bluuebunny/crossref_metadata_2025 huggingface dataset:
Low Plasma Renin Activity in Diabetes: Relation to Urine Prostaglandin Excretion
Authors: Wilson Thomas W , Tan Leonard K
Published: September 2013

Renal functional abnormalities, occurring before overt renal disease and possibly due to abnormal vascular control mechanisms, have been described in diabetes mellitus. We used intravenous (i.v.) furosemide, which stimulates renal prostaglandin (PG) synthesis and renin release, to compare these vasoactive systems in 14 diabetic and 23 normal control subjects. Using urine thromboxane B2 (TXB2) as an index of renal synthesis of the vasoconstrictor prostanoid TXA2, and urine 6keto-PGF1α for the vasodilator PGI2, we found evidence of increased renal TXA2 synthesis in diabetic subjects in response to furosemide. The increased TXA2 synthesis did not occur at the expense of PGI2 synthesis, as urine 6keto-PGF1α was not reduced. Increased TXB2 excretion in diabetic subjects was particularly marked in the first 10 min after i.v. furosemide. During this time, diabetic males excreted 31 ± 6 ng of TXB2 compared with 10 ± 1 ng for normal males (P < 0.05), while diabetic females excreted 15 ± 3 ng compared with 7 ± 1 ng for normal females (P < 0.05). Also, 6keto-PGF1α excretion at 10 min was increased in diabetic subjects: males, 29 ± 3 ng versus 19 ± 3 (P < 0.05); females, 33 ± 8 versus 16 ± 3 (P < 0.05). The ratio of TXB2 to 6keto-PGF1α tended to be higher in diabetic males. Plasma renin activity 10 min after i.v. furosemide was lower in diabetic males: 0.9 ± 0.2 ng · ml−1 · h−1 versus 3.5 ± 0.4 (P < 0.05) and tended to be lower in diabetic females: 1.5 ± 0.3 versus 2.0 ± 0.3. There was a significant negative correlation between the TXB2/6keto-PGF1α ratio and PRA at this time (r = −0.392, P < 0.05), suggesting that the relatively increased TXA2 could be a factor in the reduced early increment in plasma renin activity after i.v. furosemide.
Characterization of Prostaglandin E2 Binding to Isolated Human Adipocytes
Authors: Day Richard P , Little Sally A , Robertson R Paul
Published: September 2013

Prostaglandin (PG) E2 binding to fat cells and its consequent antilipolytic effect have been studied in experiments using laboratory animals, but no binding studies have yet been reported using adipocytes from humans. Consequently, we have characterized PGE2 binding to human isolated fat cells to compare the apparent binding constant to the IC50 for the antilipolytic effect of PGE2. Our data indicate that human fat cells contain binding sites that specifically recognize prostaglandins of the E series and demonstrate stereospecific recognition of the more potent of two 15-methyl-PGE2 analogues. There was no evidence for rapid metabolism of PGE2 by isolated adipocytes such as occurs in lung and liver tissue. A double-reciprocal plot of binding data obtained at saturation using [3H]PGE2 and increasing concentrations of PGE2 indicated a single class of binding sites with an apparent binding constant (0.54 nM) that agreed well with the IC50 (0.26 nM) for the antilipolytic effect of PGE2 we observed in human fat cells. The findings from these binding and lipolysis studies are in general agreement with published observations using adipocytes from rodents and provide evidence that the conclusions reached from previous studies of laboratory animals are relevant to adipocyte physiology in humans.
Administration of Silica Prevents Diabetes in BB-Rats
Authors: Oschilewski U , Kiesel U , Kolb H
Published: September 2013

Administration of silica to young BB-rats almost completely prevented the development of spontaneous diabetes. Only 1 of 31 silica-treated rats developed hyperglycemia, whereas 9 of 31 in the untreated group did so. Since silica is highly specific in its action against macrophages, our observations indicate an important role of these cells in the pathogenesis of the disease.
Neonatal Morbidities in Infants of Mothers with Glucose Intolerance in Pregnancy
Authors: Widness John A , Cowett Richard M , Coustan Donald R , Carpenter Marshall W , Oh William
Published: September 2013

Of 1839 pregnant women screened prospectively, 52 were identified to have glucose intolerance. Ten additional pregnant women identified as having glucose intolerance before the universal screening were also included in the study cohort. These 62 patients were followed in a perinatal high-risk clinic with weekly plasma glucose determinations. The patients were treated with diet and, in addition, 21 of 62 were treated with insulin therapeutically. By observational cohort design, the infants and a comparable number of matched controls were evaluated for evidence of neonatal morbidities and classified into percentile for birth weight. Compared with the control group, the operative mode of delivery, the mean birth weight, the birthweight percentile, the male/female ratio, the frequency of low Apgar score (≤ 6 at 1 min), and the number of infants with congenital anomalies were significantly higher in the infants born to the glucose-intolerant mothers. Although the mean maternal blood sugar was maintained within a reasonably euglycemic range, the usual neonatal morbidities were not eliminated entirely. Further understanding and management of glucose intolerance in pregnancy is necessary to further diminish or eliminate neonatal morbidities.
Metabolic and Therapeutic Assessment of Gestational Diabetes by Two-Hour and Twenty-Four-Hour Isocaloric Meal Tolerance Tests
Authors: Hollingsworth Dorothy Reycroft , Ney Denise , Stubblefield Nancy , Fell Tammy
Published: September 2013

Lean and obese women with gestational diabetes (GDM) were given two different isocaloric meal challenge tests to assess glucose and insulin responses. Forty-three pregnant women received a 400-kcal isocaloric breakfast meal tolerance test (mini-MTT). Twenty of the subjects were also given a 2000-kcal isocaloric diet with three meals and three snacks during a 24-h period (maxi-MTT). This was the first study to utilize the physiologic challenge of mixed meals to compare insulin and glucose responses of both obese and lean normal pregnant women and women with GDM around the clock.

Normal obese pregnant women had higher integrated glucose and insulin values around the 24-h clock < 0.003 and < 0.03, respectively) than lean pregnant women. Lean and obese women with GDM also responded differently to the physiologic challenge of mixed meals. Some, but not all, obese diabetic subjects were markedly hyperinsulinemic in contrast to lean diabetic women who were relatively insulin deficient. One normoglycemic massively obese 18-yr-old pregnant woman was unexpectedly found to be severely hyperinsulinemic.

The two meal tolerance tests clearly defined a delay in the release of insulin in women with GDM (lean and obese) and markedly different quantitative insulin responses to identical meal challenges in obese diabetic subjects. Maternal hyperinsulinemia was positively correlated with prepregnancy body mass index (kg/m2) and heavier infants, but not with plasma glucose levels.

These studies provide evidence that GDM is a heterogeneous syndrome. Because of the variety of tests for GDM, the not unusual occurrence of carbohydrate intolerance in lean pregnant women without risk factors, and the worldwide differences in diagnostic criteria for this syndrome, we recommend screening for diabetes in all pregnant women. Finally, we have proposed that an extension of the National Diabetes Data Group classification be applied to pregnant women. In this scheme, gestational carbohydrate intolerance would be considered type III or GDM and be subdivided into nonobese and obese women. The increasing number of patients with carbohydrate intolerance secondary to renal dialysis, transplantation, cystic fibrosis, and other disorders might be called type IV or secondary diabetes.
Impaired Insulin Biosynthetic Capacity in a Rat Model for Non-insulin-dependent Diabetes: Studies with Dexamethasone
Authors: Giddings Stephen J , Orland Matthew J , Weir Gordon C , Bonner-Weir Susan , Permutt M Alan
Published: September 2013

These studies of a rat model for non-insulin-dependent diabetes mellitus (NIDDM) were performed to determine whether hyperglycemia occurs when capacity to synthesize insulin is exceeded. The neonatal streptozocin (STZ)-treated rat has acute hyperglycemia with marked destruction of pancreatic β-cells, followed by gradual regeneration to 50–70% normal (β-cell number. At age 4 wk, fed serum glucose concentration is only mildly elevated relative to controls. With age, the rats become progressively hyperglycemic, and by 12 wk they have marked impairment of glucose-stimulated insulin release. In these studies, dexamethasone (0.125 mg/kg/day for 4 days) was administered to control and to STZ-treated animals to produce insulin resistance. The relationship between insulin biosynthesis and serum glucose concentrations was assessed. In control rats, response to dexamethasone was similar at both 4 and 12 wk. Serum glucose levels and pancreatic insulin concentration remained unchanged. Both insulin biosynthetic rates (as measured by 3Hleucine incorporation into proinsulin) and proinsulin mRNA levels increased twofold. STZ-treated rats at age 4 wk demonstrated mild hyperglycemia. Dexamethasone injection resulted in an increase in insulin biosynthesis and proinsulin mRNA in these animals, while serum glucose did not increase. STZ-treated rats at 12 wk showed more profound hyperglycemia (serum glucose 315 ± 38 mg/dl versus control, 187 ± 12 mg/dl). A marked rise in serum glucose (to 519 ± 42 mg/dl) was observed after4 days of dexamethasone injection. Pancreatic insulin content became severely depleted relative to salineinjected, STZ-treated animals, and there was no response of levels of proinsulin mRNA.

These studies demonstrate a significant correlation between mean insulin biosynthetic rates and mean proinsulin mRNA levels in 4-wk-old rats. In these rats with mild glucose intolerance related to a decrease in insulin synthesis, the rate of biosynthesis can still increase if demand for insulin is acutely increased by induction of an insulin-resistant state. If the correlation between proinsulin mRNA and biosynthesis exists in 12-wk-old animals, the data suggest that older, more hyperglycemic animals lose the capacity to increase rates of insulin biosynthesis and secretion; severe hyperglycemia ensues when capacity to synthesize insulin is exceeded.
Gliclazide Therapy Is Associated with Potentiation of Postbinding Insulin Action in Obese, Non-insulin-dependent Diabetic Subjects
Authors: Ward Glenn , Harrison Len C , Proietto Joseph , Aitken Patricia , Nankervis Alison
Published: September 2013

Six obese, non-insulin-dependent diabetic subjects were studied before and 3 mo after treatment with the sulfonylurea gliclazide, 40–80 mg b.i.d. Fasting plasma glucose fell significantly from 13.4 ±1.6 (SEM) to 8.6 ±1.2 mmol/L, accompanied by a significant reduction from 40.6 ± 3.7 to 29.8 ± 2.8 mM · h of the plasma glucose response to 75 g oral glucose. Fasting plasma insulin showed a nonsignificant increase from 24.8 ± 2.0 to 31.3 ± 2.3 mU/L. The percent specific binding of tracer 125I-insulin to erythrocytes and monocytes did not change significantly (from 9.8 ± 1.7 to 8.5 ± 0.7 for erythrocytes and 1.7 ± 0.3 to 1.6 ± 0.4 for monocytes). Glucose utilization was measured at three levels of insulin infusion (40, 100, and 300 mU/ kg/h) by the euglycemic clamp technique. Overall there was a significant (P < 0.05) increase in the disappearance rate (Rd) and metabolic clearance rate (MCRg) for glucose at the two higher insulin infusion rates (MCRg: 3.3 ± 0.7 to 5.1 ± 0.7 and 5.9 ± 0.9 to 7.9 ± 0.9 ml/kg/min), but not at the lowest infusion rate (MCRg: 3.6 ± 0.8 to 3.3 ± 0.6). Thus, the chronic hypoglycemic effect of gliclazide in obese diabetic subjects was associated with an improvement in insulin-mediated glucose utilization at high plasma insulin concentrations. This enhanced effect of insulin after gliclazide treatment was not accompanied by increased monocyte or erythrocyte insulin binding, which suggests that it was due to potentiation of postbinding insulin-sensitive pathways.
Insulin Secretion In Vitro and Insulin Binding to Isolated Hepatocytes in Congenic Mice with Different H-2 Complexes
Authors: Petersen Karl-Georg , Heidenreich Stefan , Pilot Paul-Richard , Kerp Lothar
Published: September 2013

Congenic male mice with differences in the H-2 complex have been used to investigate insulin secretion in vitro, insulin binding to isolated hepatocytes, plasma glucose, and serum insulin. Plasma glucose and serum insulin did not show consistent differences in the B10.BR, B10.D2, B10.A, B10.G, B10.M, B10.S, C57 10SCSN, and C3H.OH strains. Isolated islets of Langerhans responded to stimulation with 400 mg/dl glucose with a 3–5-fold increase in insulin secretion rates (2P < 0.01): B10.BR > B10.M > C57BL/10SCSN > B10.G > C3H.OH, B10.D2, B10.A, B10.S. The biphasic pattern of insulin secretion was less distinct in B10.M, B10.G, and C3H.OH mice.

The high-affinity constants of insulin binding to isolated hepatocytes at 37°C varied between 4.5 × 10−7 L · mol−1 and 4.5 × 108 L · mol−1 (2P < 0.01): B10.A > B10.BR > C57BL/10SCSN, B10.S, B10.D2 > B10.M, B10.G.

The glucose-stimulated insulin secretion from isolated islets of Langerhans and the binding of insulin to isolated hepatocytes correlate to the H-2 complex independently.
Insulin's Effect on Leucine Turnover Changes During Early Fasting in the Conscious Dog
Authors: Hoxworth Ben , Miller Bonnie , Radosevich Paul , Buckspan Randy , Lacy William , Abumrad Naji N
Published: September 2013

To study the effects of insulin on leucine turnover during fasting, acute insulin deficiency was induced by the simultaneous infusion of somatostatin and glucagon in conscious dogs fasted 18 h (N = 10) and 48 h (N = 11). Insulin levels during the basal period (before hormone perturbation) were similar in both groups of dogs (12 ± 3 versus 10 ± 3 μU/ml, respectively). Glucagon levels were similar in the two groups (94 ± 9 versus 106 ± 19 pg/ml). Leucine levels rose from 118 ± 9 jimol/L to 155 ± 12 fimol/L as fasting progressed (P < 0.005). Its rate of appearance also increased by 30% (P < 0.005) from 3.4 ± 0.3 to 4.3 ± 0.4 μmol/kg/min (P < 0.005), while its clearance remained unchanged. Acute insulin deficiency caused an increase in leucine levels in both 18-h and 48-h-fasted dogs by 55% (to 181 ± 10 μnol/L) and 45% (to 225 ± 20 μmol/L), respectively (P < 0.005). However, while the rate of appearance of leucine remained unchanged in dogs fasted overnight, it rose to 5.1 ± 0.3 μmol/kg/min (P < 0.01) in those fasted 48 h. The metabolic clearance rate fell in both groups, although this drop was twice as great in the 18-h group (from 28 ± 3 to 17 ± 3 ml/kg/min, P < 0.005) as in the 48-h group (from 28 ± 3 to 23 ± 2 ml/kg/min, P < 0.005).

We conclude that insulin has disparate effects on protein turnover as fasting becomes more prolonged. Its acute absence results in enhanced proteolysis in the 2-day-fasted, but not in the overnight-fasted dog. These data suggest that the response of protein turnover to fasting is quite complex and involves more than the effects of changing insulin concentrations.
Skeletal Muscle Basement Membrane in Maturity-onset Diabetes in the Young
Authors: Mauer M , Steffes M , Johnson E , Rich S , Hoogwerf B , Chavers B , Barbosa J
Published: September 2013

We have studied skeletal muscle capillary basement membrane width (CBMW) and intensity of skeletal muscle extracellular basement membrane staining for albumin and IgG in eight families with maturity-onset diabetes in the young (MODY).

Ninety-two MODY patients were identified. Sixtythree of these patients, 33 relatives with nondiagnostic oral glucose tolerance studies, and 61 normoglycemic relatives were studied for glucose and insulin. Twentysix of these MODY patients, 20 normoglycemic relatives, and 16 unrelated normal controls had skeletal muscle capillary morphologic studies.

The muscle capillary basement membrane was significantly increased in MODY patients younger than 40 yr when compared with unrelated normal subjects and relatives of the same age (P < 0.001). However, in these families, the CBMW of MODY patients showed no significant thickening with age (slope = 0.45, P < 0.14), as expected and seen in the normal subjects and in the normoglycemic relatives of the patients (slope = 1.21, P < 0.001). The slope derived from the linear regression of age and CBMW in MODY patients (0.45 ± 0.29) was significantly less (P < 0.05) than that of the nondiabetic subjects (1.21 ± 0.19). The mean intensity of skeletal muscle extracellular basement membrane staining for albumin was higher in MODY patients (1.1 ± 0.15) than in unrelated normal subjects (0.7 ± 0.1, P < 0.025) and normal MODY family members (0.6 ± 0.08, P < 0.01).

The unexpected absence of basement membrane thickening with age in the MODY patients may explain the paucity of vascular complications that have been reported by some. However, these findings may also reflect genetic heterogeneity, mild hyperglycemia, shorter duration of disease, or any combination of these factors. The immunohistochemical studies indicate that the increased localization of plasma proteins in extracellular membranes in diabetes, also seen in insulin-dependent diabetes, is a marker of hyperglycemia and is not specific for different types of diabetes.
Characteristics of Insulin Receptors and Insulin Action in Human Myelogenous Leukemia Cell Line K-562
Authors: Yamanouchi Toshikazu , Tsushima Toshio , Akanuma Yasuo , Kasuga Masato , Mizoguchi Hideaki , Takaku Fumimaro
Published: September 2013

Specific binding sites for insulin have been identified and characterized for the human erythroleukemia cell line K-562. The binding of [125I]-insulin to the cells increased as a function of time, reaching a maximum at 20 min when incubation was performed at 37°C. The binding of [125I]-insulin was dose-dependently inhibited by insulin or proinsulin. Scatchard plot of the binding data was curvilinear, and the number of insulin receptors was approximately 39,000. Insulin at concentrations of 0.05–10.0 ng/ml stimulated CO2 production and DNA and protein synthesis in K-562 cells in a dose-dependent manner, indicating that the insulin binding sites are functionally important in mediating these biochemical events induced by insulin. Maximal insulin responses were elicited at concentrations of <5 ng/ml, when (at most) 10% of the insulin receptors were occupied. After binding to the cells, [125I]-insulin was degraded in a time- and temperature-dependent manner. As reported for other types of cells, unlabeled insulin also downregulated insulin receptors in K-562 cells. When the cells were incubated with 1 × 10−7 M unlabeled insulin for 24 h, the number of insulin receptors decreased by 50% without a change of affinity. K-562 cells may be useful in studying the role of insulin receptors in cell functions induced by insulin.
Pump-induced Insulin Aggregation: A Problem with the Biostator
Authors: Brennan James R , Gebhart Suzanne S P , Blackard William G
Published: September 2013

A fall in plasma IRI despite constant C-peptide levels during prolonged insulin euglycemic clamp studies using the Biostator (Ames Division, Miles Laboratories, Elkhart, Indiana) prompted a meticulous evaluation of the Biostator's insulin delivery system. At slow infusion rates, a striking loss of immunoreactive and biologically active insulin was observed after 6 h of the Biostator run. Studies with labeled insulin indicated that the loss of insulin was not due to adsorption of insulin to the tubing since recovery of labeled insulin was close to 100%. A variety of techniques (gel filtration, polyacrylamide gel electrophoresis, centrifugation, and Coomassie Brilliant Blue protein assay) indicated that the loss of insulin activity was due to insulin coming out of solution. The insoluble nature of the immunologically and biologically inactive insulin was confirmed by centrifugation, i.e., 88% 125I-insulin precipitated into the pellet. The dependency of this loss of insulin activity on flow rate was clearly demonstrable with activity (IRI) less than 20% of expected at flow rates of 2.1 ml/h, and 30% at 4.2 ml/h. Full recovery was observed only with flow rates of 16.8 ml/h or greater. At each flow rate, IRI rose only after delivery of the effluent between the pump and exit port, demonstrating that insulin alteration occurs within the pump assembly presumably from heat-induced aggregation. Investigators employing the Biostator should carefully examine their systems for this time- and flow ratedependent alteration of insulin. The loss of IRI at low flow rates (low-dose insulin clamp or insulin delivery during basal periods) will profoundly influence data generated from the Biostator.
Insulin Action During Pregnancy: Studies with the Euglycemic Clamp Technique
Authors: Ryan Edmond A , O'Sullivan Mary Jo , Skyler Jay S
Published: September 2013

To assess the mechanisms responsible for the insulin resistance associated with both normal human pregnancy and gestational-onset diabetes, we have measured exogenous glucose disposal using sequential insulin infusions with the euglycemic glucose clamp technique and erythrocyte insulin binding. Three groups of women were studied: nonpregnant women with normal glucose tolerance (N = 7, mean age 32.9 ± 2.1 yr), pregnant women with normal glucose tolerance (N = 5, mean age 24.8 ± 3.5 yr), and pregnant women with gestational-onset diabetes (N = 5, mean age 34.6 ± 2.6 yr). Despite normal plasma glucose levels obtained during a 100-g oral glucose tolerance test, plasma insulin levels were significantly elevated in the pregnant women compared with the nonpregnant control subjects, suggesting a state of insulin resistance. Insulin binding to erythrocytes was similar in all three groups (maximum specific binding being 5.0 ± 0.6%, 5.5 ± 1.1%, and 6.0 ± 0.7% in nonpregnant, nondiabetic pregnant, and gestational-onset diabetic women, respectively). In vivo peripheral insulin action was measured using the euglycemic glucose clamp technique during an insulin infusion of 40 mil/ m2 · min, with blood glucose clamped at a concentration of 75 mg/dl using a variable glucose infusion. Glucose infusion rates were 213 ± 11 mg/m2 min, 143 ± 23 mg/m2 · min, and 57 ± 18 mg/m2 · min in nonpregnant, nondiabetic pregnant, and gestationalonset diabetic women, respectively. This demonstrates that pregnant subjects display a state of insulin resistance, and that this appears to be more marked in gestational-onset diabetic subjects. To further define the possible mechanism of insulin resistance during pregnancy, the insulin infusion rate was increased to 240 mU/m2 · min and further euglycemic clamp measurements performed. Glucose infusion rates were 372 ± 11 mg/m2 · min, 270 ± 31 mg/m2 · min, and 157 ± 26 mg/m2 · min, in nonpregnant, nondiabetic pregnant, and gestational-onset diabetic women, respectively. This demonstrates a shift to the right of the dose-response curve of insulin action and suggests that the insulin resistance of pregnancy may include a decrease in presumed “maximum” insulin responsivity. In four subjects, studies were repeated in the postpartum period, and these demonstrated that the insulin resistance of pregnancy is ameliorated shortly after delivery. These studies suggest that the insulin resistance of pregnancy results from a target cell defect in insulin action beyond the initial step of insulin binding to cellular receptors, a postreceptor (or postbinding) defect in insulin action.
Age-related Changes in Hepatic Glycogen Metabolism in the Genetically Diabetic (db/db) Mouse
Authors: Roesler William J , Khandelwal Ramji L
Published: September 2013

Hepatic glycogen metabolism was investigated in genetically diabetic C57BL/KsJ-db/db mice during their development. Initially, the development of obesity, hyperglycemia, hyperinsulinemia, and hyperglucagonemia in these mice was examined, which illustrated that the diabetes progressed normally. Little difference in hepatic glycogen concentrations was observed, averaging approximately 50 and 60 mg/g liver in diabetic (db/db) and control heterozygote (db/+) mice, respectively. Glycogen synthase activity (total and a-form) was significantly elevated by 5 wk in the diabetic mice relative to controls and reached maximum levels (twofold higher than controls) around 8–9 wk. This activity then slowly declined during the rest of the 15-wk period examined. Both phosphorylase a and total phosphorylase activities were also elevated by 5 wk, reaching levels twofold higher than controls. These activities did not decline at the end of this 15-wk period, but instead continued to slowly increase. Glycogen synthase a activity showed a positive correlation (r = 0.54, N = 144) with circulating levels of insulin, and a similar correlation was seen for phosphorylase a activity and plasma glucagon levels (r = 0.64, N = 72). Protein kinase and phosphoprotein phosphatase activities were also measured, but no differences were detected between diabetic and control mice. This longitudinal study clarifies some of the changes in hepatic glycogen metabolism that occur during the progression of diabetes in the db/db mouse and indicates a role for circulating insulin and glucagon concentrations on the steady-state activities of glycogen synthase and phosphorylase, respectively.
Insulin-like Action of Proinsulin on Rat Liver Carbohydrate Metabolism In Vitro
Authors: Probst Irmelin , Hartmann Heinz , Jungermann Kurt , Creutzfeldt Werner
Published: September 2013

Short-term effects of human proinsulin on metabolic rates and its long-term action on enzyme induction were studied in primary cultures of rat hepatocytes and in the perfused rat liver, and compared with the effects of bovine insulin. In the perfused rat liver, proinsulin decreased the glucagon-dependent increase of glycogenolysis. The action of 0.5 nM glucagon was almost completely suppressed by 100 nM proinsulin. Proinsulin and insulin showed similar potency.

In cultured rat hepatocytes, proinsulin stimulated glycolysis up to fivefold with a half-maximal effective dose of 30 nM. Proinsulin induced the key glycolytic enzymes glucokinase and pyruvate kinase by twofold and antagonized the glucagon-dependent induction of phosphoenolpyruvate carboxykinase with a halfmaximal effective dose at 3 nM. For the effects in cultured hepatocytes, about 100-fold higher concentrations of proinsulin than of insulin were required.
Aggregation of Insulin, Containing Surfactants, in Contact with Different Materials
Authors: Chawla Attar S , Hinberg Irwin , Blais Pierre , Johnson David
Published: September 2013

The aggregation of insulin and of insulin protected with surfactants was studied by shaking at 37°C in glass, in polypropylene and polystyrene vials, and in CPI and Auto-Syringe insulin syringes and infusion sets. Surfactants such as Pluronic 17R8 and 25R5 hastened the aggregation, whereas Pluronic F68 was effective in preventing it. Furthermore, there was no change in the immunoreactivity of insulin containing Pluronic F68 even after 8 days of shaking. Unprotected insulin aggregated in all the vials.

There appears to be little problem with the commercial syringes tested, but the infusion sets could cause aggregation of insulin if used over an extended period of time. Although Pluronic F68 prevented insulin aggregation in situ, it extracted more impurities from the contacting plastics. Further development in materials and design of insulin sets and insulin containers appears necessary.
In Vitro Preparation of Nonenzymatically Glucosylated Human Transferrin, α2-Macroglobulin, and Fibrinogen with Preservation of Function
Authors: Ney Kathryn A , Pasqua John J , Colley Karen J , Guthrow C Earl , Pizzo Salvatore V
Published: September 2013

Human transferrin, α2-macroglobulin, and fibrinogen were incubated with [3H]-glucose. After a 7-day, 37°C incubation at 20 mM glucose, transferrin incorporated 1.1 mol of glucose/mol protein; α2-macroglobulin, 10 mol of glucose/mol; and fibrinogen, 3.8 mol of glucose/ mol, or approximately 14 μmol of glucose/g for each protein. These results were the same for glucose labeled in the 1 or 6 position. No radiolabel was incorporated into the proteins during incubations with glucose labeled in the 2 position. The rate and extent of iron binding were identical for both glucosylated and nonglucosylated transferrin. Glucosylated transferrin bound to Wil-2 human lymphoblast cells with a Kd = 33 nM and receptor number of 3.4 × 105 receptors/cell; nonglucosylated transferrin with a Kd = 31 nM and receptor number of 3.9 × 105 receptors/cell. Glucosylated and nonglucosylated α2-macroglobulin showed the same conformational change as determined on native PAGE after reaction with trypsin, plasmin, or methylamine and had the same activity in the Ganrot assay after reaction with trypsin or plasmin. The clearance of 125I-labeled, methylamine-treatedα2-macroglobulin from the mouse circulation was identical for both glucosylated and nonglucosylated α2-macroglobulin, t1/2 = 3 min. α2-Macroglobulin that was first glucosylated then reacted with methylamine bound to mouse peritoneal macrophages with a Kd of 2.5 nM and receptor activity of 370 fmol/mg cell protein. α2-Macroglobulin that was first reacted with methylamine and then glucosylated bound with a Kd of 3 nM and receptor activity of 320 fmol/mg cell protein. Glucosylated fibrinogen had the same clotting time as control fibrinogen. The crosslinking by factor XIII and degradation by plasmin were the same for glucosylated and control fibrin as determined on SDS-PAGE. Glucosylated fibrinogen was degraded by plasmin to yield identical fragments on SDSPAGE as the control fibrinogen; however, glucosylated fibrinogen was degraded at a slightly slower rate. Platelet binding studies showed a Kd of 6.6 × 10−7 M and receptor number of 33,000 sites per cell for control fibrinogen and a Kd of 6.8 × 10−7 M and receptor number of 28,000 sites per cell for glucosylated fibrinogen.
Nonenzymatic Glycation of Fibronectin and Alterations in the Molecular Association of Cell Matrix and Basement Membrane Components in Diabetes Mellitus
Authors: Tarsio Joseph F , Wigness Bruce , Rhode Thomas D , Rupp William M , Buchwald Hēnry , Furcht Leo T
Published: September 2013

This study reports the nonenzymatic glycation of plasma fibronectin in vivo in diabetic dogs and also in vitro by incubation of human plasma fibronectin with excess glucose. Although no difference is observed in the total plasma fibronectin level, the nonenzymatic glycation of fibronectin is increased 2.3-fold in inbred male beagle dogs made diabetic with alloxan in comparison with age-matched controls. The extent of nonenzymatic glycation of fibronectin is shown to be proportional to blood glucose levels. HPLC reverse-phase analysis of the hydrolyzed amino acids and glycoamino acids from plasma fibronectin samples of normal and diabetic dogs show that nonenzymatic glycation occurs only on lysine residues.

When purified human plasma fibronectin was incubated in vitro with 500 mM glucose, the extent of nonenzymatic glycation of fibronectin was observed to increase proportionately with time. Ligand binding assays conducted in solution with varying concentrations of 3H-heparin in the presence of a constant amount of normal or nonenzymatically glycated human plasma fibronectin gave virtually identical binding curves. However, the binding of 3H-heparin to normal fibronectin could be increased fourfold by the concomitant addition of normal gelatin (denatured calfskin collagen). If in vitro glycated fibronectin and/or in vitro glycated gelatin are added under this latter condition with 3H-heparin, there is a tremendous decrease in the expected heparin binding seen with normal levels of nonenzymatic glycation. Other experiments were performed to quantitate the binding of 3H-labeled fibronectin to gelatin-coated nitrocellulose filters. Nonenzymatic glycation of fibronectin in vitro resulted in markedly decreased binding of 3H-fibronectin to collagen. Enhanced nonenzymatic glycation of fibronectin in vivo and alterations in the molecular association of fibronectin, gelatin, and heparin brought about by nonenzymatic glycation are discussed relevant to the pathophysiology of the complications of diabetes.
Products of Therapeutic Insulins in the Blood of Insulin-dependent (Type I) Diabetic Patients
Authors: Robbins David C , Shoelson Steven E , Tager Howard S , Mead Patricia M , Gaynor Deborah H
Published: September 2013

The tendency of insulin in high concentrations to selfassociate and the widespread presence of insulindegrading enzymes suggest that fragments and/or aggregates of insulin may circulate in normal and insulin-dependent diabetic (IDOM) individuals. To examine this possibility, we have analyzed, by sensitive physicochemical methods, immunoreactive insulin (IRI) taken from the blood of 9 healthy volunteers and 12 insulin-dependent diabetic patients. IRI from the blood of the normal volunteers was composed of 6000 (91.0 ± 1.4%) and 9000 (9.0 ± 1.4%) molecular weight (mol wt) material. By 10% polyacrylamide disc gel electrophoresis (PAGE) and reverse-phase, high-performance liquid chromatography (HPLC), the 6000 mol wt material was indistinguishable from human insulin standards and insulin fragments were not found. C-peptide reactivity in the 9000 mol wt material confirmed the expected presence of proinsulin and intermediates of proinsulin conversion.

IRI harvested from the blood of 12 C-peptide-negative IDDMs, using a variety of insulin preparations, also separated into 6000 (80.5 ± 3.9%) and 9000–12,000 (19.5 ± 3.9%) mol wt material. By HPLC, 6000 mol wt IRI was either pork insulin (in volunteers using pure pork insulin) or a mixture of beef (∼90%), pork (∼10%) and deamidated beef (trace) insulin in those using a beef-pork mixture. However, the 9000–12,000 mol wt material had characteristics entirely distinct from proinsulin of either human or animal origin: (1) C-peptide reactivity was undetectable using any of three sensitive radioimmunoassay systems, (2) on PAGE it migrated more rapidly than proinsulin-like material, and (3) in contrast to proinsulin, it was unaffected by proteolytic degradation.

We conclude that a covalent aggregate of therapeutic insulin circulates in diabetic patients who use a variety of insulin preparations. Insulin aggregates may account for a relatively large amount of the total IRI in IDDMs, and these aggregates may promote the formation of anti-insulin antibodies and affect the action of insulin.
Abnormal Glucose Modulation of Islet A- and B-Cell Responses to Arginine in Non-insulin-dependent Diabetes Mellitus
Authors: Dimitriadis George D , Pehling Gregory B , Gerich John E
Published: September 2013

To assess the normality of islet A- and B-cell responses to a nonglucose secretogogue as well as the modulating effect of glucose in NIDDM, we examined plasma C-peptide and glucagonresponses to arginine in eight patients with NIDDM and in six age- and weight-matched nondiabeticvolunteers under conditions of identical hypoglycemia (∼70 mg/dl), euglycemia (94 mg/dl), and hyperglycemia (∼190 mg/dl). Plasma C-peptide responses to glucose and to arginine in the diabetic subjects were both significantly reduced at all glucose concentrations studied (P < 0.01–0.005). The modulating effect of glucose on both islet A- and B-cell responses (slope of relation between plasma Cpeptide or glucagon response versus plasma glucose concentration) was reduced 80% in the diabetic subjects (P < 0.01). We conclude that islet A- and B-cell responses to nonglucose secretogogues are abnormal in patients with NIDDM and that this may result from a functional defect in the modulating effect of glucose on insulin and glucagon secretion, which in some patients may be compensated for by hyperglycemia.
Stimulation of Insulin Secretion from Isolated Rat Islets by SaRI 59–801
Authors: Hanson Ronald L , Isaacson Craig M , Boyajy Louis D
Published: September 2013

Oral administration of SaRI 59–801 (DL-α-[dimethylaminomethyl]-2-[3-ethyl-5-methyl-4-isoxazolyl]-1H-indole-3-methanol) has been reported to decrease blood glucose in several species and to elevate plasma insulin in rats and mice. In studies with isolated rat pancreatic islets incubated 1 h with 3 mM glucose, 0.05 mM 59–801 produced a significant increase in insulin secretion, and 0.3 mM produced maximum release. 59–801 (0.3 mM) stimulated insulinrelease 4–5-fold from islets incubated with 0, 3, or 5 mM glucose but had little effect on the high rates of release obtained at 10 or 20 mM glucose. Ten millimolar mannoheptulose, which inhibits phosphorylation of glucose and blocks glucose-stimulated insulin release, had little effect on the stimulation of insulin release by 0.3 mM 59–801 from islets incubated with 3 mM glucose. Stimulation of insulin release in the absence of glucose or in the presence of 3 mM glucose plus 10 mM mannoheptulose suggests that glucose metabolism is not required for the action of 59–801. The rate of conversion of 5 mM [5–3H]-glucose to 3H2O by islets, a measure of the rate of glycolysis, was not affected by 59–801. The potency, dependency on glucose concentration, lack of inhibition by mannoheptulose, and lack of effect on glycolysis of 59–801 were similar to that of tolbutamide. However, proinsulin synthesis by islets incubated with 5.55 mM glucose was not affected by 0.5 mM 59–801, but was inhibited 72% and 67% by 0.5 mM tolbutamide and 0.1 mM glibenclamide, respectively.
“Cytoplasmic” Islet Cell Antibodies: Evidence That the Target Antigen Is a Sialoglycoconjugate
Authors: Nayak R C , Omar M A K , Rabizadeh A , Srikanta S , Eisenbarth G S
Published: September 2013

We have biochemically treated (periodate, borohydride, neuraminidase, organic solvents) frozen sections of human pancreas and studied the reactivity of islet-cellantibody-positive human sera and monoclonal antibodies. The autoantigen of pancreatic sections has the properties ofsialic acid containing glycolipid.
Glucose Toxicity for Human Endothelial Cells in Culture: Delayed Replication, Disturbed Cell Cycle, and Accelerated Death
Authors: Lorenzi Mara , Cagliero Enrico , Toledo Silva
Published: September 2013

Functional and anatomical abnormalities of endothelium may represent a pathway to the increased vascular permeability and accelerated atherosclerosis characteristic of diabetes. To identify whether and how hyperglycemia may compromise the endothelial barrier, we have employed an in vitro system of human endothelial cells obtained from umbilical veins and cultured in elevated glucose concentrations (20 mM). Under these conditions, the achievement of saturation density was substantially delayed, with cell counts throughout most of the growth curve being 70–80% of control (P < 0.002). More profound suppression of cell number was present in cultures exposed to 40 mM glucose. Similar, albeit slightly lesser, effects were observed in cultures exposed to 20 mM mannitol, mimicking the hypertonicity of the high glucose media. The effect of elevated glucose and mannitol was primarily mediated by a decrease in overall rate of replication of the endothelium as documented by the lower mitotic index (P < 0.025).

Analysis of the distribution of cells along phases of the cell cycle uncovered in the high glucose cultures a decreased proportion of cells in G0-G1 (70.5 ± 5% versus 73.2 ± 4% in controls, P < 0.05) and an increased proportion of cells in S phase (16.5 ± 2.7% versus 13.5 ± 2.2% in controls, P < 0.01), suggesting that the replicative delay is likely to occur between the phase of DNA synthesis and mitosis. Increased cellular death was specifically observed in the cultures exposed to elevated glucose concentrations (P < 0.05), but it could account for only a minor portion of the deficit in cell number.

The mechanism(s) underlying the described ill effects of high glucose/hypertonicity on endothelium and the relevance of such abnormalities to diabetic angiopathy are currently under investigation.
Abnormal Insulin Secretion in a Streptozocin Model of Diabetes: Effects of Insulin Treatment
Authors: Leahy J L , Bonner-Weir S , Weir G C
Published: September 2013

We have proposed that chronic hyperglycemia causes the abnormal glucose influence on arginine-stimulated insulin secretion in the neonatal streptozocin (STZ) rat model of NIDDM and therefore studied the effect of 24h of mild insulin-induced hypoglycemia on this defect. Ultralente insulin, 20 U/kg, was given at 9 a.m. and 10 U/kg at 5 p.m., and insulin and glucagon secretion were then studied the next morning using the in vitro isolated, perfused pancreas. The fed plasma glucose concentrations decreased in the STZ rats from 191 ± 13 to 101 ± 9 mg/dl and from 133 ± 4 to 99 ± 8 mg/dl in the controls.

As expected, 10 mM arginine caused a trivial insulin response at 2.8 mM glucose in the treated and untreated control groups compared with the marked one at 16.7 mM. The response to arginine at 2.8 mM glucose in the untreated STZ rats, however, was strikingly elevated (7.65 ± 2.29 versus 0.41 ± 0.16 ng/ml in the untreated controls) and it was not potentiated by the high glucose background, but the result at 2.8 mM glucose in the treated STZ rats was similar to that of the treated controls (0.46 ± 0.12 versus 0.16 ± 0.03 ng/ml). A returh of glucose influence on IBMX-stimulated insulin secretion was also noted. Glucose-induced insulin release, however, was not restored in the treated STZ rats, but it was markedly suppressed in the controls by the insulin treatment.

Glucose influence on the glucagon response to arginine was maintained in the STZ model even though the glucagon release to a lowered glucose concentration was lost.

These data suggest that chronic hyperglycemia causes the abnormal glucose influence on argininestimulated insulin release in the STZ model of diabetes.
Variable Deterioration in Cortical Function During Insulin-induced Hypoglycemia
Authors: Herold Kevan C , Polonsky Kenneth S , Cohen Robert M , Levy Jerre , Douglas Frank
Published: September 2013

Cortical function during insulin-induced hypoglycemia was studied in 14 normal controls and 12 type I diabetic patients by measuring the reaction time to a visual stimulus. Each subject was studied on two occasions, during insulin-induced hypoglycemia and under euglycemic conditions. The mean reaction time during euglycemic conditions was 260 ± 6 ms in the controls and 309 ± 11 ms in the diabetic subjects (P < 0.001) and did not change significantly over a 2-h period. Intravenous (i.v.) insulin administration to both groups of subjects resulted in similar reductions in glucose concentrations, which were maintained below 50 mg/dl for at least 30 min. Under these conditions, the reaction time increased significantly (mean increase 104 ± 37 ms [P < 0.02] in the controls and 75 ± 28 ms [P < 0.02] in the diabetic subjects). However, significant variability in responsiveness was observed in individual subjects. Three of the 14 controls and 4 of the 12 diabetic subjects showed no significant change in reaction time during hypoglycemia, while the remainder demonstrated significant increases. Individual differences were not correlated with severity or duration of hypoglycemia or counterregulatory hormone responses. The maximum increase in reaction time occurred as long as 60 min after the nadir glucose and returned to baseline 10–40 min after normalization (> 60 mg/dl) of the plasma glucose level. Subjective awareness of hypoglycemia was unrelated to the change in reaction time.

These studies demonstrate that: (1) the reaction time to a visual stimulus is an objective, reproducible measure of cortical function during insulin-induced hypoglycemia; (2) control and diabetic subjects demonstrate variable sensitivity to the effects of hypoglycemia on cortical function, which is unrelated to the counterregulatory hormone responses; (3) the deterioration of cortical function observed during induction of hypoglycemia and the recovery of cortical function after euglycemia is restored may lag behind the changes in plasma glucose level (these findings help explain the frequently observed lack of correlation between the neurologic effects of hypoglycemia and simultaneous peripheral glucose levels); and (4) individual differences in cortical sensitivity as reflected by the reaction time response to hypoglycemia may parallel the variable clinical picture associated with hypoglycemia in insulin-treated diabetic subjects.
Growth Hormone Responses to Growth-Hormone-Releasing Hormone and Thyrotropin-Releasing Hormone in Diabetic Patients With and Without Retinopathy
Authors: Kaneko Kenzo , Komine Shingo , Maeda Tadao , Ohta Meisei , Tsushima Toshio , Shizume Kazuo
Published: September 2013

Growth hormone (GH) responses to growth-hormonereleasing hormone (GRH) and thyrotropin-releasing hormone (TRH) were studied in 17 diabetic patients. Ten patients (group 1) had retinopathy corresponding to stage III—V (Scott's classification), and the remaining seven patients (group 2) had no retinopathy despite longer duration of diabetes in comparison with the patients in group 1. There were no differences in age, percent of ideal body weight, and serum HbA1 levels between the two groups. Basal serum GH levels were 1.9 ± 0.4 ng/ml (mean ± SEM) in group 1, and not different from the values in group 2 (1.6 ± 0.7 ng/ml). However, GH responses to synthetic human GRH-44 (1 μg/kg body wt, i.v. bolus) were significantly greater in group 1, as judged by the maximal response or integrated GH secretion after the administration of GRH. There were no differences in serum insulin-like growth factor I (IGF-I) levels between group 1 (262 ± 35 ng/ml) and group 2 (232 ± 30 ng/ml), and no significant correlation was found between serum IGF-I levels and GH responses to GRH in either of the two groups. Paradoxical GH responses to TRH (500 μg, i.v. bolus) were found in only one patient in each group. We have thus demonstrated that GH responses to GRH are more pronounced in diabetic patients with retinopathy than in patients without this complication, although it remains to be determined whether or not greater GH responses to GRH are causally related to the development of diabetic retinopathy.
Sequential Studies of Glucose Tolerance and Red Blood Cell Insulin Receptors in Normal Human Pregnancy
Authors: Lerario A C , Wajchenberg B L , El-Andere W , Ohnuma L Y , Monaci J , Sankowsky M , Souza I T Toledo E , Germek O
Published: September 2013

Insulin binding to erythrocytes was sequentially studied in 12 healthy pregnant women during the anabolic (11–22 wk) and the catabolic (31–38 wk) gestational phases. For comparison, we studied 12 nonpregnant subjects at mid-luteal and mid-follicular menstrual phases. Oral glucose tolerance tests were also performed during these studies. There was a progressive worsening of the glucose tolerance from the anabolic to the catabolic phase associated with fasting hypoglycemia and hyperinsulinemia. The worsening of glucose tolerance was accompanied by a progressive increment of insulin secretion. Insulin binding to red blood cells increased progressively from the anabolic to the catabolic phase, due to an increased number of receptors per cell, associated with a reduction in the apparent affinity at the low occupancy levels. We concluded that the insulin resistance of pregnancy was not accompanied by an impaired binding of insulin to its receptors, at least in the RBC. The data suggest that the defect of insulin action lies at a site distal to the receptor.