bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Type-Specific Variability of Bacitracin's Effects on Insulin Binding and Intracellular Accumulation
Authors: Gansler Ted S , Smith Robert M , Jarett Leonard
Published: September 2013

Bacitracin is known to inhibit proteolytic degradation of insulin and several other peptide hormones. Previous work with isolated rat adipocytes showed that bacitracin blocked insulin degradation by the plasma membrane and, even in the absence of detectable insulin degradation, bacitracin increased insulin binding by decreasing the rate of insulin dissociation. The present study examined the effects of bacitracin on insulin binding and degradation and on levels of intracellular insulin in a variety of cell types. Bacitracin inhibited insulin degradation in all cell types. Maximal inhibition varied from 70% (H4IIEC3 hepatoma cells) to 95% (rat adipocytes); concentrations giving half-maximal inhibition varied from 25 μM (3T3-A31 fibroblasts) to 250 μM (H4IIEC3). Dose-response curves showed three distinctive effects on insulin binding: dose-dependent stimulation (rat adipocytes), a biphasic curve with slight stimulation at low doses and inhibition at concentrations >50 μM (human fibroblasts, H4IIEC3, and 3T3-L1 adipocytes), or dose-dependent inhibition of binding (3T3-L1 preadipocytes and 3T3-A31 fibroblasts). The intracellular accumulation of insulin rat adipocytes was not affected by bacitracin but was decreased in all other cell types. These data illustrated type-specific variability in the effects of bacitracin on insulin processing resulting from cellular heterogeneity either in processing insulin or in response to bacitracin, or both, and suggest that insulin binding studies performed in the presence of bacitracin can be biased.
Tris(hydroxymethyl)aminomethane Inhibits the Synthesis and Processing of Proinsulin in Isolated Rat Pancreatic Islets Without Affecting Release of Insulin Stores
Authors: Halban Philippe A , Amherdt Mylene , Orci Lelio , Renold Albert E
Published: September 2013

Isolated rat islets of Langerhans were pulse-labeled (5 min, [3H]leucine) and then exposed to 10 or 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 during an 85-min chase period. There was a dose-related inhibition of the conversion of labeled proinsulin to insulin by Tris. At 50 mM, Tris also inhibited the release of newly synthesized (labeled) proinsulin and insulin. These inhibitory effects of Tris were almost absent if the islets were exposed to 50 mM Tris during only the last 60 min of the 85-min chase period. Both proinsulin and total islet protein synthesis (as indexed by incorporation of [3H]leucine) were inhibited acutely by 50 mM Tris (5-min exposure); after 85 min of exposure to 50 mM Tris, the inhibition of proinsulin biosynthesis was more marked than that of total islet protein. In contrast to its effects on newly synthesized products, 50 mM Tris failed to inhibit the release of immunoreactive insulin during an 85-min incubation. However, when islets were exposed to 50 mM Tris for a longer period, a partial inhibition of immunoreactive insulin release was observed as from 120 min. Insulin released from islets consists of a mixture of older stored material and of newly synthesized products, the latter being released preferentially. These results are consistent with a selective effect of 50 mM Tris on the production of newly synthesized insulin. During the first 120 min of exposure to Tris, islet reserves of newly synthesized products will be depleted thereby leading to a new, reduced, rate of release of immunoreactive material consisting only of older insulin stores. To confirm that Tris neither inhibited the release of stored insulin nor exerted any nonspecific toxic effects on B-cells during prolonged exposure, islets were labeled with [3H]leucine in the absence of Tris for 3 days. The fate of the prelabeled insulin stores was then followed during a 24-h chase period in the presence of 50 mM Tris. The release of the labeled, stored insulin was not affected by Tris, whereas the release of immunoreactive insulin (consisting of both new and old material) was partially inhibited as expected.

We conclude that Tris inhibits proinsulin synthesis and processing. The inhibition of newly synthesized proinsulin release reflects perturbation of intracellular events before exocytosis, which, itself, is not affected by TriS.
Streptococcal Preparation (OK-432) Inhibits Development of Type I Diabetes in NOD Mice
Authors: Toyota Takayoshi , Satoh Jo , Oya Keiichiro , Shintani Shigeki , Okano Tsuyoshi
Published: September 2013

OK-432 (a streptococcal preparation) has been widely used for cancer immunotherapy in Japan. It is the most potent immunomodulator in activating both macrophages and killer T cells and in increasing interleukin 2 production. Two K.E. (Klinische Einheit, clinical unit) of OK-432 were given intraperitoneally to each of 17 female nonobese diabetic (NOD) mice every week from 4–24 wk of age. NOD mice as well as BB rats spontaneously develop type I diabetes. During administration of OK-432, the development of diabetes was inhibited in 17 of 17 mice over the 24-wk observation period, whereas 14 of 17 female NOD mice given physiological saline had developed diabetes by 24 wk of age. At the onset of diabetes, nonfasting blood glucose was 511 ± 8 2 mg/dl. Histologic examination showed that in the OK-432-treated NOD mice, 98% of total islets were intact or mildly infiltrated with mononuclear cells, whereas in saline-treated NOD mice, 79% of total islets exhibited severe insulitis. In OK-432-treated NOD mice, both the number of the mononuclear spleen cells and their natural killer cell activity was significantly increased.
Effect of Insulin and Oral Glutathione on Glutathione Levels and Superoxide Dismutase Activities in Organs of Rats With Streptozocin-Induced Diabetes
Authors: Loven Dean , Schedl Harold , Wilson Helen , Daabees Tahia T , Stegink Lewis D , Diekus Mary , Oberley Larry
Published: September 2013

The effect of insulin or glutathione treatment on glutathione content of liver and jejunal mucosa and on superoxide dismutase (SOD) activity of liver, kidney, and erythrocytes was investigated in pair-fed animals with streptozocin (STZ)-induced diabetes. Diabetes lowered hepatic glutathione concentration, but glutathione concentration of the jejunal mucosa was not affected. Insulin, but not oral glutathione, restored hepatic glutathione concentration to normal levels. Diabetes depressed activity of the cytosolic form of SOD in liver, kidney, and erythrocyte. Treatment of diabetic rats with oral glutathione or intramuscular insulin increased cytosolic SOD activity of renal cortex and liver (but not erythrocytes) to control levels. These results suggest a link between glutathione metabolism and cytosolic SOD activity in diabetes.
Mitogen Responsiveness of Lymphocytes From the BB/W Rat
Authors: Woda Bruce A , Padden Carolyn
Published: September 2013

The response of BB diabetes-prone (DP) and W-line non-diabetes-prone rats to the T-cell mitogen concanavalin A (ConA) was measured. The W line was a good responder to ConA, whereas the DP was relatively unresponsive. This unresponsiveness could not be reversed with exogenous interleukin 2 (IL-2). The response of DP rats was enhanced by removing adherent cells. To directly test the response of BB T-cells, they were isolated by flow sorting. These experiments demonstrated that BB T-cells could mount a normal ConA response. The normal function of isolated BB T-cells suggested that they were under suppression. Suppressor activity could not be found in the 0X8+ population but was found in the DP-adherent cell population. Adherent cells from the W line were not suppressive at the concentrations used. These results showed that the decreased mitogen responsiveness of BB T-cells was not due to an intrinsic T-cell abnormality but was due, in part, to suppression by adherent cells.
Immunochemical Characterization of Anti-Islet Cell Surface Monoclonal Antibody From Nonobese Diabetic Mice
Authors: Hari Joji , Yokono Koichi , Yonezawa Kazuyoshi , Amano Kazuhiko , Yaso Shinji , Shii Kozui , Imamura Yoshimichi , Baba Shigeaki
Published: September 2013

We have produced a monoclonal antibody 3A4 to the surface of islet cells by fusing spleen lymphocytes from nonobese diabetic (NOD) mice. To identify the molecular weight of specific target antigens reacting with 3A4, 125I-surface-labeled In-111 insulinoma cells were solubilized and extracts were absorbed with 3A4, and immunoprecipitates were followed by polyacrylamide gel electrophoresis and autoradiography. 3A4 recognized two major polypeptides with apparent molecular weights of radioactive 64K and inactive 28K. In order to evaluate the antibody-mediated cytotoxic mechanisms of 3A4, complement-dependent antibodymediated cytotoxicity (C'-AMC) and antibody-dependent cellular cytotoxicity (ADCC) were tested using a method of specific 51Cr release. In the study for C'- AMC, even over wide ranges of concentration of antibody and rabbit complement, purified 3A4 had no apparent cytotoxic effects on ln-111 cells. On the other hand, significant ADCC was observed at an antibody concentration of 10 μg/ml and a target:effector cell ratio of 1 40 (P < 0.01). Finally, the effects of 3A4 on glucose-stimulated insulin release were examined in isolated rat islets. At a glucose concentration of 16.7 mM, 3A4 significantly inhibited the insulin release either in the presence or absence of complement (P < 0.01).

In conclusion, 3A4 could not only bind but also be active to the target cells. Therefore, this monoclonal antibody should be a useful tool to permit a detailed analysis of the pathogenesis of type I diabetes mellitus.
Desferrioxamine Treatment Prevents Chronic Islet Allograft Damage
Authors: Bradley Brenda , Prowse Stephen J , Bauling Paul , Lafferty Kevin J
Published: September 2013

BALB/cByJ islet allografts are acutely rejected when transplanted into allogeneic mice (CBA/J). Culture of the tissue for 7 days in 95% O2 before grafting is a suboptimal treatment for the reduction of immunogenicity in this strain combination. Approximately half the animals reject these transplants in a chronic fashion. Chronic islet rejection differs from acute rejection of uncultured allogeneic islets. During chronic rejection, beta cells within the transplanted tissue degranulate but remain intact when the animal returns to the diabetic condition. Acute islet rejection is characterized by the destruction of beta cells that remain heavily granulated as long as they remain intact. We examined the effect of the iron chelating agent, desferrioxamine, on chronic islet allograft damage. Desferrioxamine inhibited chronic islet allograft damage but did not influence the process of rejection of uncultured islet tissue. This effect of desferrioxamine could not be attributed to a direct immunosuppressive effect of this agent.
Comparison of Glucose Counterregulation During Short-Term and Prolonged Hypoglycemia in Normal Humans
Authors: Feo Pierpaolo De , Perriello Gabriele , Cosmo Salvatorec De , Ventura Mariarosa M , Campbell Peter J , Brunetti Paolo , Gerich John E , Bolli Geremia B
Published: September 2013

To compare glucose counter regulatory mechanisms during short-term hypoglycemia and prolonged hypoglycemia, insulin was infused either intravenously (160 mil · M−2 · min) for 10 min or subcutaneously (15 mU · M−2 · min) for 12 h in normal volunteers. With each type of insulin infusion, hypoglycemia (∼50 mg/dl) was either allowed to develop or was prevented (control experiments) by the glucose-clamp technique. During prolonged hypoglycemia, both increased glucose production (1.55 ± 0.05 versus 0.33 ± 0.14 mg · kg−1 · min in control experiments at 12 h, P < 0.01) and suppressed glucose utilization (1.55 ± 0.06 versus 3.17 ± 0.15 mg · kg−1 · min in control studies at 12 h, P < 0.01) were involved in counterregulation. During short-term hypoglycemia, only increased glucose production (3.23 ± 0.33 versus 0.06 ± 0.03 mg · kg−1 · min in control experiments at 60 min) was involved, since glucose clearance actually increased (3.99 ± 0.20 versus 2.88 ± 0.02 ml · kg−1 · min in control experiments at 60 min, P < 0.01). Estimated portal venous insulin concentrations decreased 40% (basal 24 ± 3 versus 14 ± 1 mU/ml at 60 min, P < 0.01) in the short-term hypoglycemia experiments but remained at basal levels (basal 25 ± 1 versus ∼26 μU/min between 1 and 12 h) during prolonged hypoglycemia. Despite the fact that hypoglycemia was more gradually induced in the prolonged hypoglycemia model, peak counterregulatory hormone responses were at least as great as those during short-term hypoglycemia. Plasma free fatty acids and ketone bodies increased 150–200% above basal (both P < 0.01) with counterregulation during prolonged hypoglycemia but did not increase above basal levels with counterregulation during short-term hypoglycemia. Finally, plasma alanine remained unchanged during short-term hypoglycemia but decreased nearly 40% during prolonged hypoglycemia (basal 327 ± 20 versus 208 ± 21 μM at 12 h, P < 0.01). We conclude that glucose counterregulatory mechanisms differ during short-term and prolonged hypoglycemia. In the former, only an increase in glucose production mediated by suppression of insulin secretion and increased glucagon secretion is involved, whereas during prolonged hypoglycemia, both a suppression of glucose utilization, and an increase in glucose production are important. These latter changes in glucose production and utilization may be influenced by changes in circulating nonglucose substrates (e.g., alanine, free fatty acids, and ketone bodies) as well as by hormonal factors acting on both hepatic and extrahepatic tissues.
Exercise Adaptation Responses for Gastric Inhibitory Polypeptide (GIP) and Insulin in Obese Children: Possible Extra-Pancreatic Effects
Authors: Kahle E Bowie , O'Dorisio Thomas M , Walker Robert B , Eisenman Patricia A , Reiser Sheldon , Cataland Samuel , Zipf William B
Published: September 2013

Thirteen obese children and matched controls were fed a mixed meal, and responses were evaluated at fixed intervals for glucose, insulin, and gastric inhibitory polypeptide (GIP). The obese children were evaluated before and within 48 h after completion of a 5-mo exercise training program (ETP). The ETP included three aerobic exercise sessions per week and modest diet restrictions. Caloric expenditure was increased by ∼300 kcal/exercise session. Weight gain was minimal over the 5 mo. An unexpected increase in GIP response and improved insulin tolerance were recorded for the obese children post-ETP. GIP values were higher (P < 0.05) at 30 and 60 min and led to a highly significant elevation (P < 0.01) of the integrated GIP response for post-ETP obese versus both pre-ETP and normal-weight controls. Insulin values were lower (P < 0.05) at 30 and 60 min and led to a lower integrated insulin response (P < 0.0585) for post-ETP obese children. However, the obese children continued to secrete more insulin (P < 0.05) than normal-weight controls. Glucose tolerance, similar for pre-ETP obese subjects and controls, did not change in post-ETP children.

Exercise-induced improvement in glucose utilization in these obese children was associated with an increase in GIP secretion. This contrasts with reports that calorie restriction will improve glucose utilization with decreased insulin and GIP secretion. The study demonstrates a previously unreported uncoupling of GIP and insulin secretion and suggests shifts in peripheral tissue sensitivity to insulin-induced glucose uptake. These shifts may, in part, be influenced by GIP.
Dietary Sardine Oil Increases Erythrocyte Membrane Fluidity in Diabetic Patients
Authors: Kamada Tetsuro , Yamashita Tomoko , Baba Yasutada , Kai Motoaki , Setoyama Shiro , Chuman Yasuo , Otsuji Shogo
Published: September 2013

The effects of dietary sardine oil rich in eicosapentaenoic acid, C20:5 (EPA), on erythrocyte membrane fluidity and membrane and plasma lipids were investigated in diabetic and control subjects. Before consumption of this oil, the levels of erythrocyte membrane fluidity were lower in the diabetic subjects, as noted in our previous work (Diabetes 1983; 32:585–91). Decreased membrane polyunsaturated fatty acid contents were evident. Daily consumption of 2700 mg of sardine oil for 8 wk increased erythrocyte membrane fluidity, as determined by electron spin resonance using the 12- or 16- stearic acid label. This increase was seen after 4 wk, and the level remained elevated for 8 wk. Membrane EPA of phospholipid acyl-chains significantly increased after 4 wk and was even more apparent after 8 wk. Membrane-free cholesterol to phospholipid molar ratios significantly decreased after 8 wk. Both the diabetic and normal subjects responded to the sardine oil in the same way. After feeding with sardine oil, there no longer were differences in erythrocyte membrane fluidity between, the normal and diabetic subjects. We propose that improvement in membrane fluidity may contribute to the amelioration of altered cell membrane functions in diabetic patients.
Effects of Hyperglycemia on Glucose Production and Utilization in Humans: Measurement With [23H]-, [33H]-, and [614C]Glucose
Authors: Bell P M , Firth R G , Rizza R A
Published: September 2013

Studies with tritiated isotopes of glucose have demonstrated that hyperglycemia per se stimulates glucos utilization and suppresses glucose production in humans. These conclusions rely on the assumption that tritiated glucose provides an accurate measure of glucose turnover. However, if in the presence of hyperglycemia the isotope either loses its label during “futile” cycling or retains its label during cycling through glycogen, then this assumption is not valid. To examine this question, glucose utilization and glucose production rates were measuredin nine normal subjects with a simultaneous infusion of [23H]glucose, an isotope that may undergo futile cycling but does not cycle through glycogen; [614C]glucose, an isotope that may cycle through glycogen but does not futile cycle; and [33H]glucose, an isotope that can both undergo futile cycling and cycle through glycogen. In the postabsorptive state at plasma glucose concentration of 95 mg dl−1, glucose turnover determined with [614C]glucose (2.3 ±0.1 mg kg−1 min−1) was greater than that determined with [33H]glucose (2.1 ± 0.1 mg kg−1 min−1 P = 0.002) and slightly less than that determined with [23H]glucose (2.7 ± 0.2 mg kg−1 min−1 P = 0.08). Plasma glucose was then raised from 95 to 135 to 175 mg dl−1 while insulin secretion was inhibited, and circulating insulin, glucagon, and growth hormone concentrations were maintained constant by infusion of these hormones and somatostatin. Glucose production and utilization rates determined with [614C]-glucose continued to be less than those determined with [23H]glucose and greater than those seen with [33H]glucose. However, the decrements in glucose production and increments in glucose utilization were identical with all isotopes. Glucagon was then infused at a high rate to stimulate endogenous glucose release. This resulted in a significant (P < 0.05) increase in both [614C]- and [33H]- but not [23H]giucose, indicating release of the former two isotopes from glycogen. This resulted in a significantly lower (P < 0.04) estimate of glucose production and utilization during the glucagon infusion determined with [614C]- and [33H]glucose compared with that determined with [23H]glucose. Thus, whereas neither [23H]- nor [33H]glucose precisely reflect glucose turnover measured by [614C]glucose, all three isotopes provide an equivalent assessment of the effects of hyperglycemia on glucose production and utilization in humans. However, release of either [614C]- or [33H]glucose from glycogen may result in an underestimate Of glucose turnover.
A Method for the Mass Isolation of Islets From the Adult Pig Pancreas
Authors: Ricordi Camillo , Finke Edward H , Lacy Paul E
Published: September 2013

A method for mass isolation of islets from the pig pancreas is described. The procedure involves the use of an enzymatic and mechanical pancreatic digestion procedure followed by filtration and separation of the islets on Ficoll gradients. A remarkably high yield of purified islets has been obtained from the pig pancreas with this procedure. The islets are morphologically intact and respond to acute stimulation with glucose in Vitro.
Controlled Release of Insulin From Polymer Matrices: Control of Diabetes in Rats
Authors: Brown Larry , Munoz Christina , Siemer Linda , Edelman Elazer , Langer Robert
Published: September 2013

The controlled release of insulin from ethylene-vinyl acetate copolymer matrices wasdemonstrated for over 100 days in vivo. The matrices were designed to release sodium insulin at near-constant rates. These 0.06-cm3 implants were coated completely with an impermeable layer of polymer. An aperture was drilled in the center of one face of the matrix, restricting release through this opening. These devices were implanted into 13 streptozocin-induced diabetic rats. Plasma glucose concentrations fell from 386 ± 18 to 119 ± 35 mg/dl (mean ± SEM), and urinary glucose was eliminated. Thee parameters were controlled for up to 105 days by a single implant. Glycosylated hemoglobin concentrations measured 90 days after implantation were 3.86 ± 0.11% for the polymer-treated rats, 3.10 ± 0.18% for the normal controls, and 5.42 ± 0.33% for the diabetic controls. The average weight gain of the treated rats was similar to that of the controls, whereas the diabetic controls failed to thrive. In addition, all of the diabetic controls developed cataracts 1 mo after diabetes induction, whereas none of the treated rats developed cataracts.
Mechanism of Stimulation of Liver Glycogen Synthesis by Fructose in Alloxan Diabetic Rats
Authors: Niewoehner Catherine B , Nuttall Frank Q
Published: September 2013

In diabetic animals and humans, stimulation of liver glycogen synthesis has been reported after administration of a large parenteral fructose load. The effects of an oral fructose load have not been examined previously. In the diabetic state, glycogen synthase phosphatase activity is reduced, and synthase D (the inactive form) is a poor substrate for the phosphatase. Thus,activation of synthase to the synthase R and synthase I (R + I) (active) forms by fructose would not be expected. We have determined that oral fructose administration does stimulate glycogen synthesis and have examined the mechanism by which this is accomplished. In 24-h-fasted alloxan diabetic rats, basal liver glycogen was higher than in normal rats (8.3 ±1.8 vs. 3.0 ± 0.5 mg/g wet wt). After fructose (4 g/kg) was given, the initial rate of glycogen synthesis was normal in diabetic rats, but total glycogen synthesis was reduced. By 240 min, liver glycogen increased to 18 ± 4.0 mg/g wet wt in diabetic rats versus 30.5 ±1.5 mg/g wet wt in normal rats. Synthase R + I was low and did not increase significantly (0.063 ± 0.006 to 0.064 ± 0.010 U/g wet wt) after fructose administration to the diabetic animals. Phosphorylase a did not decrease significantly during the period of active glycogen synthesis. In the diabetic rats, glucoses-phosphate increased by 84% (0.103 ± 0.010 to 0.184 ± 0.020 μmol/g wet wt) within 10 min and remained elevated above the control level. UDPglucose decreased from 0.336 ± 0.013 to 0.271 ± 0.011 μmol/g wet wt at 10 min and remained below the control level. ATP1, P1 and cAMP were unchanged.These data are compatible with a “pull” mechanism of glycogen synthesis due to stimulation of synthase R activity by a rise in glucose-6-P, with little conversion of synthase to the more active R + I forms. They do not suggest a substrate push mechanism for stimulation of glycogen synthesis by fructose in diabetic rats.
Estrone Treatment Dissociates Primary Versus Secondary Consequences of “Diabetes” (db) Gene Expression in Mice
Authors: Prochazka Michal , Premdas Francis H , Leiter Edward H , Lipson Loren G
Published: September 2013

Feeding 0.001% estrone in a diet to C57BL/KsJ mice homozygous for the recessive obesity gene “diabetes” (db) permitted dissociation of the primary consequences of obesity gene expression from the secondary consequences of diabetes effected through interaction between the db gene and other diabetogenic genes in the inbred background. Estrone-treated db/db mice were similar to untreated mutants in exhibiting hyperphagia and marked obesity. However, estrone-treated mutants did not develop the hyperinsulinemia, hyperglycemia, and islet atrophy characteristic of untreated db/db mice. Thus, expression of the primary defect could be studied in the absence of the myriad secondary sequelae elicited by chronic hyperinsulinemia and hyperglycemia. Reduced numbers of hepatocyte plasma membrane insulin receptors (50% of normal) persisted in the estrone-treated mice in the absence of hyperinsulinemia, indicating that this deficiency was a consequence of the primary genetic defect and not merely a downregulation phenomenon secondary to hyperinsulinemia. Comparison of insulin secretion from comparably sized + / + islets versus islets from estrone-treated db/db mice showed no intrinsic defects in β-cell sensitivity to glucose. In conclusion, db-induced obesity can be dissociated from hyperinsulinemia, hyperglycemia, β-cell dysfunction, and hyperphagia but is associated with a generalized membrane defect reflected in part by the persistent deficiency of plasma membrane insulin receptors.
Islet Insulin Release and Net Calcium Retention In Vitro in Vitamin D–Deficient Rats
Authors: Chertow B S , Sivitz W I , Baranetsky N G , Cordle M B , DeLuca H F
Published: September 2013

In our previous studies, perifused islets from vitamin D-deficient (D-def) rats showed marked impairment of glucose-induced biphasic release, accounted for at least in part by a decrease in food intake. In studies reported here, we test whether D-def rat islets have an impaired response to 5.6 mM glucose or tolbutamide, (T), and if so, whether this impairment is related to a decrease in food intake or a defect in islet calcium metabolism. We isolated islets of normal rats, D-def rats, and rats pair fed (PF) to D-def rats. Biphasic insulin release from perifused islets and net 45Ca retention in lot-incubated islets were measured in response to 5.6 mM glucose, 0.37 mM T, or both.

Compared with secretion from normal islets, biphasic insulin release from islets of both D-def rats and PF rats was diminished by 50% in response to 5.6 mM glucose alone or 5.6 mM glucose plus T. Insulin secretion was not significantly different between islets of D-def rats and islets of PF rats. In contrast, net calcium retention in islets of D-def rats was decreased to 68% of retention in islets of PF rats. However, net calcium retention in islets of both PF and D-def rats increased in response to T.

The pair-feeding experiments suggest that the decrease in insulin release from islets of D-def rats is due to the decrease in food intake associated with the D-def state. On the other hand, the defect in calcium retention in islets of D-def rats raises the possibility that vitamin D may have a specific effect on islet calcium metabolism. In this case, the mechanism of impaired insulin release in islets of D-def rats would be different from that in islets of PF rats and would involve a defect in intracellular calcium handling.
Insulin-Receptor Biosynthesis in Cultured Lymphocytes From an Insulin-Resistant Patient (Rabson-Mendenhall Syndrome): Evidence for Defect Before Insertion of Receptor Into Plasma Membrane
Authors: Moncada Victoria Y , Hedo José A , Serrano-Rios Manuel , Taylor Simeon I
Published: September 2013

In some patients with genetic forms of extreme insulin resistance, there is a marked decrease in the number of insulin receptors on the cell surface. We studied an insulin-resistant patient (RM-1) with the Rabson-Mendenhall syndrome. As judged by insulin-binding studies, Epstein-Barr virus–transformed lymphocytes from patient RM-1 exhibit a 90% decrease in the number of insulin receptors. Similarly, with either lactoperoxidase-catalyzed radioiodination of cell surface receptors or biosynthetic labeling of receptors with [3H]glucos-amine, we demonstrated an 80–90% decrease in the number of insulin receptors in cells from patient RM-1.

Previous studies have shown that the marked decrease in insulin receptors of the Rabson-Mendenhall patient is not due to accelerated receptor degradation. Therefore, we investigated the possibility that a slow rate of receptor biosynthesis might account for the 90% reduction of insulin receptors in cells from this patient.

Insulin-receptor biosynthesis proceeds through a glycoprotein precursor with an apparent M, of 190,000. It undergoes endopeptidase cleavage and further post-translational processing to yield the mature 135,000- and 95,000-Mr glycoprotein subunits. We studied the biosynthesis of the 190,000-Mr precursor and mature receptor subunits by a pulse-chase labeling technique with [2-3H]mannose. The time course of insulin-receptor biosynthesis appeared normal in cells from patient RM-1, despite a 10-fold reduction in the number of receptors on the cell surface. Parallel pulse-chase experiments with either [2-3H]mannose or [35S]methionine yielded the same results regardless of which label was employed. Thus, the receptor precursor in the Rabson-Mendenhall patient seems to be synthesized at a normal rate. Moreover, once the receptor is inserted in the membrane, it appears to be degraded at a normal rate. Therefore, we propose that there may be a defect subsequent to the synthesis of the receptor but before insertion of the receptor into the plasma membrane.
Are Insulin Autoantibodies Markers for Insulin-Dependent Diabetes Mellitus?
Authors: Atkinson M A , Maclaren N K , Riley W J , Winter W E , Fisk D D , Spillar R P
Published: September 2013

Recent studies have shown that insulin autoantibodies occur in patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) before exogenous insulin treatment. Our study was designed to test the hypothesis that insulin autoantibodies, like cytoplasmic islet cell antibodies (ICAs), can identify individuals with ongoing autoimmune β-cell destruction and increased risk of IDDM development. Insulin autoantibodies detected by use of a radioligand-binding assay were found in 1.4% of normal controls, 4% of first-degree relatives of IDDM patients, and in 37% of newly diagnosed IDDM patients. A strong positive correlation between insulin autoantibodies and ICAs was observed. HLA typing of insulin-autoantibody-positive first-degree relatives of IDDM patients, as well as in the general population, revealed a strong association with HLA-DR3 and/or-DR4, suggesting that insulin autoantibodies are restricted to persons genetically susceptible to IDDM. In an ongoing study of β-cell function in ICA-positive nondiabetic individuals, the additional presence of insulin autoantibodies significantly increased the likelihood of β-cell dysfunction. After intravenous glucose stimulation, insulinopenia was present in 70% of ICA and insulin-autoantibody-positive individuals in contrast to only 23% of ICA-positive, insulin-autoantibody-negative persons. These data document a significant association between insulin autoantibodies and ICAs and support the contention that insulin autoantibodies, like ICAs, are markers of ongoing β-cell destruction.
Ca2+-Mg2+-ATPase Activity in Kidney Basolateral Membrane in Non-Insulin-Dependent Diabetic Rats: Effect of Insulin
Authors: Levy J , Gavin J R , Hammerman M R , Avioli L V
Published: September 2013

The direct effect of insulin on the high-affinity Ca2+-Mg2+-ATPase was studied in kidney proximal tubular basolateral membranes (BLM) obtained from control and streptozocin-induced non-insulin-dependent diabetes mellitus (NIDDM) rats. Plasma glucose of the diabetic animals was only mildly elevated (217 ± 9 vs. 138 ± 3 mg/dl). Both high- and low-affinity calcium-dependent Ca2+-Mg2+-ATPase activities were identified in the BLM. Enzyme activity in BLM from diabetic rats was higher at all Ca2+ concentrations tested due to a higher maximum velocity of the enzyme from NIDDM rats. The high-affinity Ca2+-Mg2+-ATPase activity was inhibited by trifluoroperzine (TFP) in both membranes. No difference in calmodulin content was found in the membranes from the diabetic and control rats.

Insulin (16–200 μU/ml) significantly increased the high-affinity Ca2+-Mg2+-ATPase activity (17–40%) in membranes from control animals but had no effect on the enzyme activity in the membranes from the NIDDM rats. The basal activity of the enzyme at 0.1 μM free Ca2+ was higher in the BLM from the NIDDM animals compared to controls (17.8 ± 0.5 vs. 14.7 ± 0.8 nM Pi mg1 · min−1; P < .02). There was no effect of insulin on the Ca2+-independent ATPase activity of BLM preparations.

These findings demonstrate a defect in the ability of insulin to regulate the high-affinity Ca2+-Mg2+-ATPase activity in BLM from diabetic rats. Such a defect in enzyme activity may play a role in the mechanism of impaired insulin action observed in these NIDDM rats.
Apolipoprotein E-Containing Lipoproteins and Lipoprotein Remnants in Experimental Canine Diabetes
Authors: Wilson D E , Chan I -F , Elstad N L , Peric-Golia L , Hejazi J , Albu D S , Cutfield R
Published: September 2013

The effects of diabetes on plasma lipoproteins were examined in a cohort of control and streptozocin-alloxan diabetic beagles fed either standard rations or an atherogenic cholesterol-supplemented diet. Lipoprotein cholesterol, triglyceride, and retinyi ester concentrations were measured in fractions separated by density gradient ultracentrifugation. Individual lipoprotein classes and apolipoproteins were assessed by electro-phoresis. Postheparin plasma lipoprotein triglyceride lipase activities were also examined.

In the absence of added dietary cholesterol, diabetic animals became hypercholesterolemic with relatively increased low-density (LDL) and decreased high-density (HDL) lipoprotein cholesterol concentrations. Apolipoprotein E-containing β- to α2-migrating HDL1 (HDLc) appeared in ρ = 1.020–1.080 g/ml subfractions, whereas α1-migrating typical HDL (ρ = 1.06–1.21 g/ml) was reduced. In comparison to nondiabetic cholesterol-fed animals, diabetic cholesterol-fed animals had increased cholesterol (but not triglyceride) concentrations in very-low- and intermediate-density classes. These classes contained retinyi esters and low-molecular-weight apolipoprotein B (components of intestinal lipoprotein remnants) as well as apolipoprotein E and high-molecular-weight apolipoprotein B. These findings could not be explained by decreased postheparin plasma lipoprotein lipolytic activities.

Increased plasma concentrations of HDLc in poorly controlled diabetic dogs may reflect a pathologic disturbance in the excretory limb of cholesterol transport from peripheral cells to the liver. In addition, exaggerated retention of lipoprotein remnants in cholesterol-fed diabetic dogs may contribute to increased delivery of cholesterol to extrahepatic tissues. This model appears to be suitable for physiologic studies of the effects of diabetes on reverse cholesterol transport.
Plasma Level of 13,14-Dihydro-15-keto-PGE2 in Patients With Diabetic Ketoacidosis and in Normal Fasting Subjects
Authors: Axelrod L , Shulman G I , Blackshear P J , Bornstein W , Roussell A M , Aoki T T
Published: September 2013

Plasma levels of 13,14-dihydro-15-keto-PGE2, a stable derivative of PGE2, are elevated in rats with diabetic ketoacidosis (DKA) and decrease in response to insulin therapy. In patients with insulin-dependent diabetes mellitus type I (IDDM) the plasma levels of this derivative also rise in response to insulin withdrawal and then fall in response to insulin replacement. We wished to determine whether the level of this substance is elevated acutely when patients present with DKA and to determine whether the levels fall during treatment. We also wished to identify the origin of the circulating 13,14-dihydro-15-keto-PGE2 in patients with DKA and in normal fasting subjects. We measured the plasma level of 13,14-dihydro-15-keto-PGE2 in five patients with DKA and in six normal subjects during a 24-h fast. In the patients with DKA before treatment, the plasma 13,14-dihydro-15-keto-PGE2 level was threefold above normal. During therapy, the 13,14-dihydro-15-keto-PGE2 level fell toward normal. There was a significant direct correlation between the plasma free fatty acid (FFA) level and the plasma 13,14-dihydro-15-keto-PGE2 level before and during treatment. In addition, the inverse correlation between the plasma free-insulin level and the plasma 13,14-dihydro-15-keto-PGE2 level approached significance (P = .06). In contrast, in the normal fasting subjects the plasma FFA level rose to values comparable to those observed in the patients with DKA, but there was no significant increase in the plasma 13,14-dihydro-15-keto-PGE2 level. There was no quantitatively important correlation between the plasma FFA level and the plasma 13,14-dihydro-15-keto-PGE2 level. We conclude that 1) the plasma level of 13,14-dihydro-15-keto-PGE2 is elevated in DKA and decreases during treatment, 2) the adipocyte is an important source of the circulating 13,14-dihydro-15-keto-PGE2 in this disorder, and 3) insulin inhibits the production of PGE2 by the adipocyte and perhaps other cells in patients with DKA. The increased production of PGE2, a potent antilipolytic substance, may moderate the accelerated lipolysis that occurs in DKA but is probably not of sufficient magnitude to produce systemic effects. The rise in the plasma FFA level without a concomitant rise in the plasma 13,14-dihydro-15-keto-PGE2 level in normal fasting subjects suggests that cells other than the adipocyte may be the source of the circulating 13,14-dihydro-15-keto-PGE2 in normal subjects.
Progressive Damage of Cultured Pancreatic Islets After Single Early Exposure to Streptozocin
Authors: Bolaffi J L , Nowlain R E , Cruz L , Grodsky G M
Published: September 2013

To examine effects of streptozocin (STZ) on pancreatic islets in the absence of a functioning immune system, we examined isolated rat islets cultured for 96 h after a single 1-h exposure to STZ in vitro. In addition to an immediate and sustained suppression of insulin secretion, STZ also induced a progressive decline in insulin content per islet as well as in total islet tissue mass, characterized by a decrease in both islet number and volume. Viability studies show that STZ-induced cell death was also progressive and was not commensurate with loss of secretory function. Furthermore, mediatransfer experiments demonstrate that decline of tissue mass is not due to accumulation of metabolite or degradation products in the media. After 96 h in culture, untreated islets showed a marked insulinogenic capacity that was inhibited more than fourfold by the initial STZ treatment. Progressive loss of glucagon content per islet suggests that STZ causes disruption of islet morphological integrity. These progressive sequelae observed in vitro indicate that several aspects of the time-delayed attack on the β-cell by STZ are independent of a functioning immune system.
Diabetes Increases Active Fraction of Branched-Chain 2-Oxoacid Dehydrogenase in Rat Brain
Authors: Brosnan M E , Lowry A , Brosnan J T
Published: September 2013

Conditions were developed to permit the extraction of branched-chain 2-oxoacid dehydrogenase from brain tissue in the state of phosphorylation in which it occurred in vivo. Tissue was cold clamped to prevent interconversion of the enzyme forms without rupturing mitochondrial membranes. Extraction was carried out in the presence of NaF to prevent dephosphorylation of the enzyme complex and in the presence of ADP to prevent phosphorylation. In adult male control rats, ∼35% of the total enzyme from brain was present in the active form. In brains of diabetic rats, the active fraction was increased to 56%. Total activities did not differ in the two groups of rats.
Growth-Promoting Effects of Diabetes and Insulin on Arteries: An In Vivo Study of Rat Aorta
Authors: Capron L , Jarnet J , Kazandjian S , Housset E
Published: September 2013

Proliferation of arterial smooth muscle cells is regarded as an important event in atherogenesis, which according to in vitro culture studies is influenced by diabetes and insulin. To assess whether this holds true in vivo, we studied the cellular kinetics of thoracic aorta in normal and streptozocin-induced diabetic rats with and without insulin treatment. We measured the incorporation of [3H]thymidine into intima-media, as well as its DNA content, 2 and 14 days after endothelial denudation. We found that the mitotic response of an injured artery is not modified by diabetes but is depressed by insulin treatment in nondiabetic rats, probably due to hypoglycemia. Our data in insulin-treated diabetic rats support but do not definitely settle the view that insulin is mitogenic as long as the treatment does not cause sustained hypoglycemia.
Increase in Insulin Antibodies During Continuous Subcutaneous Insulin Infusion and Multiple-Injection Therapy in Contrast to Conventional Treatment
Authors: Dahl-Jørgensen Knut , Torjesen Peter , Hanssen Kristian F , Sandvik Leiv , Aagenæs Øystein
Published: September 2013

Forty-five insulin-dependent diabetics were randomized to 1 yr treatment with either continuous subcutaneous insulin infusion (CSII), multiple insulin injections (MI), or continued conventional treatment. The CSII group used regular insulin only, the MI group used 4-6 premeal injections of regular insulin and intermediate insulin at night, and the conventional group used two daily injections of combined regular and intermediate insulin. Only highly purified porcine insulin was used. Near normoglycemia was obtained during CSII and MI but not during conventional treatment.

Antibodies against insulin were measured in serum samples by measuring the binding of iodinated porcine insulin to serum after removal of free and antibodybound insulin from the samples by acid charcoal. The percent binding of 125I-labeled insulin increased significantly during MI and CSII, in contrast to conventional treatment.

Nineteen patients had sufficient binding capacity for Scatchard analysis. In the CSII and MI groups, high- or low-affinity antibodies or both were induced.

When insulin was administered subcutaneously during MI or CSII for 1 yr, the insulin antibody production increased, in contrast with conventional treatment.
Effects of Fasting, Feeding, and Insulin on Enhancing Effect of GTP on cAMP Binding in Rat Hepatic Cytosol
Authors: Rosenberg Edward M , Goodman A David , Lipinski Thomas I
Published: September 2013

GTP, in physiologic concentration, enhances the binding of cAMP to a protein in the hepatic cytosol that may be the regulatory subunit of protein kinase II. Ingestion of carbohydrate suppresses hepatic gluconeogenesis and glycogenolysis, two processes that are stimulated by cAMP. In this study, we have examined the possibility that carbohydrate inhibits these processes partly by decreasing the sensitivity of the GTP-responsive cAMP-binding protein to the effect of GTP. We found that 100 μM GTP was much less effective in enhancing cAMP binding in the hepatic cytosol of rats given 15% glucose for 2 days than in the cytosol of fasted rats [21 ± 3% (mean ± SE) increase vs. 67 ± 6%, P < .01]. Corresponding results were noted in diethylaminoethyl (DEAE)-cellulose extracts of the hepatic cytosol of these rats. GTP stimulation of cAMP binding was also diminished in the hepatic cytosol of diabetic rats treated for 7 days with insulin compared with that of untreated diabetic rats (29 ± 10 vs. 81 ± 11% increase, P < .01), but this could have been due to increased food intake in the treated rats. We conclude that GTP stimulation of hepatic cAMP binding is decreased in the carbohydrate-fed state and that this effect may be mediated by the increase in plasma insulin induced by carbohydrate. Our observations suggest that some of the cellular effects of cAMP may be regulated by modulation of the stimulatory effect of GTP on the GTP-responsive cAMP-binding protein.
Oral Protein Hydrolysate Causes Liver Glycogen Depletion in Fasted Rats Pretreated With Glucose
Authors: Gannon Mary C , Nuttall Frank Q
Published: September 2013

Studies in rats indicated that the major physiologic stimulus for synthesis of liver glycogen is a rise in the portal glucose concentration after ingestion of a meal. Conversely, glycogen degradation in the liver is stimulated by a rise in portal glucagon concentration. In humans, ingestion of carbohydrate lowers the concentration of circulating glucagon, whereas protein stimulates an increase in peripheral glucagon concentration. Little is known about the effects of these nutrients on glucagon concentrations in the rat. Therefore, we studied the effects of oral protein administration to 24-h-fasted rats pretreated with glucose for 2 h to test the effect of two potent but potentially opposite signals for glycogen metabolism. An increase in liver glycogen concentration was observed in fasted rats given oral glucose, as expected. Removal of glucose by the liver could not account for the glycogen synthesized, indicating that most glycogen formed was derived from gluconeogenesis. In addition, the apparent intracellular and extracellular glucose concentrations were not in equilibrium. A small amount of glucose may have been taken up against a concentration gradient. The portal glucagon was not significantly decreased. Oral protein administration to the rats pretreated with glucose resulted in a rapid and dramatic decrease in liver glycogen concentration. This was associated with an increase in the portal glucagon concentration, no change in insulin concentration, a slight increase in liver cAMP concentration, an increase in the active form of phosphorylase, and a decrease in the active form of synthase. Glycogenolysis could account for the glucose released into the circulation from the liver after protein administration.
Interaction of Cooling Rate, Warming Rate, and Extent of Permeation of Cryoprotectant in Determining Survival of Isolated Rat Islets of Langerhans During Cryopreservation
Authors: Taylor Michael J , Benton Marcus J
Published: September 2013

Cryopreservation of islets of Langerhans offers a number of important benefits for attempts to cure diabetes by transplantation. In the published literature, a variety of cooling rates, ranging from 0.25 to 75°C/min, in conjunction with warming rates of 4–200°C/min have been proposed to give optimal preservation of islets. In view of the general importance of rates of temperature change in determining survival and because of the possibility of modulating tissue immunogenicity by freezing and thawing, we have studied the interaction of cooling rate and warming rate for isolated rat islets that had been either fully or partially equilibrated with 2 M dimethyl sulfoxide (DMSO). Batches of islets were stored at −196°C after cooling at 0.3, 3.0, 10, 30, 60, 150, or 1000°C/min and then warmed at either 10 or 50°C/min. Survival was assessed by measuring the secretion of insulin during static incubation in alternating nonstimulatory and stimulatory media. Cooling rates extending over three orders of magnitude proved not to be a major determinant of survival when the islets were equilibrated with 2 M DMSO: >50% survival was achieved at all cooling rates studied when the warming rate was at 50°C/min. Peak survival (83%) was attained at a cooling rate of 0.3°C/min, but only slightly lower recoveries were obtained at 60 and >1000°C/min. However, in islets only partially equilibrated with cryoprotectants, functional recovery was highly dependent on the cooling and warming rates, with peak survivals after slow cooling and rapid warming. Full permeation of the tissue with cryoprotectant offered maximal recovery of function.