bluuebunny/crossref_metadata_2025 huggingface dataset:
Receptor-Mediated Adenylate Cyclase—Coupled Mechanism for PGE2 Inhibition of Insulin Secretion in HIT Cells
Authors: Robertson R Paul , Tsai Phoebe , Little Sally A , Zhang Hui-Jian , Walseth Timothy F
Published: September 2013

Prostaglandin E2 (PGE2) inhibits glucose-induced insulin secretion, and inhibitors of PGE2 synthesis augment this event. However, there has been confusion regarding prostaglandin regulation of insulin secretion, partly because no mechanism has been demonstrated for the inhibitory action of PGE2 on β-cell function. These studies were performed with a clonai cell line of glucose-responsive β-cells (HIT cells) to determine whether PGE2 effects on insulin secretion are receptor mediated and, if so, whether the postreceptor effects are mediated by inhibitory regulatory components (N1) of adenylate cyclase. Saturable [3H]PGE2 binding to HIT cells was demonstrated. This binding was dissociable and specific for prostaglandins of the E series. Scatchard analyses of binding data indicated a single class of sites with a Kd of ∼1 × 10−9 M. Guinea pig islets were also demonstrated to have a single class of binding sites with a similar Kd but only 22% as many binding sites (0.060 vs. 0.013 pmol/mg protein, HIT cells vs. guinea pig islet). HIT cells were demonstrated to synthesize PGE2, and this synthesis was inhibitable by acetylsalicylic acid. Accumulation of cAMP by HIT cells was inhibited in a concentration-dependent manner by PGE2 with an IC50 of 1 × 10−9 M. Insulin secretion by HIT cells during static incubations with 11.1 mM glucose was also inhibited by PGE2 in a concentration-dependent manner with an IC50 of 1 × 10 −9 M. PGE2 was more potent than epinephrine but less potent than somatostatin in this regard. Maximum inhibition of glucose-induced insulin secretion was 26, 37, and 29% of control values for somatostatin, PGE2, and epinephrine, respectively. Incubation of HIT cell membranes with pertussis toxin and [α-32P]NAD with subsequent electrophoresis via sodium dodecyl sulfate–polyacrylamide gels revealed ADP-ribosylation of a component consistent with the N, component of adenylate cyclase. Pretreatment of intact HIT cells with pertussis toxin prevented subsequent labeling of this component when membranes were incubated with pertussis toxin and [32P]NAD. Pertussis toxin partially prevented the inhibitory effects of PGE2, but not NiCI2, on glucose-induced insulin secretion. These findings uniquely demonstrate inhibition by PGE2 of glucose-induced insulin secretion from HIT cells. The mechanism of PGE2 action appears to involve specific PGE receptors that activate the N1, component of adenylate cyclase, resulting in diminished cAMP accumulation.
Quin 2 and Manganese Define Multiple Alterations in Cellular Calcium Homeostasis in Diabetic Rat Pancreas
Authors: Korc Murray , Schöni Martin H
Published: September 2013

The effects of streptozocin-induced diabetes on Ca2+ fluxes and intracellular free-Ca2+ ([ Ca2+]i) levels were studied in isolated rat pancreatic acini. The basal rates of 45Ca influx in acini and of 45Ca efflux from acini that were preloaded with 45Ca were similar in normal and diabetic rats. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8, 0.1 nM) and the Ca2+ ionophore A23187 (2 μM) increased 45Ca influx to the same extent in both groups of acini. CCK-8 and the divalent cation manganese exerted marked stimulatory effects on 45Ca efflux. In contrast to the rapid effect of CCK-8 on 45Ca efflux, the effect of manganese was slow in onset and was greater in diabetic rat acini. The diabetic state was also associated with a significant decrease in resting [Ca2+]i levels, as determined with the Ca2+ indicator quin 2. Furthermore, the ability of CCK-8 to raise [Ca2+]i levels was significantly attenuated in these cells. Insulin did not alter either basal or CCK-8–mediated increases in [Ca2+]i levels. Our findings suggest that insulin deficiency is associated with multiple alterations in Ca2+ homeostasis in the pancreatic acinar cell. These include a decrease in basal [Ca2+]i levels, perturbations in the signal transduction system that mediates the rapid mobilization of [Ca2+]i after CCK-8–receptor binding, and enhanced sensitivity to the actions of manganese on intracellular Ca2+ pools.
Evidence of IgG Autoantibodies Against Human Proinsulin in Patients With IDDM Before Insulin Treatment
Authors: Kuglin Bernd , Gries F Arnold , Kolb Hubert
Published: September 2013

IgG proinsulin autoantibodies (IgG-PAAs) have been found in a fraction of sera from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) before onset of insulin treatment. Only sera lacking insulin antibodies have been analyzed, to avoid interference. Competitive inhibition studies provide specificity for human proinsulin but not for insulin. IgG-PAAs largely cross-react with human C-peptide. Precursors of insulin thus are involved in the immune process of IDDM.
Additive Hypoglycemic Effects of Drugs That Modify Free-Fatty Acid Metabolism by Different Mechanisms in Rats With Streptozocin-Induced Diabetes
Authors: Reaven Gerald M , Chang Helen , Hoffman Brian B
Published: September 2013

In this study the effect of two drugs [etomoxir and nicotinic acid (NA)] on plasma glucose, free–fatty acid (FFA), and triglyceride (TG) concentrations was determined in rats with streptozocin (STZ)-induced diabetes. The two compounds modify FFA metabolism by different mechanisms, etomoxir (ethyl-2-[6-(4-cholorophenoxyl)-hexyl]oxirane-2-carboxylate) by inhibiting hepatic fatty acid oxidation, and NA by inhibiting lipolysis in adipose tissue. Diabetes was induced in male Sprague-Dawley rats, weighing ∼ 400 g, by STZ injection (30 mg/kg i.v.), and the metabolic effects of the two drugs were studied 7–10 days later. The acute administration of either etomoxir or NA lowered plasma glucose concentrations in diabetic rats by ∼150 mg/dl (P < .001) in 4 h. However, the two drugs differed dramatically in their effects on plasma FFA and TG concentrations. Specifically, etomoxir produced striking increases in plasma FFA and TG concentrations, whereas NA administration caused a marked decrease. However, when NA was given in conjunction with etomoxir, NA prevented the increase in plasma FFA and TG concentration seen with etomoxir; the combination of NA and etomoxir approximately doubled the decrease in plasma glucose concentration produced by NA or etomoxir when given alone. Because plasma insulin concentrations did not change in response to either drug, whether administered singly or in combination, these metabolic effects do not result from a change in insulin secretion. These results suggest that modulation of FFA metabolism at the level of the adipocyte or the liver can have dramatic effects on carbohydrate and lipid metabolism. Also, NA prevented the potentially adverse effects that etomoxir had on FFA and TG. The observation that the two drugs given in combination were capable of lowering plasma glucose from <400 mg/dl to essentially normal levels within 4 h raises the possibility that this pharmacologic approach may have relevance to the treatment of diabetes.
Central Versus Peripheral Effect of Clonidine on Hepatic Venous Plasma Glucose Concentrations in Fasted Rats
Authors: Gotoh Minehiro , Iguchi Akihisa , Sakamoto Nobuo
Published: September 2013

To evaluate whether clonidine exerts its action within the central nervous system or outside the central nervous system to induce hyperglycemia, we compared the effects of clonidine injected into the third cerebral ventricle or intravenously on hepatic venous plasma glucose concentrations in fasted rats. Clonidine administration produced a dose-dependent hyperglycemia in each case. At all tested doses (5, 50, and 100 nmol) the hyperglycemic responses to clonidine injected intravenously were not significantly different from those to clonidine injected into the third cerebral ventricle. Intraperitoneal pretreatment with yohimbine (50 nmol) or phentolamine (50 nmol), both α-adrenergic antagonists, reduced the hyperglycemic response to clonidine (100 nmol) given intravenously, but these antagonists preinjected into the third cerebral ventricle did not reduce the response. Moreover, no significant differences in venous plasma clonidine concentrations were observed when intravenous and third cerebral ventricle injections of clonidine (100 nmol) were compared. These results suggest that clonidine-induced hyperglycemia is mainly mediated by the peripheral mechanism rather than through the central mechanism. To gain an insight into the peripheral mechanism for the hyperglycemic action of clonidine, we measured the plasma immunoreactive glucagon and insulin concentrations after intravenous clonidine (100 nmol). We found that plasma immunoreactive glucagon concentrations significantly increased, whereas plasma immunoreactive insulin concentrations did not change significantly despite hyperglycemia. Furthermore, the clonidine-induced hyperglycemia and hyperglucagonemia were completely abolished by constant intravenous infusion of somatostatin (1.0 μg. kg− · min−1). These results suggest that clonidine-induced hyperglycemia is associated with not only the suppression of insulin release but also the stimulation of glucagon release. Additionally, clonidine may not act directly on the liver to produce hyperglycemia in fasted rats.
Lipogenesis From Ketone Bodies in Perfused Livers From Streptozocin-Induced Diabetic Rats
Authors: Freed Lisa E , Endemann Gerda , Tomera John F , Gavino Victor C , Brunengraber Henri
Published: September 2013

Production of ketone bodies and their contribution to lipogenesis were measured in isolated livers from normal and streptozocin-induced diabetic (STZ-D) rats perfused with tracer amounts of 3H2O and (R)-3-hydroxy[3-14C]butyrate. Diabetes decreased by 80–95% the total rates of fatty acid and 3-β-hydroxysterol synthesis in perfused livers and livers of live rats. The activity of cytosolic acetoacetyl-CoA synthetase was slightly (17%) decreased in livers from STZ-D rats. The incorporation of ketone bodies into fatty acids and sterols was markedly inhibited in perfused livers from STZ-D rats despite the stimulation of ketogenesis by diabetes and the presence of oleate. Treatment of the rats with insulin before liver perfusion led to a normalization of the rates of ketogenesis and fatty acid synthesis. The rates of sterol synthesis were only partially normalized by insulin treatment. We conclude that in STZ-D, ketosis does not stimulate hepatic lipogenesis via cytosolic activation of acetoacetate.
Relationship of Glycosylated Hemoglobin to Oral Glucose Tolerance: Implications for Diabetes Screening
Authors: Little Randie R , England Jack D , Wiedmeyer Hsiao-Mei , McKenzie Edith M , Pettitt David J , Knowler William C , Goldstein David E
Published: September 2013

The oral glucose tolerance test (OGTT) for diagnosis of diabetes is inconvenient and requires a great deal of patient cooperation. Glycosylated hemoglobin (GHb), an index of long-term glycemic control, could offer several practical advantages over the OGTT for diabetes screening. We evaluated GHb as a screen for diabetes in 381 adults from a population with a high prevalence of non-insulin-dependent diabetes (Pima Indians). All individuals underwent a standard OGTT (75 g) and were separated into one of three groups: normal (N), impaired glucose tolerance (IGT), or diabetes mellitus (D) based on World Health Organization criteria. HbA1c, a GHb, was measured by highly precise high-performance liquid chromatography (interassay C.V. <4%). The normal range for HbA1c was 4.07–6.03% based on the 95% confidence interval for a nondiabetic, mostly Caucasian population. Compared with OGTT, HbA1c was highly specific (91%); an elevated HbA1c usually indicated D or IGT (sensitivity = 85 and 30%, respectively). A normal HbA1c did not, however, exclude a diagnosis of D or IGT. Based on previous epidemiological studies relating plasma glucose to chronic diabetic complications, GHb as measured in this study would properly identify the vast majority of subjects at risk. Long-term studies are necessary to determine the actual risk of complications in individuals with persistently normal HbA1c and D or IGT (based on OGTT).
Epinephrine Impairs Insulin Release by a Mechanism Distal to Calcium Mobilization: Similarity to Lipoxygenase Inhibitors
Authors: Metz Stewart A
Published: September 2013

The mechanisms that enable epinephrine (EPI) and lipoxygenase inhibitors to impede insulin secretion are unknown. We examined the possibility that EPI inhibits Ca2+ fluxes as its major mechanism by studying 45Ca efflux from prelabeled, intact rat islets. EPI (2.5 × 10−7 to 1 × 10−5 M) inhibited insulin release induced by the influx of extracellular Ca2+ (46 mM K+) or the mobilization of intracellular Ca2+ stores (2 mM Ba2+), but it did not reduce the 45Ca efflux stimulated by either agonist. EPI also nullified insulin release induced by isobutylmethylxanthine or dibutyryl cAMP, with minimal or no effects on 45Ca efflux, and blocked the insulinotropic effects of 12-O-tetradecanoylphorbol-13-acetate (a direct activator of protein kinase C), which is believed primarily to sensitize the exocytotic apparatus to Ca2+ without mobilizing additional Ca2+. Previously we reported that similar effects were induced by inhibitors of pancreatic islet lipoxygenase. In this study, however, pretreatment with either the α2-adrenergic antagonist yohimbine or pertussis toxin did not block the effects of lipoxygenase inhibitors, although either agent did block the effects of EPI. Thus, EPI, via an α2-receptor mechanism, is able to reduce exocytosis largely distal to, or independent of, changes in Ca2+ flux, cAMP formation or its Ca2+-mobilizing action, or generation of protein kinase C activators. Therefore, EPI may reduce the sensitivity of the exocytotic apparatus to Ca2+. Inhibition of islet lipoxygenase may have a similar effect; however, in this case, the effect would have to be unrelated, or distal, to stimulation of α2-receptors.
Insulin Effects on Pantothenic Acid Uptake in Isolated Perfused Working Hearts From Diabetic Rats
Authors: Lopaschuk Gary D
Published: September 2013

Pantothenic acid uptake was studied in isolated working hearts from spontaneously diabetic BB Wistar and streptozocin-induced diabetic (STZ-D) rats. If insulin treatment was stopped for a 24-h period from spontaneously diabetic rats, a significant decrease in the rate of pantothenic uptake was noted (from 147.3 ± 5.0 to 110.8 ± 10.6 nmol · g−1 dry wt · 30 min−1). Pantothenic acid uptake rates were also reduced in 48-h STZ-D rats (118.0 ± 6.1 nmol · g−1 dry wt · 30 min1, compared to 158.2 ± 5.3 in control rats). The decrease in pantothenic acid uptake in all diabetic animals occurred whether hearts were perfused with 1.2 m M palmitate or 1.2 m M palmitate and 11 mM glucose. If insulin (500 μU/ml) was added to the perfusion medium of hearts from spontaneously diabetic rats perfused with palmitate and glucose, a significant increase in pantothenic acid uptake was noted (from 110.8 ± 10.6 to 167.0 ± 9.4 nmol · g−1 dry wt · 30 min−1). Insulin had no significant effect on pantothenic acid uptake in hearts from spontaneously diabetic rats perfused with palmitate alone. In STZ-D rats, insulin added to hearts perfused with palmitate and glucose resulted in a small but significant increase in pantothenic acid uptake (from 118.0 ± 6.1 to 130.6 ± 4.0 nmol · g−1 dry wt · 30 min−1). Insulin had no effect on pantothenic acid uptake in control hearts perfused either in the presence or absence of glucose. These data suggest that insulin, in the presence of glucose, can increase pantothenic acid uptake in diabetic rats.
ATPase Activity Defects in Alloxan-Induced Diabetic Sciatic Nerve Recovered by Ganglioside Treatment
Authors: Bianchi Roberto , Marini Paolo , Merlini Serena , Fabris Michele , Triban Chiara , Mussini Emilio , Fiori Mario G
Published: September 2013

ATPase activities were measured in sciatic nerves from rats with alloxan-induced diabetes (ALX-D) of various duration (2 wk, 5 wk, 9 wk, and 6 mo). Our data confirm that sciatic nerve Na+-K+-ATPase abnormalities are present very early in ALX-D rats, similar to results previously described in streptozocin-induced diabetic rats, spontaneously diabetic BB Wistar rats, and ALX-D rabbits. Na+-K+-ATPase activity decreased by 26–47% in ALX-D rats compared with age-matched controls. Ganglioside treatment (10 mg/kg i.p. for 10 or 30 days starting 1 wk after ALX injection) completely impeded the enzyme reduction. The effect observed at the end of either 10 or 30 days of treatment lasted ≥1 mo. Chronic diabetic groups treated for 30 days before killing also presented normal ATPase activity at the end of treatment. Therefore, gangliosides are effective on Na+-K+-ATPase even in animals with a longer duration of diabetes. The maintenance of fairly normal ATPase activity by ganglioside treatment could mirror a more general recovery from early metabolic dysfunction and/or late structural abnormalities in diabetic nerve fibers.
Glucose-Regulated Proinsulin Processing in Isolated Islets From Rat Pancreas
Authors: Nagamatsu Shinya , Grodsky Gerold M
Published: September 2013

We demonstrated previously that the conversion rate of proinsulin to insulin in pancreatic islets progressively increased after prolonged prior exposure to glucose (11 mM) and that this effect could be blocked by cycloheximide. This study was designed to characterize further the time course and regulation of the proinsulin conversion process. The effects of prior exposure to glucose on proinsulin conversion were dose dependent (Km, ∼7 mM glucose) and time dependent, taking ∼3 h to reach the maximum rate. Glucose added at or after the subsequent [3H]leucine pulse was ineffective. Mannoheptulose, added during a 3-h exposure with glucose (11 mM), prevented glucose-induced activation of the proinsulin conversion process. L-Leucine (20 mM) was as effective as 11 mM glucose in activating conversion, whereas 2-α-ketoisocaproic acid (20 mM) or phorbol ester (50 nM) had little effect. Activation of proinsulin conversion by a 24-h exposure to glucose (11 mM) was reversed by a subsequent 3-h prior exposure to cycloheximide. α-Amanitin, an inhibitor of mRNA synthesis, did not influence the glucose-induced activation of proinsulin conversion when present during a 3-h exposure to glucose; however, it completely inhibited glucose-stimulated conversion when present during 24 h exposure. Results suggest that activation of the proinsulin conversion process is regulated by glucose metabolism rahier than the glucose molecule per se and that other, but not all, secretagogues are effective. Conversion may require prior synthesis of a pool of converting enzyme(s) or other regulatory proteins whose turnover is relatively rapid (∼33 h) and whose mRNA is more stable (to 24 h).
Metabolic Effects of Low-Dose Insulin Therapy on Glucose Metabolism in Diabetic Ketoacidosis
Authors: Luzi Livio , Barrett Eugene J , Groop Leif C , Ferrannini Eleuterio , DeFronzo Ralph A
Published: September 2013

The effect of low-dose insulin treatment (5–10 U/h) on hepatic glucose production (HGP) and peripheral glucose disposal was determined in 5 insulindependent diabetes mellitus (IDDM) subjects who were admitted with diabetic ketoacidosis (DKA; plasma glucose 598 ± 50 mg/dl, blood pH 7.20 ± 0.06, plasma bicarbonate 12 ± 2 meq/L). Basal hepatic glucose production (4.3 ± 0.5 mg · kg1 min1) in the DKA patients was 1.5- to 2-fold greater (P < .01) than in controls (2.1 ± 0.1 mg · kg−1 · min−1) and nonketotic IDDM subjects (2.9 ± 0.3 mg · kg−1 · min−1), whereas tissue glucose disposal was significantly reduced (1.7 ± 0.1 vs. 2.1 ± 0.1 mg kg−1 min−1 P < .05). After the institution of insulin therapy (1 mU · kg−1 · min−1), the plasma glucose concentration fell at the rate of 60 ± 5 mg· dl−1 h−1 to reach a value of 220 ± 10 mg/dl, which was maintained constant for 2 h (insulinclamp technique). Blood pH (7.21 ± 0.06 to 7.35 ± 0.05) and plasma bicarbonate (12 ± 3 to 18 ± 2 meq\L) both increased during insulin therapy (P < .01). The decline in plasma glucose concentration during insulin therapy primarily resulted from a suppression of HGP (from 4.3 ± 0.5 to 1.7 ± 0.2 mg · kg−1 min−1 P < .01) and to a lesser extent from the stimulation of tissue glucose disposal (1.7 ± 0.2 to 2.6 ± 0.3 mg kg−1 min−1 P < .01). At this time, urine glucose excretion decreased from 2.6 ± 0.2 to 0.6 ± 0.1 mg kg−1 min−1. During the 2-h period of the insulin clamp (steady-state plasma glucose 220 ± 1 0 mg/dl, plasma free insulin 85 ± 5 (ill/ml), HGP in the DKA patients was further suppressed (1.70 ± 0.20 to 1.00 ± 0.20 mg · kg−1 min−11) but remained well above (P < .01) rates observed in control subjects (0.1 ± 0.02 mg · kg−1 · min−1) and nonketotic diabetic subjects (0.2 ± 0.1 mg · kg−1 · min−1). At the end of the 2-h insulin-clamp period, the rate of tissue glucose uptake (3.2 ± 0.3 mg kg−1 · min−1) had not increased significantly above the rate observed at the start of the clamp (2.6 ± 0.2 mg/kg). Urinary glucose losses during the insulin-clamp period averaged 0.10 ± 0.01 mg · kg−1 · min−1. In summary, in DKA, 1) hyperglycemia results from both a markedly increased rate of HGP and a modest decrease in tissue glucose uptake, 2) the ability of insulin to suppress HGP and to stimulate tissue glucose uptake is severely impaired, and 3) during the initial 6-h period of insulin therapy, urinary glucose losses (0.4 mg kg−1 min−1), stimulation of tissue glucose uptake (increment 1.5 mg kg−1 min−1), and suppression of HGP (decrement 3.3 mg · kg−1 · min−1) all contributed to the decline in plasma glucose. However, from the quantitative standpoint, inhibition of HGP is the most important determinant in the decline in plasma glucose concentration after institution of insulin therapy.
Choline Turnover in Phosphatidylcholine of Pancreatic Islets: Implications for CDP-Choline Pathway
Authors: Hoffman Joanne M , Laychock Suzanne G
Published: September 2013

The CDP-choline pathway is the major route of phosphatidylcholine (PC) biosynthesis in mammalian cells. The incorporation of [14C]choline into PC of isolated pancreatic islets of the rat was time dependent, glucose stimulable, and inhibited by mannoheptulose. Removal of extracellular Ca2+ enhanced glucose-stimulated choline incorporation without affecting basal levels. Glucose stimulated PC synthesis in islets labeled to equilibrium with 32PO4 in the presence or absence of extracellular Ca2+. The water-soluble intermediates of the CDP-choline pathway, phosphorylcholine and CDP-choline, accumulated to a lesser extent under Ca2+-free conditions; however, glucose enhanced the levels of these intermediates in the presence and absence of Ca2+. Thus, glucose stimulates CDP-choline-pathway activity. Ca2+-free conditions may promote flux of choline intermediates through the pathway and retard the hydrolysis of PC. The phospholipase A2-activating agents Δ-9-tetrahydrocannabinol and melittin enhanced [3H]choline incorporation into PC and potentiated incorporation in response to a submaximal secretagogic concentration of glucose (8.5 mM); insulin release paralleled the changes in PC. p-Bromophenacyl bromide and mepacrine reduced islet glucose utilization and glucose-stimulated [3H]choline levels in PC. An inhibitor of CTP: phosphorylcholine cytidylyltransferase, 5'-deoxy-5'-isobutylthioadenosine, reduced glucose-stimulated [14C]choline incorporation into PC; insulin release was inhibited in a parallel fashion. Thus, islet PC turnover and CDP-choline pathway activity appear to be modulated by glucose metabolism and membrane phospholipid hydrolysis. PC turnover and insulin release appear to be related.
Stimulation of Glucose Production Through Hormone Secretion and Other Mechanisms During Insulin-Induced Hypoglycemia
Authors: Frizzell R Tyler , Hendrick Grant K , Brown Laurel L , Lacy D Brooks , Donahue E Patrick , Carr R Keith , Williams Phillip E , Parlow Albert F , Stevenson Ralph W , Cherrington Alan D
Published: September 2013

To assess the role of counterregulatory hormones per se in the response to continuous insulin infusion, overnight-fasted dogs were given 5 mil · kg−1 · min−1 insulin intraportally either alone (INS, n = 5), with glucose to maintain euglycemia (INS + GLU, n = 5), or with glucose and hormone replacement [i.e., glucagon, epinephrine, norepinephrine, and cortisol infusions (INS + GLU + HR, n = 6)]. The increases in counterregulatory hormones that occurred during insulin-induced hypoglycemia were simulated in the latter group. In this way, it was possible to separate the effects of hypoglycemia per se from those due to the associated counterregulatory hormone response. Glycogenolysis and gluconeogenesis were measured with a combination of tracer ([3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous (AV) difference techniques during a 40-min control and a 180-min experimental period. Insulin levels increased similarly in all groups (to ≃250 μU/ml), whereas plasma glucose levels decreased in INS (115 ± 3 to 41 ± 3 mg/dl; P < .05) and rose slightly in both INS + GLU (108 ± 2 to 115 ± 4 mg/dl; P < .05) and INS + GLU + HR (111 ± 3 to 120 ± 3 mg/dl; P < .05) due to glucose infusion. Glucagon, epinephrine, norepinephrine, and cortisol were replaced in INS + GLU + HR so that the increments in their levels were 102 ± 6, 106 ± 14, 117 ± 9, and 124 ± 37%, respectively, of their increments in INS. At no time was there a significant difference between the hormone levels in INS and INS + GLU + HR. The rise in the counterregulatory hormones per se accounted for only half (53 ± 9% by the AV difference method and 54 ± 10% by tracer method) of the glucose production associated with hypoglycemia resulting from insulin infusion. The rate and efficiency of alanine conversioto glucose in the hormone-replacement studies were only 29 ± 10 and 50 ± 27% of what occurred during hypoglycemia induced by insulin infusion. In conclusion, the counterregulatory hormones alone (i.e., without accompanying hypoglycemia) can account for only 50% of the glucose production that is present during insulin-induced hypoglycemia. The remaining 50%, therefore, must result from effects of hypoglycemia other than its ability to trigger hormone release.
Autoantibodies in Nonobese Diabetic Mice Immunoprecipitate 64,000-Mr Islet Antigen
Authors: Atkinson Mark A , Maclaren Noel K
Published: September 2013

In insulin-dependent diabetes mellitus (IDDM) in humans and BB rats, islet cell autoimmunities associated with autoantibodies to a β-cell protein of 64,000 Mr (64K) have been described. We report that sera from newly diagnosed nonobese diabetic (NOD) mice similarly contain an autoantibody that immunoprecipitates 64K autoantigen from detergent lysates of [35S]methionine-labeled murine islet cells. The autoantibody was detectable by weaning; it disappeared within weeks after diabetes onset and was absent in older nondiabetic NOD mice as well as all of three non-diabetes-prone control strains tested. The 64K β-cell autoantigen may be a critical target in the immunopathogenesis of IDDM.
Association of HLA-DR Phenotypes and T-Lymphocyte–Receptor β-Chain–Region RFLP With IDDM in Japanese
Authors: Ito Masafumi , Tanimoto Mitsune , Kamura Hiromi , Yoneda Masahiro , Morishima Yasuo , Takatsuki Kensuke , Itatsu Takeharu , Saito Hidehiko
Published: September 2013

Fifty Japanese patients with insulin-dependent diabetes mellitus (IDDM) and 94 normal subjects were genotyped for Bg/II restriction-fragment-length polymorphism (RFLP) of the T-lymphocyte-receptor β-chain (TLRβ)-region gene and analyzed in relation to HLA-DR phenotypes. The antigen frequencies of DR4 and DR9 in the IDDM population were significantly higher than those in the normal population, with relative risks of 1.87 (P < .02) and 2.42 (P < .01), respectively. Hybridization of digested DNA with the TLRβ probe revealed two alleles of 9.3 and 8.6 kilobases (kb). The allele frequency of 8.6 kb in patients with IDDM (79%) was significantly (P < .05) higher than that in normal subjects (64%). When TLRβ-region RFLP in IDDM was further analyzed with respect to the HLA-DR phenotypes, the frequency of 8.6 kb was significantly increased in patients with DR4 but not DR9 (DR4/X) and those with DR9 but not DR4 (DR9/X) compared with the frequency found in normal subjects (P < .05); the relative risks of 8.6 kb in DR4/X and DR9/X were 2.77 and 4.98, respectively. Although the frequencies of HLA-DR phenotypes and of TLRβ-region RFLP in IDDM and normal subjects were apparently different from those reported for Caucasians, this population-association study indicates that in the Japanese, genes conferring susceptibility to IDDM exist near or at the HLA-DR and the TLRβ loci, as has been demonstrated in Caucasians.
Phenotypic Expression of Collagen Types in Mesangial Matrix of Diabetic and Nondiabetic Rats
Authors: Abrass Christine K , Peterson Carl V , Raugi Gregory J
Published: September 2013

This study was performed to evaluate the composition of the extracellular matrix of the mesangium in both diabetic and nondiabetic rats. Four groups of rats (n = 10 each) were studied. Nondiabetic rats were injected with saline (group 1) or insulin (3.5 U NPH daily) (group 2). Streptozocin-induced diabetic rats were similarly injected with saline (group 3) or insulin (group 4). Six weeks after initiation of study, glomerular diameter (µm) was increased in groups 2 (147 ± 21), 3 (144 ± 22), and 4 (150 ± 7) compared with group 1 (104 ± 12) (P < .01). Glomerular hypertrophy was associated with an increase in the relative amount of mesangial matrix as determined by staining for fibronectin. By immunofluorescence microscopy (0–4+ scale), type I collagen antigen was not detected in the mesangium of any of the experimental groups. Staining for type V collagen and thrombospondin was similar between the experimental groups. Type III collagen antigen was not detected in the mesangium of control (group 1) or insulin-deficient diabetic rats (group 3); however, it was detected (2–3+) in the mesangium of both insulin-treated diabetic and nondiabetic rats (Mann-Whitney, P < .01). Comparable intensity of staining (1+) for type IV collagen antigen was detected in the mesangium of animals from groups 1, 2, and 4; however, the staining intensity was markedly increased (3+) in insulindeficient diabetic rats (group 3; Mann-Whitney, P < .01). These studies demonstrate that glomerular hypertrophy and an increase in mesangial matrix occur in both hyperglycemic and insulin-treated rats; however, the mesangial matrix composition varies with the metabolic environment. Hyperglycemia is associated with a significant increase in the content of type IV collagen, and insulin treatment is associated with a switch in the matrix composition with the new expression of type III collagen. The significance of these findings in the evolution of diabetic glomerulosclerosis is discussed.
Intact Adipocyte Insulin-Receptor Phosphorylation and In Vitro Tyrosine Kinase Activity in Animal Models of Insulin Resistance
Authors: Truglia Joseph A , Hayes Gary R , Lockwood Dean H
Published: September 2013

We evaluated the possibility that impaired insulin-receptor kinase activity contributes to insulin resistance by examining in vitro receptor tyrosine kinase activity and in situ receptor phosphorylation in four models of insulin resistance. Adipocytes from streptozocin-induced nonketotic diabetic (STZ-D), glucocorticoid-treated, fasted, and chronically uremic rats showed reduced basal and maximally insulin-stimulated 2-deoxy-d-glucose transport compared with matched controls. Adipocytes from these models were also resistant to stimulation of hexose transport by hydrogen peroxide, a postbinding insulin mimicker. Changes in the number of insulin receptors per cell could not account for these alterations in transport. Cell surface 125I-labeled insulin binding was 142% of control in STZ-D and 129% with fasting and unchanged in glucocorticoid excess and chronic uremia. Insulin-stimulated tyrosine kinase was measured by means of a synthetic substrate, Glu80Tyr20. Partially purified receptors from these resistant models had unaltered kinase activity when normalized to soluble 125I-insulin binding. In situ stimulation of receptor phosphorylation by 7 and 100 nM insulin was determined after equilibration of adipocytes with 32PO4. Compared with matched controls, these intact cells, from all four resistant models, had insulin-stimulated receptor phosphorylation that was unchanged per unit of cell surface binding. Similar to results with insulin, hydrogen peroxide stimulation of in situ receptor phosphorylation was unchanged in each model. Thus, both in vitro and in situ measures of receptor phosphorylation suggest that the cellular alterations leading to insulin resistance in these adipocytes resides beyond phosphorylation of the insulin receptor.
Persistent Grossly Elevated Plasma Immunoglobulin A Levels in Untreated Streptozocin-Induced Diabetic Rats
Authors: Miller Leon L , Izzo Mary Jane , Wemett Drusilla
Published: September 2013

Experimental diabetes mellitus was induced in adult male and female rats by injecting streptozocin (STZ; 60 mg/kg i.p.) in preparation for a screening survey of changes in the pattern of undenatured plasma proteins, as revealed by two-dimensional (2-D) gel electrophoresis followed by silver staining. As early as 8–12 days later, the 2-D gels revealed three high-molecular-weight plasma protein spots, which persisted for 150 days in the blood of untreated diabetic rats. Such spots were not seen in plasma of normal control rats. Evidence is presented for the presumptive characterization of these proteins as oligomers of immunoglobulin A (IgA). Specific measurement of total IgA content of diabetic plasma samples by single-radial immunodiffusion, after reduction with dithiothreitol and alkylation with iodoacetamide, reveals that IgA content increases linearly from control values of 11.1 ± 4.6 to 358 ± 249 mg/dl (means ± SE) 21 days after STZ and persists at these high levels for as long as 150 days. Diabetic rats injected daily with insulin showed IgA levels only two to four times higher than normal. Neither experiments designed to quantitate the rates of clearance (catabolism plus excretion) of 125I-labeled secretory IgA from the circulation of normal and diabetic rats nor measurement of total IgA in the bile from diabetic and normal bile fistula rats supports the view that slowed clearance from the circulation or impaired biliary excretion in the diabetic rat causes observed gross hyperimmunoglobulinemia A.
Morphometry of Rambourg-Positive and Rambourg-Negative β-Cell Granules After Culture With Low and High Glucose Concentrations
Authors: Norlund Robert , Norlund Lena , Täljedal Inge-Bert
Published: September 2013

Dispersed islet cells from noninbred ob/ob mice were cultured for 3 days with 3 or 20 mM D-glucose and silver stained according to Rambourg et al. Two tinctorial subsets of dark and light intracellular granules were analyzed by morphometry at the ultrastructural level. The two types of granules were similar in size and shape. However, with 3 mM glucose the dark granule cores were surrounded by larger vesicles than the light granules. With 20 mM glucose, both types of granule vesicles and cores became smaller and dark-granule cores became more rounded, compared with cultures with 3 mM glucose. The higher glucose concentration also induced a marked decrease in the number (−84%) and volume density (−90%) of dark granules. In contrast, the number of light granules increased (+60%) with maintenance of their volume density. We suggest that the dark Rambourg-positive and the light Rambourg-negative β-cell granules are functionally distinct subsets. The dark granules are probably engaged in insulin discharge. We discuss the unclear role of the light granules with a view to previously postulated heterogeneities of the insulin granule pool and their significance for exocytosis and intracellular hormone degradation.
Interferon-γ Induces Class II MHC Antigens on RINm5F Cells
Authors: Varey Anne-Marie , Lydyard Peter M , Dean Betty M , Van Der Meide Peter H , Baird Joyce D , Cooke Anne
Published: September 2013

The ability of recombinant interferon-γ (rIFN-γ) to induce major histocompatibility complex (MHC) antigen expression in the rat insulinoma cell line RINm5F was investigated. The cells were stained with monoclonal antibodies specific for rat class I and class II MHC antigens. RINm5F cells endogenously expressed class I antigens; this was enhanced by rIFN-γ. Class II antigens could not be detected on RINm5F cells, but both I-A and I-E were induced by rIFN-γ.
Discordance of Exocrine and Endocrine Growth After 90% Pancreatectomy in Rats
Authors: Brockenbrough J Scott , Weir Gordon C , Bonner-Weir Susan
Published: September 2013

As we have previously shown, by 8 wk after 90% pancreatectomy (PX) in the rat, there is considerable regeneration of both exocrine and endocrine pancreas. In this study we examine the growth of both the exocrine and endocrine tissue 3, 7, 14, and 21 days after PX by following the pancreatic content of insulin, glucagon, and amylase as well as the mitotic indices for exocrine and islet β-cells. By 7 days the pancreatic remnant weighed more than the anatomically equivalent tissue in the sham, the remnant equivalent. The growth of the exocrine tissue and the endocrine β-cells was discordant during these initial weeks after PX, as shown by the mitotic index. The mitotic index, measured as accumulated mitotic figures after a 4-h colchicine treatment, for both the exocrine and β-cells in the sham animals was low and unchanging at the different time points (∼0.5%). At 3 and 7 days after PX, both the exocrine and β-cells had mitotic indices three- to fourfold that of the sham animals. At 14 days after PX, the exocrine cells had a slightly, albeit significantly, elevated mitotic index, whereas that of the β-cells was still double that of the shams. By 21 days there was no difference in mitotic index for exocrine tissue, but the β-cells on the PX animals had a mitotic index still double that of the shams. Another index of growth, the cell birthrate, was estimated at 7 days from the slope of regression lines of the mitotic frequency accumulated 1, 2, 3, and 4 h after colchicine. The cell birthrates of both the exocrine cells (PX 3.8 ± 0.96%, sham 0.96 ± 0.72%) and the β-cells (PX 7.2 ± 1.7%, sham 0.72 ± 0.96%) were significantly elevated in the pancreatic remnant. Thus, both exocrine cells and endocrine β-cells undergo considerable growth in the initial 3 wk after PX. However, there is discordance in their growth with respect to each other. In addition, these adult β-cells have a greater and more sustained growth than previously thought possible. The PX model could therefore become a useful model for studying the mechanisms of both exocrine and endocrine regeneration.
Splanchnic Amino Acid and Glucose Metabolism During Amino Acid Infusion in Dogs
Authors: Ferrannini Eleuterio , DeFronzo Ralph A , Gusberg Richard , Tepler Jeffery , Jacob Ralph , Aaron Michael , Smith Douglas , Barrett Eugene J
Published: September 2013

With the organ-balance technique, we studied amino acid and glucose metabolism by hepatic and extrahepatic splanchnic tissues in awake dogs in the postabsorptive state and during a 3-h intravenous amino acid infusion. Dogs received a high (1.4 g/kg body wt, n = 5) or low (0.7 g/kg body wt, n = 8) dose of amino acids. In four of the latter dogs, the dose was delivered into a mesenteric vein. During the basal period there was a net removal of gluconeogenic amino acids (particularly alanine), but not branched-chain amino acids, and a net production of glucose by the liver in all dogs. During this time there was a net removal of glucose and production of alanine by the extrahepatic splanchnic tissues. During either high- or low-dose amino acid infusion, net hepatic glucose release increased; despite this, arterial plasma glucose declined due to an increase in tissue glucose uptake at extrasplanchnic sites. The net amount of glucogenic amino acids removed by the liver during high-dose (9.1 ± 1.0 mmol · kg−1 · 3 h−1) and low-dose (4.8 ± 0.6 mmol · kg−1 · 3 h−1) infusion equaled or exceeded the infused load of these amino acids. In addition, the liver contributed to the net disposal of branched-chain amino acids during high-dose (536 ± 147 μmol · kg−1 · 3 h−1) and low-dose (341 ± 70 μmol · kg−1 · 3 h−1) infusion. During high-dose infusion, extrahepatic splanchnic tissues participated in the net removal of branched-chain amino acids (436 ± 162 ¼mol · kg−1 · 3 h−1) but not glucogenic amino acids, and net alanine production continued (410 ± 91 μmol · kg−1 · 3 h−1). The route (peripheral or mesenteric) did not appear to influence the subsequent disposal of the amino acid load. We conclude that during intravenous amino acid administration in the dog, the splanchnic bed is the major site of net amino acid disposal. The liver contributes to the net disposal of both glucogenic and branched-chain amino acids, whereas extrahepatic splanchnic tissue contributes to the net disposal of only the branched-chain amino acids. Overall, only 10–15% of the total infused amino acids was removed by extrasplanchnic (peripheral) sites, despite increased concentrations of plasma amino acids and elevated plasma insulin concentrations. These tissues may replete nitrogen stores during feeding by mechanisms other than removal of circulating amino acids.
Induction of Nesidioblastosis Will Reverse Diabetes in Syrian Golden Hamster
Authors: Rosenberg Lawrence , Duguid William P , Brown Rea A , Vinik Aaron I
Published: September 2013

Nesidioblastosis, which is the formation of new islets and the differentiation of cells within the islets, represents part of the spectrum of hyperfunctioning states of the islets of Langerhans at the clinical level. Nesidioblastosis in the Syrian golden hamster can be induced by wrapping the head of the pancreas with cellophane tape. Ligation of the duct is not involved, and acinar cell atrophy does not occur. The purpose of this study was to determine whether the induction of nesidioblastosis could be used as a means of reversing streptozocin-induced diabetes. Outbred hamsters (n = 32), 8 wk of age, were rendered diabetic by treatment with 40 mg/kg i.p. streptozocin, administered daily for 3 days. Four days later, 16 animals chosen at random underwent laparotomy with cellophane wrapping of the pancreas. Before surgery, the serum glucose and insulin levels (means ± SE) in the unoperated control animals (389.0 ± 18.6, 33.9 ± 3.8) did not differ from those in the animals awaiting the operation (373.2 ± 18.6, 37.9 ± 3.8). After 7 wk, 50% of the operated animals had serum glucose and insulin levels that were normal, compared to only 12% of the unoperated control animals (χ2 = 5.53, P < .05). Islets from normoglycemic operated animals were characterized by increased numbers, including many small islets, positive immunoreactive insulin staining, and minimal vacuolation of cells. Islets from hyperglycemic operated hamsters and from the unoperated control animals were decreased in number and generally larger in size, demonstrated little or no immunoreactive insulin staining, and exhibited marked vacuolation of cells. It is concluded that cellophane wrapping of the pancreas induces the formation of islets and endocrine cells that are functionally capable of reversing streptozocin-induced diabetes.
Eicosanoids as Pluripotential Modulators of Pancreatic Islet Function
Authors: Robertson R Paul
Published: September 2013

Eicosanoids both negatively and positively modulate glucose-induced insulin secretion. Although the identity of the positive modulator is uncertain, the negative modulator appears to be prostaglandin E2 (PGE2), because 1) glucose stimulates PGE2 synthesis from islet cells; 2) exogenous PGE2 inhibits glucose-induced insulin secretion; 3) inhibition of β-cell PGE2 synthesis increases glucose-induced insulin secretion, and this increase is reversed by exogenous PGE2; and 4) PGE2 binds to specific β-cell receptors that are coupled to inhibitory regulatory components of adenylate cyclase whose activation decreases cAMP levels. Other possible regulatory effects of eicosanoids on islet function include modulation of islet blood flow and its immune responsiveness. From these considerations, the perspective is offered that eicosanoids are pluripotential modulators of islet function.
Evidence for Insulinotropic Effect From Rat Parotid Glands
Authors: Leonora John , Tieche Jean-Marc , Cook Denise S
Published: September 2013

Previous observations have suggested that the salivary glands exercise a regulatory role on insulin secretion and/or glucose metabolism. We have challenged the issue by studying in unanesthetized, unrestrained rats the short- and long-term effect of selective sialoadenectomy on the animals' ability to meet an intra-arterial glucose challenge. Ten-minute intra-arterial glucose tolerance tests were carried out in chronically catheterized adult rats before and after sialoadenectomy. Eighty-five to 100 days postsurgery, the parotidectomized rats experienced a 45% reduction in plasma immunoreactive insulin output (P < .001) compared with the sham-operated animals; the plasma glucose levels of the test subjects remained 19% higher (P < .001) than those of the control group. In younger rats, similar observations were made; however, the difference in insulin and glucose responses between treatments was less than in the adult rats. Our findings suggest that the insulinotropic effect resides primarily with the parotids, and the role of the submandibular glands seems to be permissive at best. We hypothesize that parotidectomy deprives the β-cells of a humoral principle that appears to be essential for optimizing the immediate insulin response to a glucose challenge. These results suggest that the insulinotropic effect of the parotids is of particular importance when aging changes insulin secretion and action.
Deficient Axonal Transport of Substance P in Streptozocin-Induced Diabetic Rats: Effects of Sorbinil and Insulin
Authors: Tomlinson David R , Robinson Jean P , Willars Gary B , Keen Peter
Published: September 2013

This study measured the accumulation of substance P–like immunoreactivity (SPLI) proximal and distal to 12-h constricting ligatures applied to rat sciatic nerves. There were three separate experiments, and the baseline for each consisted of control and age-matched rats with 3 wk of untreated streptozocin-induced diabetes. We compared the effects of twice-daily insulin treatment, daily sorbinil (25 mg · kg−1 · day−1 p.o.), and a combination of both treatments. In untreated diabetic rats the anterograde accumulation of SPLI was reduced by 30–40%. This deficit was unaffected by sorbinil alone but was attenuated by insulin and prevented completely by insulin and sorbinil combined. There were also indications that diabetes caused reductions in retrograde accumulation of SPLI and its content in unconstricted nerve and the L4 dorsal root ganglion. The fraction of SPLI undergoing net anterograde or retrograde movement and the velocities of accumulation were unaffected by diabetes or the treatment regimens. These findings indicate a reduction in the amount of substance P moved by axonal transport in diabetic rats that is related partly to aldose reductase activity and partly to some other insulin-correctable consequence of experimental diabetes.