bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis and Processing of the Human Insulin Receptor
Authors: Forsayeth John , Maddux Betty , Goldfine Ira D
Published: September 2013

We investigated the biosynthesis of the human insulin receptor in IM-9 lymphocytes and HEP-G2 hepatoma cells. Cells were first pulse labeled for 15 min with [35S]methionine and then chased for up to 4 h. At each time, the cells were solubilized in 1% Triton X-100; the insulin receptor was immunoprecipitated and then analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6%) and fluorography. At 15 min, a major precursor protein of 190,000 Mr was precipitated. During the chase period, two smaller proteins became apparent, which evolved into two major species of 130,000 and 95,000 Mr, the mature α- and β-subunits, respectively. When IM-9 cells were trypsinized after pulse chase, the α- and β-subunits were completely digested, whereas the 190,000-Mr precursor was unaffected. 125I-surface labeling of cells, followed by immunoprecipitation, revealed the presence of only the α- and β-subunits, indicating that only these two species were on the cell surface. To study this biosynthetic pathway, several inhibitors were used (tunicamycin, monensin, and swainsonine). These inhibitors revealed the following. The receptor is first synthesized as a 170,000-Mr protein that is cotranslationally N-glycosylated to yield a high-mannose 190,000-Mr precursor. This precursor is rapidly transported from the endoplasma reticulum to the Golgi apparatus where it is cleaved into two subunits of 120,000 Mr (α) and 90,000 Mr (β). These subunits then increase in molecular weight by processing of the high-mannose oligosac-charides to the low-mannose complex type. The two subunits then migrate to the cell surface where they function to transmit the insulin signal.
Transfer of Autoimmune Diabetes Mellitus with Splenocytes from Nonobese Diabetic (NOD) Mice
Authors: Wicker Linda S , Miller Beverly J , Mullen Yoko
Published: September 2013

The nonobese diabetic (NOD) mouse, a model of human type I diabetes, develops insulitis beginning at 4–6 wk of age. By 30 wk of age, 72% of females and 39% of males develop spontaneous diabetes, apparently because of an overwhelming autoimmune response to the insulin-producing β-cells within the islets. To identify the immune mechanism responsible for destruction of β-cells in the NOD mouse, we developed an adoptive transfer protocol that induces diabetes in NOD mice at an age when spontaneous diabetes is rarely observed.

Splenocytes from overtly diabetic NOD mice were unable to transfer diabetes to very young (≤6 wk) irradiated NOD mice but effectively transferred diabetes to irradiated NOD mice 6 wk of age. In such transfers, overt diabetes was induced within 12–22 days in >95% (79/82) of the recipients. Thus, transfer of splenocytes to young mice induces them to become diabetic at a higher frequency and at a younger age than their untreated littermates. Equally successful transfers with as few as 5 × 106 spleen cells have been performed in male and female NOD mice, even though males display a lower spontaneous incidence of diabetes than females. Splenocytes obtained from diabetic mice maintained on insulin for up to 2 mo also transferred diabetes.

Because NOD mice display increasing levels of insulitis with age, spleen cells obtained from nondiabetic NOD mice of different ages were tested for their ability to transfer diabetes. Spleen cells obtained from 7-wk-old nondiabetic donors were unable to transfer disease, suggesting that high numbers of effector cells are not yet present in the spleens of young NOD mice. In contrast, spleen cells obtained from most nondiabetic NOD mice >15 wk of age were able to transfer diabetes, indicating that the donor need not be overtly diabetic at the time of transfer.

Our study provides direct demonstration of the immunological nature of the diabetic disease process in the NOD mouse and provides a method to determine which cells are the effectors of β-cell destruction.
Development of a Predictive Model for Symptomatic Neuropathy in Diabetes
Authors: Weinberg C R , Pfeifer M A
Published: September 2013

In evaluating therapeutic interventions aimed at preventing diabetic neuropathy, choosing a suitable measure of neural function is difficult, partly because the relation between most available objective measures and the development of symptomatic neuropathy (SN) is unclear. Using data from 67 diabetic patients, we developed a linear logistic regression model to assess the relationship between SN and a set of neural measures, including dark-adapted pupil size; pupillary latency; heart rate; a measure of respiratory sinus arrhythmia (); the Valsalva ratio; and conduction velocities for the peroneal, median motor, and median sensory nerves. Models allowed for possible effects related to age, sex, duration and type of diabetes, glyco-sylated hemoglobin, and adiposity. Thirty-two of the patients reported SN (autonomie and/or sensorimotor). The best-fitting model for predicting the presence of any SN included only heart rate, duration of disease, and . Exclusion of duration (P < .01), or heart rate (P < .05), or (P < .001) significantly impaired the fit of the model.

To evaluate the temporally predictive power of the model, nine of the asymptomatic patients were reinter-viewed 2 yr later by the same interviewer, who was unaware of the results of the modeling. Four of five to whom the model had assigned high probability of symptoms had indeed developed SN during the follow-up period, whereas none of the four assigned low probability had developed SN (P < .05). Thus it seems that a measure of respiratory sinus arrhythmia provides an index of neural function strongly related to SN, and our follow-up data suggest that diminished can be used to predict the development of SN in diabetes.
Efficacy of Pulsatile Versus Continuous Insulin Administration on Hepatic Glucose Production and Glucose Utilization in Type I Diabetic Humans
Authors: Bratusch-Marrain P R , Komjati M , Waldhäusl W K
Published: September 2013

To evaluate the role of pulsatile insulin administration, hepatic glucose production (HGP) and utilization were studied in type I diabetic patients in the fasting state and during a euglycemic insulin (1 mU · kg−1 · min−1 i.v.) clamp with continuous and pulsatile insulin administration. In the latter study, insulin was infused at twice the continuous rate with 3-min-on/7-min-off intervals, thereby reducing total insulin delivery by 40%. The restraining effect of pulsatile insulin on basal HGP (1.91 ± 0.35 mg · kg−1 · min−1) was equipotent to continuous insulin exposure (1.80 ± 0.17 mg · kg−1 min−1). During the insulin-clamp studies, HGP was equally suppressed by pulsed (0.62 ± 0.12 mg · kg−1 min−1) as by continuous insulin infusion (0.63 ± 0.12 mg · kg−1 · min−1). Insulin-stimulated glucose utilization was not significantly altered in either study (2.55 ± 0.27 vs. 2.92 ± 0.23 mg · kg−1 min−1). When in further studies the total insulin dose given during the pulsatile study was infused continuously (0.6 mU · kg−1 · min−1), HGP in the basal state and residual HGP during the insulin-clamp study were 25–30% higher than in the pulsatile experiments, whereas glucose utilization was not significantly different.

In conclusion, by reducing total hormone delivery by up to 40%, but given in a pulsatile fashion, insulin is equally potent in controlling HGP as continuous insulin administration. This greater efficacy of pulsatile exposure in suppressing HGP is accompanied by an equipotent effect on glucose utilization. Application of pulsatile insulin substitution in intravenous-pump users may reduce systemic hyperinsulinemia and, in the long run, insulin resistance by reversing downregulation of insulin receptors.
Cyclosporin-Induced Inhibition of Insulin Secretion in Isolated Rat Islets and HIT Cells
Authors: Robertson R P
Published: September 2013

The effects of cyclosporin, an immunosuppressive agent used in diabetic and nondiabetic patients, on in vitro glucose-induced insulin secretion were evaluated in isolated islets and in a glucose-responsive clonal pcell line (HIT cells). Cyclosporin inhibited glucose-induced insulin secretion in a dose-response manner at concentrations commonly found in human blood. With isolated islets, four time periods (0–5, 5–15, 15–30, and 30–60 min) were examined after stimulation with 300 mg/dl glucose. Inhibition of insulin secretion by cyclosporin was evident by 5–15 min as well as during later times with progressively smaller drug concentrations. With HIT cells, longer-term effects were examined after 16 h of incubation with various drug concentrations. Inhibition of insulin secretion was again observed, and these inhibitory effects were not reversed by drug washout. A cyclosporin concentration of 0.1 μg/ml, which is a therapeutic blood level in humans, was sufficient to inhibit insulin secretion in both in vitro models. Patients using this drug should be carefully monitored for signs of deficient insulin secretion.
Direct Effects of Cyclosporin A on Human Pancreatic β-cells
Authors: Nielsen J H , Mandrup-Poulsen T , Nerup J
Published: September 2013

Cyclosporin A (CyA) may induce clinical remission in newly diagnosed insulin-dependent diabetes mellitus patients. Recently, however, adverse effects of high doses of CyA on rodent islets have been reported in vivo and in vitro. The possible direct effects of CyA on the human endocrine pancreas were therefore evaluated. Islets isolated from eight necrokidney donors were cultured in the presence of a therapeutically relevant dose of CyA, i.e., 100 ng/ml. During a 5-day culture period the release of insulin was reduced by 36% (range 7–61%), whereas the islets' content of insulin was increased by 59% (range 3–268%). The glucagon content was not affected. Cyclosporin G inhibited the insulin release, whereas dihydrocyclosporin D had no consistent effects. Glucose-stimulated insulin release from perifused islets was markedly depressed in CyA treated islets. This effect was not fully reversed 48 h after removal of the drug. We concluded that CyA has a direct inhibitory effect on insulin release from human pancreatic islets with a concomitant increase in the residual insulin content. If applicable to the in vivo condition, CyA may therefore, in addition to its immunosuppressive effect, have direct effects on the endocrine pancreas, which may be relevant for clinical application Of the drug.
Increased Susceptibility to Ischemic Damage in Steptozocin-Diabetic Nerve
Authors: Nukada H
Published: September 2013

Nerve ischemia, both acute and chronic, may contribute to diabetic neuropathies, but the pathogenesis remains obscure. The vulnerability of diabetic nerve to ischemia was assessed by ligating arteries of supply of rat sciatic nerve after 20 wk of streptozocin-induced diabetes. In the first 24 h after the arterial ligations, severe and more rapidly occurring clinicopathological abnormalities were invariably seen in diabetic nerves, but these findings were less severe in nondiabetic nerves. The results imply that peripheral nerve in diabetes mellitus is more subject to ischemia than normal nerve. This phenomenon may ensue from endoneurial hypoxia and play an important role in the development of diabetic neuropathies.
Effects of Weight Loss on Mechanisms of Hyperglycemia in Obese Non-Insulin-Dependent Diabetes Mellitus
Authors: Henry R R , Wallace P , Olefsky J M
Published: September 2013

To quantitate the effects of weight loss on the mechanisms responsible for hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM), eight obese subjects with NIDDM were studied before and after weight reduction with posttreatment assessment after 3 wks of isocaloric (weight maintenance) refeeding.

After weight loss of 16.8 ± 2.7 kg (mean ± SE), the fasting plasma glucose level decreased 55% from 277 ± 21 to 123 ± 8 mg/dl. The individual fasting glucose levels were highly correlated with the elevated basal rates of hepatic glucose output (HGO) (r = 0.91, P < .001), which fell from 138 ± 11 to 87 ± 5 mg · m2 · min1 after weight loss. The change in fasting plasma glucose also correlated significantly with the change in the basal rates of HGO (r = 0.74, P < .05). This was associated with reduced fasting serum levels of glucagon (from 229 ± 15 to 141 ± 12 pg/ml), reduced free fatty acids (from 791 ± 87 to 379 ± 35 μeq/L), and unchanged basal insulin levels (17 ± 4 to 15 ± 2 fill/ml).

Peripheral glucose disposal, assessed by the euglycemic glucose-clamp technique, at insulin infusion rates of 120 and 1200 mU · m2 · min1 increased between 135 and 165%, from 128 ± 17 to 288 ± 24 mg · m2 · min1 during the 120-mU · m2 · min1 studies and from 159 ± 19 to 318 ± 24 mg · m2 · min1 during the 1200-mU · m2 · min1 clamp studies, despite comparable steady-state serum insulin levels at each infusion rate before and after weight loss. HGO during the 120-mU · m2 · min1 clamp studies increased from 85% to complete (100%) suppression after treatment. Adipocyte size was reduced 44% (851 ± 91 to 475 ± 48 pi), whereas surface area decreased by 32% (4.30 × 104 to 2.92 × 104 μm2cell). Insulin binding to isolated adipocytes was unchanged, whereas basal in vitro rates of 3-O-methylglucose transport increased from 0.21 ± 0.13 to 0.53 ± 0.24 pmol/(2 × 109 μm2) × (10 s−1) and maximal glucose transport rates increased from 0.64 ± 0.29 to 1.18 ± 0.48 pmol/(2 × 105 cells) × (10 s−1) and 0.42 ± 0.20 to 1.04 ± 0.30 pmol/(2 × 109 μm2) × (10 s−1).

Finally, absolute serum insulin levels during oral glucose-tolerance tests and meal-tolerance tests were unchanged by weight reduction, whereas plasma glucose levels were markedly reduced.

We conclude that in obese NIDDM, weight loss results in improved glucose homeostasis with 1) reducedbasal HGO predominently responsible for the lowering of fasting glucose levels; 2) improved postprandial glucose excursions with marked amelioration of peripheral insulin resistance due to improved postreceptor insulin action, which is at least partly due to enhanced glucose transport system activity; and 3) unchanged absolute insulin levels in the face of markedly reduced glycemia, indicative of enhanced p-cell sensitivity to insulinogenic stimuli.
Trapped Immunoglobulins on Peripheral Nerve Myelin From Patients With Diabetes Mellitus
Authors: Brownlee M , Vlassara H , Cerami A
Published: September 2013

Diabetic peripheral neuropathy is characterized by endoneurial capillary closure and by segmental demyelination and axonal degeneration in a spatial pattern consistent with ischemic damage. The increased permeability of human diabetic endoneurial capillaries to plasma proteins may contribute to the pathogenesis of these structural changes in peripheral nerve by further accelerating the rate at which plasma proteins are trapped by reactive nonenzymatic glycosylation products on long-lived proteins such as myelin.

We have measured trapped immunoglobins (Ig)G and M on peripheral nerve myelin from diabetic and nondiabetic patients by an enzyme-linked immunosorbent assay to determine whether plasma proteins accumulate on nerves as they do in the glomerular matrix of diabetics. The amount of trapped IgG on brain myelin from these subjects was also determined.

Peripheral nerve myelin from diabetics had on average >14 times the amount of trapped IgM found in identically prepared samples from nondiabetics (0.90 ± 0.2 vs. 0.06 ± 0.004 OD/μ.g myelin protein) and >4 times the amount of trapped IgG (6.40 ± 1.92 vs. 1.5 ± 0.25 OD/μg myelin protein). In contrast, no significant trapping of IgG was detected in any samples of brain myelin. This most likely reflects effective exclusion of IgG by the blood-brain barrier.

These data suggest that excessive trapping of Igs and other plasma proteins by diabetic peripheral nerve myelin may contribute to the development of peripheral nerve damage, whereas the lack of such trapping by brain myelin may partly explain the rarity of diabetic central neuropathy.
Effect of Aldose Reductase Inhibitor ONO 2235 on Reduced Sympathetic Nervous System Activity and Peripheral Nerve Disorders in STZ-lnduced Diabetic Rats
Authors: Yoshida Toshihide , Nishioka Hitoshi , Yoshioka Keiji , Nakanoa Koji , Kondo Motoharu , Terashima Hiroshi
Published: September 2013

To test the hypothesis that inhibitors of aldose reductase, a key enzyme for polyol synthesis, prevent the decrease of sympathetic nervous system (SNS) activity and improve motor nerve conduction velocity (MNCV) through the reduction of sorbitol accumulation in streptozocin (STZ)-induced diabetic rats, norepinephrine (NE) turnover (reliable indicator of SNS activity in the interscapular brown adipose tissue (IBAT), heart, and pancreas), and MNCV were measured in untreated STZ-induced diabetic rats, STZ-induced diabetic rats treated with an aldose reductase inhibitor (ARI) (ONO 2235, 50 mg · kg−1 · day−1, orally), STZ-induced diabetic rats treated with insulin therapy, control rats, and control rats treated with ARI. As expected, results from studies with the inhibition of NE biosynthesis with α-methyl-p-tyrosine or radiolabeled NE to measure NE turnover demonstrated significant reductions in SNS activity in IBAT, heart, and pancreas of untreated STZinduced diabetic rats, compared with those in control rats. MNCV determined with the tail nerve was significantly reduced in untreated STZ-induced diabetic rats, compared with that of the controls. Both daily ARI treatment and insulin therapy in STZ-induced diabetic rats prevented partially but significantly the decrease of NE turnover in IBAT, heart, and pancreas, ameliorated MNCV, and reduced sorbitol accumulation in the nerve tissue and red blood cells. ARI treatment in control rats had no effect on NE turnover, MNCV, or sorbitol content.

These results suggest that STZ-induced diabetic rats had not only motor neuropathy but also sympathetic nervous dysfunction and that ARI treatment might prevent these as well as insulin therapy does through the reduction of sorbitol accumulation.
Identification and Characterization of Insulin Receptors in Basolateral Membranes of Dog Intestinal Mucosa
Authors: Gingerich Ronald L , Gilbert William R , Comens Patricia G , Gavin James R
Published: September 2013

Little is known about hormonal regulation of substrate transport and metabolism in the mucosal lining of the small intestine. Because insulin regulates these functions in other tissues by binding to its receptor, we have investigated the presence of insulin receptors in canine small intestinal mucosa with basolateral membranes (BLM) and brush border membranes (BBM) prepared by sorbitol density centrifugation. A14-[125l]iodoinsulin was used to study binding and structural characteristics of specific insulin receptors in BLM. Analysis of receptors in BLM identified binding sites with high affinity (Kd 88 pM) and low capacity (0.4 pmol/mg protein) as well as with low affinity (Kd 36 nM) and high capacity (4.7 pmol/mg protein). Binding was time, temperature, and pH dependent, and 125l-labeled insulin dissociation was enhanced in the presence of unlabeled insulin. Cross-reactivity of these receptors to proinsulin, IGF-II, and IGF-I was 4,1.8, and <1%, respectively. Covalent cross-linking of labeled insulin to BLM insulin receptors with disuccinimidyl suberate revealed a single 135,000-Mr band that was completely inhibited by unlabeled insulin. There was a 16-fold greater specific binding of insulin to BLM (39.0 ± 2.4%) than to BBM (2.5 ± 0.6%). These results demonstrate the presence of a highly specific receptor for insulin on the vascular, but not the luminal, surface of the small intestinal mucosa in dogs, and suggest that insulinmay play an important role in the regulation of gastrointestinal physiology.
Mechanism of Exercise-Induced Hypoglycemia During Sulfonylurea Treatment
Authors: Kemmer Friedrich W , Tacken Michael , Berger Michael
Published: September 2013

Exercise-induced hypoglycemia in diabetic patients on sulfonylurea treatment is not uncommon. However, its pathophysiology has not been examined. We studied 9 postabsorptive nondiabetic subjects after oral administration of 1.75 mg glyburide (glibenclamide) (protocol A), during60 min of leg exercise on a bicycle ergometer at a work load of 80 ± 10 W (protocol B), and during a combination of these conditions (protocol C). Serum glibenclamide levels rose to similar levels (160 ng/ml) with protocols A and C. Heart rate, blood pressure, and blood lactate levels increased immediately after onset of exercise and were comparable under conditions of protocols B and C. Serum insulin levels fell during protocol B from 6.1 ± 0.6 to 4.0 ± 0.3 μU/ml (P < .001) but increased from 6.5 ± 0.6 to 12.3 ± 2.8 μU/mlduring protocol A and from 6.6 ± 0.6 to 12.7 ± 4.3 μU/ml during protocol C, P < .001. Maximal levels were reached at 80 min under both conditions. Comparable responses were seen for serum C-peptide concentrations. Blood glucose concentrations did not change during exercise alone. Glycemia decreased markedly after administration of the drug reaching a nadir of 49 ± 3 mg/dl with protocol A and a nadir of 46 ± 3 mg/dl under protocol C. However, the nadir was reached 80 min after oral ingestion of the drug when exercise and glyburide were combined, compared to 110 min (P < .01) after ingestion of the drug alone. In conclusion, the hypoglycemic action of orally administered glyburide is enhanced by physical exercise in nondiabetic subjects, because exercise does not blunt the glyburide-stimulated insulin secretion. Because the same mechanism may be operative in normoglycemic type II diabetic patients on sulfonylureas, we suggest that such patients should be advised to reduce the dose of the drug before endurance exercise.
Physiological Role of Cholecystokinin in Meal-Induced Insulin Secretion in Conscious Rats: Studies With L 364718, A Specific Inhibitor of CCK-Receptor Binding
Authors: Rossetti Luciano , Shulman Gerald I , Zawalich Walter S
Published: September 2013

It has been suggested that the gut hormone cholecystokinin (CCK), by modulating insulin output from pancreatic β-cells, plays an important role in the enteroinsular axis. To investigate this hypothesis, eight rats were studied on two different occasions: after injection of L 364718, a specific antagonist of CCK binding to its membrane receptor, and after vehicle injection. In both studies a mixture of casein (11%) and glucose (9%) was infused through a chronic indwelling intraduodenal catheter to evoke CCK secretion. Plasma was analyzed for insulin, glucose, glucagon, and tyrosine many times during the procedure. Prior administration of the CCK antagonist significantly attenuated the increase in plasma insulin and glucagon after casein infusion. These results support the concept that cholecystokinin plays an important physiologic role in the in vivo regulation of postprandial plasma insulin and glucagon concentrations after protein ingestion.
Different Effects of Glyburide and Glipizide on Insulin Secretion and Hepatic Glucose Production in Normal and NIDDM Subjects
Authors: Groop Leif , Luzi Livio , Melander Arne , Groop Perc-Henrik , Ratheiser Klaus , Simonson Donald C , DeFronzo Ralph A
Published: September 2013

Glyburide (GB) and glipizide (GZ) differ in their pharmacokinetics, but it is not known whether they also differ in mode of action. To examine this question, 10 young healthy subjects and 6 non-insulindependent diabetic (NIDDM) patients participated in each of three studies: 1) infusion of saline for 120 min followed by a 100-min hyperglycemic (125 mg/dl) clamp; 2) 120-min primed continuous infusion of GZ followed by a 100-min hyperglycemic clamp; and 3) 120-min primed continuous infusion of GB followed by a 100-min hyperglycemic clamp. The GB and GZ infusions were continued throughout the hyperglycemic clamp. Similar plasma concentrations of GB and GZ were obtained in both groups. All studies were performed with [3-3H]glucose to allow quantification of hepatic glucose production. When administered under basal conditions of glycemia, the acute phase (0-10 min) of plasma insulin and C-peptide increase in both control and NIDDM subjects was twice as great with GZ compared with GB (P < .01). During the hyperglycemic-clamp studies performed in normal subjects, both GB and GZ increased the first- (1.6-fold) and second- (2.2-fold) phase plasma insulin responses more than hyperglycemia alone. During the hyperglycemic clamp in NIDDM subjects, the first-phase plasma insulin response was absent, and the second-phase insulin response was markedly impaired. Neither GB nor GZ improved first-phase insulin secretion in the NIDDM patients. In both NIDDM and control subjects, the effects of hyperglycemia and sulfonylurea drugs (both GB and GZ) on the first- and second-phase plasma insulin responses were simply additive. These results challenge the concept that sulfonylurea agents, when given acutely, potentiate glucose-stimulated insulin secretion. In NIDDM patients, GB decreased basal hepatic glucose production (2.30 ± 0.30 mg · kg−1 · min−1) more effectively than GZ (to 0.40 ± 0.12 vs. 0.82 ± 0.11 mg · kg−1 · min−1 P < .01). These results indicate that the acute administration of GB and GZ to NIDDM subjects has quantitatively different results on hepatic glucose production and acute plasma insulin response to hyperglycemia.
Role of Lipid Oxidation in Pathogenesis of Insulin Resistance of Obesity and Type II Diabetes
Authors: Felber Jean-Pierre , Ferrannini Eleuterio , Golay Alain , Meyer Hans U , Theibaud Daniel , Curchod Bernard , Maeder Evelyn , Jequier Eric , DeFronzo Ralph A
Published: September 2013

Increased lipid oxidation is generally observed in subjects with obesity and diabetes and has been suggested to be responsible for the insulin resistance associated with these conditions. We measured, by continuous indirect calorimetry, lipid and glucose oxidation and nonoxidative glucose disposal in 82 obese subjects during a 100-g oral glucose tolerance test (OGTT) and in 26 during a euglycemic insulin (40 mU · min−1 · m−2) clamp. The obese subjects were subdivided into those with normal glucose tolerance (group A), those with impaired glucose tolerance (group B), and those with overt diabetes (group C). Forty-five healthy nonobese subjects were subdivided into a young and an older control group, which were age-matched to the nondiabetic obese (groups A and B) and diabetic obese (group C) subjects, respectively. In the postabsorptive state, as well as in response to insulin stimulation (both OGTT and insulin clamp), lipid oxidation was significantly increased in all three obese groups in comparison with either young or older controls. Basal glucose oxidation was significantly decreased in obese nondiabetic and obese glucose— intolerant subjects (groups A and B) compared with age-matched controls. During the OGTT and during the insulin clamp, insulin-stimulated glucose oxidation was decreased in all three obese groups. In contrast, nonoxidative glucose disposal was markedly inhibited in nondiabetic and diabetic obese patients during the euglycemic insulin clamp but not during the OGTT. After glucose ingestion, nonoxidative glucose uptake was normal in nondiabetic obese and glucoseintolerant obese subjects and decreased in diabetic obese subjects. Statistical analysis revealed that lipid and glucose oxidation were strongly and inversely related in the basal state, during euglycemic insulin clamp, and during OGTT. The negative correlation between lipid oxidation and nonoxidative glucose uptake, although significant, was much weaker. Fasting and post-OGTT hyperglycemia were the strongest (negative) correlates of nonoxidative glucose disposal in both single and multiple regresson models. We conclude that 7) reduced glucose oxidation and reduced nonoxidative glucose disposal partake of the insulin resistance of nondiabetic obese and diabetic obese individuals; 2) hyperglycemia provides a compensatory mechanism for the defect in nonoxidative glucose disposal in nondiabetic obese subjects; however, this compensation is characteristically lost when overt diabetes ensues; and 3) increased lipid oxidation may contribute, in part, to the defects in glucose oxidation and nonoxidative glucose uptake in obesity.
Effect of Physical Training on Insulin Action in Obesity
Authors: DeFronzo Ralph A , Sherwin Robert S , Kraemer Nancy
Published: September 2013

We evaluated insulin secretion and insulin action with the hyperglycemic (125 mg/dl above basal) and euglycemic insulin (40 mU · m2 · min−1) clamps in seven moderately obese subjects before and after a 6-wk exercise training program. Thirty-nine normalweight, age-matched subjects served as controls. Both fasting plasma insulin concentration and glucosestimulated (hyperglycemic clamp) insulin secretion were significantly (P < .001) increased in the obese subjects. After the training program fasting insulin levels decreased by 26% (P < .01). Insulin secretion in response to hyperglycemia decreased by a similar percentage (P < .01). Nonetheless, total-body glucose metabolism increased significantly (P < .05) during the hyperglycemic clamp. With the euglycemic insulin clamp, obese subjects were shown to be significantly (P < .001) insulin resistant compared with controls. The decrease in total-body glucose uptake resulted from defects in both peripheral glucose disposal and suppression of hepatic glucose production. After the 6-wk training program, insulin-mediated total-body glucose metabolism increased due to significant improvements in peripheral glucose uptake (P < .01) and more effective suppression of hepatic glucose production (P < .05). These results indicate that a moderate-intensity physical conditioning program is capable of ameliorating the insulin resistance and reducing glucose-stimulated hyperinsulinemia observed in obese subjects with normal glucose tolerance.
Tissue Culture of Human Fetal Pancreas: Effects of Human Serum on Development and Endocrine Function of Isletlike Cell Clusters
Authors: Sandler Stellan , Andersson Arne , Landström Annika Schnell , Tollemar Jan , Borg Håkan , Petersson Birger , Groth Carl-Gustav , Hellerström Claes
Published: September 2013

The human fetal pancreas represents a source of insulin-producing β-cells with a potential for transplantation to diabetic patients. It has previously been shown that such cells can be viably maintained in tissue culture media containing fetal calf serum (FCS) and that these explants continue to synthesize and release insulin. In this study the effects of human serum (HS) on the growth and function of human fetal pancreatic explants have been compared with those of FCS. For this purpose, pancreatic glands, obtained after prostaglandin-induced abortions, were briefly exposed to collagenase, and the digest was cultured in RPMI-1640 medium plus 10% pooled HS or FCS. The outgrowth of isletlike cell clusters (ICCs) was monitored. In 31 of 58 consecutively explanted glands, development of ICCs was observed. In the presence of FCS the outgrowth of ICC took place on top of a fibroblast monocellular cell layer; HS effected less growth of fibroblasts and increased the formation of ICCs about sevenfold compared with explants from the same glands maintained in FCS. However, in the explant cultures with HS, the cell number per ICC, expressed as DNA content, was reduced by 50%. In both FCS and HS the insulin content of the medium showed great variability and progressively declined from day 2 to day 5. The medium glucagon concentration also decreased but not to the same extent as that of insulin. Immunocytochemical-stained ICCs showed insulin- and glucagon-positive cells scattered among most nonstained, presumably nonendocrine cells. The ultrastructure of the cultured cells was well preserved, with frequent granulated cells, some of which were identified as β-cells. Measurements of [3H]thymidine incorporation into ICCs indicated a high rate of DNA replication. ICCs maintained in HS showed significant rates of insulin biosynthesis and release. We conclude that in comparison with explants cultured with FCS, the yield of ICCs is markedly increased by HS. The size and geometry of these ICCs may make them particularly suited for transplantation.
Stable Insulin Preparation for Implanted Insulin Pumps: Laboratory and Animal Trials
Authors: Grau Ulrich , Saudek Christopher D
Published: September 2013

The stability and longevity of the polyethylenepolypropylene glycol-stabilized insulin have been tested in vitro and in vivo in an implanted insulininfusion device, the programmable implantable medication system (PIMS). In vitro tests demonstrated long-term compatibility with refill cycles of up to 3 mo, with a preparation of 400 U/ml. Total test period in vitro was 3.2 device-yr (combined time of device use). Insulin retained 88-93% native structure. A major modification, which was biologically active and nonimmunogenic, was identified and partially characterized. Examination of one device by scanning electron microscopy and X-ray microanalysis after 1 yr of insulin infusion revealed surfaces clean of insulin precipitate or other material along the entire insulindelivery pathway. Surface analysis of the silicone-lined polyethylene catheters after 6 mo of infusion also showed no evidence of major insulin precipitate. In vivo stability trials were accomplished with PIMS implanted in diabetic dogs with an intraperitoneal delivery site. There has been no insulin blockage of the catheter of active pumps after 5.1 dog-yr (combined time of trials) of trials (up to 5 mo between refills in a single dog). Structural stability of insulin was analyzed by high-performance liquid chromatography. On average, 90.8% of the insulin sampled from the reservoir in vivo was native insulin, with an average of 96.2% retention of active forms.
Effect of Glyburide on Glycemic Control, Insulin Requirement, and Glucose Metabolism in Insulin-Treated Diabetic Patients
Authors: Simonson Donald C , Delprato Stefano , Castellino Pietro , Groop Leif , DeFronzo Ralph A
Published: September 2013

Glycemic control and glucose metabolism were examined in 5 patients with insulin-dependent diabetes mellitus (IDDM) and 8 insulin-treated non-insulin-dependent diabetes mellitus (NIDDM) patients before and after 2 mo of therapy with glyburide (20 mg/day). Glycemic control was assessed by daily insulin requirement, 24-h plasma glucose profile, glucosuria, and glycosylated hemoglobin. Insulin secretion was evaluated by glucagon stimulation of C-peptide secretion, and insulin sensitivity was determined by a two-step euglycemic insulin clamp (1 and 10 mU ·kg−1 · min−1) performed with indirect calorimetry and [3−3H]glucose.

In the IDDM patients, the addition of glyburide produced no change in daily insulin dose (54 ± 8 vs. 53 ± 7 U/day), mean 24-h glucose level (177 ± 20 vs. 174 ± 29 mg/dl), glucosuria (20 ± 6 vs. 35 ± 12 g/day) or glycosylated hemoglobin (10.1 ± 1.0 vs. 9.5 ± 0.7%). Furthermore, there was no improvement in basal hepatic glucose production (2.1 ± 0.2 vs. 2.4 ± 0.1 mg · kg−1 · min−1), suppression of hepatic glucose production by low- and high-dose insulin infusion, or in any measure of total, oxidative, or nonoxidative glucose metabolism in the basal state or during insulin infusion. C-peptide levels were undetectable (<0.01 pmol/ml) in the basal state and after glucagon infusion and remained undetectable after glyburide therapy.

In contrast to the IDDM patients, the insulin-treated NIDDM subjects exhibited significant reductions in daily insulin requirement (72 ± 6 vs. 58 ± 9 U/day), mean 24-h plasma glucose concentration (153 ± 10 vs. 131 ± 5 mg/dl), glucosuria (14 ± 5 vs. 4 ± 1 g/day), and glycosylated hemoglobin (10.3 ± 0.7 vs. 8.0 ± 0.4 %) after glyburide treatment (all P ≤ .05). However, there was no change in basal hepatic glucose produc-tion (1.7 ± 0.1 vs. 1.7 ± 0.1 mg kg−1 min−1), suppression of hepatic glucose production by insulin, or insulin sensitivity during the two-step insulin-clamp study. Both basal (0.14 ± 0.05 vs. 0.32 ± 0.05 pmol/ ml, P < .05) and glucagon-stimulated (0.24 ± 0.07 vs. 0.44 ± 0.09 pmol/ml) C-peptide levels rose after 2 mo of glyburide therapy and both were correlated with the decrease in insulin requirement (basal: r = .65, P = .08; glucagon stimulated: r = .93, P < .001).

These data indicate that in IDDM subjects, the addition of glyburide to insulin does not affect insulin requirement, glycemic control, or insulin sensitivity. In contrast, in insulin-treatedNIDDM patients, glyburide produces a modest decrease in insulin dose and improves glycemic control without altering insulin sensitivity. This improvement in glucose metabolism primarily reflectsan increase in endogenous insulin secretion.
Plasma Insulin Response Among Nauruans: Prediction of Deterioration in Glucose Tolerance Over 6 Yr
Authors: Sicree Richard A , Zimmet Paul Z , King Hilary O M , Coventry Janice S
Published: September 2013

A longitudinal study of 266 randomly selected nondiabetic Nauruans [215 with normal tolerance and 51 with impaired glucose tolerance (IGT)] over 6 yr showed that deterioration in glucose tolerance status had occurred in 61 subjects. Of the subjects with initially normal tolerance, 34 (16%) progressed to IGT and 14 (6.5%) progressed to diabetes. Thirteen of the subjects with IGT (25%) progressed to diabetes.

Subjects were examined in 1975 through 1976, and follow-up examinations were performed in 1982. After age, a high 2-h plasma insulin response to a glucose load was the factor most predictiveof progression from normal tolerance to both diabetes (P < .001) and IGT (P < .01). Both a high 2-h glucose level and greater obesity independently predicted progression from IGT, and a diminished 2-h insulin response just failed to significantly improve the model (P < .06). The negative parameter of the insulin response associated with deterioration from IGT differed significantly (P < .01) from the positive-parameter estimate of the response associated with progression to diabetes from normal tolerance (P < .01), implying a qualitative difference between these nondiabetic subgroups. The use of a glucose-insulin interaction term to predict (P < .01) progression to diabetes for all nondiabetic subjects confirmed this difference; this term's addition improved the model (P < .01), and progression to diabetes was associated with a high insulin response for 2-h glucose <7.8 mM but a low response for 2-h glucose >7.8 mM.
Effects of Experimental Diabetes, Uremia, and Malnutrition on Wound Healing
Authors: Yue D K , McLennan S , Marsh M , Mai Y W , Spaliviero J , Delbridge L , Reeve T , Turtle J R
Published: September 2013

The strength of linear wounds was studied in normal and diabetic rats in the first 8 wk after wounding. The strength of wounds from diabetic animals was found to be reduced compared with normal controls but could be improved by insulin treatment, especially when excellent metabolic control was achieved. There appeared to be both quantitative and qualitative defects in the formation of wound tissues in diabetic animals, because wound strength was not normalized when the thinner skin of diabetic animals was takeninto consideration. This was different from the findings in rats with renal failure or malnutrition: in these two conditions, wound strength appeared reduced but was normalized when adjusted for skin thickness. Increased activity of aldose reductase did not appear to be an important factor in the impairment of wound healing in diabetes, because wound strength was not corrected by treatment with sorbinil, an aldose reductase inhibitor. The precise mechanism of abnormal wound strength in diabetes remains to be studied further, but careful control of diabetes, maintenance of nutrition, and treatment of systemic illness are important factors in the promotion of wound healing.
Urine C-Peptide As Index of Integrated Insulin Secretion in Hypocalpric States in Obese Human Subjects
Authors: Yale Jean-François , Leiter Lawrence A , Marliss Errol B
Published: September 2013

To determine the effects of different hypocaloric diets on insulin secretion, 24-h urine C-peptide was measured in 11 obese subjects on a weight-maintaining baseline diet, and the results were compared with values obtained during 14-day periods of diets containing 400 kcal/day of only protein (n = 6) or glucose (n = 5), followed by 14 days of fasting and 14 days of refeeding on 800-1000 kcal/day. A significant positive correlation between total caloric intake and urine C-peptide excretion was found once the C-peptide excretion reached steady state after several days on each diet. Multiple regression analysis showed no contribution of body weight to urine C-peptide during the different diets. In contrast, a significant correlation was found between body weight and urine C-peptide in the fasting state. A marked and identical decrease of ∼75% in urine C-peptide occurred over the first 5-7 days of the two 400-kcal diets, followed by a further decrease during fasting to 5% of baseline values. Refeeding was associated with a progressive increase. Plasma insulin and C-peptide followed the same trends as found for urine C-peptide, although the magnitude of change was much smaller. C-peptide clearance was not assessed because of the variation in plasma levels on eating meals. However, the same responses were found when C-peptide excretion was factored for creatinine excretion. Thus, the major determinant of urine C-peptide excretion appears to be food intake, and adaptations take 5-7 days to reach steady state. This probably accounts for the significant correlation between body mass and urine C-peptide seen only afterfasting, when the effect of ingested secretagogues is removed. Glucose andprotein appear to be equipotent insulin secretagogues in the hypocaloric diets studied.
Tolrestat, an Aldose Reductase Inhibitor, Prevents Nerve Dysfunction in Conscious Diabetic Rats
Authors: Notvest Ronald R , Inserra John J
Published: September 2013

The effect of 4 wk of streptozocin (STZ)-induced diabetes on the transmission time of the auditory-evoked brain stem response (ABR) was examined in conscious male Sprague-Dawley rats. Distal nerve transmission time of the auditory pathway (latency of peak II), which includes conduction along the 8th cranial nerve, increased in diabetic rats (n = 9) relative to nondiabetic rats (n = 17). The difference in peak II latency between diabetic and control rats was significant beginning 2 wk after the induction of diabetes (P < .05). In contrast, 4 wk of STZ-induced diabetes had no effect on the central transmission time of the auditory pathway (interpeak latency between peaks II and IV). Oral administration of tolrestat, a structurally novel aldose reductase inhibitor (n = 8; 20 mg/kg twice daily 1 wk before and 4 wk after STZ injection), prevented the diabetes-induced increase in distal nerve transmission time. These findings indicate that experimentally induced diabetes can result in a nerve dysfunction as measured by the increased latencies of the early components of the ABR. Furthermore, because tolrestat prevents these changes in the ABR, aldose reductase and the polyol pathway are implicated in this neuropathy.
Insulin-Stimulated Glucose Transport and Insulin Internalization Share a Common Postbinding Step in Adipocytes
Authors: Jochen Albert L , Berhanu Paulos
Published: September 2013

We recently demonstrated that chymotrypsin substrate analogues inhibit receptor-mediated insulin internalization in isolated rat adipocytes. In this study, the effect on glucose transport of inhibiting insulin internalization with these agents was examined. Glucose transport was assayed by measuring [3H]-2-deoxyglucose uptake, and internalized insulin was measured after rapidly dissociating surface-bound insulin with an acidic buffer. The chymotrypsin substrate analogue N-acetyl-Tyr ethyl ester inhibited insulin internalization by 85% while increasing surface-bound insulin by 80-110%. Under these conditions, ATP levels were minimally altered, and basal glucose transport was unchanged; however, insulin-stimulated glucose transport was decreased by 86%. The inhibition of insulinstimulated glucose transport was not overcome by supramaximal concentrations (400 ng/ml) of insulin. When insulin internalization and insulin-stimulated glucose transport were measured in the presence of increasing concentrations of N-acetyl-Tyr ethyl ester (0.1-1 mM), a strong and highly significant correlation (r = .97, P < .001) was found between inhibition of insulin internalization and inhibition of insulin-stimulated glucose uptake. Fragments of N-acetyl-Tyr ethyl ester that do not inhibit insulin internalization were also without effect on insulin-stimulated glucose transport. In addition to N-acetyl-Tyr ethyl ester, four other chymotrypsin substrate analogues that are effective inhibitors of insulin internalization also markedly inhibited insulin-stimulated glucose transport. These results indicate that insulin internalization and insulin-stimulated glucose transport share a common postbinding step in adipocytes and that this step is inhibitable by chymotrypsin substrate analogues.
Direct and Indirect Effects of Insulin to Inhibit Hepatic Glucose Output in Obese Subjects
Authors: Prager Rudolf , Wallace Penny , Olefsky Jerrold M
Published: September 2013

The effects of small increases in plasma insulin on hepatic glucose production are incompletely understood. To partially elucidate this issue we have studied seven obese subjects with the euglycemic clamp technique with a low-dose insulin infusion rate of 15 mU · m−2 · min−1 over 3 h. Basal insulin levels were 24 ± 7 μU/ml and increased to steady-state levels of 35 ± 3 μU/ml during insulin infusion. Endogenous insulin secretion, quantitated by C-peptide measurements, decreased by 58% of the basal value after peripheral insulin infusion. Based on C-peptide measurements and the contribution of the peripheral insulin infusion to the circulating insulin concentrations, calculated portal insulin levels either decreased or remained unchanged during the clamp studies. Basal glucagon levels were 165 ± 18 and did not change during the insulin infusion. The basal glucose disposal rate was 86 ± 2 mg · m−2 · min−1 and did not increase significantly during the clamp studies. In contrast, hepatic glucose output (HGO) was suppressed by 82 ± 5% of the basal value. In summary, in a group of insulin-resistant obese subjects, glucose-clamp studies were performed at peripheral insulin levels of 35 ± 3 μU/ml; glucose disposal did not increase, whereas HGO was suppressed by 82%. At the same time, glucagon levels remained constant and estimated portal insulin levels either decreased or remained unchanged. These findings suggest that insulin can suppress HGO through indirect extrahepatic actions.