bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin Resistance of Fat Cells from Spontaneously Diabetic KK Mice: Analysis of Insulin-sensitive Phosphodiesterase
Authors: Makino H , Kanatsuka A , Suzuki T , Kuribayashi S , Hashimoto N , Yoshida S , Nishimura M
Published: September 2013

In an attempt to determine the mechanism of insulin resistance in the presence of obesity, we examined effects of insulin on insulin-sensitive phosphodiesterase (PDE) in spontaneously diabetic KK mice. Isolated fat cells prepared from epididymal adipose tissue were incubated, with or without insulin, for 10 min. In the caseof subcellular fractionation, only membrane-bound PDE was activated by insulin, as was noted in the case of rat fat cells. The specific activity was decreased in KK mice compared with control C57BL/6 mice. The doseresponse curve, expressed as a percent of the maximal insulin effect, shifted to the right and the increase of ED50 indicated a decreased insulin sensitivity in the KK mice. The maximal insulin effect did not change, either when expressed as a percent of the basal enzyme activity or when expressed on a per cell basis. Specific binding of [125I]-insulin in fat cells increased in KK mice and curvilinear Scatchard plots showed an increase of the high-affinity sites. These data indicate that impairment of PDE activation in fat cells of KK mice relates to postreceptor defects and the uncoupling may result in a decreased sensitivity.
Insulin, via Its Own Receptor, Regulates Growth and Amylase Synthesis in Pancreatic Acinar AR42J Cells
Authors: Mössner Joachim , Logsdon Craig D , Williams John A , Goldfine Ira D
Published: September 2013

Previous in vivo studies have suggested a long-term regulatory role for insulin in the exocrine pancreas. To directly study the long-term effects of insulin on the pancreas in vitro, we have used cultured AR42J cells, a rat cell line that is derived from a transplantable tumor of the acinar pancreas. Hormone-binding experiments with 125I-labeled hormones indicated that AR42J cells have insulin receptors, relatively fewer receptors for insulin-like growth factor II (IGF-II), and no detectable receptors for insulin-like growth factor I (IGF-I). Insulin at concentrations as low as 1 nM stimulated the growth of these cells, as measured by an increase in DNA and protein content, and in cell number. At 100 nM, where insulin had a maximal effect, the growth of AR42J cells was stimulated by 46.1 ± 10.9% (mean ± SEM, N = 11).Insulin increased the amylase activity of AR42J cells over the same concentration range that it stimulated growth; at 100 nM, insulin increased amylase by 91.0 ± 15.4% (mean ± SEM, N = 23). Immunoprecipitation of [35S]methionine-labeled proteins revealed that insulin induced a selective increase of amylase synthesis over general protein synthesis. These studies indicate, therefore, that insulin stimulates both growth and amylase synthesis of AR42J cells.
Structure, Function, and Immune Properties of Reassociated Islet Cells
Authors: Shizuru Judith , Trager David , Merrell Ronald C
Published: September 2013

Rat islets were mechanically dissociated to single cells and allowed to form aggregates by rotation-mediated cell-cell interaction. The aggregates, or neoislets, demonstrated insulin release in response to 20 mM glucose and 10 mM theophylline that was comparable to that of intact islets cultured for a similar time. However, basal insulin release was considerably greater than that from freshly isolated islets. The microscopic structure of the neoislets revealed sortinginto a B-cell domain at the surface with A-cells interior to the aggregate. The neoislets generated no mitogenic response in allogeneic lymph node lymphocytes. Reassociation of single islet cells provides stable, functional endocrine units with substantial reduction of immunogenicity.
Time Course of Islet Cell Antibodies in Diabetic and Nondiabetic BB Rats
Authors: Laborie C , Sai P , Feutren G , Debray-Sachs M , Quiniou-Debrie M C , Poussier P , Marliss E B , Assan R
Published: September 2013

The BB rat develops a spontaneous type I diabetic syndrome with anti-islet autoimmunity. Sera from diabetic and nondiabetic BB rats (from diabetes-prone litters), nondiabetic BB rats (from low-risk lines), and nondiabetes-prone Sprague-Dawley rats were collected twice a week from age 40 days to 160 days. Sera were tested for: (1) complement-dependent toxicity to 51Crlabeled islet cells in vitro; (2) immunoglobulin binding to RIN-5 F insulinoma cells; and (3) ability to selectively suppress insulin secretion from normal islets in vitro. All sera from rats that subsequently became diabetic or glucose-intolerant were toxic to islet cells from various rat strains in the presence of complement. They were toxic neither to hepatocytes nor to fibroblasts. The toxic potency was associated with the globulin fraction. It was, in most cases, maximal either before or immediately after the onset of the disease. Sera from the nondiabetes-susceptible BB rats and the rats which, in diabetes-prone litters, died too early to be classified tended toward greater toxicity to islets. Immunoglobulins from diabetic sera bound to RIN-5 F cells more than did the serum globulins from other groups, their maximal binding capacity occurring afterthe onset of diabetes. Furthermore, BB diabetic sera were capable of selectively inhibiting the insulin secretion from normal rat islets in vitro either in the presence or, in some cases, in theabsence of complement. The A- and D-cell functions were not suppressed. The combination of such results suggests the presence of one or more antibodies capable of binding to beta cells, inhibiting their function, and inducing their lysis. These antibodies may contribute to the beta cell disruption in this model of diabetes.
Insulin-induced Desensitization at the Receptor and Postreceptor Level in Mitogen-activated Human T-Lymphocytes
Authors: Ercolani Louis , Lin Hsin Lee , Ginsberg Barry H
Published: September 2013

Human T-lymphocytes activated by phytohemagglutin acquire insulin receptors in culture. Saturation analysis of insulin-binding activity in the presence of competing ligand revealed curvilinear Scatchard plots. Insulin receptorswere not regulated by insulin before mitogen activation and culture of T-lymphocytes. However, insulin-induced downregulation of insulin receptors was: (1) demonstrable in receptor-positive cells, (2) dependent on insulin concentration, (3) temporally unrelated to insulin internalization, and (4) prevented by culture at 4°C but not by cycloheximide at 37°C. Recovery of insulin receptors required further culture of cells in media depleted of insulin for 24 h. Scatchard analysis revealed loss of receptor number without changes in receptor affinity.

Insulin-induced increases in glucose transport and oxidation were demonstrable in receptor-positive cells but not in receptor-negative cells. However, these effects were extremely time-dependent. After a 2-h exposure of cells to 10−8 M insulin, increases in glucose transport were no longer demonstrable. Elution of bound insulin from these cells followed by re-exposure to insulin depressed glucose transport in them. Recovery from this hyporesponsive, desensitized state required a 6-h culture in insulin-depleted media. Glucose oxidation of desensitized cells couldbe stimulated by spermihe but not by insulin. These studies demonstrate the activated human T-lymphocyte is an insulinsensitive tissue that is capable of limiting its physiologic response to insulin by receptor- and postreceptor-mediated mechanisms.
Immunochemical Studies of an Insulin-like Material in the Parotid Gland of Diabetic BB Rats
Authors: Smith Phillip H , Leone John P , Stearns Susan B
Published: September 2013

Immunocytochemical and radioimmunoassay studies were performed on pancreatic and parotid tissues from diabetic BB and control Wistar rats. Compared with those of normoglycemic controls, the pancreata of diabetic BB rats generally lacked insulin-containing Bcells. Extracts from the parotid glands of diabetic rats contained less immunoassayable insulin-like material than was present in parotid extracts of controls. However, the parotid glands of both groups of animals contained numerous cells displaying insulin-like immunoreactivity. These insulin-immunoreactive cells, located mainly in the intercalated portion of the duct system, were comparable to those we reported recently in the parotid glands of normal and streptozocin-diabetic Sprague-Dawley rats. The presence of an insulin-like material in the parotid salivary gland of two types of diabetic animals suggests that such cells may be spared, in part, from the effects of both chemical and hereditary diabetogenic factors.
Islet Allografts Are Destroyed by Disease Occurrence in the Spontaneously Diabetic BB Rat
Authors: Prowse Stephen J , Bellgrau Donald , Lafferty Kevin J
Published: September 2013

Transplantation of cultured islet and pituitary tissue from PVG (RT1C) donors to major histocompatibility complex-incompatible BB/D recipients (RT1U) results in tissue-specific destruction of the grafted islets but not of the pituitary. We interpret this response as disease occurrence in the MHC-incompatible islet graft. Islet damage is associated with eosinophil and mast cell accumulation in and around the grafted tissue. Antibody deposition is also present in tissues of the BB rat. This is suggestive of an antibody-mediated allergic reaction.
Sex Steroid Dependency of Diabetes-induced Changes in Polyol Metabolism, Vascular Permeability, and Collagen Cross-Linking
Authors: Williamson Joseph R , Rowold Edwin , Chang Katherine , Marvel Joyce , Tomlinson Martha , Sherman William R , Ackermann Karen E , Berger Richard A , Kilo Charles
Published: September 2013

The effects of castration on diabetes-induced increases in collagen cross-linking and vascular permeability and on polyol levels in new granulation tissue formed after induction of streptozocin (STZ) diabetes were examined in male Sprague-Dawley rats. New granulation tissue formation was induced by implanting sterile polyester fabric subcutaneously (s.c.) at the time of STZ injection 3 wk before assessment of vascular permeability and collagen cross-linking. Castration was performed 10 days before implanting the fabric. The characteristic increases in collagen cross-linking (manifested by decreased solubility in 0.5 M acetic acid) and in albumin permeation of the vasculature seen in intact diabetic rats were completely prevented by castration. Net collagen accumulation was not affected by diabetes or castration. Castration also markedly diminished diabetes-induced increases in tissue levels of sorbitol and completely prevented the decreases in tissue levels of myo-inositol and scyllo-inositol observed in intact diabetic rats, but had no effect on serum glucose levels, nonenzymatic glycosylation of plasma and granulation tissue proteins, or plasma somatomedin-C levels.

The demonstration that castration prevents diabetesinduced increases in vascular permeability and collagen cross-linking as well as imbalances in tissue levels of sorbitol, myo-inositol, and scyllo-inositol in this model indicates that all of these changes are sex steroid-dependent phenomena. While the pathogenesis of these vascular permeability and collagen cross-linking changes is clearly multifactorial, these new findings: (1) indicate that the role of sex steroids in the development of late complications of diabetes may be far more important than hitherto suspected, and (2) suggest an explanation for the clinical observation that diabetic complications are uncommon in prepubertal diabetic subjects regardless of duration of diabetes.
Effects of Tolbutamide Pretreatment on the Rate of Conversion of Newly Synthesized Proinsulin to Insulin and the Compartmental Characteristics of Insulin Storage in Isolated Rat Islets
Authors: Gold Gerald , Pou Jose , Gishizky Mikhail L , Landahl Herbert D , Grodsky Gerold M
Published: September 2013

Toibutamide (1 g/kg body wt) was administered to male rats for 3 days to determine the effects of this pretreatment on subsequent insulin biosynthesis and compartmental storage characteristics of freshly isolated islets. Islets were isolated 16 h after the last toibutamide administration, at a time when fed plasma glucose concentrations were normal. Islet glucagon was unchanged but insulin content was significantly reduced (38 ± 1.2 ng IRI/islet from seven untreated rats versus 7.9 ± 1.2 ng IRI/islet from eight treated rats). After toibutamide pretreatment, the rate of incorporation of 3Hleucine into islet proinsulin was unchanged, but the t1/2 of labeled proinsulin-to-insulin conversion was significantly (P < 0.001) decreased from 36 to 20 min. After treatment, actual rates of glucose-stimulated insulin secretion were 50% lower, however, because due to the proportionately greater depletion of islet insulin content, the fractional rate of secretion was increased twofold. After treatment, there was evidence of compartmental, heterogeneous insulin storage, and glucose still marked newly synthesized insulin for preferential release; however, the differential release of new and old insulin converged rapidly with time. Mathematical integration of the data suggested dilution of the newly synthesized insulin compartment with unlabeled insulin during the chase period, but additionally indicated more rapid mixing of newly synthesized with previously stored, unlabeled insulin. Thus, tolbutamide-treated rats partially compensated for acute insulin depletion by (1) increasing the rate of proinsulin-to-insulin conversion, but not increasing the rate of proinsulin biosynthesis; (2) doubling the glucose-stimulated fractional secretory rate of the depleted cellular insulin storage compartment; and (3) retaining compartmental storage characteristics but mixing newly synthesized insulin more rapidly with the compartment of previously stored, unlabeled insulin.
Chlorpropamide Raises Fructose-2,6-Bisphosphate Concentration and Inhibits Gluconeogenesis in Isolated Rat Hepatocytes
Authors: Monge Luis , Mojena Marina , Ortega Jose L , Samper Begoña , Cabello M Angeles , Feliu Juan E
Published: September 2013

The addition of chlorpropamide to hepatocytes isolated from fed rats raised the cellular concentration of fructose-2,6-bisphosphate (F-2,6-P2), a regulatory metabolite that plays a relevant role in the control of hepatic glucose metabolism. The effect of chlorpropamide was dose dependent; a statistically significant increase was already seen at 0.2 mM of the sulfonylurea. The accumulation of F-2,6-P2 caused by chlorpropamide (1 mM) was parallel to the stimulation of L-lactate production (36.6 ± 4.8 versus 26.1 ± 2.6 μmol of lactate/g of cells × 20 min; N = 5, P < 0.05) and to the inhibition of gluconeogenesis (0.57 ± 0.1 versus 0.94 ± 0.09 μmol of [U-14C]pyruvate converted to glucose/g of cells × 20 min; N = 5, P < 0.05). In addition, chlorpropamide enhanced the inhibitory action evoked by insulin on glucagon-stimulated gluconeogenesis. This combined effect of chlorpropamide and insulin seems to be correlated with the synergistic accumulation of F-2,6-P2provoked by the simultaneous action of these two agents on glucagon-treated hepatocytes. Finally, neither 6-phosphofructo-2-kinase activity nor hepatocyte cyclic AMP levels were significantly changed by the presence of the sulfonylurea in the incubation medium. Our results support the concept that chlorpropamide, by a cyclic AMP-independent mechanism, increases the hepatic content of F-2,6-P2 and, in this way, enhances the glycolytic flux and inhibits glucose output by the liver.
Lower Prevalence of Diabetes in Female Former College Athletes Compared With Nonathletes
Authors: Frisch R E , Wyshak G , Albright T E , Albright N L , Schiff I
Published: September 2013

The prevalence rate of diabetes was determined for 5398 living college alumnae (2622 former college athletes and 2776 nonathletes) from data on medical history, athletic training, and diet. For all ages, the prevalence rate among the former athletes was 0.57% (15/2622) and among the nonathletes was 1.3% (37/2776). The former college athletes and the nonathletes had similar percentages in family history of diabetes, i.e., 12.0 and 13.5%, respectively.

For cases occurring at age ≥20 yr (thus assessing the effects of college athletic training), 0.5% (13/2622) of the former college athletes had diabetes compared to 1.2% (32/2776) of the nonathletes; the relative risk is 2.24 [95% confidence limits (CL), 1.19 and 4.74, respectively]. Omitting cases of gestational diabetes, the relative risk of diabetes in nonathletes versus athletes is 3.41 (95% CL, 1.33 and 8.70). The percentages of former athletes and nonathletes that are insulin-using, non-insulin-using, and gestational diabetics did not differ significantly. The athletes were leaner than the nonathletes at all ages up to 70 yr. Of the former college athletes, 82% had been on precollege teams, compared to 25% of the college nonathletes; 74% of the former athletes were exercising regularly, compared to 57% of the nonathletes. We conclude that long-term athletic training is associated with a lower risk of the development Of diabetes.
Effect of Choline and Methionine Treatment on Cardiac Dysfunction of Diabetic Rats
Authors: Heyliger C E , Rodrigues B , McNeill J H
Published: September 2013

There is an abnormal lipid accumulation in the myocardium that might be involved in the congestive heart failure frequently associated with individuals with diabetes mellitus. Because choline and methionine have been reported to modify the incidence of myocardial necrosis in rats fed various fat diets, we decided in our study to assess their effect on the cardiac dysfunction of diabetic rats. Female Wistar rats were made diabetic with streptozocin (STZ, 55 mg/kg i.v.). One week after diabetes induction, one group received choline (0.3 mg/ml), another received methionine (0.25 mg/ml), a third received a combination of the same doses of choline and methionine, and a fourth received neither of these agents; all were administered in the drinking water. Animals were treated for 7 wk. Insulin levels were lower and glucose values higher in the serum of STZ rats relative to controls. Serum cholesterol and triglycerides were significantly elevated in all diabetic animals, and they were also elevated (P < .05) in the hearts of untreated diabetics. In contrast, the myocardial values of the same lipids were drastically reduced in the treated diabetic animals. Cardiac performance was depressed in all STZ animals, but there was a significant improvement in heart function in treated diabetics relative to untreated ones. Thus, it appears that the buildup of cholesterol and triglycerides in the myocardium are important contributors to the cardiac dysfunction that frequently accompanies diabetes mellitus.
Novel Gene Activated in Rat Insulinomas
Authors: Takasawa S , Yamamoto H , Terazono K , Okamoto H
Published: September 2013

Insulinomas can be induced in experimental animals by the combined administration of diabetogenic agents with polyadenosine diphosphate (polyADP)-ribose synthetase inhibitors. A complementary DNA (cDNA) library that was constructed from streptozocin-nicotinamide-induced rat insulinomas has been found to contain a novel gene encoding a basic protein of 145 amino acids. The gene was expressed in alloxan-nicotinamide–induced insulinomas as well as in streptozocin-nicotinamide–induced insulinomas but not in normal pancreatic islets or in regenerating islets. This indicates that the activation of the gene designated rig, i.e., rat insulinoma gene, may be a general feature of pancreatic β-cell transformation.
Insulin Receptors in Embryo and Extra Embryonic Membranes of Early Somite Rat Conceptus
Authors: Unterman T , Goewert R R , Baumann G , Freinkel N
Published: September 2013

Intact rat conceptuses were cultured from day 9.5 of gestation on. Individual components of the conceptus, including the embryo and the extraembryonic membranes (consisting of yolk sac, amnion, and allantoic placenta), were isolated and examined for insulin receptors at two time points during organogenesis: 1) day 10.4 of gestation (∼10–12 somites) when the yolk sac had become vascularized and just before closure of the anterior neuropore and 2) day 11.6 (∼27–31 somites) when vascularization of the chorioallantoic placenta had been established and the neural tube was closed completely. The studies were designed to provide inferential insights about the possible role of insulin in embryogenesis during different phases of nutrient delivery.

Active insulin degradation occurred with embryo as well as membrane homogenates during incubation at 37°C. Degradation was markedly reduced at 4°C, and binding of 125I-labeled insulin by embryo or membrane homogenates prepared on day 10.4 or 11.6, respectively, of gestation approached equilibrium after a 20-h incubation at this temperature. Values for the specific binding of tracer (0.4 ng/ml) or carrier (10.4 ng/ml) insulin by embryo and membrane homogenates were the same on days 10.4 and 11.6; specific binding was significantly greater with preparations of membranes than embryo at both time points. Full binding curves on day 11.6 showed similar affinities for insulin by embryo and membranes (Ke = 1.2 × 108/M and 4.6 × 108/M, respectively). Specificity of the binding was confirmed by comparison with proinsulin, desoctapeptide insulin, and insulin-like growth factor I and by the demonstration that maximally effective amounts of human antiinsulin receptor antiserum inhibited insulin binding ∼75% in embryos and 92% in membranes on both days. Photoaffinity cross-linking studies confirmed the presence of a major insulin-binding moiety in embryos and membranes similar to that found in rat liver plasma membranes (L) but different in apparent molecular weight (liver plasma membranes > membranes > embryo).

Our studies indicate that specific insulin receptors are present in the embryo and membrane components of the early postimplantation rat conceptus at two different stages in the development of nutrient delivery systems. However, the embryos and extraembryonic membranes display some differences with regard to the number and binding characteristics of their specific insulin receptors at both time points. The functional significance of these differences remains to be elucidated.
Fine Structural Abnormalities of the Placenta in Diabetic Rats
Authors: Gewolb I H , Merdian W , Warshaw J B , Enders A C
Published: September 2013

In the streptozocin-induced diabetic rat, the placenta is larger and the fetus is smaller than normal. To study cellular differences that might contribute to the size and functional disparity between diabetic and control placentas, a light- and electron-microscopic analysis was performed on 14-, 18-, and 22-day (term) control and diabetic placentas.

Diabetic placentas, especially later in gestation, were marked by the presence of large numbers of glycogendistended cells in the basal zone. Within the placental labyrinth, the trophoblastic layers of the interhemal membrane were significantly thicker in the diabetic placentas on days 18 and 22, and large accumulations of lipid droplets were present, especially in the inner two trophoblastic layers. In normal placentas there is a marked thinning of the placental barrier in the labyrinth by day 22 of gestation, making the thickness of the labyrinthine layers in age-matched diabetic placentas even more impressive. Finally, the labyrinth of 22-day diabetic placentas contained more glycogen and rough endoplasmic reticulum in the inner trophoblastic layer, a feature that is found in less-mature (18-day) control placentas.

Thus, the diabetic placentas have a number of features that are consistent with functional immaturity/ dysmaturity. Cytologic evidence confirms the presence of increased glycogen and lipid reserves in both the junctional zone and the cellular area between maternal and fetal blood. The thickening and modification in the interhemal membrane of the labyrinth of the diabetic placentas increase the diffusion distance between the maternal and fetal circulations and thus may adversely affect placental exchange functioning and result in the fetal growth retardation seen in this model of the diabetic pregnancy.
Regulation of Ketogenesis, Gluconeogenesis, and Glycogen Synthesis by Insulin and Proinsulin in Rat Hepatocyte Monolayer Cultures
Authors: Agius L , Chowdhury M H , Davis S N , Alberti K G M M
Published: September 2013

The metabolic actions of porcine insulin and biosynthetic human proinsulin on fatty acid and glucose metabolism were studied in rat hepatocytes cultured in monolayer for 24 h. Our aim was to establish whether proinsulin action in the liver is similar to insulin action and whether the relative potencies of the two hormones are the same for different metabolic processes. Proinsulin and insulin exerted a similar maximal inhibitory effect on ketone body formation from palmitate and on gluconeogenesis from pyruvate. The half-maximal effective concentration of proinsulin was 11–13 times that of insulin. The antiketogenic effects of insulin and proinsulin were associated with an increased glycerol 3-phosphate content and a decreased affinity of carnitine palmitoyltransferase for its substrate palmitoyl-CoA. When the basal rate of ketogenesis was increased with isobutyl methylxanthine, the half-maximal effective concentrations of both proinsulin and insulin were decreased, but the relative potency of the two hormones was unchanged. Proinsulin and insulin exerted similar maximal stimulatory effects on glycogen synthesis and on the activities of pyruvate kinase, glucose 6-phosphate dehydrogenase, phosphogluconate dehydrogenase, and malic enzyme. The half-maximal effective concentration of proinsulin was 10–30 times that of insulin. These findings are consistent with receptor binding studies on liver membranes that suggest that proinsulin interacts with insulin-specific and not proinsulin-specific receptors. Our findings also suggest that proinsulin action does not differ from insulin action at a postreceptor site.
Maturation of Immune Response to Insulin: Recruitment of New Epitopes by Antibodies
Authors: Petersen K -G , Schuler G , Hösl W , Storch M-J , Kerp L
Published: September 2013

Guinea pigs were immunized with regular insulin in Freund's adjuvant weekly (N = 6) or with NPH insulin daily (N = 10). The concentrations, the affinities, and the sizes of the insulin-immunoglobin G (IgG) antibody complexes were determined by ultracentrifugation. During the immune response to insulin, 7-S complexes were observed after 3 wk, and 10-S complexes were observed after 4 wk (2P < .05 in each group). “7-S” antibodies were transferred into immunized guinea pigs before the formation of measurable antibody concentrations. The development of 10-S structures was enhanced in outbred Pirbright white guinea pigs (2P < .05, N = 6) and in inbred strain II l.b.m. guinea pigs (2P < .05, N = 7). Insulin may act as a monovalent antigen (7 S) after 3 wk and become bivalent after 4 wk (10 S). The change in valency is enhanced by antibody transfer. It reflects the formation of antibodies recognizing new epitopes on insulin.
Reversal of Diabetes in Outbred Mice by Islet Allotransplantation
Authors: Simeonovic C J , Prowse S J , Lafferty K J
Published: September 2013

The combination of donor pretreatment with cyclophosphamide, organ culture in 95% O2:5% CO2 for 7–10 days, and short-term immunosuppression of recipients with cyclosporin A (CsA) were necessary to obtain 100% survival of single-cluster BALB/c islet allografts in outbred mice. In vivo and in vitro pretreatment of the donor tissue alone resulted in the acceptance of 45% of the islet allografts in nonimmunosuppressed outbred mice. CsA treatment of recipients alone yielded 40% survival of the untreated allografts. CsA treatment played an important role in maintaining the capacity of islet allografts to function in outbred mice. During CsA treatment, 88% of streptozocin-treated mice showed graft-dependent reversal of diabetes; the remainder showed no evidence of graft function, and CsA treatment failed to prevent acute graft rejection. After withdrawal of CsA immunosuppression, 38% of this total group remained normoglycemic. These findings suggest that modulation of both donor-tissue immunogenicity and recipient responsiveness will be required for successful pancreatic islet transplantation in diabetic humans.
Mechanism of Abnormal Oral Glucose Tolerance of Genetically Obese fa/fa Rats
Authors: Rohner-Jeanrenaud F , Proietto J , Ionescu E , Jeanrenaud B
Published: September 2013

The genetically obese fa/fa rat is glucose intolerant when tested in a conscious state after the spontaneous ingestion of a glucose solution. The aim of this study was to investigate the mechanism(s) underlying the abnormal oral glucose tolerance test of obese animals with the non-steady-state measurement of glucose turnover proposed by Steele et al. in 1968. Our results show that the total rate of glucose appearance is enhanced in obese compared with lean animals. This abnormality is not due to an increased gut glucose absorption but to a lack of suppression and even a transient stimulation of hepatic glucose production after the ingestion of glucose. The rate of glucose utilization by the obese animals is somewhat increased compared with controls or unchanged when expressed as glucose metabolic clearance rate, thus excluding this parameter from the factors contributing to the observed glucose intolerance. The results obtained with genetically obese rats agree with those reported for type II diabetes in humans. The observed defect of the obese group could be related to an abnormal regulation of insulin counterregulatory hormone(s) or of hepatic innervation as well as to other defects of hepatic glycogen handling.
Increased Accumulation of Sorbitol in Offspring of Manifest Diabetic Rats
Authors: Eriksson U J , Naeser P , Brolin S E
Published: September 2013

The effects of maternal diabetes on somatic development and activity of the polyol pathway were investigated during early and late gestation in a rat model for diabetic pregnancy. We studied embryo-fetal growth, mortality, and malformation rate in the offspring of nondiabetic rats and in the offspring of diabetic rats either treated with an aldose reductase inhibitor during gestation or left untreated. The numbers of embryo-fetal resorptions and malformations were significantly increased in the diabetic groups compared with the controls despite maternal treatment with the aldose reductase inhibitor. The sorbitol content of embryos and membranes from the diabetic rats in early gestation was increased 3–5 times over the control values. Similarly, elevated sorbitol levels were observed in the fetal livers and placentas of the diabetic rats in late gestation. Administration of the aldose reductase inhibitor to the pregnant diabetic rats normalized the sorbitol levels in the embryos and their membranes, whereas the sorbitol contents of the fetal livers and placentas were significantly lowered but not completely corrected. Furthermore, in the diabetic groups, no differences in sorbitol levels could be demonstrated between malformed and nonmalformed offspring. The results of this study suggest that enhanced polyol metabolism leading to increased sorbitol accumulation is present in the embryos of diabetic mothers as early as organogenesis. This accumulation is apparently not a major factor in the early developmental disturbances (e.g., growth perturbations and congenital malformations) of diabetic pregnancy.
Metabolic Consequences of Prolonged Hyperinsulinemia in Humans: Evidence for Induction of Insulin Insensitivity
Authors: Marangou A G , Weber K M , Boston R C , Aitken P M , Heggie J C P , Kirsner R L G , Best J D , Alford F P
Published: September 2013

Hyperinsulinemia is frequently associated with a variety of insulin-resistant states and has been implicated causally in the development of insulin resistance. This study examines the metabolic consequences of prolonged hyperinsulinemia in humans. Basally and 1 h after cessation of a 20-h infusion of insulin (0.5 mU · kg−1 · min−1, aimed at elevating plasma insulin levels to ∼30 mU/L) or normal saline, subjects were assessed for 1) glucose turnover with 3-[3H]glucose; 2) insulin sensitivity, as measured by either the euglycemic glucose-clamp technique or the intravenous glucose tolerance test (IVGTT) minimal model method of Bergman; and 3) monocyte insulin-receptor binding. Hepatic glucose production (Ra) was suppressed by >95% during each euglycemic clamp and during the 20-h insulin infusion. After the insulin infusion, Ra and glucose utilization rate returned to the initial basal level within 1 h, as did insulin levels. At that time, insulin sensitivity was significantly decreased, as measured by the “insulin action” parameter during the 40- to 80-min phase of the clamp (0.049 ± 0.003 vs. 0.035 ± 0.007 min−1 P < .05) and during the 80- to 120-min phase (0.047 ± 0.005 vs. 0.039 ± 0.007 min−1, .05 < P < .1). The IVGTT minimal model analysis revealed a fall in the rate of glucose disposal (KGTT) (2.8 ± 0.6 vs. 1.9 ± 0.2 min−1 P < .05), which was entirely explained by a decrease in insulin sensitivity (SI, 9.4 ± 0.3 vs. 3.8 ± 0.2 min−1 · μU−1 · ml−1, P < .02); there was no change in glucose-mediated glucose disposal (SG, 0.029 ± 0.004 vs. 0.029 ± 0.004 min−1) or pancreatic Ybgr;-cell responsiveness (ø1, 2.7 ± 0.4 vs. 2.6 ± 0.5 μU · ml−1 · min mg−1 · dl−1; ø2, 7.8 ± 2.4 vs. 7.8 ± 2.4 μU · ml−1 · min−2 – mg−1 · dl−1). Monocyte insulin-receptor binding was unaffected by the prolonged hyperinsulinemia.

Our studies indicate that modest sustained hyperin- sulinemia may lead to decreased insulin action in the presence of normal monocyte insulin-receptor binding and normal pancreatic insulin secretion. If the monocyte reflects insulin binding in the key insulin-sensitive tissues, this defect in insulin action probably lies at a postreceptor level.
Functional Characteristics of Decreased Insulin Receptors on Fibroblasts Obtained from a Subject with Severe Insulin Resistance and Acanthosis Nigricans
Authors: Prince Melvin J , Smith Fannie E , Peters Edward J , Stuart Charles A
Published: September 2013

In a patient with severe insulin resistance and acanthosis nigricans, a decrease in the number of insulin receptors has been found on freshly isolated monocytes and cultured fibroblasts compatible with a primary or genetic decrease in cell-surface insulin receptors. To determine the functional characteristics of the remaining receptors on these cells, insulin-stimulated glucose uptake, insulin internalization, and insulin-induced receptor loss were evaluated in monolayer fibroblasts obtained from this subject. Maximal insulin stimulation of 2-deoxyglucose was markedly blunted, compatible with abnormal insulin responsiveness due to a functional impairment of the remaining receptors. In the presence of chloroquine, the acanthotic subject's fibroblasts internalized more insulin per available receptor compared with the normal cell line, suggesting an accelerated rate of insulin internalization. When the rate of insulin internalization was more directly determined by assessing the rate of appearance of acid-resistant, cell-associated radioactivity at 37°C, a similar increase in insulin internalization rate was evident. When downregulation was assessed, insulin's ability to induce receptor loss in the acanthotic subject's cell line was augmented.

Thus, a primary or genetic decrease in insulin receptors on cultured fibroblasts from a patient with acanthosis nigricans and insulin resistance is associated with functional impairment of the remaining receptors leading to significant alterations in ligand processing and subsequent insulin action.
Islet Cell Antibodies Identify Latent Type I Diabetes in Patients Aged 35–75 Years at Diagnosis
Authors: Groop Leif C , Bottazzo Gian Franco , Doniach Deborah
Published: September 2013

One hundred fifty-four selected patients with nonketotic diabetes diagnosed between the ages of 35 and 75 yr and treated with diet or oral hypoglycemie agents for at least 1 yr were investigated for parameters of glycemie control (weight loss, blood glucose, and glycosylated hemoglobin), islet cell function (fasting and glucagon-stimulated C-peptide responses), and immunologie markers of insulitis (total ICA and CF-ICA) or autoimmunity (thyroid and gastric antibodies). These parameters were all repeated in 9 of 22 ICA-positive patients after a 2-yr follow-up and correlated with secondary drug failure. The antibody tests were also done on 51 nondiabetic controls matched for age and body weight.

The 22 (14%) diabetic subjects having positive islet cell antibodies (ICA) included more women than men with a shorter duration of symptoms, lower body weight, more associated thyroid autoimmunity, and a tendency to have more type I diabetes in their families, although glycemic control, age at onset, and family history of type II diabetes were the same as in the 132 ICA-negative cases.

Patients with ICA had lower initial C-peptide levels and showed little rise after glucagon stimulation. Beta cell function deteriorated significantly during the 2-yr follow-up in 9 of 22 positive patients and more ICA-positive patients required insulin. It is suggested that these latent type I diabetic patients are characterized by persistent ICA, progressive loss of beta cells, and a high frequency of thyrogastric autoimmunity. The determination of ICA may be of clinical value in the diagnosis and treatment of nonketotic diabetes with onset in later life.
Quantitative Autoradiographic Evidence for Insulin Receptors in the Choroid Plexus of the Rat Brain
Authors: Baskin Denis G , Brewitt Barbara , Davidson David A , Corp Eric , Paquette Thomas , Figlewicz Dianne P , Lewellen Thomas K , Graham Michael K , Woods Stephen G , Dorsa Daniel M
Published: September 2013

Techniques of in vitro receptor autoradiography were used to visualize binding of 125I-insulin on slices of frozen rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.05 nM porcine 125I-monoio-doinsulin, alone or mixed with 1 μM unlabeled porcine insulin, ribonuclease, or glucagon, for 2 h at 22°C. The labeled brain slices were apposed to LKB Ultrofilm to generate autoradiograms. The method permitted equal access of labeled insulin to both sides of the blood-brain barrier and localization of insulin binding sites in small anatomic regions. Quantitative estimates of specific iodoinsulin binding were made by computer digital image densitometry of the autoradiographic film images. High concentrations of specific binding sites for iodoinsulin were present in the choroid plexus of the lateral (26.9 ± 2.0 × 103 fmol/mm2), fourth (18.3 ± 3.0 × 10-3 fmol/mm2), and third (13.2 ± 1.5 × 103fmol/mm2) ventricles (insulin binding is expressed per unit area of autoradiographic image). Binding to the third ventricular choroid plexus was similar to the concentrations observed for liver slices and the external plexiform layer of the olfactory bulb. Specific binding of iodoinsulin in the cingulate cortex and other surrounding regions was less than in choroid plexus. Ribonuclease or glucagon had no measurable effect on binding when mixed with labeled insulin. The results support the hypothesis that the choroid plexus has a high density of receptors for insulin, and suggests that the choroid plexus may be a target of CSF insulin action and/or a site of insulin transport into the CSF.
The Effect of Immediate Polyethylene Glycol Precipitation on Free Insulin Measurements in Diabetic Patients with Insulin Antibodies
Authors: Rudkowski Roman , Antony Gabriel
Published: September 2013

Free insulin (FI) measurements obtained by polyethylene glycol (PEG) precipitation within 3 min of drawing the blood sample (FI3) from four insulin-treated diabetic subjects with a wide range of insulin antibodies were compared with published methods of FI estimation. Comparison of FI values obtained by PEG precipitation in assays of replicate samples of the same specimens (N = 9) stored at 4°C for 24 h (EFI) and FI3 were 4.76 ± 1.5 μU/ml (mean ± SEM) and 17.13 ± 4.7 μU/ ml, respectively (P < 0.005). Comparison of FI values obtained in six groups of replicate samples (N = 12, 18 per group, a total of 91 specimens) from these four patients assayed immediately after thawing to 18°C, and incubated at 37°C for 30, 60, and 120 min, and FI3 showed a significant difference in at least oneof these four comparisons (mean ± SEM) in each of these six sample groups. In 18 of 24 comparisons there was a loss of FI when stored samples were used with or without incubation (12 of these were significant at the P < 0.05–0.001 level), but in 6 of the 24 comparisons there was an increase in the FI against FI3 (3 of these 6 significant at the P < 0.05–0.01 level). There was a trend toward a greater loss of FI in stored samples with higher FI3 content. Loss of FI during incubation occurred in all groups irrespective of the FI3 content. The three comparisons showing a significant increase in FI during incubation were all from the same patient with high levels of insulin antibodies. We conclude that, in the presence of insulin antibodies, only immediately processed fresh serum samples can give a best approximation of in vivo FI. This observation may account, in part, for the conflicting data and views in the literature on the usefulness of FI measurements in IDDM.
Putative Hypothalamic Glucoreceptors Play No Essential Role in the Response to Moderate Hypoglycemia
Authors: Cane Patricia , Artal Raul , Bergman Richard N
Published: September 2013

The response to insulin-induced hypoglycemia includes increased plasma levels of glucagon, epinephrine, norepinephrine, cortisol, and growth hormone. This integrated response is thought to be mediated via sympathetic afferent pathways emanating from the ventromedial hypothalamus. However, the precise loci of the receptors that sense glucopenia are not known. In this study, we investigated the importance of putative forebrain glucoreceptors to the systemic response to hypoglycemia. Three protocols were performed. Protocol 1: the systemic response was observed in conscious dogs to hypoglycemia induced by infusion of insulin at a high rate (150 mU/min). Protocol 2: the effect of concomitant bilateral, intracarotid glucose infusion on the response to intravenous insulin was examined. Intracarotid glucose infusion rates were chosen to yield central euglycemia in the face of systemic hypoglycemia. Protocol 3: as a control for protocol 2, glucose was infused at low rates into the systemic circulation, yielding hypoglycemia in both central and systemic blood. When insulin was infused at 150 mU/ min, without glucose replacement (protocol 1; N = 6), plasma insulin increased from 14 ± 3 to 335 ± 35 μU/ ml at 60 min (P < 0.001). Glucose fell from basal (104 ± 3 mg/dl) to 38 ± 3 mg/dl (P < 0.001). Significant increases were observed in epinephrine (basal, B: 63 ± 8; steady state, SS: 1762 ± 582 pg/ml; P < 0.007), norepinephrine (B: 209 ± 33, SS: 650 ± 133 pg/ml; P < 0.01), and glucagon (B: 256 ± 35, SS: 467 ± 35 pg/ml; P < 0.03). In addition, endogenous glucose production (Ra) increased from 72 ± 4 to 108 ± 9 mg/min (P < 0.02) despite frank hyperinsulinemia. Infusing glucose into the carotid arteries at 204 ± 10 mg/min (protocol 2; N = 7) during a 4-h systemic insulin infusion was sufficient to prevent jugular hypoglycemia [jugular glucose, Gj, B: 100 ± 3, SS (90–160 min): 101 ± 3 mg/ dl; P > 0.70], but not peripheral hypoglycemia (Gp, B: 102 ± 2, SS: 55 ± 3 mg/dl; P < 0.001). Despite carotid glucose replacement, counterregulatory responses were still observed in epinephrine (B: 98 ± 14, SS: 466 ± 127 pg/ml; P < 0.04) and norepinephrine (B: 213 ± 19, SS: 474 ± 133 pg/ml; P < 0.05). Moreover, the magnitude of counterregulatory responses was not different when the glucose was given peripherally rather than centrally [protocol 3: epinephrine (B: 84 ± 18, SS: 523 ± 105 pg/ml; P < 0.004), norepinephrine (B: 277 ± 65, SS: 445 ± 71 pg/ml; P < 0.02)].

Counterregulatory responses to modest systemic hypoglycemia are not altered by the concomitant prevention of central hypoglycemia. Apparently, hypothalamic glucoreceptors are not essential for the sympathetic response to hypoglycemia, implying that important glucose-sensitive receptors are located elsewhere in the body.
Effects of Obesity, Hyperinsulinemia, and Glucose Intolerance on Insulin Action in Adipose Tissue of Sixty-Year-Old Men
Authors: Bolinder Jan , Lithell Hans , Skarfors Einar , Arner Peter
Published: September 2013

The relative effects of obesity alone, and in combination with fasting hyperinsulinemia and glucose intolerance, on the peripheral action of insulin in adipose tissue were investigated in twenty-four 60-yr-old men, who had been followed for 10 yr. They were divided into four groups of six subjects each on the basis of the following criteria: (1) normal body weight, normal fasting insulin level, and normal glucose tolerance; (2) moderate obesity, normal fasting insulin level, and normal glucose tolerance; (3) moderate obesity, fasting hyperinsulinemia, and normal glucose tolerance; and (4) moderate obesity, fasting hyperinsulinemia, and newly developed, moderate, untreated fasting hyperglycemia and/or glucose intolerance (i.e., mild type II diabetes mellitus). Specific adipocyte insulin binding and the effects of the hormone on adipose tissue lipolysis and glucose oxidation were determined. Insulin receptor binding per cell and per cell surface area were similar in all four groups. Regarding antilipolysis, the insulin sensitivity was the same in all groups and the maximum effect was significantly increased in the three obese groups, as compared with the normal-weight control group. In groups 1–3, insulin stimulated adipose tissue glucose oxidation in a dose-dependent way, and the sensitivity and responsiveness to insulin were comparable. In contrast, in the obese glucose-intolerant subjects (4) there was no significant effect of insulin on glucose oxidation when the hormone was added in increasing concentrations of ≤35 nmol/L. The basal glucose oxidation was similar in all four study groups. The in vivo insulin tolerance was gradually reduced in groups 2–4, as compared with the normalweight control group.

It is concluded that, in 60-yr-old men, obesity per se,or in combination with fasting hyperinsulinemia, does not inhibit insulin action on glucose metabolism in adipose tissue, although the in vivo insulin action is reduced. Only when obesity is accompanied by glucose intolerance does insulin reduce the glucose utilization of adipose tissue. In this situation the resistance to insulin is solely due to postreceptor defects of insulin action. The antilipolytic effect of insulin is enhanced in obese 60-yr-old men, whether or not the overweightness is accompanied by hyperinsulinemia and/or glucose intolerance.
Prevention of Basement Membrane Thickening in Retinal Capillaries by a Novel Inhibitor of Aldose Reductase, Tolrestat
Authors: Robison W Gerald , Kador Peter F , Akagi Yoshio , Kinoshita Jin H , Gonzalez Ramona , Dvornik Dushan
Published: September 2013

A diabetic-like thickening of retinal capillary basement membranes induced in rats fed for 207 consecutive days a diet containing 50% galactose was prevented by the addition to the diet of tolrestat, a potent, structurally novel inhibitor of aldose reductase. Analysis of electron micrographs (× 25,000) of capillaries from the outer plexiform layer of the retina by computer planimetry showed that the basement membranes were approximately twofold thicker in rats fed galactose than in those fed either a standard diet or a diet containing galactose and tolrestat in doses of 43 or 57 mg/kg/day. The thickening of basement membranes in galactosefed rats was accompanied by other ultrastructural alterations mimicking changes typical of diabetic microangiopathy, such as multilamination and the formation of vacuoles and dense inclusions. Therefore, the galactosemic rat represents a useful model for studying basement membrane-related complications of diabetes and their possible prevention by aldose reductase inhibitors.