblasts with expression plasmids that did and did not contain the gene for glutamine:fructose-6-phosphate amidotransferase and measured glycogen synthase activity at varying insulin concentrations. Insulin stimulation was blunted in the glutamine:fructose-6-phosphate amidotransferase-transfected cells, resulting in decreased insulin sensitivity reflected by a rightward shift in the dose-response curve for activation of synthase (ED50 = 7.5 nM vs. 3.4 nM insulin, in glutamine:fructose-6-phosphate amidotransferase and control cells, respectively). Rat-1 fibroblasts incubated with 5.0 mM glucosamine for 3 days exhibited a similar shift in the dose-response curve. The rightward shift in the dose-response curve is seen as early as 2 days after poration. Overexpression of glutamine:fructose-6-phosphate amidotransferase also induced an increase in basal activity of glycogen synthase. The increase in basal glycogen synthase activity in glutamine:fructose-6-phosphate amidotransferase cells developed later than the shift in the dose-response curve; by 4 days after transfection, a 61 ± 14.5% increase in basal glycogen synthase activity had been reached. Overexpression of glutaminerfructose-6-phosphate amidotransferase did not affect total glycogen synthase activity or glucose uptake in these cells. Furthermore, no change occurred in the number or affinity of insulin receptors in the glutamine:fructose-6-phosphate amidotransferase–transfected cells, indicating that the insulin resistance induced by glutamine:fructose-6-phosphate amidotransferase occurs distal to insulin binding.
Pancreatic Islet Function in Nondiabetic and Diabetic BB Rats
Authors: Teruya Masanori , Takei Shinichiro , Forrest Luette E , Grunewald Axel , Chan Eve K , Charles M Arthur
Published: September 2013

A decreased acute insulin response to glucose in islet cell antibody positive humans predicts diabetes. Because the dominant mechanism leading to decreased in vivo acute insulin response to glucose remains unclear, perifused islets were examined before and after diabetes onset in BB rats to assess the role of glucose sensitivity on insulin secretion in individual islets. Islets from normal WF rats, diabetes-prone rats without inflamed islets, diabetes-prone rats with inflamed islets, and diabetic rats were studied at 2.0, 8.3, and 16.7 mM glucose. Immunoreactive insulin from WF islets at 16.7 mM glucose was 0.15 ± 0.02 ng · 0–7 min−1 · islet−1 for the first phase and 1.00 ± 0.05 ng · 7–20 min−1 · islet−1 for the second phase of biphasic secretion, compared with basal secretion of 0.10 ± 0.03 ng · 20 min−1 · islet−1 at 2 mM glucose. Diabetes-prone noninflamed islets showed a 0.20 ± 0.03 ng first-phase secretion, a 1.32 ± 0.13 ng second-phase secretion after 16.7 mM glucose, and 0.093 ± 0.02 ng · 20 min−1 · islet−1 at 2 mM glucose, indicating no intrinsic BB rat strain secretion abnormality. Diabetes-prone inflamed islets had secretions of 0.35 ± 0.02 ng during the first phase (P < 0.05 vs. WF) and 1.78 ± 0.29 ng during the second phase (P < 0.05 vs. WF) after 16.7 mM glucose, with 0.24 ± 0.08 ng · 20 min−1 · islet−1 at 2 mM glucose. Islets derived from diabetic rats had a first-phase secretion of 0.63 ± 0.14 ng (P < 0.05 vs. diabetes-prone noninf lamed and WF) and a second phase of 1.48 ± 0.28 ng (P < 0.05 vs. WF) with 0.30 ng · 20 min−1 · islet−1 at 2 mM glucose. Dose-response studies showed enhanced insulin secretion to each concentration of glucose when compared with controls (WF or diabetes-prone noninf lamed). These data indicate that 1) BB inflamed islets can be isolated for dynamic insulin secretion studies in nondiabetic and diabetic rats; 2) β-cell glucose sensitivity appears enhanced in islets derived from nondiabetic rats with inflamed islets or rats soon after diabetes onset; and 3) the reported impairment of in vivo acute insulin response to glucose in the BB rat model does not result primarily from a decrease in residually functioning β-cell sensitivity to glucose, but rather the data suggest a more important role for decreased residual β-cell mass.
Predictive Value of Lymphocyte Antibodies for the Appearance of Diabetes in BB Rats
Authors: Bertrand Sylvie , Vigeant Claire , Yale Jean-François
Published: September 2013

Lymphocyte antibodies have been described in autoimmune disorders, including insulin-dependent diabetes mellitus (IDDM). We have developed a quantitative method to measure autoantibodies directed against T-lymphocytes, based on two-color fluorescence labeling of Wistar mononuclear cells and analysis of fluorescence by flow cytometry. The lymphocyte antibody levels were determined retrospectively in the serum of 73 BB and 18 Wistar rats. We demonstrated the binding of the lymphocyte autoantibodies of both CD4+ and CD8+ T-cells. Lymphocyte antibodies were present in 90% of the BB rats at diabetes onset, compared with 11% of the Wistar rats. At 75 days of age, 83% of the BB rats, which later became diabetic, were positive for the lymphocyte antibodies, compared with 15% of their litter mates who maintained a normal glucose tolerance. In all cases, the antibodies were of the immunoglobulin M isotype. We conclude that lymphocyte antibodies are present before diabetes onset and, using this method, that their presence can predict the development of diabetes with a sensitivity of 83% and a specificity of 85% in BB rats.
Effects of Calcium Antagonists on Insulin-Mediated Glucose Metabolism in Skeletal Muscle
Authors: Foot Elizabeth A , Leighton Brendan
Published: September 2013

The effect of three calcium antagonists (verapamil, diltiazem, and nifedipine) on insulin effects was investigated in isolated rat soleus muscles. Soleus muscles were incubated in the presence of insulin (100 μU/ml), a concentration that stimulates the rates of lactate formation and glycogen synthesis half-maximally and with and without a calcium antagonist. A decrease (48%; P < 0.001) was noted in the insulin-mediated rate of glycogen synthesis by verapamil at 100 μM; no effect was observed at lower concentrations of verapamil. Diltiazem decreased the insulin-mediated rates of glycogen synthesis by 36 (P < 0.001), 64 (P < 0.001), and 73% (P < 0.001) at 1,10, and 100 μM, respectively. Nifedipine decreased the insulin-mediated rates of glycogen synthesis by 37% at 0.1 μM (P < 0.001), 36% at 1 μM (P < 0.001), 21% at 10 μM (P < 0.05), and 72% at 100 μM (P < 0.001). Verapamil at 100 μM decreased lactate formation by 48% (P < 0.001). However, diltiazem increased the rate of lactate formation by 22 (P < 0.01), 43 (P < 0.001), and 61% (P < 0.001) at 1,10, and 100 μM, respectively. In contrast, nifedipine increased the insulin-mediated rate of lactate formation by 45% only at 100 μM (P < 0.01). The increased rate of lactate formation was probably caused by an increased rate of glycogenolysis, because high concentrations of all the calcium antagonists significantly decreased muscle glycogen content. The insulin-stimulated rate of 3-O-methyl-D-glucose transport or cAMP content was not affected by diltiazem at 1 or 10 μM. The results suggest that the calcium antagonists work by a mechanism, possibly by activating a calcium channel or an extracellular receptor, to influence markedly insulin-mediated intracellular glucose metabolism in skeletal muscle.
Thyroid Hormone Increases Basal and Insulin-Stimulated Glucose Transport in Skeletal Muscle: The Role of GLUT4 Glucose Transporter Expression
Authors: Weinstein Steven P , O'Boyle Elixabeth , Haber Richard S
Published: September 2013

In skeletal muscle, the main site of insulin-mediated glucose disposal, the major muscle glucose transporter GLUT4 is induced by thyroid hormone. To test the hypothesis that thyroid hormone alters muscle glucose transport, we examined the effect of L-triiodothyronine (T3) on glucose transport and GLUT4 protein content in isolated rat skeletal muscles. Euthyroid rats were treated with or without T3 for 3 days, and [3H]2-deoxy-D-glucose (2-DG) uptake in soleus and extensor digitorum longus (EDL) muscles was measured under conditions in which transport was rate limiting for uptake in the absence or presence of 10 nmol/l insulin. In control animals, insulin stimulated 2-DG uptake sevenfold in soleus and fivefold in EDL. T3 treatment increased basal 2-DG uptake in soleus and EDL by 115 ± 29% and 136 ± 23%, respectively, and increased insulin-stimulated 2-DG uptake in soleus and EDL by 55 ± 9 and 42 ± 12%, respectively. Immunoblot analysis revealed that T3 treatment increased GLUT4 protein content in soleus by 43 ± 6% and in EDL by 56 ± 13%. These data demonstrate that thyroid hormone increases basal and insulin-stimulated glucose transport in skeletal muscle. The percentage increase in insulin-stimulated transport in T3-treated muscles is similar to the increase in GLUT4 protein content, whereas the percentage change in basal transport greatly exceeds the change in GLUT4. Thus, increased insulin-stimulated glucose transport in T3-treated muscle can be accounted for by the induction of GLUT4 protein. However, increased basal glucose transport in T3-treated muscle must reflect additional mechanisms, such as increased subcellular partitioning of GLUT4 to plasma membrane.
Capillary Pressure in Patients With NIDDM
Authors: Shore Angela C , Jaap Alan J , Tooke John E
Published: September 2013

The hemodynamic hypothesis suggests that raised capillary pressure may play a role in the pathogenesis of diabetic microangiopathy. Although patients with non-insulin-dependent diabetes mellitus (NIDDM) and insulin-dependent diabetes NIDDM) develop a similar range of microvascular complications, differences in their expression and prevalence suggest that different pathogenic mechanisms may be operational. Capillary pressure is elevated in IDDM; the aim of this study was to assess whether capillary pressure was also elevated in NIDDM. Twenty-one patients with NIDDM (15 men) and 21 healthy control subjects matched for age, sex, and skin temperature were investigated supine with the hand at heart level. Finger nailfold capillary pressure was measured after direct cannulation at the summit of the capillary loops using glass micropipettes. The groups were matched for skin temperature (30.4 [24.2–33.8]°C, median [95% confidence interval], NIDDM patients vs. 30.0 [23.4–33.6] degrees C control subjects), age (62.0 [39.4–72.7] years NIDDM patients vs. 62.0 [39.4–72.0] years control subjects), and both systolic (sBP) and diastolic (dBP) blood pressures (133.0 [111.0–167.3]/78.0 [57.0–89.5] mmHg NIDDM patients vs. 133.0 [114.1–158.9]/80.0 [68.2–88.9] mmHg control subjects). Capillary pressure did not differ in the two groups (17.6 [13.1–21.2] mmHg NIDDM patients vs. 19.1 [14.1–23.6] mmHg control subjects [NS]). There was no correlation of capillary pressure with either HbA1c or glucose; however, there was a negative association between capillary pressure and diabetes duration (Rs = −0.50, P = 0.020). Although these data provide no evidence of capillary hypertension in patients with NIDDM, at least under resting conditions, they raise the possibility that diabetes of long duration might modify the normal mechanisms controlling capillary pressure.
Quantitation of Glutamic Acid Decarboxylase Autoantibody Levels in Prospectively Evaluated Relatives of Patients With Type I Diabetes
Authors: Yu Liping , Gianani Roberto , Eisenbarth George S
Published: September 2013

In this study, we demonstrate that levels of glutamic acid decarboxylase (GAD) autoantibodies (GAAs) by radioassay differ between relatives with GAD-absorbable and GAD-nonabsorbable islet cell antibodies (ICAs). Extremely high levels of GAAs are often found in relatives with GAD-absorbable ICAs (>1,800 cpm, >9 SD above normal control subjects; mean = 1,991 cpm), and lower levels (mean = 1,078 cpm) of GAAs were present in relatives with nonabsorbable ICAs (P < 10−5). The serum levels of GAAs were remarkably constant for relatives of both groups over time. The levels of GAAs were found to be inversely related to both the levels of insulin autoantibodies and the rate of loss of intravenous glucose-stimulated insulin secretion (P < 10−5 and P < 0.01, respectively). Relatives with low positive levels of GAAs had more rapid loss of insulin secretion and were at high risk to become diabetic (50% diabetic at 4 years) compared with relatives with higher levels (1,800 cpm) of GAAs (10% diabetic at 4 years; P < 0.05). These data suggest that high levels of GAAs are associated with a decreased risk of progression to type I diabetes and extend the hypothesis that distinct subsets of ICAs and GAAs with differing prognostic significance can be identified.
Sequence of the Human Glycogen-Associated Regulatory Subunit of type 1 Protein Phosphatase and Analysis of Its Coding Region and mRNA Level in Muscle From Patients With NIDDM
Authors: Hua Chen Yu , Hansen Lars , Xiang Chen Mao , Bjørbæk Christian , Vestergaard Henrik , Hansen Torben , Cohen Paticia T W , Pedersen Oluf
Published: September 2013

Impaired insulin-stimulated glycogen synthesis of peripheral tissues is a characteristic feature of many patients with non-insulin-dependent diabetes mellitus (NIDDM) and their first-degree relatives with normal glucose tolerance, suggesting putative inherited defects in this metabolic pathway. In previous studies, we have failed to reveal mutations in the coding regions of the muscle-specific glycogen synthase gene and the three genes that encode the catalytic subunits of protein phosphatase 1 (PP1) as frequent causes of insulin resistance. Because the glycogen-associated regulatory subunit of protein phosphatase 1 (PP1 G-subunit) plays a key role in the insulin stimulation of glycogen synthesis and the activity of PP1 is decreased in insulin-resistant subjects, we have now cloned the human G-subunit cDNA to search for abnormalities in the corresponding gene (designated PPP1R3 in the human genome nomenclature) in patients with NIDDM. The human cDNA was isolated from a skeletal muscle cDNA library and was found to encode a 126-kDa protein, which shows 73% amino acid identity to the rabbit PP1 G-subunit. The human G-subunit cDNA from 30 insulin-resistant NIDDM patients was analyzed for genetic variations in the G-subunit by using single-stranded conformation polymorphism (SSCP) scanning of reversely transcribed mRNA. One variant SSCP profile was detected in the region encoding the COOH-terminal part of the PP1 G-subunit in only one NIDDM patient, and subsequent nucleotide sequencing showed a C to A transversion on one allele at base position 2792. This change predicts an amino acid substitution from alanine to glutamic acid. The carrier of this mutation was characterized by reduced insulin-stimulated nonoxidative glucose metabolism when examined with the euglycemic hyperinsulinemic clamp. SSCP scanning of the 2584–2844 nucleotide fragment of PP1 G-subunit cDNA from an additional 22 NIDDM patients and 29 control subjects did not reveal additional genetic variants. To indirectly screen for abnormalities in PP1 G-subunit gene regulation, we measured the mRNA level of the G-subunit in skeletal muscle. However, no difference in the abundance of mRNA of PP1 G-subunit was found between patients with diabetes and control subjects.
Antibodies to Islet 37k Antigen, But Not to Glutamate Decarboxylase, Discriminate Rapid Progression to IDDM in Endocrine Autoimmunity
Authors: Christie Michael R , Genovese Stefano , Cassidy David , Bosi Emanuele , Brown Thomas J , Lai Manuelita , Bonifacio Ezio , Franco Bottazzo Gian
Published: September 2013

Apart from islet cell antibodies (ICAs), antibodies to glutamate decarboxylase (GAD), insulin autoantibodies (IAAs), and a novel islet antigen (37k antigen) are potential markers for insulin-dependent diabetes mellitus (IDDM). GAD is also an antigen in stiff-man syndrome (SMS), and both SMS and IDDM are associated with ICAs and autoimmunity to other endocrine organs. We investigated possible links between antibody responses to islet antigens with autoimmunity to other endocrine organs and determined which specific antibodies can identify individuals who progress to IDDM. Antibodies to GAD were detected in ≥90% of both diabetic and nondiabetic patients with ICAs and other endocrine autoimmunity, in 59% of ICA-positive IDDM patients without endocrine autoimmunity, in all patients with SMS, but in only 1–3% of healthy (nondiabetic) and autoimmune disease control subjects. GAD antibody levels were increased in ICA-positive IDDM patients with polyendocrine autoimmunity compared with those without. In contrast, antibodies to 37k antigen were only detected in patients who developed acute-onset IDDM. IAAs were also associated with IDDM. Thus, certain factors enhance antibody responses to GAD in polyendocrine autoimmunity, but this does not necessarily lead to development of IDDM or SMS. Antibodies to 37k antigen are strongly associated with acute-onset IDDM and are useful serological markers for disease.
Importance of Substrate Changes in the Decrease of Hepatic Glucose Cycling During Insulin Infusion and Declining Glycemia in the Depancreatized Dog
Authors: Qing Z Shi , Giacca Adria , Fisher Simon , Vidal Hubert , Werve Gerald Van De , Vranic Mladen
Published: September 2013

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G⇆G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6–9 h at a starting dose of 45–150 pmol-kg−1-min−1 to normalize plasma glucose from 23.9 ± 1.4 to 5.0 ± 0.4 mmol/1 and gradually decreased to and maintained at a basal rate (1.7 ± 1.0 pmol · kg−1 · min−1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 ± 2.7 and normalized at 1.3 ± 0.6 μmol · kg−1 · min−1 (P < 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 ± 3.1 to 11.6 ± 1.2) than did glucose production (from 25.9 ± 1.9 to 10.3 ± 1.0 μmol · kg−1 · min−1) (both P < 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 ± 24 vs. 71 ± 13 nmol/g, P < 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 ± 0.8 to 19.6 ± 2.6 U/g) and glucokinase (from 1.1 ± 0.2 to 1.0 ± 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P < 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content. We therefore conclude that in the depancreatized dog, 1) acute insulin infusion with a concurrent correction of hyperglycemia can markedly reduce and normalize GC, and 2) the acute reduction in GC is primarily mediated by reduced substrate fluxes through glucokinase and G6Pase and does not require stable changes in maximal activities of the two enzymes. However, in vivo allosteric changes in enzyme activities (particularly G6Pase), immeasurable in vitro, cannot be excluded. In addition to enzyme measurements, the importance of tracer methods that allow for in vivo measurements of fluxes through glucokinase and G6Pase is emphasized.
Combined Analysis of Autoantibodies Improves Prediction of IDDM in Islet Cell Antibody-Positive Relatives
Authors: Bingley Polly J , Christie Michael R , Bonifacio Ezio , Bonfanti Ricardo , Shattock Marion , Fonte Maria-Teresa , Bottazzo Gian-Franco , Gale Edwin A M
Published: September 2013

Prediction of insulin-dependent diabetes mellitus (IDDM) is still largely based on islet cell antibodies (ICAs), but it may be improved by combined analysis with other humoral markers. We examined autoantibodies to insulin (IAAs), glutamic acid decarboxylase (GAD), and Mr 37,000 and Mr 40,000 fragments of islet antigens (37 and 40 kDa) together with ICA subtypes in 101 family members with ICAs ≥10 Juvenile Diabetes Foundation units (JDF U) followed for up to 14 years, of whom 18 have developed IDDM. Life-table analysis showed a 43% risk of IDDM within 10 years for those with ICAs ≥10 JDF U, rising to 53% for those with ICAs ≥20 JDF U. The risk for ICAs ≥10 JDF U was 62% in the family members in the youngest age quartile (<13.2 years) and fell with increasing age to 4% in those >40.7 years of age (P = 0.03). ICAs ≥10 JDF U combined with IAAs gave a risk of 84% (P = 0.03 compared with IAA−), and ICAs ≥10 JDF U combined with GAD antibodies gave a risk of 61% (P = 0.018). The risk for ICAs >10 JDF U with antibodies to 37-kDa antigen was 76% (P < 0.0001). Risk increased with the number of autoantibodies, from 8% for ICAs alone to 88% with >3 autoantibodies (14 cases detected) (P < 0.0001). The increased risk associated with multiple antibodies was observed independent of age. The median time to diagnosis in those with antibodies to 37- and/or 40-kDa antigen was 1.5 years, compared with 7.2 years in those with IAAs and GAD antibodies in the absence of antibodies to 37/40 kDa. The intensity and range of the autoantibody response offers better overall prediction of diabetes than any single autoantibody specificity, although antibodies to 37-/40-kDa antigens may prove to be useful markers of early clinical onset. We found that 78% of future cases of IDDM in ICA+ relatives came from the 27% with multiple autoantibodies and estimate that 88% of individuals within this category will need insulin treatment within 10 years. We propose a simple predictive strategy based on these observations.
Effect of Hyperketonemia and Hyperlacticacidemia on Symptoms, Cognitive Dysfunction, and Counterregulatory Hormone Responses During Hypoglycemia in Normal Humans
Authors: Veneman Thiemo , Mitrakou Asimina , Mokan Marian , Cryer Philip , Gerich John
Published: September 2013

The brain usually depends almost exclusively on glucose for its energy requirements. During hypoglycemia associated with prolonged fasting or strenuous exercise, circulating ketone-body and lactate levels increase severalfold; in both situations, certain signs and symptoms of hypoglycemia are diminished. Therefore, to test the hypothesis that hyperketonemia or hyperlacticacidemia of the magnitude seen during certain clinical situations can substitute for glucose as an energy source for the brain and alter physiological responses to hypoglycemia, we assessed autonomic and neuroglycopenic symptoms, counterregulatory hormone responses, and cognitive function during standardized insulin-induced hypoglycemia in normal volunteers with and without infusion of (β-hydroxybutyrate (BOHB) or lactate designed to reproduce circulating levels of these substrates seen during prolonged fasting and strenuous exercise. Compared with paired control experiments, infusion of BOHB and lactate increased the glycemic threshold (required greater hypoglycemia for initiation) and reduced the magnitude of autonomic and neuroglycopenic symptoms, counterregulatory hormone responses, and cognitive dysfunction (all P < 0.05). The hypoglycemic threshold for autonomic symptoms increased from 3.8 ± 0.1 to 3.1 ± 0.2 mmoM during BOHB infusion and from 3.7 ± 0.1 to 2.8 ± 0 . 1 mmol<1 during lactate infusion, and the threshold for neuroglycopenic symptoms increased from 2.8 ± 0.1 to 2.4 ± 0.1 and 2.3 ± 0.1 mmol/1, respectively. The magnitude for autonomic symptoms decreased from 12 ± 2 and 11 ± 1 to 6 ± 2 and 4 ± 1 during BOHB and lactate infusion, respectively. Neuroglycopenic symptoms decreased from 11 ± 2 to 3 ± 1 during both series of experiments. Infusion of BOHB and lactate-reduced responses for all counterregulatory hormones, the reduction being the greatest for epinephrine (∼57 and 73%, during BOHB and lactate infusion, respectively) and least for cortisol (7sim;28 and 29%, respectively). These results indicate that under certain clinical conditions, BOHB and lactate may substitute for glucose as a fuel for the brain and alter physiological responses to hypoglycemia.
HLA-DQ-Restricted, Islet-Specific T-Cell Clones of a Type I Diabetic Patient: T-Cell Receptor Sequence Similarities to Insulitis-Inducing T-Cells of Nonobese Diabetic Mice
Authors: Durinovic-Bellò Ivana , Steinle Alexander , Ziegler Anette G , Schendel Dolores J
Published: September 2013

We established a T-cell line and 20 CD4+ T-cell clones from the peripheral blood lymphocytes of a type I diabetic patient using a membrane preparation of a rat insulinoma cell line (beta membrane antigen [BMA]) as a source of antigen. The T-cell line and three selected clones proliferated specifically to stimulation with BMA and to membranes prepared from human islets, rat pancreas, and NOD pancreas, but not to control antigens. Proliferationinhibition studies using human leukocyte antigen (HLA)-specific monoclonal antibodies revealed HLA-DQw1-restricted recognition of BMA. An analysis of the T-cell receptor (TCR) repertoire of the T-cell line after 8 and 40 days of culture showed a prominent usage of the Val and Vα12 gene families. Although sequencing of the TCR Vα and Vβ chains of the three clones demonstrated that each used different Va and V(J gene segments, structural similarities were found in complementary-determining region 3 (CDR3), the region that is postulated to interact with the peptide component of the TCR ligand. When we compared these TCR sequences with published sequences of disease-inducing T-cells of NOD mice, highly related TCR Vβ families were detected. Furthermore, stretches of identical amino acids within the CDR3 region were found between two pairs of human and mouse T-cells. If one considers the species differences in TCR genes and sequence differences in the restriction elements for these cells (HLA-DQ vs. H-2I-Anod), these sequence similarities are striking and may be useful for pinpointing T-cells of primary importance in the development of disease.
Regulation of Skeletal Muscle Blood Flow During Acute Insulin-Induced Hypoglycemia in the Rat
Authors: Hickner Robert C , Ungerstedt Urban , Henriksson Jan
Published: September 2013

The mechanism behind hyperemia in skeletal muscle during insulin-induced hypoglycemia was investigated in 42 anesthetized male Wistar rats using the microdialysis ethanol technique of monitoring nutritive blood flow. Microdialysis probes were inserted bilaterally into the gastrocnemius muscle and perfused with a modified Krebs-Henseleit buffer containing 20 mmol/l ethanol and one or more of the following compounds: propranolol (10−6 molA), phentolamine (10−6 mol/1), and calyculin A (1.0 (xmol/1). Muscle blood flow increased, as indicated by a decrease in the ethanol outflow:inflow ratio (P < 0.001, n = 6), during hypoglycemia induced by a bolus intravenous infusion of insulin (680 mU/kg body wt). This increase was not present during normoglycemia or during hypoglycemia and local (β-adrenergic blockade via propranolol. However, the hyperemic response was potentiated during hypoglycemia and local β-adrenergic blockade via phentolamine. A normal hyperemic response to hypoglycemia was detected during simultaneous α- and β-adrenergic blockade. This response was eliminated on further supplementation of the microdialysis perfusion medium with calyculin A. Therefore, although stimulation of the α- and β-adrenergic receptors does occur during insulin-induced hypoglycemia, it is not essential for the induction of hyperemia in this state. It may be concluded that hyperinsulinemia results in vasodilatation during hypoglycemia, although hyperinsulinemia does not have an effect on skeletal muscle blood flow under normoglycemic conditions.
Glomerular Structure in Nonproteinuric IDDM Patients With Various Levels of Albuminuria
Authors: Fioretto Paola , Steffes Michael W , Mauer Michael
Published: September 2013

Although microalbuminuria is known to foretell the later development of overt proteinuria in patients with insulindependent diabetes mellitus (IDDM), different investigators have reported different levels of albuminuria as being predictive. However, whether different levels of albuminuria reflect differences in glomerular structure is not well known. In this study, we divided a cohort of 66 nonproteinuric long-standing (duration 20 ± 7 years) IDDM patients, who had both renal functional and structural studies performed, into four groups according to their urinary albumin excretion rate (AER). The several different levels of microalbuminuria previously reported to be predictive served to demarcate these groups: group I, AER ≤22 mg/24 h (upper limit for normal in our laboratory) (33 patients); group II, AER 23–45 mg/24 h (11 patients); group HI, AER 46–100 mg/24 h (13 patients); and group IV, AER 101–220 mg/24 h (9 patients). Creatinine clearance was similar in groups I, II, and III but was lower in group IV. Systemic hypertension was present in five patients in group I, one in group II, seven in group III, and five in group IV. Mean values for glomerular basement membrane (GBM) width and volume fraction of the mesangium [Vv(mes/glom)] were greater in all groups than in a group of 52 age-matched normal kidney donors (P < 0.0001). Also, filtration surface density [Sv(PGBM)], inversely related to Vv(mes/glom) (r = 0.61, P < 0.0001), was reduced in all diabetic groups compared with the normal group (P < 0.0001). Structural measures were identical in group I and II. GBM width, Vv(mes/ glom), and Sv(PGBM) were more abnormal in groups III and IV than either group I or II (P < 0.05). Hypertension was unrelated to the values for any of these structural measures. However, AER and blood pressure had an interactive effect on Vv(mes/glom) (P = 0.002); in patients with AER <45 mg/24 h and hypertension, Vv(mes/ glom) was higher than in normotensive patients in the same AER category (P < 0.03). Thus, morphometric measures characteristic of diabetic glomerulopathy are present in normoalbuminuric IDDM patients, albeit in some of these patients renal structure is in the normal range. Lesions, on average, are more advanced when albuminuria exceeds 45 mg/24 h. In patients with AER >45 but <220 mg/24 h, a further division based on AER (< and >100) does not discriminate groups with different glomerular lesions. Therefore, albuminuria >45 mg/24 h indicates more advanced diabetic glomerulopathy and is frequently associated with other functional abnormalities such as reduced glomerular filtration rate and increasing blood pressure. These results are consistent with the majority of studies that have found the higher ranges of microalbuminuria to predict progression to overt nephropathy with greater specificity.
Hypoglycemia: The Limiting Factor in the Management of IDDM
Authors: Cryer Philip E
Published: September 2013

Iatrogenic hypoglycemia is the limiting factor in the management of insulin-dependent diabetes mellitus (IDDM). It causes recurrent physical morbidity, some mortality, and recurrent or even persistent psychosocial morbidity. The principles of glucose counterregulation, the physiological mechanisms that normally very effectively prevent or correct hypoglycemia, are now known. Decrements in insulin, increments in glucagon, and, in the absence of the latter, increments in epinephrine stand high in the hierarchy of redundant glucose counterregulatory factors. Iatrogenic hypoglycemia in IDDM is the result of the interplay of absolute or relative therapeutic insulin excess and compromised glucose counterregulation. Syndromes of compromised glucose counterregulation include defective glucose counterregulation (the result of combined deficiencies of the glucagon and epinephrine responses to falling glucose levels), hypoglycemia unawareness (loss of the warning, neurogenic symptoms of developing hypoglycemia), and elevated glycemic thresholds (lower glucose levels required) for autonomic activation and symptoms during effective intensive therapy. These have been conceptualized as examples of hypoglycemiaassociated autonomic failure, a functional disorder distinct from classical diabetic autonomic neuropathy, in IDDM. Recent antecedent iatrogenic hypoglycemia appears to be a major factor in the pathogenesis of hypoglycemia unawareness; there is increasing evidence that this syndrome is reversible with scrupulous avoidance of hypoglycemia. It probably also contributes substantially to the syndrome of elevated glycemic thresholds during intensive therapy. However, factors in addition to recent antecedent hypoglycemia play an important role in the pathogenesis of the syndrome of defective glucose counterregulation. Pending the prevention and cure of IDDM, we need to learn to replace insulin in a much more physiological fashion and/or to prevent, correct, or compensate for compromised glucose counterregulation if we are to eliminate hypoglycemia from the lives of people with IDDM without compromising glycemic control. In the meantime, we must continue to seek better insight into the fundamental mechanisms of compromised glucose counterregulation and to develop practical preventive clinical strategies and practice hypoglycemia risk factor reduction with our patients.
Clonal Insulinoma Cell Line That Stably Maintains Correct Glucose Responsiveness
Authors: Knaack David , Fiore Deborah M , Surana Manju , Leiser Margarita , Laurance Megan , Fusco-DeMane David , Hegre Orion D , Fleischer Norman , Efrat Shimon
Published: September 2013

A number of pancreatic β-tumor cell (β TC) lines have been derived from insulinomas arising in transgenic mice expressing the SV40 T antigen gene under control of the insulin promoter. Some of these lines secrete insulin in response to physiological glucose concentrations. However, this phenotype is unstable. After propagation in culture, these nonclonal lines become responsive to subphysiological glucose levels and/or manifest reduced insulin release. Here we report the use of soft-agar cloning to isolate single-cell clones from a β TC line, which give rise to sublines that maintain correct glucose responsiveness and high insulin production and secretion for > 55 passages (over a year) in culture. One of these clonal lines, denoted β TC6-F7, was characterized in detail. β TC6-F7 cells expressed high glucokinase and low hexokinase activity, similarly to normal islets. In addition, they expressed mRNA for the GLUT2 glucose transporter isotype and no detectable GLUT1 mRNA, as is characteristic of normal β-cells. These results demonstrate that transformed β-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous β-cell lines for transplantation therapy of diabetes.
Pancreatic Expression and Secretion of Human Islet Amyloid Polypeptide in a Transgenic Mouse
Authors: D'Alessio David A , Verchere C Bruce , Kahn Steven E , Hoagland Vicki , Baskin Denis G , Palmiter Richard D , Ensinck John W
Published: September 2013

Islet amyloid polypeptide (IAPP) is a secretory product of the pancreatic β-cell, which is the primary constituent of the islet amyloid that develops in type II diabetes. To study the role the inherent amyloidogenicity of human IAPP (hIAPP) plays in the formation of islet amyloid deposits and to investigate a possible hormonal role for IAPP, transgenic mice expressing hIAPP were developed. The transgene was composed of a fragment of an hIAPP cDNA linked to the rat insulin II promoter. One line of transgenic mice expressed the transgene and synthesized hIAPP in their pancreatic islets. IAPP-like immunoreactivity in pancreatic extracts and plasma were two- to threefold greater in the transgenic mice compared with nontransgenic control mice. Although plasma concentrations of immunoreactive insulin (IRI) and glucose were equal in transgenic and control mice, the pancreatic content of IRI was nearly twofold greater in the transgenic animals, and proinsulin mRNA was significantly elevated, suggesting increased rates of insulin biosynthesis. Pancreatic samples obtained from transgenic mice up to 19 months of age had no evidence of islet amyloid. These results indicate that an increased level of synthesis of the amyloidogenic hIAPP is not sufficient to cause islet amyloid deposition. However, the increased synthesis and storage of insulin in the islets of the transgenic mice are consistent with either a direct regulatory effect of IAPP on the β-cell or indirect stimulation of insulin production through IAPP-induced insulin resistance.
Insulin Resistance, Body Fat Distribution, and Sex Hormones in Men
Authors: Haffner Steven M , Karhapää Pauli , Mykkänen Leena , Laakso Markku
Published: September 2013

Although many studies have suggested that increased androgenicity is associated with insulin resistance and hyperinsulinemia in both pre- and postmenopausal women, relatively few data are available on this relationship in men. We examined the association of body mass index (BMI), waist-to-hip ratio (WHR), sex hormone–binding globulin (SHBG), total and free testosterone, dehydroepiandrosterone sulfate (DHEA-SO4), and estradiol with insulin concentrations and whole-body glucose disposal in 87 men from a population-based study in Kuopio, Finland. BMI was significantly correlated with fasting insulin (r = 0.46), total whole-body glucose disposal (r = −0.30), glucose oxidation (r = −0.21), and nonoxidative glucose disposal (r = −0.25). WHR also was significantly associated with fasting insulin (r = 0.61), total whole-body glucose disposal (r = −0.54), glucose oxidation (r = −0.23), and nonoxidative whole-body glucose disposal (r = −0.50). SHBG and total and free testosterone were significantly associated with insulin concentrations and total and nonoxidative glucose disposal but not with glucose oxidation. DHEA-SO4 and estradiol were not associated with insulin, glucose concentrations, or whole-body glucose disposal in univariate analysis. In multivariate analysis, total whole-body glucose disposal was associated negatively with WHR and positively associated with total testosterone and SHBG; nonoxidative whole-body glucose disposal was associated negatively with WHR and positively associated with total and free testosterone. Glucose oxidation was significantly associated only with WHR. In conclusion, higher WHR and lower testosterone were strongly associated with a decrease in total and nonoxidative whole-body glucose disposal in men.
Mutational Analysis of the NH2-Terminal Glycosylation Sites of the Insulin Receptor α-subunit
Authors: Caro L Heleen , Ohali Anat , Gorden Phillip , Collier Elaine
Published: September 2013

The insulin receptor is synthesized as a single chain of 190 kiloDaltons, which is processed to disulfide-linked mature α- and β-subunits, containing N- and O-linked oligosaccharides and fatty acids. Previously (Collier E, Carpentier J-L, Beitz L, Caro LHP, Taylor SI, Gorden P: Biochemistry 32:7818–23, 1993), site directed mutagenesis of the asparagine in the first four sites of N-linked glycosylation to glutamine resulted in a receptor that was retained in the endoplasmic reticulum and not processed past the proreceptor form. In this study, mutation of these sites individually and in various combinations is studied. Mutation in the first or second glycosylation site does not significantly impair processing of the receptor; the receptor is found on the cell surface and binds insulin normally. If both the first and second sites are mutated, a significant reduction occurs in the amount of receptor found on the cell surface and in insulin binding. There is some processing of the receptor in cells expressing this mutant compared with the four-part mutant. If only the third and fourth sites are mutated, processing is impaired < in the mutant with the first and second sites mutated. However, the amount of receptor found on the cell surface is < in the mutant of only the first or only the second site. In all of these glycosylation mutants, the amount of receptor on the cell surface correlates with the level of 125I-labeled insulin binding on the cell surface. N-linked glycosylation of the NH2-terksminal of the α-subunit of the insulin receptor is necessary for normal processing and cell surface expression of the insulin receptor but not for the function of the receptor. Specific sites of glycosylation and the number of sites glycosylated both determine the amount of receptor processed.
Mortality and Treatment Side-Effects During Long-Term Intensified Conventional Insulin Treatment in the Stockholm Diabetes Intervention Study
Authors: Reichard Per , Pihl Margareta
Published: September 2013

Altogether, 102 patients were randomized to intensified conventional treatment (ICT) (n = 48) or standard treatment (ST) (n = 54). After 7.5 years, 89 patients remained, and it was shown that microangiopathy was retarded by the lower blood glucose concentrations seen in the patients in the ICT group. HbA1c was reduced from (means ± SE) 9.5 ± 0.2% to 7.1 ± 0.1% in the ICT group and from 9.4 ± 0.2% to 8.5 ± 0.1% in the ST group (P < 0.001). Of the patients, 4 in the ICT group and 3 in the ST group died. Mortality was predicted by albuminuria, the amplitude of the sural nerve action potential, and the test of arm blood flow during contraction of the contralateral hand (sympathetic nerve function) at baseline (P < 0.05). Weight increased by 4.4 ± 1.1 kg in the ICT group and 1.8 ± 0.7 kg in the ST group (P = 0.05). Atherosclerosis, measured with digital pulse plethysmography, was ∼ the same in the groups at baseline and after five years. In each group, 3 patients had myocardial infarctions, and 2 from each group had ketoacidosis once. There was a mean of 1.1 episodes per patient and per year of serious hypoglycemia in the ICT group and 0.4 episodes per patient and per year in the ST group. No adverse incidents or accidents were observed in either group, and there were no differences between the groups with regard to cognitive function measured with a battery of tests. retarded the microvascular complications, caused some weight gain and an almost threefold increase of the frequency of serious hypoglycemia, but brain function did not deteriorate.
Effect of NIDDM on the Kinetics of Whole-Body Protein Metabolism
Authors: Gougeon Réjeanne , Pencharz Paul B , Marliss Errol B
Published: September 2013

We postulated that dietary protein utilization and body protein metabolism are altered in hyperglycemic individuals with non-insulin-dependent diabetes mellitus (NIDDM). This was tested by estimating the kinetics of protein metabolism in obese NIDDM patients in the hyperglycemic state of isoenergetic feeding and in the normoglycemic state induced by the prolonged use of a very-low-energy diet (VLED) and comparing them with results in obese nondiabetic subjects studied previously. Seven obese subjects with NIDDM (one male, six females, body mass index = 35.8 ± 2.0 kg/m2) were given a 1.7 MJ (410 kcal) all protein (93 g/day) diet derived from hydrolyzed collagen and supplemented with tryptophan and methionine, which provides 16% of its amino acids as essential, a multivitamin and mineral supplement, and 16 mmol KCl for 42 days. During the seven-day isoenergetic diet and at weeks 4 and 6 of the VLED, amino nitrogen (N) flux rate was calculated from the urine [15N]urea enrichment by using the 60-h oral [15N]glycine method to obtain the integrated feeding-fasting metabolism. Rates of synthesis (S) and breakdown (B) were calculated from N flux. At day 7 of the isoenergetic diet, whole-body N flux, S, B, and resting metabolic rate (RMR) were 12–24% greater (P < 0.05) in the NIDDM subjects than observed in nondiabetic obese subjects. Mean plasma glucose decreased (P < 0.05) from the isoenergetic period (14.9 ± 2.4 mM) to 7.2 ± 1.2 mM at week 4 and 6.5 ± 1.1 mM at week 6 of the VLED. RMR declined progressively by 25% at week 5 of the VLED. Corresponding significant (P < 0.05) decreases from isoenergetic feeding to weeks 4 and 6 of the VLED occurred in whole-body N flux (from 51 ± 2 to 42 ± 1 g N/day), in S (from 38 ± 3 to 24 ± 1 g N/day), and in B (from 39 ± 3 to 26 ± 1 g N/day) resulting in net losses (S – B). S – B was significantly more negative (P < 0.05) in NIDDM than in the nondiabetic obese subjects at week 4 (−1.5 ± 0.5 vs. 0.9 ± 0.3 g N/day) but not at week 6 (−1.3 ± 0.4 vs. −0.9 ± 4 g N/day). During the VLED, N balance became less negative with time but never reached equilibrium in NIDDM. Thus, abnormal protein metabolism is present in NIDDM in the isoenergetic fed state with moderate hyperglycemia and persists during a VLED that restores glycemia to near normal.