In vitro and in vivo performance of porcine islets encapsulated in interfacially photopolymerized poly(ethylene glycol) diacrylate membranes.

The usefulness of interfacial photopolymerization of poly(ethylene glycol) (PEG) diacrylate at a variety of concentrations and molecular weights to form hydrogel membranes for encapsulating porcine islets of Langerhans was investigated. The results from this study show in vitro and in vivo function of PEG-encapsulated porcine islets and the ability of PEG membranes to prevent immune rejection in a discordant xenograft model. Encapsulated islets demonstrated an average viability of 85% during the first week after encapsulation, slightly but significantly lower than unencapsulated controls. Encapsulated porcine islets were shown to be glucose responsive using static glucose stimulation and perifusion assays. Higher rates of insulin release were observed for porcine islets encapsulated in lower concentrations of PEG diacrylate (10-13%), not significantly reduced relative to unencapsulated controls, than were observed in islets encapsulated in higher concentrations (25%) of PEG diacrylate. Perifusion results showed biphasic insulin release from encapsulated islets in response to glucose stimulation. Streptozotocin-induced diabetic athymic mice maintained normoglycemia for up to 110 days after the implantation of 5,000-8,000 encapsulated porcine islet equivalents into the peritoneal cavity. Normoglycemia was also confirmed in these animals using glucose tolerance tests. PEG diacrylate-encapsulated porcine islets were shown to be viable and contain insulin after 30 days in the peritoneal cavity of Sprague-Dawley rats, a discordant xenograft model. From these studies, we conclude that PEG diacrylate encapsulation of porcine islets by interfacial photopolymerization shows promise for use as a method of xenoprotection toward a bioartificial endocrine pancreas.

Immunoisolation of adult porcine islets for the treatment of diabetes mellitus. The use of photopolymerizable polyethylene glycol in the conformal coating of mass-isolated porcine islets.

Functional porcine islets, free of known pathogens, can serve as a source of insulin producing cells for the treatment of experimentally induced insulin dependent Diabetes Mellitus. Porcine islets can be conformally coated (microencapsulated) with a covalently linked, stable permselective membrane while maintaining islet viability and function. The PEG conformal coating is immunoprotective in a discordant xenograft animal model (porcine islets to rat).

Endothelial dysfunction in a model of hyperglycemia and hyperinsulinemia.

Insulinopenic diabetes is known to produce endothelial dysfunction. This dysfunction could arise from either hyperglycemia or inadequate insulin. It is not known whether endothelial dysfunction occurs when hyperglycemia is present with elevated insulin levels. In this study, we utilized an experimental model of hyperglycemia with hyperinsulinemia to investigate latent endothelial dysfunction. Rats were continuously infused with glucose or saline for 72 h to achieve peak plasma glucose concentrations of approximately 25 mM. Plasma insulin rose by 12-fold in glucose-infused rats. No significant differences in serum electrolyte concentration were noted between control and glucose-infused rats after 72 h. Blood pressure was not altered by this intervention. Aortic rings taken from control rats relaxed to the endothelium-dependent vasodilators, acetylcholine and A-23187, and to the endothelium-independent vasodilator, nitroglycerin. Relaxation to acetylcholine but not to A-23187 or nitroglycerin was impaired in glucose-infused rat aortic rings. Incubation in vitro with either indomethacin or superoxide dismutase did not restore the impaired relaxation to acetylcholine in rings taken from glucose-infused rats. Thus hyperglycemia with hyperinsulinemia selectively impairs receptor-dependent, endothelium-dependent relaxation. These studies suggest that elevated glucose may be a common pathway leading to endothelial dysfunction in insulin-dependent diabetes mellitus and hyperglycemia-induced insulin resistance.

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats.

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.

Quantitation of GLUT1 and GLUT4 mRNA using a solution hybridization assay.

The development of a solution hybridization assay for detecting GLUT1 and GLUT4 mRNA is described. The details of this assay are described in which copy RNA is used to quantitate messenger RNA in total RNA samples. This solution hybridization assay is highly specific and reproducible and is significantly more sensitive than Northern blotting. Since GLUT mRNAs can be quantitated in as little as 25 mg tissue, this technique is essential when the supply of tissue is limited. Furthermore, the elimination of gel-based separation techniques allows for mRNA quantitation in several hundred samples within two days following isolation of samples.

Insulin resistance in normal rats infused with glucose for 72 h.

Insulin resistance is accentuated during periods of poor metabolic control in human non-insulin-dependent diabetes mellitus. The role of hyperglycemia in this suppression of insulin action is not clear. If glucose impairs insulin action, then the effect should be reproducible in vivo in tissues of normal intact rats. To test this possibility, normal rats were continuously administered 50% glucose in water (60-66 mg.kg-1.min-1) via an indwelling jugular catheter. After 72 h, these animals were hyperglycemic, hyperinsulinemic, and glucosuric compared with control rats infused for 72 h with normal saline (P less than 0.01). Basal glucose uptake in vivo was greater in muscle of glucose-infused rats. Insulin-stimulated glucose uptake in vivo and in vitro (by perfused hindquarters and isolated adipocytes) were suppressed in the glucose-infused group (P less than 0.01). Glycogen synthase activity was reduced 40% in extracts of muscle and adipose tissue of hyperglycemic rats. Basal and isoproterenol-stimulated lipolysis were increased, whereas insulin suppression of lipolysis was blunted in adipocytes from glucose-infused animals (P less than 0.01). Glucose infusion did not alter insulin binding by isolated adipocytes or solubilized skeletal muscle insulin receptors. These results suggest that a 72-h in vivo glucose infusion impaired insulin action in muscle and adipose tissue of normal rats by inducing postbinding defects similar to those observed in human diabetes mellitus during intervals of deteriorated metabolic control.

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat.

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin resistance of uremia.

Glucose intolerance is a nearly universal finding in patients with chronic renal failure and in animal models of uremia. The glucose intolerance results from impaired insulin-mediated glucose disposal by muscle, adipose, and liver tissue. Insulin binding by these tissues is not reduced. Rather, several defects exist in the postreceptor cascade of insulin action. Although impaired insulin-mediated glucose uptake and metabolism occur, the primary defect and causative agent are not established. The purpose of the present article is to review recent literature on the potential mechanisms underlying the insulin resistance of chronic renal failure.

Effects of parenteral testosterone administration on insulin action in perfused hindlimb muscle of female rats.

The effects of 14 days of testosterone (T) administration (1 mg/kg/d) on insulin-regulated phenylalanine release and glucose uptake by perfused hindquarters of intact and ovariectomized (OVX) female rats were studied. Results were compared with control groups that did not receive T. In the absence of exogenous insulin, T treatment significantly suppressed phenylalanine release (an index of net protein degradation) from hindquarters of intact and OVX rats below that of the intact control group (P less than .01). In perfusions containing insulin (100 or 500 microU/mL), phenylalanine efflux from intact and OVX control hindquarters was significantly lower as compared with hindquarters perfused without exogenous insulin (P less than .01). Insulin also reduced the phenylalanine release by T-treated groups to the efflux rates of control rats perfused at the same insulin concentration. Basal glucose uptake by perfused hindquarters was similar in all four treatment groups. In perfusions containing 100 or 500 microU insulin/mL, glucose uptake by hindlimbs of intact and OVX control rats was increased 2.7-fold and 5-fold, respectively, above that observed in perfusions without insulin (P less than .01). T treatment did not alter glucose uptake by perfused hindquarters in the absence or presence of insulin under these experimental conditions. The results of these studies suggest that T has anabolic effects on skeletal muscle that are independent of exogenous insulin action.

Hormones and the barnacle muscle fiber as a preparation.

This minireview discusses the use of single barnacle muscle fibers as a model system for studying hormonal actions. The response of barnacle muscle fibers to serotonin, proctolin, octopamine, aldosterone and insulin is described. Recent data relating to the actions of these hormones on other invertebrate and vertebrate preparations is touched upon. The use of the barnacle muscle fiber as a preparation to investigate hormone-stimulated protein phosphorylation is emphasized.

Bicarbonate-carbon dioxide buffer system: a determinant of the mitochondrial pH gradient.

The influence of the bicarbonate-carbon dioxide buffer system on the pH gradient (delta pH) across the inner membrane of mitochondria from rabbit renal cortex was studied with and without phosphate in the medium. delta pH with bicarbonate buffer or phosphate in the medium was greater at low than at high medium pH so that the difference (delta delta pH) between delta pH at pH 7.1 and at 7.6 was positive. Varying the concentration of phosphate from 0 to 10 mM had little effect on delta delta pH produced by bicarbonate buffer. Inhibition of the phosphate-hydroxyl carrier with N-ethylmaleimide abolished delta delta pH when phosphate was present in non-bicarbonate-containing media. With bicarbonate buffer present, N-ethylmaleimide increased delta delta pH. Similar effects were observed in mitochondria from liver and heart as well as from kidney. The effects of the bicarbonate buffer system on delta pH may result either from an inner membrane permeable to carbon dioxide but not to bicarbonate ion or from an active carrier for bicarbonate ion in the inner membrane. In intact kidney cells, the influence of the bicarbonate buffer system on delta pH may provide a mechanism for regulating substrate metabolism in response to acid-base changes. It may also serve in many organs to reduce fluctuations in matrix pH when alterations in cytoplasmic pH occur.

Myosin light chain and membrane protein phosphorylation in various muscles.

Phosphorylation of myosin light chain was compared in various muscles. In 32P-labeled chicken anterior latissimus dorsi and posterior latissimus dorsi the [32P]phosphate content of the 19,000-dalton and 18,000-dalton light chains, respectively, was 1.8-fold higher in muscles tetanized for 5 or 15 s than in the contralateral resting muscles. Similar results had been previously obtained with frog sartorius and semitendinosus muscles. In addition to the radioactive technique, the extent of light chain phosphorylation was also measured by densitometry after separating the phospho and dephospho forms of the light chain with two-dimensional gel electrophoresis. The extent of light chain phosphorylation in tetanized chicken muscles and caffeine-treated frog muscles was not greater than 50%. The extent of phosphorylation of the 19,000-dalton light chain in hearts from several animals (turtle, rat, frog, chicken, dog, and cat) showed major differences. At the extremes it was 76% in turtle and 10% in chicken. the polymorphism of heart light chain was also demonstrated. Not only myosin light chains but also membrane proteins were detected to be phosphorylated. Some of the membrane proteins exhibited increases in phosphorylation during muscle contraction.

pH and bicarbonate effects on mitochondrial anion accumulation. Proposed mechanism for changes in renal metabolite levels in acute acid-base disturbances.

Mitochondria from rabbit and dog renal cortex were incubated with 1 mM (14)C-weak acid anions in media containing low (10 mM) or high (40 mM) concentrations of bicarbonate and the steady-state accumulation of labeled anion in the matrix was measured. In the absence of an energy source, no concentration of (14)C-anion in the mitochondrial matrix space was present, but the anion concentration was significantly higher at low- than at high-bicarbonate concentration. Addition of an energy source, usually ascorbate plus tetramethyl-p-phenylenediamine, led to increases in matrix space anion levels and to accentuation of the difference in anion uptake between low- and high-bicarbonate media, so that two to four times as much anion was present at low- than at high-bicarbonate concentrations. The anions affected included substrates for which inner membrane carriers are present in mitochondria, such as citrate, alpha-ketoglutarate, malate, and glutamate, as well as substances which diffuse passively across the inner membrane such as acetate and formate. When a nonbicarbonate medium buffered with Hepes was used, pH change did not alter anion uptake although anion concentrations exceeding those in the medium still developed when an energy source was present. The difference in mitochondrial anion accumulation between low- and high-bicarbonate levels diminished with decreasing temperature or with increasing anion concentration in the medium. Estimation of intramitochondrial pH with [(14)C]5,5-dimethyl-oxazolidine-2,4-dione showed that the pH gradient across the inner mitochondrial membrane was significantly greater with 10 than with 40 mM bicarbonate in the medium.A hypothesis is described that relates this effect of pH and bicarbonate on mitochondrial anion accumulation to the very rapid changes in substrate levels in renal cortex, which develop when acute metabolic acidosis or alkalosis is produced in the intact animal. It is suggested that an abrupt fall in systemic pH and bicarbonate is associated with a shift in substrate in renal cortex out of the cytoplasm and into mitochondria, where some of the added substrate is metabolized. Reduction in the size of the cytoplasmic pool of substrate occurs with relatively little accompanying change in the size of the mitochondrial pool, thus causing a net reduction in the total tissue pool. This mechanism accounts for the reduction in tissue levels of many mitochondrial substrates observed acutely in metabolic acidosis. In metabolic alkalosis, reversal of these effects leads to expansion of the cytoplasmic pool, thereby resulting in the rise in tissue levels of substrates which occurs in this condition.