bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Analysis of Pancreatic Proinsulin mRNA in Genetically Diabetic (db/db) Mice
Authors: Orland Matthew J , Permutt M Alan
Published: September 2013

C57BL/KsJ db/db mice develop hyperphagic obesity and nonketotic diabetes similar to non-insulin-dependent diabetes mellitus in humans. Initially the mice demonstrate an abundant β-cell mass and hyperinsulinemia, which is followed by apparent β-cell loss. As an index of insulin synthesis, this study assesses pancreatic proinsulin mRNA, measured by dot hybridization to cloned cDNA, during the development of diabetes in the mice. Changes in proinsulin mRNA from 5 to 13 wk of age are compared with serum insulin, pancreatic insulin content, and blood glucose. In control (+ /db) mice, total proinsulin mRNA and pancreatic insulin content increased with age. Both changes were proportional to an increase in body weight. Obesity, hyperglycemia, and hyperinsulinemia were evident in diabetic (db/db) mice at 5 wk of age. Although pancreatic insulin content was comparable to that in the + /db controls at 5 wk, a fourfold relative elevation of proinsulin mRNA was observed. Despite an increase in body weight, proinsulin mRNA concentration and total proinsulin mRNA fell to levels similar to those of the control mice at 10 and 13 wk, associated with a loss of hyperinsulinemia, a mild decrease in pancreatic insulin content, and a marked increase in fasting blood glucose. A separate group of db/db mice was pair fed with the + /db controls from 4 to 13 wk. These diet-restricted diabetic mice were heavier than control mice and gained weight with age, but they weighed less than the unrestricted mice at all ages. Compared with the unrestricted db/db mice, a more modest fasting hyperglycemia was apparent, and a persistent hyperinsulinemia was observed. This group also preserved a high proinsulin mRNA concentration and showed a less marked fall in total proinsulin mRNA. Pancreatic insulin content was lower than that of controls, but a modest increase occurred with age.

A strong correlation between serum insulin and total proinsulin mRNA is apparent in this study. Hyperinsulinemia in young diabetic mice is associated with abundant proinsulin mRNA, but occurrence of hyperglycemia and reduced pancreatic insulin content suggests that the ability to augment insulin synthesis is limited and insufficient to meet metabolic needs. The deterioration of hyperinsulinemia with age, accompanying more profound fasting hyperglycemia, is associated with a reduction of insulin synthesis reflected by a decreased total proinsulin mRNA. The loss of insulin synthetic capacity in these diabetic mice can be lessened by diet restriction, suggesting that the maintenance of total proinsulin mRNA in diet-restricted db/db mice may be important in ameliorating their diabetic state.
Glucose Tolerance, Insulin Release, and Insulin Sensitivity in Normal-Weight Women With Previous Gestational Diabetes Mellitus
Authors: Efendić Suad , Hanson Ulf , Persson Bengt , Wajngot Alexandre , Luft Rolf
Published: September 2013

Out of 57 women with previous histories of gestational diabetes (GD), 23 were of normal weight postpartum and willing to participate in three studies characterizing oral glucose tolerance (OGTT), insulin responsiveness to intravenous glucose (glucose infusion test, GIT), and insulin sensitivity (somatostatin, insulin, and glucose infusion test, SIGIT). The experiments were performed 6-36 mo after cessation of breast-feeding. The control group comprised 10 healthy women with normal OGTT matched for age and weight. Among subjects with previous histories of GD, 9 had normal, 8 borderline, and 6 decreased OGTT. As a group, women with previous histories of GD have significantly decreased insulin response and insulin sensitivity. Furthermore, all 14 with borderline and decreased OGTT demonstrated a low early insulin response during GIT (5-min value below the upper border of the lower quartile of normals), whereas insulin sensitivity was normal in 6 and low in 8 (glucose values attained during SIGIT were lower di higher, respectively, than the lower border of the upper quartile of controls). The women with previous histories of GD and normal OGTT exhibited normal (n = 4) and low (n = 5) insulin responses. Three of the former subjects had low and the remaining 6 had normal insulin sensitivity. In conclusion, as many as 60% of normal-weight women with previous histories of GD had borderline or decreased OGTT 6- 36 mo postpartum. This derangement could be due to impaired early insulin response, which in some subjects was combined with low insulin sensitivity. Follow- up of women with previous histories of GD might enlighten the pathogenesis of non-insulin-dependent diabetes mellitus.
Glyburide Sensitizes Perfused Rat Liver to Insulin-Induced Suppression of Glucose Output
Authors: McGuinness Owen P , Green Donald R , Cherrington Alan D
Published: September 2013

Glyburide, a second-generation sulfonylurea, is used in the treatment of NIDDM because of its hypoglycemic action. However, the site and mechanism of action of this sulfonylurea remain unclear. We examined the ability of glyburide to enhance insulin's inhibitory effect on glucagon-stimulated hepatic glucose production.

The livers of fed male rats were perfused with a Krebs-Henseleit buffer containing washed human red blood cells. After a 60-min control period during which the liver was exposed to both insulin and glucagon (10 μU/ml and 11 pg/ml, respectively), the glucagon concentration was increased to 88 pg/ml in the presence of 0, 10, 40, and 240 μU/ml of insulin. Hepatic glucose output and phosphorylase a activity were monitored during the control and elevated-glucagon periods. The glyburide-infused group received glyburide (1.6 μg/ml) during both the control and elevated-glucagon periods. As expected, high levels of insulin suppressed glucagon- stimulated glucose production and phosphorylase activation. Insulin at a concentration of 10 μU/ml was unable to suppress glucagon's stimulation of glucose production or its activation of phosphorylase. However, in the presence of glyburide it was able to decrease stimulated hepatic glucose production and phosphorylase activation by 40 and 50%, respectively. In the absence of insulin, glyburide was unable to suppress glucagon's glycogenolytic action, suggesting that the drug potentiates insulin's action on the liver rather than exerting an inhibitory effect directly. Insulin at a concentration of 240 μU/ml completely suppressed glucagon action, and glyburide had no additional effect. Therefore, glyburide is able to enhance the sensitivity of the perfused rat liver to insulin with-out altering maximal insulin responsiveness.
Fuel-Stimulated Insulin Secretion by Clonal Hamster β-Cell Line HIT T-15
Authors: Meglasson Martin D , Manning Carol D , Najafi Habiba , Matschinsky Franz M
Published: September 2013

Insulin secretion by monolayer cultures of HIT T-15 cells was measured in response to various fuel molecules (glucose, dihydroxyacetone, lactate, glutamine, α-ketoisocaproic acid, α-ketoisovaleric acid) and a nonmetabolized glucose analogue (3-O-methylglucose). HIT cells secreted insulin in response to fuel molecules, but 3-O-methylglucose was ineffective. Stimulation of insulin release by fuels was increased by isobutylmethylxanthine and blocked by antimycin A. lodoacetate selectively inhibited glucose-stimulated insulin release but had little effect on α-ketoisocaproic acid-stimulated insulin secretion. These results indicate that HIT cells retain the capacity of normal β-cells to act as fuel sensors. Thus, HIT cells may provide a well-defined and relatively abundant tissue source in studies of stimulus-secretion coupling in β-cells stimulated by fuels.
Relationshjp of Organ Lipoprotein Lipase Activity and Ketonuria to Hypertriglyceridemia in Starved and Streptozocin-lnduced Diabetic Rats
Authors: Wilson Dana E , Zeikus Regina , Chan Ing-Fong
Published: September 2013

Although lipoprotein lipase (LPL) is believed to be rate limiting in the catabolism of triglyceride-rich lipoproteins, LPL activity has not correlated with plasma triglyceride concentrations in experimental rat diabetes. To gather more information about this enzyme system in diabetes, LPL activities were measured in representative tissues from control and streptozocin-induced diabetic rats fed fat-free chow and in 48-h-starved animals. The DNA content of each tissue was determined so that LPL activity could be expressed in a way that was unaffected by tissue wasting. Diabetic animals lost ∼20% of their body mass. Adipose tissue and soleus muscle cell masses were reduced, and there was marked fat atrophy at necropsy. Adipose tissue LPL was decreased in both starved and diabetic animals, whereas skeletal muscle activities were variably affected. Lipase content and distribution among the individual organs were calculated with published data for rat carcass composition. In diabetic rats, total LPL (adipose tissue, muscle, and parenchymal organs) was reduced by nearly two-thirds so that skeletal muscle became the predominant source of LPL. Ketonuria was less frequent in diabetic than in starved rats (P < .018) despite their severe wasting. Serum triglyceride concentrations were higher in ketonuric than nonketonuric diabetic animals, and severe hypertriglyceridemia was seen exclusively in heavily ketonuric animals. These observations together with published information suggest that plasma triglyceride concentrations in the rat model are determined by a complex interplay between very-low-density lipoprotein synthesis, the capacity of the LPL removal system, properties of the lipoprotein substrate, and other unidentified factors.
Evidence for Presence of Proinsulin Immunoreactive Glycoprotein(s) in Fetal Bovine Pancreatic Extracts
Authors: Tung A K , Siu K P
Published: September 2013

A large-molecular-weight proinsulin-immunoreactive protein fraction was obtained from an extract of fetal bovine pancreases by gel filtration in 6 M guanidine- 1 M acetic acid. Concanavalin A-Sepharose-affinity column chromatography of the large-molecular-weight fraction yielded a discrete α-methyl-mannoside-displaceable immunoreactive peak that also displayed Nacetylglucosamine- specific binding to wheat germ lectin- Sepharose. Chemically tritiated and radioiodinated lectin-reactive proteins interacted specifically with antibodies to insulin and bovine proinsulin. Immunochemically purified (reaction with antibodies followed by separation of antigen-antibody complexes on protein A-Sepharose) radiolabeled lectin-reactive proteins were analyzed by gel filtration in guanidine-acetic acid and by sodium dodecyl sulfate polyacrylamide gel electrophoresis after disulfide bond-cleavage treatments. Results from these studies suggest the existence of an ∼67,000-Mr glycoprotein that contains antigenic domains common to proinsulin and insulin.
Preventive Effect of Monoclonal Anti-L3T4 Antibody on Development of Diabetes in NOD Mice
Authors: Koike Takao , Itoh Yohjiro , Ishii Tatsuko , Ito Isao , Takabayashi Katsuhiko , Maruyama Naoki , Tomioka Hisao , Yoshida Sho
Published: September 2013

We analyzed the surface phenotypes of infiltrated cells in pancreases of nonobese diabetic (NOD) mice with monoclonal antibodies to mouse lymphocytes. Most of the infiltrated cells were Thy1+ and Ly1+ T-lymphocytes, and most of them were L3T4+ helper T-lymphocyte subsets. To elucidate the role of L3T4+ T-lymphocytes in the development of insulitis and diabetes in NOD mice, we treated the animals with injections of monoclonal anti-L3T4 antibody. Administration of this antibody prevented the insulitis and diabetes in NOD mice. These results lead to conclusions that the L3T4+ helper T-lymphocytes may play an essential role in the pathogenesis of type I diabetes and that the manipulation of the OKT4+ (Leu3+) T-lymphocyte subset, the human homology of L3T4, with monoclonal antibodies may provide effective therapy for human type I diabetes.
Functional Nature of Glomerular Injury in Progressive Diabetic Glomerulopathy
Authors: Tomlanovich Stephen , Deen William M , Jones Henry W , Schwartz Herbert C , Myers Bryan D
Published: September 2013

We describe in physiological terms the increasing glomerular capillary wall (GCW) dysfunction of 20 patients with diabetic glomerulopathy and heavy proteinuria. The clearances of uncharged polysaccharide markers of graded size were used to probe the glomerular filter on three occasions over a 24-mo period. The findings were analyzed with a theoretical model of solute transport that depicts most of the GCW as an isoporous membrane and the minor portion as a nondiscriminatory shunt pathway. Initially, the mean glomerular ultrafiltration coefficient Kf is computed to have been 3-5 times lower and mean pore radius of the major membrane component (r0) 2 Å smaller than normal control values. In contrast, the model computes the fraction of filtrate volume permeating the nondiscriminatory shunt pathway (ω2) to have been sixfold elevated above control values and to have correlated strongly in individual patients with the fractional clearances of albumin (r = .72) and of IgG (r = .73). Sequential studies after 12 and 24 mo revealed an invariable decline in glomerular filtration rate (GFR). Fractional clearances of albumin and IgG increased with time in most patients but declined in a few instances (20-25%). Change in ω2 tended to occur in parallel with fractional protein clearance, regardless of its direction. We conclude that in progressive diabetic glomerulopathy 1) GFR declines because of a loss by glomerular capillaries of ultrafiltration capacity, 2) proteinuria is largely a consequence of increasingly impaired barrier-size selectivity, and 3) the foregoing injuries reflect damage to different parts of the GCW and may become dissociated from one another with the passage of time.
Involvement of Endogenous Opiates in Glucose-Stimulated Hyperinsulinism of Canine Endotoxin Shock: Inhibition by Naloxone
Authors: Merrill Gerald A , Anderson James H
Published: September 2013

Hyperinsulinism has been associated with infection and endotoxin shock in rodents, dogs, and humans. In dogs with Escherichia coli-induced endotoxin shock, this hyperinsulinism was in response to glucose administration.

To determine the role of endogenous opiates in endotoxin-induced glucose-stimulated hyperinsulinism, plasma β-endorphin, Met-enkephalin, Leu-enkephalin, insulin, and glucose concentrations were measured for 6 h in fasted, anesthetized dogs given 1) LD70 of E. coli endotoxin; 2) endotoxin and glucose; 3) endotoxin, glucose, and naloxone (an opiate antagonist); 4) glucose and naloxone; or 5) glucose alone.

Plasma endogenous opiate immunoreactivity was elevated in dogs that received endotoxin, regardless of the presence of glucose or naloxone. The elevation of plasma Met-enkephalin and β-endorphin preceded the onset of hyperinsulinism, but the elevation of plasma Leu-enkephalin did not. Plasma insulin was elevated 100-fold by 360 min in dogs given endotoxin and glucose. The magnitude of this hyperinsulinism was markedly reduced by naloxone, supporting the hypothesis that endogenous opiates are involved in the development of the glucose-stimulated hyperinsulinism associated with endotoxin shock. Interestingly, naloxone, given in conjunction with glucose, appeared to have a stimulatory effect on insulin secretion.
Insulin-Receptor Kinase Activity of Adipose Tissue From Morbidly Obese Humans With and Without NIDDM
Authors: Sinha Madhur K , Pories Walter J , Flickinger Edward G , Meelheim Diane , Caro José F
Published: September 2013

We have determined glucose transport, insulin binding, and insulin-receptor kinase activity in adipose tissue from morbidly obese patients with and without non-insulin-dependent diabetes mellitus (NIDDM). The insulin sensitivity and responsiveness of glucose transport in freshly isolated adipocytes were significantly reduced in NIDDM subjects compared with nondiabetics. This was due in part to decreased insulin binding in adipocytes. Reduced specific 125I-labeled insulin binding was also observed in crude detergent extracts and partially purified insulin receptors from adipose tissue. In addition, the basal and insulin-stimulated tyrosine-specific protein kinase activity per milligram of protein was significantly decreased in NIDDM patients compared with nondiabetics. The differences between maximally insulin-stimulated and basal kinase activities expressed by insulin-binding activity were also significantly reduced in NIDDM subjects. We conclude that insulin resistance in morbidly obese patients with NIDDM is due to both insulin-binding and postbinding defects. One of the postbinding defects in NIDDM appears to be impaired insulin-receptor kinase activity of fat tissue.
Treatment With Artificial β-Cell Decreases Very-Low-Density Lipoprotein Triglyceride Synthesis in Type I Diabetes
Authors: Dunn Fredrick L , Carroll Patricia B , Beltz William F
Published: September 2013

The effect of restoration of euglycemia with the artificial β-cell (Biostator GCIIS) on triglyceride metabolism was studied in seven normolipidemic patients with type I diabetes mellitus. Very-low-density lipoprotein triglyceride (VLDL-TG) transport was determined with [3H]glycerol as an endogenous precursor of VLDL-TG; the resultant kinetic data were evaluated by multicompartmental analysis. Studies of triglyceride metabolism were performed in diabetic patients taking their usual dose of subcutaneous insulin (control study) and after 72 h of euglycemia with the artificial β-cell (Biostator study). Treatment with the artificial β-cell resulted in a decrease in mean (±SE) 24-h plasma glucose levels from 199 ± 9 to 123 ± 7 mg/dl and an increase in mean plasma freeinsulin levels from 12.3 ± 1.9 to 27.6 ± 4.2 μU/ml (P < .05). These changes were accompanied by a decrease in mean plasma TG levels from 134 ± 29 to 88 ± 15 mg/dl (P < .05). Kinetic studies demonstrated that the change in plasma triglyceride levels was primarily due to a decrease in VLOL-TG transport (i.e., synthesis), which fell from 11.7 ± 2.5 mg · h−1 · kg−1 ideal wt during the control study to 7.5 ± 2.0 mg · h−1 · kg−1 ideal wt during the Biostator study (P < .05). There was no significant change in fractional catabolic rates of VLDL-TG between the two studies (0.35 ± .05 vs. 0.38 ± .07 h−1). Thus, our data suggest that the decrease in plasma TG levels after short-term restoration of euglycemia with the artificial β-cell is due to a decrease in VLDL-TG synthesis.
Disturbances in Glomerular Basement Membrane Glycosaminoglycans in Experimental Diabetes
Authors: Wu Van-Yu , Wilson Bryan , Cohen Margo Panush
Published: September 2013

Glycosaminoglycans (GAGs) were purified from basement membranes isolated from glomeruli of control and streptozocin-induced diabetic rats and were quantitatively analyzed with a recently described competitive binding assay that is specific for and sensitive to microgram amounts of chondroitin and heparan sulfate. Total GAG content in glomeruli from diabetic rats and in the basement membranes prepared from these samples (17.22 ± 1.45 and 6.56 ± 0.49 μg/105 glomeruli, respectively) was significantly less than that found in comparable control preparations (43.71 ± 3.35 and 16.05 ± 1.41 μg/105 glomeruli, respectively). The portion of total GAG in the water-soluble fraction recovered after osmotic lysis of isolated glomeruli was also markedly decreased in diabetic samples (26.11 ± 4.55 vs. 3.30 ± 0.32 μg/105 glomeruli, control vs. diabetic). Treatment of lysed glomeruli with the ionic detergent deoxycholate, required for liberation of the extracellular matrix from plasma membrane lipoproteins and purification of the insoluble glomerular basement membrane (GBM), resulted in solubilization of ∼10% of the waterinsoluble GAG in control samples but >50% in diabetic membranes. Heparan sulfate comprised >90% of the GAGs in both control and diabetic GBM, defined as the water- and detergent-insoluble matrix. The findings clearly demonstrate that the GAG content of GBM is diminished in experimental diabetes and provide evidence that the reduction in GBM anionic sites associated with diabetes derives from a decrease in the constituent GAGs of this extracellular matrix. The results further suggest that the interaction between GBM and populations of GAG associated with the surface of plasma membranes of adjacent cells is disturbed in diabetes.
Lack of Rapid Enhancement of Insulin Action After Oral Glucose Challenge in Myotonic Dystrophy
Authors: Moxley Richard T , Kingston William J , Griggs Robert C , Livingston James N
Published: September 2013

Oral glucose administration to normal humans stimulates insulin release and simultaneously enhances the action of insulin by producing a rapid increase in tissue insulin sensitivity by a mechanism separate from the amount of hormone released. We determined whether insulin-resistant patients with myotonic dystrophy lose the ability to produce the normal rapid increase in tissue insulin action after oral glucose. Nine ambulatory, nonobese men with myotonic dystrophy were studied with 120-min euglycemic insulin infusions (20 mU · M−2 · min−1) given before and after glucose ingestion (4 and 5 patients received 15- and 25-g loads, respectively). Identical studies were performed in nonobese normal volunteers (16 and 13 patients received 15- and 25-g oral glucose loads, respectively). Glucose infusion rates at 20-120 min (GIR20-120) during euglycemic insulin infusions without prior glucose were 2.87 ± 0.6 mg · kg−1 · min−1 in patients with myotonic dystrophy compared to 4.70 ± 0.3 mg · kg−1 · min−1 in normal subjects. Euglycemic insulin infusions after glucose ingestion were begun after arterialized blood glucose values had returned to baseline. After glucose ingestion by normal subjects, GIR20-120 increased by 44.4 ± 7.1% (P < .0001) and by 46.8 ± 8.6% (P < .0002) with 15- and 25-g glucose loads, respectively. GIR20-120 in the nine patients with myotonic dystrophy showed no significant increase after glucose ingestion. These results confirmed the existence of a decrease in whole-body insulin sensitivity in myotonic dystrophy and indicated that the patients lack the normal mechanism that enhances insulin action after oral glucose. This deficiency in insulin action may diminish the normal anabolic effect of insulin and may be a contributory factor for the muscle wasting seen in these patients.
Direct Stimulation of Na+-K+-ATPase and Its Glucosylated Derivative By Aldose Reductase Inhibitor
Authors: Garner Margaret H , Spector Abraham
Published: September 2013

In the presence of 10−8 M concentrations of the aldose reductase inhibitor AL 1576, there is a 20-30% increase in the rate of hydrolysis of near-saturating concentrations of ATP by bovine renal Na+-K+-ATPase. When bovine renal Na+-K+-ATPase is reacted with glucose 6-phosphate in the presence of 10−8 M concentrations of AL 1576 or 10−6 M concentrations of a second aldose reductase inhibitor, sorbinil, glucosylation occurs. Whereas sorbinil has no effect on ATP hydrolysis by the glucosylated Na+-K+-ATPase, 10−8 M AL 1576 causes a shift in the kinetics of hydrolysis of ATP from substrate inhibition to normal substrate activation. The aldose reductase inhibitors interact with the enzyme at the low-affinity ATP-binding site.
Regulation of Insulin-Receptor mRNA Levels by Glucocorticoids
Authors: McDonald Alex R , Maddux Betty A , Okabayashi Y , Wong K Y , Hawley D M , Logsdon Craig D , Goldfine Ira D
Published: September 2013

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulinreceptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.
β-Cell Function and Insulin Sensitivity in Nondiabetic HLA-identical Siblings of Insulin-Dependent Diabetics
Authors: Johnston Colin , Raghu Prema , McCulloch David K , Beard James C , Ward W Ken , Klaff Leslie J , McKnight Barbara , Bergman Richard N , Palmer Jerry P
Published: September 2013

Insulin secretion and insulin sensitivity were compared in 12 HLA-identical siblings of insulin-dependent diabetics and nondiabetic controls. Only the maximum acute insulin response to intravenous arginine was lower in the siblings than in the matched controls (P < .05); other measures of insulin secretion, including the acute insulin response to glucose or arginine, the second-phase insulin response to glucose, and the slope of glucose potentiation, were not significantly different. Insulin sensitivity, derived from an intravenous glucose tolerance test with a minimalmodeling technique, was lower in the siblings (P < .01). In a large group of nondiabetic controls of various adiposity, insulin secretion and insulin sensitivity were inversely related. In view of the difference in insulin sensitivity between siblings and matched controls, a direct comparison of β-cell function tests may be inappropriate, and the measures of insulin secretion were compared with those of nondiabetics when adjusted for differences in insulin sensitivity. This analysis revealed that all measures of insulin secretion were significantly lower in the siblings. We conclude that HLA-identical siblings of insulin-dependent diabetics show evidence of both insulin resistance and impaired β-cell function and that analysis of β-cell function in relation to insulin sensitivity shows a greater frequency of β-cell secretory abnormalities than previously appreciated.
Antibodies to Covalent Aggregates of Insulin in Blood of Insulin-Using Diabetic Patients
Authors: Robbins David C , Cooper Sheldon M , Fineberg S Edwin , Mead Patricia M
Published: September 2013

A covalent aggregate twice the size of insulin accounts for ∼28% of total circulating insulin immunoreactivity in type I diabetic patients. These aggregates are probably covalent dimers of insulin and should contain unique epitopes distinct from the parent molecule. Therapeutic insulin contains a similar material and is the source of the circulating aggregate. Anti-aggregate antibodies were detected by binding-inhibition techniques in 9 of 29 long-term diabetic patients. These antibodies were directed against structures distinct from those of the parent molecule insulin monomer. All antibody-positive patients were men whose blood also contained antibodies to insulin monomer. We conclude that the blood of ∼30% of insulin-using diabetic patients contains antibodies directed against epitopes unique to the insulin aggregates. Because insulin monomer and aggregates probably share a common primary amino acid sequence, the anti-aggregate antibodies are probably directed against conformational determinants. Further work is needed to determine whether such aggregates promote or accentuate the development of anti-insulin antibodies in certain genetically predisposed individuals.
Hypersecretion of Gastric Somatostatin in Spontaneously Diabetic BB Rats
Authors: Ruggere M Doris , Patel Yogesh C
Published: September 2013

We previously reported that the BB diabetic rat is characterized by a reduction in pancreatic immunoreactive somatostatin (SLI) content, δ-cell mass, and δ-cell secretory reserve. Despite this, portal plasma SLI levels are elevated in diabetic animals and normalized by insulin therapy. These findings comprise indirect evidence for SLI hypersecretion by the gut in untreated BB rats. This study was undertaken with isolated stomach perfusions to investigate directly the secretory status of gastric δ-cells in this diabetic model. Isolated stomachs of three groups of insulin-treated diabetic, untreated diabetic, and nondiabetic control rats were perfused in situ under basal and glucagon-stimulated (5 nM) conditions. Untreated diabetic BB rats exhibited significant enhancement of basal and glucagonstimulated gastric SLI release. Insulin treatment reduced gastric SLI release to significantly subnormal levels. More than 95% of basal and stimulated SLI released in diabetic BB and normal control rats coeluted with synthetic somatostatin-14 on Sephadex G-50 columns. We conclude that 1) basal and stimulated gastric SLI release is increased in untreated BB rats and is suppressed with insulin therapy, 2) gastric δ-cell hyperfunction accounts for portal vein hypersomatostatinemia characteristic of untreated diabetic BB rats, and 3) somatostatin-14 is the main molecular form of SLI released from normal and diabetic stomachs.
Helper-Inducer T-Lymphocytes Mediate Diabetes in EMC-Infected BALB/c By J Mice
Authors: Haynes Mark K , Huber Sally A , Craighead John E
Published: September 2013

Diabetes mellitus developing in BALB/c ByJ mice infected with the M variant of encephalomyocarditis (EMC) virus is dependent on thymic immune mechanisms. We treated mice with the anti-Tlymphocyte monoclonal anti-L3T4 and anti-Lyt2.2 antibodies before virus inoculation and monitored the infection and occurrence of diabetes thereafter. Mice depleted of L3T4+ cells exhibited a reduced incidence and severity of diabetes compared with both untreated and anti-Lyt2.2-treated animals. All mice sustained pancreatic infections, but islet lesions with β-cell degranulation only occurred in control infected and anti-Lyt2.2-treated animals. These data support the conclusion that the induction of diabetes in EMC-infected BALB/c mice is immune mediated and controlled by the L3T4+ T-lymphocyte subpopulation.
Relationships Between Plasma Lipoprotein Concentrations and Insulin Action in an Obese Hyperinsulinemic Population
Authors: Abbott William G H , Lillioja Stephen , Young Andrew A , Zawadzki Joanna K , Yki-Järvinen Hannele , Christin Laurent , Howard Barbara V
Published: September 2013

Relationships have been observed between lipoprotein concentrations and insulin action. These relationships may be important in explaining the association of insulin resistance and abnormalities of lipoprotein metabolism found in obesity, diabetes, and hypertriglyceridemia. We have measured plasma lipoprotein concentrations and indices of insulin action in 85 men and 56 women, all of whom were normolipidemic and had normal glucose tolerance. The subjects were obese Southwestern American Indians (body mass index 34 ± 1). Insulin action was measured via the hyperinsulinemic clamp with simultaneous indirect calorimetry. Triglyceride concentrations were inversely related to rates of total insulin-mediated glucose disposal (in men and women, respectively, r = –.37, P < .01; r = –.24, P < .10), glucose storage (r = –.31, P < .01; r = –.25, P < .10), increase in glucose oxidation (r = –.29, P < .01; r = –.24, P < .10), and, in men only, suppression of endogenous glucose production (r = –.32, P < .01). High-density lipoprotein (HDL) cholesterol concentration was positively related to rates of total insulin-mediated glucose disposal (r –.35, P < .01; r = .33, P < .05), increase in carbohydrate oxidation (r = .40, P < .001; r = .39, P < .001), suppression of endogenous glucose production (r = .24, P < .05; r = .29, P < .05), and, in men only, glucose storage (r = .35, P < .001). The relationships between lipoprotein concentrations and insulin action were independent of adiposity and insulin concentrations (with the exception of HDL cholesterol in women), and the relationships of Triglyceride and HDL cholesterol with insulin action were independent of each other. These relationships indicate that there may be a link between the development of insulin resistance and the abnormalities in lipoprotein metabolism associated with obesity, non-insulin-dependent diabetes mellitus, and hypertriglyceridemia.
Morphometric Studies of Secretory Granule Distribution and Association With Microtubules in β-Cells of Rat Islets During Glucose Stimulation
Authors: Yorde Donald E , Kalkhoff Ronald K
Published: September 2013

Morphometric studies of β-cell granule positions and interactions with microtubules in areas of exocytosis were performed on islets of rats injected with tolbutamide for 72 h. This treatment depleted granules 85% below normal control values and rendered mapping of their positions and visualization of microtubules more feasible during electron microscopy. Islets were perifused with 2.8 or 16.7 mM glucose and were fixed at 4 min (acute phase) or 20 min (second phase). Photomicrographs of β-cell sections were digitized, and computer replicas were compared to computer-simulated cells of similar size and shape into which an identical number of granules was inserted randomly. Margination of granules into a zone within 1.5 μm of the plasma membrane was examined. At both 2.8 and 16.7 mM glucose, real cells marginated significantly more secretory granules than did simulated cell pairs. Within the zones of margination during 16.7 mM glucose perifusion, the number of secretory granules associated with microtubules was <6% in the acute phase and <1% in the second phase. These data suggest that glucose stimulation promotes movement of secretory granules into the β-cell periphery to a greater extent than can be attributed to chance alone. The role of microtubules in peripheral granule movement into regions of exocytosis appears to be minimal.
Effects of Insulin and Glucose on Cultured Rat Hepatocyte Gene Expression
Authors: Mooradian Arshag D , Mariash Cary N
Published: September 2013

We previously used isolated adult rat hepatocyte cultures to study the ability of glucose to induce several hepatic mRNAs. However, we found that the optimal insulin concentration required to obtain the glucose effect was > 10,000 μU/ml. To test the hypothesis that the requirement for high concentrations of insulin in the culture was due to rapid loss of insulin in hepatocyte cultures, serial measurements of insulin were made at different media insulin concentrations (0–500,000 μU/ml) and glucose concentrations (5.5 and 2.75 mM). In addition, a dose-response relationship was established between media insulin concentrations and the pattern of mRNAs present in the hepatocytes determined by two-dimensional gel electrophoresis of in vitro translation products. We found that at low insulin concentrations (1000 μU/ml), 80% of the insulin was lost to the glassware, whereas at high initial insulin concentrations, ∼23% of the insulin was lost to the glassware. Placement of media into the hepatocyte culture led to further insulin disappearance with a half-life for insulin of 41.5 h at 10,000 μU/ml and 13.8 h at 100 μU/ml. We found 16 mRNAs were altered by insulin at 5.5 mM glucose and 9 mRNAs were changed by insulin at 27.5 mM glucose. After taking into consideration the distributional and metabolic losses of insulin, all but one mRNA responded to insulin within the physiologic range of portal insulin (<1–94 μU/ml). Our data indicate that the hepatocyte culture is an excellent model to study the physiologic effects of insulin on hepatic gene expression.
Insulin Treatment Improves Glucose-Induced Insulin Release in Rats With NIDDM Induced by Streptozocin
Authors: Kergoat Micheline , Bailbe Danielle , Portha Bernard
Published: September 2013

Insulin-deficient diabetes in humans, as well as in the neonatal streptozocin-induced rat model of non-insulin-dependent diabetes mellitus (NIDDM), are associated with islet β-cell insensitivity to glucose. We hypothesized that the chronic hyperglycemia- -hypoinsulinemia pattern causes this impairment of the glucose influence on insulin secretion. This study was designed to determine whether the glucose defect could be counteracted by normalizing the diabetic state in rats with NIDDM after insulin therapy. Mixte lente insulin (5 U · kg−1 · day−1) was given daily at 1700 h over 24 h or 5 consecutive days. Insulin secretion was studied the morning after the last insulin injection with the isolated perfused pancreas preparation. Fed basal plasma glucose levels decreased in diabetic rats from 183 ± 8 to 136 ± 10 mg/dl after the 1-day insulin treatment and to 135 ± 5 mg/dl after the 5-day insulin treatment (vs. 116 ± 3 mg/dl in control rats). Pancreatic insulin stores were not affected by insulin therapy.

Although the 1-day insulin treatment did not modify the lack of glucose response in the diabetic rats, the 5-day insulin treatment improved their glucose-induced insulin secretion. Moreover, insulin therapy improved the priming effect of glucose on a second stimulation with glucose. The return of this glucose effect was hardly detectable after the 1-day insulin therapy but was clearly present after the 5-day treatment. The hyperresponse to arginine, characteristic of the untreated diabetic rat, returned similar to that in controls after a 1-day insulin therapy, and it was again amplified at high glucose levels, although amplification remained lower than that of control rats. This indicates that the potentiating effect of glucose on the response to arginine was regained more precociously than the acute insulin response to glucose after insulin therapy. These data agree with the hypothesis that the chronic hyperglycemia-hypoinsulinemia in the NIDDM rat causes abnormal glucose influence on glucose- and arginine-stimulated insulin release.
Expression of Cytoplasmic Islet Cell Antigens by Rat Pancreas
Authors: Dib Sergio A , Colman Peter G , Dotta Francesco , Tautkus Maura , Rabizadeh Albert , Eisenbarth George S
Published: September 2013

A major problem in standardization of the islet cell cytoplasmic antibody (ICA) assay is variation in sensitivity of the different human pancreas substrates used in individual laboratories. To circumvent this problem, we have developed an assay that utilizes Wistar-Furth rat pancreas as substrate, an anti-islet monoclonal antibody (A2B5) to identify islets and fluorescein-conjugated protein A to identify patient autoantibodies. Sera from 85 control subjects, 27 type I diabetics, and 17 subjects at high risk for developing type I diabetes were assayed in parallel with our standard ICA assay on human pancreas substrate and with Wistar-Furth rat pancreas as substrate. Two sera from control subjects (2 of 85) were ICA positive with rat pancreas compared to 1 of 85 with human pancreas substrate. Sera from 11 of 27 type I diabetics and 15 of 17 sera from high-risk subjects were ICA positive with either rat or human pancreas substrate. A correlation between the specific islet fluorescence readings on human and rat pancreas sections was found with sera from high-risk and control subjects. Furthermore, end-point titers of an ICA-positive serum were identical with both assays. Finally, incubation of an ICA-positive serum with glycolipids, extracted from either human or Wistar-Furth rat pancreas, blocked subsequent ICA binding. These findings suggest that Wistar-Furth rat pancreas expresses an identical or similar autoantigen to human pancreas.
Sorbinil Partially Prevents Decreased Erythrocyte Deformability in Experimental Diabetes Mellitus
Authors: Robey Claude , Dasmahapatra Amita , Cohen Margo P , Suarez Sarah
Published: September 2013

The ability of red blood cells (RBCs) to undergo an adaptation in shape that permits passage through the smallest vessels is reportedly impaired in diabetes. Several hypotheses have been proposed to explain decreased erythrocyte deformability, which has been implicated in the pathogenesis of microvascular complications, but the mechanisms responsible for this change have not been clearly delineated. In view of the fact that sorbitol accumulates in RBCs in diabetes and the postulate that increased sorbitol could alter deformability properties, we examined the influence of the aldose reductase inhibitor sorbinil on erythrocyte deformability.

Erythrocyte deformability, determined as the volume of RBCs (VRBC) filtered per minute through 4.7-μm pore size filters, was significantly reduced in samples from diabetic rats compared with samples from controls (0.76 ± 0.03 vs. 0.97 ± .02 ml RBC/min; P < .001). In contrast, deformability of RBCs from diabetic animals treated with sorbinil was significantly greater than in untreated diabetes, although not completely normalized (0.88 ± 0.02; P < .01 vs. diabetic, and P < .02 vs. control). The reduced deformability characterizing cells from diabetic rats and its partial prevention by sorbinil persisted even when RBCs were washed to eliminate hyperglycemie and hyperviscous plasma. Thus, hyperviscosity per se is not responsible for the decreased deformability, and sorbinil can partially prevent this change despite persistent hyperglycemie. This effect may derive from sorbinil's action as an aldose reductase inhibitor and/or its ability to influence physicochemical properties of the erythrocyte membrane.
Prolonged Release of Insulin by Cholesterol-Matrix Implant
Authors: Wang Paul Y
Published: September 2013

Insulin dispersed in a pellet disk made by compressing an admixture with cholesterol was found to reduce hyperglycemia in streptozocin-induced diabetic Wistar rats. I evaluated the effect of insulin content. At 3% insulin, two pieces of quarter-disk implant (∼50 mg each) lowered blood glucose levels from >22 mM to 17.5 mM for only 3 days. Normalization of hyperglycemia was sustained from ∼10 days for the implant with 6% insulin content and to 24 ± 5 days for 8–10% insulin in the composition. Insulin content up to 50% resulted in hypoglycemia and shorter implant service life. Not all of the pellet disks prepared were active, especially those with 3% insulin content. However, when broken further into 1-mm3 chips, all became active, with onset of action in <2 h, and the extent of hyperglycemia reduction was reproducible. Three successive subcutaneous insertions of ∼80 mg chips with 10% insulin maintained normoglycemia for 2.5 mo. Because the implant materials are constituents of tissue, no biocompatibility problem is expected.
Sciatic Nerve ATPase Activity Is Unaffected in Diabetic Mutant C57BI/Ks (db/db) Mice
Authors: Bianchi Roberto , Boccasavia Emanuela , Vittadello Maurizio , Schiavinato Antonella , Gorio Alfredo
Published: September 2013

We investigated composite (total), Mg2+-(ouabain-resistant), and Na+-K+-(ouabain-inhibited) ATPase in sciatic nerves of diabetic mutant C57BI/Ks (db/db) and age-related littermate (db/ +) control mice at various ages (16, 26, and 40 wk). This is the first report indicating that nerve ATPase activities measured in vitro showed no deficit in mice with spontaneous diabetes. Thus, diabetic neuropathy in the genetically diabetic mouse may occur in the absence of Na+-K+-ATPase abnormalities, which were previously described in association with polyol pathway impairment in experimental diabetic and BB Wistar rats. Control and diabetic groups treated with ganglioside mixture for 30 days before death presented statistically insignificant differences. Therefore, the beneficial effect of gangliosides described in C57BI/KS (db/db) mice on electrophysiological and morphometrical parameters must be due to different pharmacological activities rather than to prevention of the decay or better maintenance of ATPase activity.
Prolongation of Islet Xenograft Survival by Cryopreservation
Authors: Coulombe Marilyne G , Warnock Garth L , Rajotte Ray V
Published: September 2013

Our attempt to reduce islet immunogenicity by slow cooling to −40°C, storage at −196°C, and rapid thawing is based on the differential susceptibility of various cell types to a freeze-thaw process. Five hundred rat islets (≥10 μm) were immediately implanted or cryopreserved and then implanted beneath the renal capsule of streptozocin-induced diabetic mice with or without an injection of anti-lymphocyte serum at the time of transplantation. Thirteen days after transplantation, all fresh xenografts had rejected, whereas 37.5% of cryopreserved grafts were still functioning. In immunosuppressed mice, 6.2% of fresh xenografts and 54.5% of cryopreserved grafts were functioning 19 days after transplantation. These results show that cryopreservation can extend xenograft survival.
Synergistic Effects of Adjuvants, Endotoxin, and Fasting on Induction of Diabetes With Multiple Low Doses of Streptozocin in Rats
Authors: Wright James R , Lacy Paul E
Published: September 2013

Three weekly intraperitoneal injections of complete Freund's adjuvant (CFA) and, 1 day later, low-dose streptozocin (STZ; 25 mg/kg i.p.) have been reported to cause immune destruction of β-cells and a gradual onset of diabetes mellitus. In this study, male Lewis rats were injected intraperitoneally with CFA and 1 day later with low-dose STZ; these were repeated at weekly intervals for 3 wk. The incidence of diabetes mellitus (nonfasted plasma glucose >200 mg/dl) in wk 1, 2, 3, and 4 was 50, 80, 93, and 100%, respectively. Rats receiving either CFA or STZ only did not develop diabetes. Injections of either the components of CFA (incomplete Freund's adjuvant and Mycobacterium butyricum), another granuloma-inducing organism (Listeria monocytogenes), or endotoxin before STZ induced diabetes, but the onset was slower and the diabetes was less severe than with CFA and STZ. Because intraperitoneal CFA injections caused peritoneal irritation, acute weight loss, and hypoglycemia on the day after injection, we examined whether fasting alone potentiated low-dose STZ. Fasting for 24 h before and 24 h after low-dose STZ caused diabetes that was similar in rapidity of onset and severity to that induced with CFA and STZ. Administration of CFA subcutaneously before STZ did not cause hypoglycemia or weight loss but did cause diabetes. Thus, the fasting effect of intraperitoneal CFA was not responsible for the induction of diabetes with CFA and STZ. These data indicate that immunologic adjuvants, endotoxin, and fasting all potentiate the diabetogenic action of low-dose STZ. The 50% incidence of diabetes in rats within 48 h of a single injection of CFA and low-dose STZ suggests that the immune system does not mediate diabetes in this model. Furthermore, transplants of islet isografts were not rejected by eight rats made diabetic with CFA and low-dose STZ.
Is Protein Kinase C Required for Physiologic Insulin Release?
Authors: Metz Stewart A
Published: September 2013

Extant data suggest that a Ca2+- and phospholipid-dependent protein kinase C (PKC) exists (as a single enzyme or possibly a family of related enzymes) in rodent β-cells. PKC activators probably induce secretion primarily through phosphorylation of key proteins, thereby sensitizing the exocytotic apparatus to Ca2+. PKC can be activated by several pharmacologic probes and by endogenous diacylglycerol (and possibly arachidonic acid) released by nutrient-activated phospholipases. Several nonspecific pharmacologic agents inhibit both PKC and physiologic insulin release. However, when a more specific inhibitor of PKC, H 7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], was studied, it did not reduce glucose-induced insulin secretion. Moreover, prolonged preexposure of islets to a phorbol ester (believed to induce selective depletion of PKC) also failed to substantially reduce the subsequent secretory response to glucose. Thus, indisputable evidence for an obligatory physiological role of PKC in the islet is still missing, and the enzyme's status as a critical coupling signal should be viewed as putative only.