Nancy R. Voyles

Naltrexone reduces weight gain, alters "β-endorphin", and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of beta-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of beta-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4-20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary beta-endorphins rose 4-6 fold in ob/ob compared with +/?. While naltrexone reduced the levels of ob/ob pituitary towards normal, no effect on beta-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets). These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.

Decreased Glucagon Receptors in Diabetic Rat Hepatocytes: EVIDENCE FOR REGULATION OF GLUCAGON RECEPTORS BY HYPERGLUCAGONEMIA

The effects of endogenous and exogenous hyperglucagonemia on the specific binding of glucagon to hepatocyte receptors was studied, as was the response of cAMP to glucagon. In streptozotocin diabetic rats, blood glucose and plasma glucagon increased and plasma insulin decreased as compared with controls. Insulin treatment in diabetic rats restored blood glucose and plasma glucagon toward normal and elevated plasma insulin. Specific binding of (125)I-glucagon to isolated hepatocytes (10(6) cells) decreased in diabetic rats (8.17+/-0.38%) compared to controls (14.05+/-0.87%) and was restored by insulin treatment (12.25+/-0.93%). Specific binding of (125)I-insulin in controls was 7.30+/-10.16%; it increased in diabetic rats to 12.50+/-0.86%, and decreased in diabetic rats after insulin treatment (9.08+/-0.87%). Scatchard analysis and the competition plots of the data indicate that decreased glucagon binding and increased insulin binding in diabetes were due to change in the number of receptors rather than a change in their affinity. Hepatocyte cAMP response to glucagon (0.25-5.0 ng/ml) was almost abolished in diabetic rats and was restored with insulin treatment. Specific glucagon binding by hepatocytes from chronically hyperglucagonemic (glucagon injected) rats was decreased (P < 0.005) to 8.76+/-0.61% compared with controls (13.20+/-0.74%) and acutely hyperglucagonemic animals (13.53+/-1.33%). The decreased binding was associated with a 70% decrease in hepatocyte cAMP response to glucagon compared with a normal response in acutely hyperglucagonemic rats.These data appear to support the concept of receptor regulation by ambient hormone level. In both endogenous and exogenous hyperglucagonemia, however, there was a disproportionately large decrease in cAMP response to glucagon compared to the decrease in glucagon binding.

Insulin, Glucagon, and Somatostatin Receptors on Cultured Cells and Clones from Rat Islet Cell Tumor

SUMMARY Cells grown in culture from rat islet cell tumor (parent cells) and clones obtained from them were used in this study. Parent cells secreted primarily insulin and somatostatin with very small quantities of glucagon. The clones, based on hormone content and secretion, were divided into three phenotypic groups: insulin secreting, somatostatin secreting, and nonsecreting clones. Specific receptors for insulin, glucagon, and somatostatin were demonstrated on parent cells and clones. Parent cells bound 4.12 ± 0.46% insulin, 2.11 ± 0.29% glucagon, and 2.49 ± 1.24% somatostatin per 2 × 106 cells. Characteristic hormone binding patterns were observed in insulin secreting versus somatostatin secreting clones. Insulin secreting clones bound less insulin than somatostatin and other noninsulin- secreting clones (P < 0.02). In contrast, somatostatin secreting clones bound more somatostatin than non-somatostatin-secreting clones (P < 0.05). Somatostatin-secreting clones had a significantly greater number of receptors for all three hormones. The difficulties involved in the interpretation of the quantitative aspects of binding in the presence of continued hormone secretion are discussed. Nonetheless, the presence of receptors on the cells for hormones secreted by the same cells strongly suggests autoregulation. The apparent low affinity of some of these receptors and the presence of receptors for all three islet cell hormones on all islet cells supports the likelihood of paracrine controls.

Opioid Peptides in Rat Islets of Langerhans: Immunoreactive Met- and Leu-Enkephalins and BAM-22P

Previous studies have shown that met- and leu-enkephalins are present in extracts of whole pancreas obtained from guinea pigs and human cadavers. The present studies demonstrate that immunoreactive methionine5 (met)- and leucine5 (leu)-enkephalins present in rat pancreas are localized in islets of Langerhans. Immunohistochemical staining of fixed, whole pancreas indicated that only islet endocrine cells were heavily stained when any of four different met- and leuenkephalin-directed antisera or an anti-BAM-22P (bovine adrenal medulla docosapeptide) antiserum was used. The peptides were characterized by a combination of gel-filtration chromatography, high-performance liquid chromatography (HPLC), and specific radioimmunoassay. Free met-enkephalin content in extracts of rat islets was 90-fold enriched over content in extracts of whole pancreas (1.72 ± 0.35 versus 0.019 ± 0.007 pmol/mg protein). Treatment with trypsin and carboxypeptidase-B of high-molecular-weight peptides extracted from pancreas or islets resulted in release of additional met-enkephalin immunoreactivity, which was 39-fold enriched in islets compared with pancreas (5.90 ± 0.58 and 0.153 ± 0.032 pmol/mg protein, respectively). Total islet content (per milligram protein) of met-enkephalin-containing peptides was similar to that reported elsewhere for bovine hypothalamus. The immunohistochemical data as well as the enrichment of extractable enkephalins in islets compared with whole pancreas indicate that essentially all the met-enkephalin present in pancreas is localized in islets, while the presence of BAM-22P immunoreactivity in islets is consistent with biosynthesis of enkephalins in islet cells via a preprohormone, such as that described in the bovine adrenal medulla and rat brain.

Interaction of Various Stimulators and Inhibitors on Insulin Secretion in vitro

The effect of starvation on pancreatic insulin release was studied in vitro. A marked decrease in response to a glucose stimulus was present. Theophylline, tolbutamide, and dibutyrylcyclic AMP (DBcAMP

Cytosolic Insulin-degrading Activity in Islet-derived Tumor Cell Lines and in Normal Rat Islets

RIN-m cells, cultured from a rat insulinoma, not only bind and secrete but also degrade insulin (Diabetes 1982; 31:521–31). The insulin-degrading activity resides in the cytosol and is similar to the insulin-specific proteases previously described in muscle and other tissues. It has an apparent Km of 0.15 μM for porcine insulin in crude cell-free extracts, a competitive inhibition constant for proinsulin that is close to the Km, and a lower but measurable affinity for glucagon. The enzyme is inactive at pHs below 6.0, indicating that it is not lysosomal, is completely inhibited by Nethylmaleimide, and exhibits apparent competitive inhibition constants (μM) for the following peptides: desoctapeptide insulin, 0.043; guinea pig insulin, 0.048; proinsulin, 0.64; insulin B-chain, 1.17; glucagon, 7.0; and cyclic somatostatin, 8.6. Highly active insulin-degrading activity was found using cell suspensions of 22 cloned and 8 subcloned cell lines derived from RINm as well as 11 other continuous cell lines derived from a variety of nonislet tissues of rat, mouse, and human origin. Homogenates of the original rat islet tumor and cytosol of normal rat islets also contained insulin-degrading activity. Although insulin protease is present in a variety of tissues, it may have an additional regulatory function in cells that are actively synthesizing, storing, and secreting insulin.

Extracellular matrix composition influences insulinlike growth factor I receptor expression in rat IEC-18 cells

BACKGROUND The composition of the extracellular matrix (ECM) as well as insulinlike growth factor I (IGF-I) receptor density vary along the crypt-villus axis. We determined whether components of the ECM influence IGF-I receptor expression in IEC-18 rat small intestine crypt cells. METHODS IEC-18 cells were cultured on plastic, collagen type IV, Matrigel, and laminin at the plateau and proliferative growth phases. Receptor affinity (Kd) and number (Bmax) were determined by competitive binding of 125I-IGF-I in the presence of increasing concentrations of unlabeled IGF-I. Receptor isolation was performed by affinity cross linking. Messenger RNA (mRNA) for IGF-I receptor was quantified by Northern analysis. RESULTS Specific binding of IGF-I > IGF-II > insulin was observed. A 130,000-molecular weight protein was identified by cross-linking, consistent with the alpha subunit of the IGF-I receptor. Scatchard analysis revealed no effect of ECM on IGF-I binding affinity. In contrast, the Bmax was 18% lower for plateau-phase cells cultured on Matrigel vs. plastic and was 42% lower for cells cultured on laminin vs. collagen type IV. The Bmax for proliferative growth phase cells was decreased when cultured on Matrigel vs. plastic and was 10-fold less than for cells cultured at the plateau growth phase. Northern analysis revealed that IEC-18 cells cultured on Matrigel had less mRNA for IGF-I receptor than cells cultured on plastic. CONCLUSIONS The rate of cell proliferation and the composition of the ECM influence IGF-I receptor expression in IEC-18 cells.

Multiple Alterations in Insulin Responses to Glucose in Islets From 48-h Glucose-Infused Nondiabetic Rats

To examine the biochemical mechanisms by which hyperglycemia produces insulin secretory abnormalities, we studied isolated islets from control rats and rats infused for 48 h with a 50% glucose solution. To preserve the effects of in vivo hyperglycemia during in vitro handling for islet isolation, our standard isolation procedure utilized buffers containing 16.8 mM glucose. Islets from infused rats released similar amounts of insulin in low or high glucose during first incubations at 37°C (92.4 ± 7.0 ng 10 islets1 45 min1 at 2.8 mM, 84.4 ± 4.1 ng · 10 islets−1 · 45 min−1 at 16.8 mM) in contrast with control (uninfused) islets (18.6 ± 2.8 ng 10 islets1 · 45 min1 at 2.8 mM and 109.8 ± 8.0 ng 10 islets1 45 min1 at 16.8 mM glucose) (P < 0.01). Secretion by islets of glucose-infused rats was lower during 60-min second incubations at 28 mM glucose than in first incubations of the same islets in low glucose (P < 0.01). This phenomenon is comparable to the paradoxical hypersecretion observed during the first 10–15 min of exposure of glucose-infused pancreas to low-glucose perfusions. Paradoxical secretion in low glucose waned rapidly, so that during second incubations at 37°C, little immunoreactive insulin release occurred at 2.8 mM glucose, despite the persistence of two additional lesions. The glucoseinsulin dose-response curves in second incubations showed a leftward shift for glucose-infused islets, with two-to threefold higher secretion at 5.6–8.4 mM glucose than control islets. This is termed sensitization to glucose. Furthermore, infused islets showed an impaired insulin release (42–73% of control islets) at high glucose (16–28 mM); the decreased maximum response to glucose is related to the previously described desensitization to glucose. This abnormality was reversed by isolation of islets in glucose-free buffer, whereas paradoxical hypersecretion in first incubations was partially reversed, and sensitization to glucose remained undiminished. Studies of secretory responses to nonglucose secretagogues indicated that the maximum response to D-glyceraldehyde was impaired in infused islets, whereas the responses to α-ketoisocaproate and tolbutamide were not significantly different from control. Both monooleoylglycerol, an inhibitor of the turnover of intracellular diacylglycerol, and 12-O-tetradecanoylphorbol-13-acetate were able to restore the decreased maximal secretion in high glucose to control levels. However, monooleoylglycerol had little effect on the low response to glyceraldehyde. Insulin secretion by infused islets was resistant to both mannoheptulose (0.75–3.0 mM) and verapamil (5 μM) inhibition in the presence of high glucose compared with control islets. The data on reversal support the existence of three separate lesions in insulin secretory responses to glucose and suggest that multiple intracellular regulatory mechanisms have been altered. Decreased maximal response to high glucose (desensitization) is the lesion that is most clearly related to and maintained by hyperglycemia, whereas the other two lesions may be related to hypersecretion per se or to extraislet effects in vivo.

Comparison of insulin secretory patterns in obese nondiabetic LA/N-cp and obese diabetic SHR/N-cp rats. Role of hyperglycemia.

Obese diabetic SHR/N-(cp/cp) rats are a genetic model for non-insulin-dependent diabetes mellitus. When SHR/N-cp rats are overtly diabetic, they are hyperinsulinemic and hyperglycemic in the fed state when consuming commercial chow or semipurified high-carbohydrate diets. Obese SHR/N-cp rats were hyperinsulinemic by 4 wk of age, although hyperglycemia did not appear until 3–4 wk later and was exacerbated by a high-sucrose diet (mean ± SE 1488 ± 238 (μ/ml insulin and 425 ± 51 mg/dl glucose). The control SHR/N-cp rats (+ /?) on the sucrose diet remained lean and normoglycemic. The obese diabetic SHR/N-cp rats showed three alterations in pancreas perfusion data (not present in control rats): 7) paradoxically high insulin secretion at low glucose levels (2.5 mM), 2) secretion of insulin in response to arginine (10 mM) in the absence of glucose, and 3) impaired response of insulin secretion to high glucose (16.7 mM). To determine whether hyperglycemia was responsible for the abnormalities of insulin secretion, perfusion studies were conducted in obese nondiabetic LA/N-cp rats and compared with the SHR/N-cp rats. The obese LA/N-cp rats resembled the corpulent SHR/N-cp rats in every way, except that they were normoglycemic on the sucrose diet. The obese LA/N-cp rats had two of the three alterations in insulin secretion shown by obese SHR/N-cp rats, lacking only the impaired response to high glucose, suggesting that hyperglycemia was required for that defect to occur. This finding is supported by the partial reversal of this defect in the obese SHR/N-cp rats after fasting for 48–72 h, when plasma glucose levels had been close to normal for >24–48 h. Additional evidence was obtained in younger, prediabetic obese SHR/N-cp rats that also showed the first two of the three defects and showed only a slightly abnormal response to high glucose. Paradoxically, high insulin secretion at low glucose levels and hypersecretion of insulin in response to arginine without glucose are early abnormalities associated with the development of obesity in these models, whereas decreased maximal response to high glucose develops later and is associated with hyperglycemia.

Is Submaxillary Gland Immunoreactive Glucagon Important in Carbohydrate Homeostasis

Abstract Previous investigators have shown rat submaxillary gland extracts to contain large amounts of material immunologically similar to pancreatic glucagon (SM-IRG). The present studies were designed to assess any physiologic contribution of this material to plasma immunoreactive glucagon (IRG) levels and to overall carbohydrate homeostasis in the rat. Bilateral submaxillary glandectomy or sham surgery was performed on adult male rats. After 2–4 wk, these animals were subjected to arginine or epinephrine infusion, fasting, insulin-induced hypoglycemia, or oral glucose loads. No measurable differences could be found between animals with or without submaxillary glands with respect to basal plasma levels of IRG, immunoreactive insulin (IRI), blood glucose (BG), or to the response of these parameters to any of the conditions tested. Furthermore, when eviscerated animals (without gastrointestinal tract or pancreas, but with intact liver and kidneys) were studied with or without submaxillary glands, no significant differences were found in either basal or stimulated levels of plasma IRG and BG. We conclude that SM-IRG makes no significant contribution to either plasma IRG or the metabolism of carbohydrates in the rat, even when other known sources of IRG have been surgically removed. While being unable to document any apparent role for SM-IRG in the maintenance of carbohydrate homestasis, we did observe dynamic changes in the glandular content of SM-IRG that occurred with the onset of puberty in both male and female weanling rats. The possibility, thus, remains that SM-IRG may be physiologically important, but in areas not necessarily related to carbohydrate metabolism.