bluuebunny/crossref_metadata_2025 huggingface dataset:
Endoneurial Microvessels in Human Diabetic Neuropathy: Endothelial Cell Dysjunction and Lack of Treatment Effect by Aldose Reductase Inhibitor
Authors: Sima Anders A F , Nathaniel Virgil , Prashar Ashok , Bril Vera , Greene Douglas A
Published: September 2013

Endoneurial microvascular abnormalities have been invoked in the pathogenesis of diabetic distal symmetric polyneuropathy. Detailed morphometric analysis of the endoneurial microvasculature was correlated with previously published data on nerve fiber morphometry and teased fiber analysis obtained from the same sural nerve biopsies. Biopsy specimens from neuropathic diabetic patients were obtained before and after 12 mo of aldose reductase inhibitor (ARI) treatment and compared to 15 carefully age-matched control subjects. Diabetic microvessels showed basement membrane thickening and loss of endothelial cell tight junctions. Microvascular density and the frequency of microvessels closed by endothelial cells increased with age in diabetic and control nerves and were unaffected by diabetes. The density of microvessels showing patent lumina did not differ between control and diabetic subjects and was not related to age or diabetes. Closed microvessels were composed of postcapillary venules that were otherwise devoid of ultrastructural abnormalities. We suggest that microvascular closure by endothelial cells may be a physiological condition and is unlikely to have any pathogenetic significance in diabetic neuropathy. Based on the current limited biopsy material, we conclude that 12 mo of ARI treatment that induced significant fiber repair and regeneration had no detectable effect on endoneurial microvascular abnormalities. These data suggest that endoneurial vascular pathology is not a rate-limiting factor in fiber damage or repair at this stage of diabetic neuropathy.
Decreased Bronchial Response to Methacholine in IDDM Patients With Autonomic Neuropathy
Authors: Bertherat Jérôme , Lubetzki Jean , Lockhart Alain , Regnard Jacques
Published: September 2013

This study was designed to evaluate the involvement of airways innervation during diabetic autonomic neuropathy. Bronchial response to methacholine was assessed by inhalation of serially doubling doses in 22 insulin-dependent diabetes mellitus (IDDM) patients and 11 nondiabetic control subjects selected for their nonsmoking habits. Cardiovascular autonomic control was studied by four standardized tests, i.e., blood pressure and heart-rate variations during orthostatism, heart-rate variation during Valsalva maneuver, and deep breathing. Magnitude and time-course of response to methacholine were similar in nondiabetic subjects and IDDM patients without any abnormal result on cardiovascular tests. Conversely, bronchial response to methacholine was markedly reduced in IDDM patients with one or more abnormal results by cardiovascular assessment of autonomic control. In the IDDM patients, bronchial response to methacholine was significantly correlated to indexes of cardiovascular autonomic control. These results suggest that, during diabetic neuropathy, innervation of the airways likewise involves cardiac autonomic control and leads to impairment of defense reflexes of the airways.
Differential Regulation of Bladder β-Adrenergic and Muscarinic Cholinergic Receptors in Experimental Diabetes
Authors: Latifpour Jamshid , Nishimoto Tadashi , Marian Moazez J , Yoshida Masaki , Weiss Robert M
Published: September 2013

To determine the contribution of diuresis-induced bladder hypertrophy, which accompanies the diabetic state, on the biochemical and functional alterations observed in the diabetic bladder, we compared three experimental groups: 8-wk streptozocin (STZ)-induced diabetic rats, 8-wk sucrose-fed diuretic rats, and age-matched controls. Diabetic and sucrose-fed rats had higher water intake, higher urine output, and larger bladders than controls. Diabetic rats had lower serum insulin levels, lower body weights, and higher serum glucose levels than either control or sucrose-fed animals. Receptor binding studies with [3H]quinuclidinyl benzilate in bladder dome demonstrated an upregulation of muscarinic receptors in diabetic and sucrose-fed rats compared with controls. Parallel binding studies with [3H]dihydroalprenolol and [125I]iodopindolol showed an upregulation of β-adrenergic receptors in diabetic but not in sucrose-fed bladder domes. Carbachol induced larger contractile responses in diabetic and sucrose-fed than in control bladder dome muscle strips. isoproterenol relaxed KCl-contracted detrusor strips from both diabetic and sucrose-fed rats to a greater degree and with a higher affinity than detrusor strips from controls. Our data show that overdistension and increased workload per se contributed to the upregulation of muscarinic but not to the upregulation of β-adrenergic receptors in STZ-induced diabetes. Furthermore, the magnitude of carbachol-induced contractions correlated with muscarinic receptor upregulation, whereas the magnitude of isoproterenol-induced relaxation did not correlate with changes in the density of the β-adrenergic receptors. Thus, it appears that different regulatory mechanisms are involved in diabetes-induced alterations in muscarinic and β-adrenergic receptors in bladder dome.
T-Lymphocyte Lines Specific for Glutamic Acid Decarboxylase (GAD) the 64K β-Cell Antigen of IDDM
Authors: Diaz Jose-Luis , Ways Judy , Hammonds Peter
Published: September 2013

Insulin-dependent diabetes mellitus (IDDM) is viewed as a thymus-dependent autoimmune disease, although the specific β-cell autoantigen or autoantigens remain unknown. The recent identification of the β-cell 64K antigen as the enzyme glutamic acid decarboxylase (GAD) permits investigation of GAD as a candidate for the autoantigen associated with β-cell destruction, mediated by T-lymphocytes, in susceptible individuals. In this study, we describe the isolation of GAD-specific T-lymphocyte lines from BB rats, an animal model of IDDM. GAD (Escherichia coli) was inoculated into the footpads of diabetes-resistant BB rats, and after 10 days, a popliteal lymph node cell culture suspension was prepared. GAD-specific T lymphocytes were obtained by culture with interleukin 2 and repeated stimulation with GAD in the presence of BB rat thymic antigen-presenting cells. Four stable, CD4+, MHC (RT1u)-restricted T-lymphocyte lines were isolated. They proliferate selectively in the presence of GAD and secrete interleukin 2 and interferon-γ. T-lymphocyte lines such as these could be important in the definition of pathogenetic epitopes associated with GAD.
Pentosidine Formation in Skin Correlates With Severity of Complications in Individuals With Long-Standing IDDM
Authors: Sell David R , Lapolla Annunziata , Odetti Patrizio , Fogarty John , Monnier Vincent M
Published: September 2013

Pentosidine is an advanced glycosylation end product and protein cross-link that results from the reaction of pentoses with proteins. Recent data indicate that long-term glycation of proteins with glucose also leads to pentosidine formation through sugar fragmentation. In this study, the relationship between the severity of diabetic complications and pentosidine formation was investigated in collagen from skin-punch biopsies from 25 nondiabetic control subjects and 41 IDDM patients with diabetes duration >17 yr. Pentosidine was significantly elevated in all IDDM patients versus control subjects (P < 0.0001). It correlated strongly with age (P < 0.0001) and weakly with duration (P < 0.082). Age-adjusted pentosidine levels were highest in grade 2 (severe) versus grade 1 and 0 complication in all four parameters tested (retinopathy, proteinuria, arterial stiffness, and joint stiffness). Significant differences were found for retinopathy (P < 0.014) and joint stiffness (P < 0.041). The highest degree of association was with the cumulative grade of individual complication (P < 0.005), determined by summing indexes of all four parameters. Pentosidine also was significantly elevated in the serum of IDDM patients compared with control subjects (P < 0.0001), but levels were not significantly correlated with age, diabetes duration, complication, or skin collagen pentosidine (P > 0.05). A high correlation between pentosidine levels and long-wave collagen-linked fluorescence also was observed, suggesting that pentosidine is a generalized marker of accelerated tissue modification by the advanced glycosylation/Maillard reaction, which is enhanced in IDDM patients with severe complications.
Further Defects in Counterregulatory Responses Induced by Recurrent Hypoglycemia in IDDM
Authors: Davis Maris R , Mellman Michael , Shamoon Harry
Published: September 2013

We evaluated the effect of previous experimental hypoglycemia on counterregulatory responses to hypoglycemia in 13 IDDM patients. These patients had defects in counterregulatory responses to hypoglycemia compared with 7 nondiabetic control subjects. Plasma EPI and glucagon responses to hypoglycemia in IDDM patients were ∼60% of levels in nondiabetic subjects (P < 0.02 and P < 0.001, respectively). Hepatic glucose output ([3-3H]glucose) was reduced by ∼60% of normal (P < 0.005), and the glucose infusion rate required to maintain plasma glucose was correspondingly greater in people with IDDM (P < 0.001). With a modified glucose clamp (plasma insulin ∼330 pM), the diabetic subjects underwent two sequential 120-min periods of hypoglycemia (∼3.0 mM) with an intervening 60-min euglycemic recovery period. In the IDDM patients, there were 30–50% decreases in plasma GH (P < 0.005) and cortisol (P < 0.001) responses during the second hypoglycemic period compared with the first. In addition, glucose output, already defective compared with that in nondiabetic subjects, was further reduced by 33% (P = 0.03) during the second period of experimental hypoglycemia. There was no effect of repeated hypoglycemia on the responses of plasma glucagon, EPI, or NE, though plasma EPI was correlated directly with glucose output (P < 0.001) and inversely with glucose uptake (P < 0.05). There was no correlation between the rise in glucose output during hypoglycemia and antecedent glycemic control as measured by HbA1. We conclude that in IDDM patients with preexisting defects in counterregulatory responses to hypoglycemia, recurrent, mild hypoglycemia is associated with additional reductions in pituitary-adrenocortical hormonal secretion and further impairment of hepatic glucose production.
Metabolic Effects of Suppression of Nonesterified Fatty Acid Levels With Acipimox in Obese NIDDM Subjects
Authors: Fulcher G R , Walker M , Catalano C , Agius L , Alberti K G M M
Published: September 2013

NEFAs characteristically are elevated in obese NIDDM patients in both the basal state and after insulin. This elevation might aggravate glycemic control both by decreasing peripheral glucose disposal (glucose-fatty acid cycle), and by increasing HGO. Thus, lowering plasma NEFA levels might improve carbohydrate metabolism. We therefore measured HGO and fuel use (by indirect calorimetry) both in the basal state and during the last 30 min of a hyperinsulinemic clamp (0.025U · kg−1 · h−1) in 8 obese NIDDM patients (BMI 34.8 ±1.0 kg/m2) after complete overnight suppression of plasma NEFA levels with acipimox, a new nicotinic acid analogue. After acipimox, mean basal plasma NEFA and glycerol levels were lower than control values (0.11 ± 0.02 vs. 0.65 ±0.04 mM, P < 0.001; and 16 ± 3 vs. 68 ± 7 μM, P = 0.004, respectively) and were accompanied by a fall in lipid oxidation (acipimox vs. placebo: 16.1 ±1.2 vs. 38.8 ± 2.4 mg · m−2 · min−1; P < 0.001) and a rise in glucose oxidation (91.1 ± 6.2 vs. 54.1 ± 9.0 mg · m−2 · min−1; P = 0.002). Basal HGO and fasting plasma glucose levels were lower (94.1 ± 9.2 vs. 118.5 ± 9.5 mg · m−2 · min−1 P = 0.01; and 8.3 ± 1.2 vs. 9.8 ± 1.2 mM; P < 0.001), respectively. Serum insulin levels were similar during the clamps (44.2 ± 4.9 vs. 48.2 ± 5.7 μU/L; NS), but despite this, HGO was suppressed more after acipimox (18.2 ± 7.6 vs. 49.7 ± 12.9 mg · m−2 · min−1; P < 0.01), and the metabolic clearance rate for glucose was higher (101.98 ± 19.34 vs. 75.43 ± 9.94 ml · m−2 · min−1; P < 0.05). In conclusion, prolonged overnight suppression of lipolysis and lipid oxidation in obese NIDDM lowers fasting blood glucose and HGO and increases peripheral and hepatic sensitivity to insulin in obese NIDDM patients.
Intrathymic Islet Cell Transplantation Reduces β-Cell Autoimmunity and Prevents Diabetes in NOD/Lt Mice
Authors: Gerling Ivan C , Serreze David V , Christianson Sherri W , Leiter Edward H
Published: September 2013

Intrathymic transplantation of syngeneic islets into adolescent NOD/Lt mice was performed to establish whether the thymus would serve as an immunoprivileged site for β-cell engraftment, and whether this treatment would prevent the development of diabetes by eliciting tolerance to islet antigens. Intrathymic injection of cells from 200 NOD islets into 4-wk-old female NOD/Lt mice produced a significant reduction in the severity of insulitis at 24 wk of age. Furthermore, diabetes development was strongly suppressed (11% incidence) compared with controls (100% incidence). Both thymus histology and thymic insulin content revealed a rapid loss of the implanted β-cells with < 1% remaining 1 wk posttransplantation. Despite the rapid loss of thymus-implanted islet cells, evidence for tolerance induction to islet cell antigens was obtained by adoptive transfer of splenic leukocytes from these mice into NOD-scidlscid recipients. After adoptive transfer of splenic leukocytes from 24-wk-old untreated prediabetic donors, 4 of 5 NOD-scid/lscid recipients developed diabetes within 4 wk, and none of the recipients became diabetic after transfer of splenocytes from intrathymic islet-implanted donors. Intrathymic islet transplantation did not lead to reduction of sialitis in females with reduced severity of insulitis, indicating that the protective effect was tissue specific. This also was reflected in adoptive transfer experiments, because equal severity of sialitis was observed in NOD-sc/d/sc/d recipients of spleen cells from either islet transplanted or control NOD/Lt mice. In conclusion, the data suggest that intrathymic injection of islet cells prevents diabetes by stimulating immunological tolerance to β-cells.
Glucose Transporter Levels in Tissues of Spontaneously Diabetic Zucker fa/fa Rat (ZDF/drt) and Viable Yellow Mouse (Avy/a)
Authors: Slieker Lawrence J , Sundell Karen L , Heath William F , Osborne H Edward , Bue Juliana , Manetta Joseph , Sportsman J Richard
Published: September 2013

We used antibodies to the fat/muscle glucose transporter (GLUT4) and the liver glucose transporter (GLUT2) to measure levels of these proteins in various tissues of two rodent models of non-insulin-dependent (type II) diabetes mellitus: the obese spontaneously diabetic male Zucker fa/fa rat (ZDF/drt) and the male viable yellow Avy/a obese diabetic mouse. The ZDF/drt strain generally develops overt diabetes associated with decreased plasma insulin levels. Depending on the age of the animals, the ZDF/drt rats can be arbitrarily segregated into age-matched obese, mildly diabetic (blood glucose < 11 mM) and obese, and severely diabetic (blood glucose > 20 mM) groups. Avy/a mice are comparably hyperglycemic but unlike the ZDF/drt rats are severely hyperinsulinemic. In both groups of diabetic animals, GLUT4 in adipose tissue, heart, and skeletal muscle was reduced 25–55%, and GLUT2 in liver was increased 30–40%, relative to lean, age-matched controls. However, when the mildly diabetic ZDF/drt rats were compared to the lean controls, the only significant difference was a 25% reduction of GLUT4 in heart. Within all of the ZDF/drt rats (excluding the lean controls), GLUT2 in liver and GLUT4 in adipose tissue, heart, and skeletal muscle correlated significantly with glycemia. These data suggest that, in these two models of type II diabetes, glucose transporter levels in muscle, adipose tissue, and liver are regulated in a tissue-selective manner in response to changes in insulin and glucose. Furthermore, at least in the ZDF/drt rat, alterations in GLUT2 and/or GLUT4 protein levels appear not to be associated with obesity per se but appear to be secondary to the severely diabetic state. In both rodent models, similar alterations in GLUT2 and/or GLUT4 levels were observed under comparable glycemia, but quite distinct insulin levels, consistent with the possibility that glucose itself may be an important contributing regulator of glucose transporter expression.
Insulin Stimulates Accumulation and Efflux of Macromolecules in Isolated Nuclei From H35 Hepatoma Cells
Authors: Soler Alejandro Peralta , Smith Robert M , Jarett Leonard
Published: September 2013

This study used 10-nm gold particles with 5–7 insulin molecules attached (Au10-Ins) to investigate the site of interaction of insulin with the nuclear envelope during insulin uptake into intact isolated nuclei. Despite its size, and in the absence of ATP, Au10-Ins entered nuclei through the nuclear pore and associated with the heterochromatin. Because Au10-Ins is essentially gold-bovine serum albumin (Au-BSA) with a few insulin molecules attached, the effect of insulin and other growth factors on the nuclear accumulation of BSA coupled to 10-, 15-, and 24-nm-diam colloidal gold particles (Au10-BSA, Au15-BSA, and Au24-BSA) was determined. The Au-BSA complexes were excluded from nuclei in the absence of insulin. Insulin (0.5–100 ng/ml) caused a dose-dependent accumulation of Au10-BSA in the nucleus. The nuclear membrane was shown to be intact by several criteria, therefore, accumulation of Au-BSA occurred via the nuclear pore and was not due to leakage across or through the membrane. Uptake of 15- and 24-nm Au-BSA molecules was not affected by insulin, suggesting the hormone had a limited effect in increasing the functional diameter of the nuclear pores. Glucagon, epidermal growth factor, platelet-derived growth factor, insulinlike growth factor I, and insulin A or B chains did not stimulate the accumulation of Au10-BSA. The insulin-stimulated accumulation of Au10-BSA was blocked by concanavalin A, mimicked by wheat-germ agglutinin, and did not require ATP. The Au10-BSA in the nucleus was associated with heterochromatin, suggesting it bound to a nuclear element. Efflux of Au10-BSA from the nucleus was studied in nuclei previously loaded with Au10-BSA in the presence of insulin and reincubated with or without insulin in the absence or presence of 3 mM ATP or a nonhydrolyzable ATP analogue. Insulin stimulated the efflux of Au10-BSA from the nucleus only in the presence of ATP, possibly indicating ATP freed bound Au10-BSA, allowing the molecule to diffuse through the insulin-dilated nuclear pore. These data describe an insulin-sensitive route for macromolecular exchange through nuclear pores that could serve a role in insulin's control of cell growth and gene expression.
Duration of Obesity Increases the Incidence of NIDDM
Authors: Everhart James E , Pettitt David J , Bennett Peter H , Knowler William C
Published: September 2013

The effect of duration of obesity on incidence of non-insulin-dependent diabetes mellitus (NIDDM) was determined among Pima Indians. Duration of obesity was defined as the time since body mass index (BMI) was first known to be at least 30 kg/m2. Among 1057 participants eligible for study, there were 224 incident cases of NIDDM in 5975 person-yr of follow-up. The association of duration of obesity with incidence of diabetes adjusted for age, sex, and current BMI was highly significant (P < 0.0001). This adjusted incidence of diabetes in cases/1000 person-yr of obesity was 24.8 for people with less < 5 yr of obesity, 35.2 for people with 5–10 yr of obesity, and 59.8 for people with at least 10 yr of obesity. There was no apparent excess risk of diabetes for people who had a BMI of at least 30 kg/m2 and then lost weight. They had a slightly nonsignificantly higher rate than people who had not attained a BMI of at least 30 kg/m2 and a lower rate than people whose BMI remained 30–35 kg/m2. The relationship of duration of obesity with serum insulin concentrations among nondiabetic people was determined controlling for sex and age, BMI, and plasma glucose concentrations at the time of a glucose tolerance test. Duration of obesity was inversely associated with fasting serum insulin concentration through most of the range of fasting plasma glucose concentrations (P < 0.001) and tended to be inversely associated with 2-h postload serum insulin concentration through the entire range of postload plasma glucose concentrations (P = 0.058).
Platelet Abnormalities in Diabetes Mellitus
Authors: Winocour Peter D
Published: September 2013

Although platelets can contribute to atherosclerosis and its thromboembolic complications in the nondiabetic population, the role of platelets in enhanced vascular disease in the diabetic population remains unclear. Most studies indicate that platelet function in vitro is enhanced in platelets from people and animals with diabetes, and the mechanisms are being identified. There remains some controversy about whether platelet changes occur before, and therefore could contribute to, vascular complications or whether they are secondary to vascular disease. It is possible that only intervention trials to determine if inhibiting platelet function limits the progression of vascular disease in diabetic patients will definitively answer this question. The earlier premise that enhanced activity of the arachidonate pathway is responsible for the hypersensitivity of platelets from diabetic humans needs to be modified to recognize that additional mechanisms are involved in platelet activation and are modified in people with diabetes and also that altered activity of the arachidonate pathway may reflect changes in earlier pathways involved in platelet activation. Clearly, alterations in these nonarachidonate pathways need to be taken into account when considering the appropriate antiplatelet agents to use in intervention trials. Information about whether hypersensitivity of platelets from people with diabetes persists in vivo and, if so, how this influences platelet-vessel wall interactions and thrombotic tendencies needs to be pursued more intensely in suitable animal models so that the theories developed from studies in vitro can be tested in the more complex environment in vivo. These are important areas for research in the future.
Insulin and Insulinlike Growth Factors in Embryonic Development: Effects of a Biologically Inert Insulin (Guinea Pig) on Rat Embryonic Growth and Development In Vitro
Authors: Travers James P , Exell Lindsay , Huang Beatrice , Town Elena , Lammiman Michael J , Pratten Margaret K , Beck Felix
Published: September 2013

Congenital anomalies occur up to four times more frequently in diabetic pregnancy than in the nondiabetic population. Although past work has shown that maternal hyperglycemia and hyperketonemia may increase embryonic abnormalities, recent experimental evidence suggests that low insulin levels may also contribute to diabetic embryopathy. This study investigated the effects of guinea pig serum (whose insulin is inactive in rat systems) on rat embryonic growth and development in culture. Supplementation of guinea pig serum with pork insulin at low (1 ng/ml) and high (5 ng/ml) physiological concentrations and insulinlike growth factors (IGF) I and II were also studied. Culture of rat embryos from the early headfold stage in guinea pig serum resulted in poor embryonic growth and development with a 92% rate of anomalies. Supplementation of guinea pig serum with zinc-binding pork insulin significantly improved rat embryonic growth and development (46% anomaly rate) especially between the first 5 and 21 h of the period of organogenesis. This evidence supports our most recent findings that low insulin levels, as encountered in untreated diabetic pregnancy, may contribute to the increased risk of congenital abnormality. Insulin at low physiological concentrations improved growth, whereas higher physiological concentrations were required to increase growth and development. IGF-I or IGF-II supplementation improved rat embryonic growth and development but failed to match that of the controls, indicating that other growth factors including insulin may also be required.
Tolbutamide Inhibits cAMP-Dependent Phosphorylation of Liver 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase
Authors: Aoki Minoru , Kaku Kohei , Inoue Hiroshi , Matsutani Akira , Kaneko Toshio
Published: September 2013

The activity of a bifunctional enzyme, liver 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (F-2,6-Pase), which regulates the level of liver fructose-2,6-bisphosphate (F-2,6-P2), the most potent activator of PFK, is modulated by its phosphorylation rate mainly catalyzed by cAMP-dependent protein kinase A (PKA). To elucidate the action mechanism of sulfonylurea on liver F-2,6-P2 production, effects of tolbutamide on PKA-dependent phosphorylation of purified liver PFK-2/F-2,6-Pase protein and on kinase and phosphatase activities of the purified enzyme were examined in vitro. The purified enzyme was phosphorylated in the presence of the catalytic subunit of PKA, and tolbutamide inhibited the enzyme phosphorylation catalyzed by PKA in a dose-dependent manner. By adding the same dosages of tolbutamide used in the phosphorylation experiment, reduced activity of PFK-2 and increased activity of F-2,6-Pase in the presence of PKA were restored to the levels observed in the absence of PKA. On the other hand, carboxytolbutamide, an inactive metabolite of tolbutamide, had little effect on enzyme phosphorylation and activity. Our results indicate that tolbutamide inhibits a phosphorylation of the liver PFK-2/F-2,6-Pase catalyzed by PKA along with an activation of PFK-2 and an inactivation of F-2,6-Pase, leading to liver F-2,6-P2 production.
Impaired Activation of Glycogen Synthase in People at Increased Risk for Developing NIDDM
Authors: Schalin-Jäntti Camilla , Härkönen Matti , Groop Leif C
Published: September 2013

To study whether impaired activation of muscle glycogen synthase represents an early defect in the pathogenesis of insulin resistance in non-insulin-dependent diabetes mellitus (NIDDM), we quantitated rates of nonoxidative glucose metabolism and measured activities of glycogen synthase and phosphorylase and concentrations of free glucose and glucose-6-phosphate in muscle biopsies, obtained before and after a euglycemic insulin clamp, in 16 NIDDM patients, 18 first-degree relatives of NIDDM patients, and 16 nondiabetic control subjects. Insulin-stimulated glucose storage (20.1 ± 1.5 and 11.6 ± 1.7 vs. 27.9 ± 1.7 βmol·kg−1 lean body mass [LBM]·min−1 P < 0.01–0.001 [3.6 ± 0.3 and 2.1 ± 0.3 vs. 5.0 ± 0.3 mg·kg−1 LBM·min−1] and glycogen synthase activity, measured at 0.1 mM glucose-6-phosphate concentration (11.3 ± 1.3 and 11.6 ± 1.3 vs. 18.3 ± 2.0 nmol·min−1·mg−1 protein, P < 0.01), were impaired in relatives and diabetic subjects compared with control subjects. Glycogen synthase activity correlated with the rate of glucose storage (r = 0.53, P < 0.001). Glycogen phosphorylase fractional activity did not differ among the groups. Apart from increased intramuscular basal glucose concentrations in NIDDM patients, no consistent differences were observed in free glucose and glucose-6-phosphate concentrations between the groups. We conclude that impaired activation of muscle glycogen synthase by insulin is observed in patients with a genetic risk of developing NIDDM and may represent an early defect in the pathogenesis of NIDDM.
Role of Glucose Transporters in Glucocorticoid-Induced Insulin Resistance: GLUT4 Isoform in Rat Skeletal Muscle is Not Decreased by Dexamethasone
Authors: Haber Richard S , Weinstein Steven P
Published: September 2013

The diabetogenic effects of glucocorticoid excess are due in part to peripheral resistance to insulin. To test the hypothesis that glucocorticoid-induced peripheral insulin resistance might be attributable to a decreased number of glucose transporters, we examined the effects of dexamethasone treatment on the expression of the GLUT4 (insulin regulatable) glucose transporter in skeletal muscle, the major site of insulin-mediated glucose uptake. Dexamethasone treatment of rats (1 mg/day for 1 wk) induced hyperglycemia and hyperinsulinemia. At dosages of either 0.1 or 1 mg/day, insulin-stimulated 2-deoxyglucose uptake in isolated soleus muscle was reduced by ≥ 50%, demonstrating the presence of insulin resistance in skeletal muscle. Immunoblots of crude membranes from deep quadriceps muscle showed that dexamethasone treatment (1 mg/day) increased the amount of GLUT4 protein by 84%. GLUT4 mRNA abundance was similarly increased when expressed per unit RNA but was unchanged when expressed on a DNA basis because the tissue RNA content was decreased by dexamethasone. In contrast to quadriceps, GLUT4 protein concentration in soleus and extensor digitorum longus extracts was not significantly increased by dexamethasone treatment. Because glucocorticoids cause selective atrophy of type IIb muscle fibers, which express relatively less GLUT4 protein, the apparent increase in GLUT4 content in quadriceps muscle from dexamethasone-treated animals may have resulted from inadvertent increased sampling of GLUT4-enriched type I and IIA fibers, caused by a glucocorticoid-induced decrease in the relative mass of the GLUT4-poor type IIb fibers. We conclude that glucocorticoids do not decrease GLUT4 content in skeletal muscle and that glucocorticoid-induced insulin resistance in this tissue is not due to suppression of glucose transporter gene expression.
Concordant Glucose Induction of Glucokinase, Glucose Usage, and Glucose-Stimulated Insulin Release in Pancreatic Islets Maintained in Organ Culture
Authors: Liang Yin , Najafi Habiba , Smith Robert M , Zimmerman Elisabeth C , Magnuson Mark A , Tal Michael , Matschinsky Franz M
Published: September 2013

Using cultured islets as the experimental system, this study established dosage-response and time-dependency curves of the inductive glucose effect on glucose-stimulated insulin release, glucose usage, and glucokinase activity. Glucose-stimulated insulin release in islets cultured for 1, 2, or 7 days was increased as a function of glucose concentration in the culture medium and as a function of time. Glucose usage in the cultured islets showed a close relationship with glucose concentration in the culture medium at both 2 and 7 days of culture. Glucokinase activity increased in islets cultured for 1, 2, or 7 days as a function of increasing glucose concentrations in the culture medium and as a function of time. The Vmax of glucokinase in islets cultured for 7 days in medium containing 30 mM glucose was twice the value of freshly isolated islets and was almost fivefold higher than that in islets cultured for 7 days in 3 mM glucose. The glucose induction of glucose-stimulated insulin release, of glucose usage, and of glucokinase activity were tightly correlated. The biochemical mechanisms of glucose induction of islet glucokinase were further studied. Immunoblotting with an antibody against C-terminal peptide of glucokinase showed that densities of a 52,000-kD protein band from tissue extracts of islets cultured for 7 days in 3, 12, and 30 mM glucose were 25, 44, and 270% compared with that of extract from freshly isolated islets (100%). RNA blot analysis of glucokinase mRNA demonstrated virtually the same levels in fresh islets and islets after 7 days of culture in 3 or 30 mM glucose. The adaptive response of glucokinase to glucose appears therefore to be occurring at a translational or posttranslational site in cultured islets. These data greatly strengthen the concept that glucose is the regulator that induces the activity of glucokinase, which in turn determines the rate change of glucose usage as well as glucose-stimulated insulin release from β-cells. Thus, the hypothesis that glucokinase is the glucose sensor of β-cells is strengthened further.
Increased Expression of Tissue Plasminogen Activator and Its Inhibitor and Reduced Fibrinolytic Potential of Human Endothelial Cells Cultured in Elevated Glucose
Authors: Maiello Michele , Boeri Daria , Podesta Francesca , Cagliero Enrico , Vichi Maurizio , Odetti Patrizio , Adezati Luciano , Lorenzi Mara
Published: September 2013

In diabetic patients, elevated plasma levels of t-PA and PAI-1 accompany impaired fibrinolysis. To identify mechanisms for these abnormalities, we examined whether vascular endothelial cells exposed to high glucose upregulate t-PA and PAI-1 production and whether ambient PA activity is decreased concomitantly. In 17 cultures of human umbilical vein endothelial cells grown to confluency in 30 mM glucose, the t-PA antigen released to the medium in 24 h was (median) 52 ng/106 cells (range 10–384) and the PAI-1 antigen was 872 ng/106 cells (range 217–2074)—both greater (P < 0.02) than the amounts released by paired control cultures grown in 5 mM glucose—29 ng/106 cells (range 7.5–216) and 461 ng/106 cells (range 230–3215), respectively. In the presence of high glucose, the steady-state levels of t-PA and PAI-1 mRNAs were increased correspondingly (median 142 and 183% of control, respectively, P < 0.05); high glucose per se and hypertonicity contributed to the upregulation in additive fashion. The PA activity of conditioned medium from cultures exposed to high glucose was 0.4 IU/ml (range 0.2–0.6), which was significantly lower (P < 0.02) than the PA activity of control medium (0.5 IU/ml, range 0.2–0.9). No difference was observed when comparing the PA activities of acidified conditioned media, expected to be depleted of inhibitors. Thus, high glucose coordinately upregulates endothelial t-PA and PAI-1 expression through effects exerted at the pretranslational level and enhanced by even mild degrees of hypertonicity. The decrease in ambient fibrinolytic potential may reflect an overwhelming effect of the increased availability of PAI-1. These findings propose a contributory mechanism for the fibrinolytic abnormalities of diabetes and the thrombotic tendency of the hyperglycemic hyperosmolar state.
Coordinate Control of Lipolysis by Prostaglandin E2 and Prostacyclin in Rat Adipose Tissue
Authors: Chatzipanteli Katina , Rudolph Sheila , Axelrod Lloyd
Published: September 2013

PGE2 is a potent antilipolytic agent produced by adipose tissue, but its role as a physiological regulator of triglyceride lipolysis is controversial because inhibitors of prostaglandin synthesis have not enhanced hormone-stimulated lipolysis in adipose tissue consistently. Adipose tissue also produces PGI2, but this eicosanoid has not had a demonstrated effect on lipolysis under physiological conditions previously. We investigated both PGE2 and PGI2 production and their effects on lipolysis in rat adipose tissue. We found that 1) EPI-stimulated PGE2 production (like PGI2 production) requires the cooperation of adipocytes and endothellal cells, 2) adipose tissue produces PGE2 and PGI2 at comparable rates, 3) indomethacin inhibits EPI-induced PGE2 and PGI2 production and has no effect on EPI-stimulated lipolysis when added to a mixture of adipocytes and endothelial cells or to intact epididymal fat pads, 4) PGI2 is a potent lipolytic agent when added to isolated adipocytes in the absence of endothelial cells under physiological conditions, 5) the magnitudes and the ED50s of the antllipolytic effect of PGE2 and the lipolytic effect of PGI2 in isolated adipocytes in the absence of endothelial cells are comparable, 6) PGI2 antagonizes the antllipolytic effect of PGE2 in isolated adipocytes in the absence of endothelial cells in a dosage-related manner, and 7) the antllipolytic effect of added PGE2 in isolated adipocytes is greater in the absence of endothelial cells than in their presence, suggesting that endogenous eicosanoid production reduces the effectiveness of added PGE2. These studies demonstrate that catecholamine-induced lipolysis is under the coordinate control of PGE2, a potent antllipolytic agent, and PGI2, a potent lipolytic agent. The failure of PGH synthase inhibition to affect lipolysis can be explained by concurrent PGE2 and PGI2 inhibition. The existence of this mechanism for coordinate control of lipolysis may have broad implications for the function of adipose tissue in health and disease.
Phosphoinositide Metabolism in the Developing Conceptus: Effects of Hyperglycemia and Scyllo-Inositol in Rat Embryo Culture
Authors: Strieleman P J , Connors M A , Metzger B E
Published: September 2013

Culture of the postimplantation rat conceptus in hyperglycemic medium causes developmental abnormalities and is associated with a diminished water-soluble myo-inositol content. We investigated the effect myo-inositol depletion has on lipid-soluble phosphoinositides, precursors, and water-soluble inositol phosphates. Rat conceptuses were cultured from gestational day 9.5 (presomite, early head fold) to day 10.5 (7–15 somites) in 6.7–73.3 mM D-glucose. Significant decreases in the phosphoinositides of the embryo were observed with increased culture D-glucose concentrations. PI was reduced 15–34%, PIP 18–46%, and PIP2 26–46%. Yolk sac phosphoinositides also were reduced but to a lesser degree. Culture in hyperglycemic media also mediated significant reductions of conceptus inositol phosphates. To investigate whether effects similar to those induced by D-glucose could be mediated by another agent capable of decreasing myo-inositol content, we used scyllo-inositol, a transported but nonmetabolized isomer of myo-inositol. Conceptuses cultured in medium containing scyllo-inositol (0.06–16.7 mM) had dose-dependent decreases of myo-[3H]inositol in water-soluble and lipid-soluble fractions. Incorporation of myo-[3H]inositol into phosphoinositides and inositol phosphates was decreased concomitantly. Developmental effects of D-glucose and scyllo-inositol were assessed in rat conceptuses cultured from day 9.5 (presomite, early head fold) to day 11.5 (22–28 somites). Culture in 40.0–73.3 mM glucose and 0.06–33.3 mM scyllo-inositol impaired growth while increasing dysmorphogenesis in a dose-dependent manner. The results suggest that decreases in conceptus myo-inositol and associated diminution of phosphoinositides, which are the inositol/lipid cycle precursors, are dysmorphogenic and may contribute to the etiology of diabetic embryopathy.
Role for Protein Kinase C in the Mediation of Increased Fibronectin Accumulation by Mesangial Cells Grown in High-Glucose Medium
Authors: Studer Rebecca K , Craven Patricia A , DeRubertis Frederick R
Published: September 2013

The fibronectin content of RMC cultures grown for 8–14 days in medium containing 30 mM (540 mg/dl) D-glucose was increased 30–60% relative to that of control cells cultured in 10 mM (180 mg/dl) glucose. Addition of equiosmolar concentrations (20 mM, 360 mg/dl) of L-glucose, 3-O-methylglucose, or mannitol to 10 mM glucose media did not alter fibronectin accumulation compared with values observed in 10 mM glucose alone. The basal phosphorylation of the 80,000-Mr MARCKS protein, which is a substrate for PKC, and the phosphorylation induced by acute (15-min) exposure of cells to 15% FCS or to 0.1 μM (50 ng/ml) PDBu were all increased in cells grown in 30 mM compared with 10 mM glucose. By contrast, phosphorylation of the 80,000-Mr protein in response to a maximal concentration of PDBu (1 μM, 500 ng/ml) was not different in cells grown in 30 mM compared with 10 mM glucose. The acute increases in phosphorylation of the 80,000-Mr protein were blocked by the PKC inhibitor calphostin C. Chronic (7-day) exposure of mesangial cells grown in 10 mM glucose to 0.1 μM of the PKC agonist PDBu also resulted in a sustained 40% increase in 80,000-Mr phosphorylation and a 20–30% increase in fibronectin accumulation. As assessed by [35S]methionine incorporation, mesangial cell fibronectin synthesis was increased by exposure to PDBu, a finding consistent with earlier observations with 30 mM glucose. The increase in 80,000-Mr proteimesangial cells grown in either 0.1 μM PDBu or 30 mM glucose was abolished in cells whose PKC activity had been downregulated by exposure to PMA. Culture of mesangial cells with the thromboxane-endoperoxide analogue U-46619 in 30 mM, but not 10 mM, glucose increased fibronectin accumulation compared withvalues obtained in 30 mM glucose alone. These results demonstrate a sustained activation of PKC in mesangial cells cultured in a high concentration of glucose and support a role for PKC activation in the mediation of enhanced fibronectin accumulation induced by glucose. Because nonmetabolizable glucose analogues and mannitol were ineffective, metabolism of glucose is implicated in expression of these actions. The results also suggest that a high ambient glucose concentration may interact with thromboxane and/or prostaglandin endoperoxide in the activation of PKC and the enhancement of fibronectin accumulation by mesangial cells.
Effect of High Glucose on Type IV Collagen Production by Cultured Glomerular Epithelial, Endothelial, and Mesangial Cells
Authors: Danne Thomas , Spiro Mary Jane , Spiro Robert G
Published: September 2013

Immunochemical and metabolic radiolabeling procedures revealed that homogeneous cultures of calf glomerular epithelial, endothelial, and mesangial cells actively synthesize type IV collagen (primarily as α1(Iv)3) which is secreted into the medium and incorporated into the extracellular matrix. Exposure of confluent cultures of the three cell types to a high glucose concentration (30 mM) for 60 h resulted in a pronounced increase (two- to threefold) in type IV collagen production over that observed at a physiological level (5 mM) of this sugar, as determined by either immunoblotting or fluorography of electrophoretically separated media or cell-matrix components. The elevated glucose did not bring about a change in the rate of cell proliferation or fibronectin production. Moreover, studies with mannitol indicated that the stimulation of type IV collagen synthesis was not a function of hyperosmolarity. In contrast to the glomerular cells, glucose-induced enhancement of formation of this collagen was not observed in 3T3 cells despite a substantial acceleration in the consumption of this sugar. Time studies indicated that the response of the glomerular cells to high glucose occurs over an extended period (maximal at ∼78 h) and, furthermore, that the stimulatory effect on type IV collagen production is only slowly reversed after restoration of the glucose to a normal level. We believe that these findings are relevant to an understanding of the sequence of events that lead to the development of diabetic glomerular lesions.
Glucose Sensitivity of ATP-Sensitive K+ Channels Is Impaired in β-Cells of the GK Rat: A New Genetic Model of NIDDM
Authors: Tsuura Yoshiyuki , Ishida Hitoshi , Okamoto Yoshimasa , Kato Seika , Sakamoto Kimihiko , Horie Minoru , Ikeda Hitoshi , Okada Yasunobu , Seino Yutaka
Published: September 2013

In the Goto-Kakizaki rat, a new genetic model of NIDDM, insulin response to glucose is selectively impaired. To elucidate the mechanism of this abnormality, we studied the properties of ATP-sensitive K+ channels, the inhibition of which is a key step of insulin secretion induced by fuel substrates, using the patch-clamp technique. The glucose-sensitivity of KATP channels was considerably reduced in GK rats. However, the inhibitory effects of ATP on channel activity and unitary conductance were not significantly different between control and GK rats. Thus, it appears that the impaired insulinotropic action of glucose in β-cells of GK rats is attributable to insufficient closure of the KATP channels, probably because of deficient ATP production by impaired glucose metabolism. KATP-channel activities in both control and diabetic β-cells were found to be equally suppressed by glyceraldehyde and 2-ketoisocaproate. These results strongly suggest that the step responsible for the metabolic dysfunction of diabetic β-cells is located within the glycolytic pathway before glyceraldehyde-3-phosphate or in the glycerol phosphate shuttle.
Inhibition of Lipolysis Decreases Lipid Oxidation and Gluconeogenesis From Lactate But Not Fasting Hyperglycemia or Total Hepatic Glucose Production in NIDDM
Authors: Puhakainen Ilpo , Yki-Järvinen Hannele
Published: September 2013

We determined whether overnight inhibition of lipolysis by a long-acting nicotinic acid derivative (acipimox) decreases gluconeogenesis from lactate in NIDDM patients. For this purpose, 250 mg of acipimox or placebo was administered in a double-blind crossover study at 2400, 0400, and 0800 to 8 NIDDM patients (54 ± 4 yr of age, body mass index 29.5 ± 1.3 kg/m2, fasting plasma glucose 11 ± 1 mM). The next morning, total hepatic glucose production (glucose Ra) and gluconeogenesis from lactate were determined using primed, continuous infusions of [3-3H]glucose and [U-14C]acetate. Glucose and lipid oxidation rates were measured using indirect calorimetry. Mean overnight serum free fatty acid concentrations averaged 242 ± 8 μM after acipimox and 721 ± 30 μM after placebo (P < 0.001). Inhibition of lipolysis decreased lipid oxidation from 33 ± 3 to 22 ± 2 J · kg−1 · min−1 (P < 0.001) and increased carbohydrate oxidation from 15 ± 3 to 23 ± 2 μmol · kg−1 · min−1 (P < 0.005). Gluconeogenesis from lactate decreased by ∼ 40%, from 6.2 ± 0.6 to 3.8 ± 0.5 μmol · kg−1 · min−1 (P < 0.005); lactate oxidation increased from 5.6 ± 0.8 to 7.9 ± 1.1 μmol · kg−1 · min−1 (P < 0.005), with no change in plasma lactate concentrations or total lactate Rd. Fasting plasma glucose concentrations were comparable at 2400 (10.0 ± 1.1 vs. 10.6 ± 1.3 mM, acipimox vs. placebo) and between 0900 and 1000 (10.6 ± 1.3 and 11.3 ± 1.3 mM, respectively). Also, total glucose production rates remained unchanged (14.0 ± 1.2 vs. 14.9 ± 1.3 mol · kg−1 · min−1, respectively). Serum growth hormone (2.8 ± 0.9 vs. 0.5 ± 0.2 μg/l, P < 0.05) and cortisol (444 ± 19 vs. 352 ± 28 nM, P < 0.05) concentrations were higher after acipimox than placebo administration. We conclude that inhibition of lipolysis decreases lipid oxidation and gluconeogenesis from lactate, but fails to lower fasting plasma glucose concentrations and total hepatic glucose production in NIDDM patients.
Expression of Functional Nerve Growth Factor Receptors in Pancreatic β-cell Lines and Fetal Rat Islets in Primary Culture
Authors: Scharfmann Raphael , Tazi Abdelali , Polak Miche , Kanaka Christina , Czernichow Paul
Published: September 2013

Previous data demonstrated that one rat insulinoma cell line, RINm5F cells, which is a rat β-cell line derived from a pancreatic tumor, express mRNA coding for both the low- and the high-affinity nerve growth factor receptors. Goals of this study were to extend our data to other β-cell lines and fetal islets in primary culture and to study further the binding characteristics of nerve growth factor receptors on β-cells. Northern blot analysis revealed that not only a panel of endocrine β-cell lines (RINm5F, INS-1, β-TC3) but also fetal rat islets in primary culture express mRNA coding for trk-A, which has been proposed to be the neuronal high-affinity nerve growth factor receptors. Reverse polymerase chain reaction followed by sequencing revealed that the sequence of trk-A receptor in RINm5F cells is identical to that of trk-A expressed in PC12 cells. The expression of the low-affinity nerve growth factor receptor was examined by Northern blot analysis that showed low-affinity nerve growth factor receptor to be expressed in RINm5F and INS-1 cell lines, in fetal rat islets in primary culture, but not in β-TC3-cells. Binding experiments revealed the presence of low- and high-affinity nerve growth factor binding sites, identical to those described for PC12 cells, on RINm5F and INS-1 cells and only high-affinity binding sites on β-TC3 cells. Exposure of all three β-cell lines to nerve growth factor increased NGFI-A and c-fos mRNA steady-state levels, showing that these receptors are functional. These data demonstrate that the entire machinery required for nerve growth factor action is present in β-cells in culture.