Kim I. Timmers

Altered β-Endorphin, Met- and Leu- Enkephalins, and Enkephalin-Containing Peptides in Pancreas and Pituitary of Genetically Obese Diabetic (db/db) Mice During Development of Diabetic Syndrome

We have recently shown that in addition to β-endorphin the opioid peptides Met- and Leu-enkephalin and their apparent precursors are localized in islet endocrine cells of the rat pancreas. To begin evaluating a possible role for these pancreatic opiates in the pathophysiology of genetic diabetes in rodents, immunoreactive β-endorphin and Met- and Leu-enkephalins were measuredin acetic acid extracts of pancreas and pituitary of C57BL/KsJ db/db mice and their lean littermates. Groups of animals were studied during three phases of development of the diabetic syndrome in the mutant mice: at 4 (hyperinsulinemic and prediabetic); 6, 9, and 12 (frankly obese and diabetic); and 30 (hypoinsulinemic) wk of age. Elevations or decreases (P < .05) were found in db/db mice (vs. lean littermates) as follows: pituitary content of Met-enkephalin was twofold higher at all ages studied; pituitary free Leu-enkephalin was lower at 4 wk and reversed to higher at 6–30 wk; pancreatic β-endorphin was 30% lower at 4 wk and reversed to threefold higher at 6–12 wk; Met- and Leu-enkephalin-containing larger peptides were elevated at one or more points between 6 and 12 wk in both the pancreas and the pituitary. Thus, the onset of overt obesity between 4 and 6 wk of age was accompanied by a marked rise in both pancreatic β-endorphin and pituitary Leu-enkephalin; similar elevations in these parameters have been reported previously in C57BL/6J ob/ob mice at ∼12 wk of age. Pancreatic Met-enkephalin content, although not significantly different from controls of any age group, was threefold higher in db/db mice at 30 wk as compared with that at 4 wk and increased with age in dbldb (r = .501, P < .01) but not in control (r = −.041) mice. This difference between correlations by genotype was statistically significant (F = 32.8, P < .001). Pancreatic Met-enkephalin was also negatively correlated with plasma insulin concentration (r = −.404, P < .01) in all animals studied. These correlations suggest a relationship between increasing pancreatic Met-enkephalin content and the declining ability of the dbldb pancreas to secrete insulin. Because the pancreatic content of Met-enkephalin-containing precursor peptides decreased with age in both dbldb and control mice, the rising free Met-enkephalin in the dbldb pancreas suggests an increasing rate of processing of precursor to free enkephalin between 4 and 30 wk of age. We conclude that the content and processing of opioid peptides in the pancreas and pituitary are regulated abnormally in C57BL/KsJ db/db mice during development of the diabetic syndrome. The known ability of these peptide neurohormones to modulate the secretion of other pituitary and pancreatic hormones suggests the possibility that altered pancreatic free Met-enkephalin content or altered rates of enkephalinprecursor processing may be involved in the pathophysiology of the hyperinsulinemia that declines with age in these mice.

Reversible Impairment of Glucose-Induced Insulin Secretion in SHR/N-cp Rats: Genetic Model of Type II Diabetes

The SHR/N-cp rat is a new genetically obese model for non-insulin-dependent diabetes mellitus. Expression of the diabetes is enhanced by a high-sucrose (54%) diet. After 4 wk on the diet, the cp/cp rats weigh significantly more than their +/? controls, have postprandial hyperglycemia (> 400 mg/dl), and are hyperinsulinemic, with immunoreactive insulin (IRI) levels 10- to 20-fold > controls. Total pancreatic IRI tends to be increased 1.6-fold in the cp/cp rats (although not significantly). There is no increase in pancreatic proinsulin content as a percent of total IRI. Studies of in vitro pancreatic function were carried out with the isolated nonrecirculating perfused pancreas method. The cp/cp rats (n = 10) showed impaired or absent IRI responses to 16.5 mM glucose, whereas +/? rats (n = 9) responded with classic biphasic curves. Comparison of insulin secreted in 20 min revealed a > 53% decrease in IRI secretion in cp/cp rats (P < .05). A paradoxical hypersecretion of IRI at glucose concentrations of 0–2.7 mM was noted in cp/cp but not lean rats, i.e., 1.8 ± 0.2 mU/min IRI in cp/cp rats vs. 0.04 ± 0.007 mU/min in +/? rats. Perfusion of pancreases for 45 min with buffers containing no glucose resulted in restoration of a normal biphasic IRI response to 16.5 mM glucose in the cp/cp rats, whereas response in the lean rats was markedly reduced. Brisk IRI responses to 10 mM arginine in buffers with no glucose also occurred in cp/cp but not +/? rats. Glucagon secretion was relatively suppressed in the cp/cp rats. These findings are similar to those reported in glucose-infused normal rats and suggest that hyperglycemia per se may be responsible for the impaired β-cell responses to glucose in cp/cp rats.

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.

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.

Neuropeptide content in pancreas and pituitary of obese and diabetes mutant mice: Strain and sex differences☆

The nature of the primary genetic defects in ob/ob and db/db mice are unknown. Both the obese (ob) and diabetes (db) mutations produce similar, multicomponent obese-hyperinsulinemic syndromes when maintained in the same strain of mouse. In an attempt to find differences between these mutations in neuroendocrine function affecting the islets of Langerhans or the pituitary, tissue content of four neuropeptides that are known to be capable of influencing the rate of insulin secretion was examined in obese (ob/ob) and diabetes (db/db) mice. In the first study, C57BL/6Job/ob and control males were studied at 3, 4, and 11 weeks of age. In the second study, db/db mice of both sexes and two inbred strains (C57BL/6J and C57BL/KsJ), which differ markedly in the severity of expression of the diabetes phenotype, were studied at 3 weeks of age, before the development of hyperglycemia and secondary consequences thereof. Immunoreactive peptides were measured in acetic acid extracts of pancreas and pituitary. No differences between male ob/ob and db/db mice of the C57BL/6J strain were found. Marked sex differences in lean control mice were found at 3 weeks of age in pancreatic Met-enkephalin-LI and galanin-LI (with two- to threefold higher content in males). Low pancreatic content (50% to 70% lower than in control mice) of galanin-LI, Met-enkephalin-LI and Leu-enkephalin-LI was associated with hyperinsulinemia in male B6 ob/ob and db/db mice at 3 weeks of age, though not in B6 db/db females and not in BKs db/db mice of either sex.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetically obese rats with (SHR/N-cp) and without diabetes (LA/N-cp) share abnormal islet responses to glucose

To assess the effect of hyperglycemia on the function of islets obtained from obese rats, the behavior of isolated islets from LA/N-corpulent (nondiabetic obese) and SHR/N-corpulent (diabetic obese) male rats was examined and compared. Islets from both genetic models showed a left-shifted glucose dose-response curve for insulin release (concentrations for half-maximal release, 5 to 6 mmol/L v 12 to 13 mmol/L in LA/N lean littermates and 3 mmol/L v 10 mmol/L in lean SHR/N). When insulin release was expressed per unit islet volume, the fourfold to fivefold enlarged islets from both obese diabetic and obese nondiabetic rats showed decreased insulin secretory response in high (16.5 to 28 mmol/L) glucose concentrations, although the decrease was more severe in the diabetic rats. Glucose-stimulated insulin release by islets from both models was relatively resistant to inhibition by 1.2 mmol/L mannoheptulose (eg, 82% +/- 3% inhibition in LA/N lean v 16% +/- 8% in LA/N obese), although nearly complete inhibition was observed with 16 mmol/L mannoheptulose (96% v 85%, NS). Islets of obese diabetic rats were also resistant to the calcium-channel blocker, verapamil, suggesting an abnormal pathway of stimulus-secretion coupling for glucose. Glucose oxidation to carbon dioxide was increased in both obese models at all glucose concentrations when expressed per islet. In data expressed per unit volume, the larger islets from the obese-nondiabetic rats showed a left-shifted dose-response curve with an unchanged maximum rate of glucose oxidation at high (16.5 mmol/L) glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Unique alterations of neuropeptide content in median eminence, amygdala, and dorsal vagal complex of 3- and 6-week-old diabetes mutant mice.

The nature of the genetic defects which define the obese (ob) and diabetes (db) loci in mice remain unknown, but both produce similar syndromes when maintained in the same strain of mice. There is some evidence suggesting a lesion in the central nervous system (CNS) in db/db mice, while ob/ob mice appear to have a primary lesion outside the CNS. In a search for further evidence of a unique central lesion in db/db mice, we have examined neuropeptide content in selected, microdissected brain areas in both of these mutants and lean controls. In order to rule out possible interactions of the db mutation with the genetic background, diabetes mice of both C57BL/KsJ and C57BL/6J strains were studied. When concentrations of nine neuropeptide immunoreactivities were examined in up to seven microdissected areas of the brain, C57BL/6J ob/ob mice showed only one reproducible alteration, a lower content of beta-endorphin-like immunoreactivity (LI) in the preoptic area at both 3 and 6 weeks of age as compared with lean littermates. In contrast, db/db mice of both C57BL/6J and C57BL/KsJ strains exhibited alterations in a total of four peptides in three brain areas: lower concentration of somatostatin-LI in median eminence, higher Met-enkephalin-LI in dorsal vagal complex of the medulla oblongata, higher substance P-LI and lower vasoactive intestinal polypeptide (VIP)-LI in amygdala. The concentrations of the peptides studied in medial basal hypothalamus, lateral hypothalamus, substantia nigra, and preoptic area were not reproducibly altered in db/db mice. These data provide preliminary evidence for unique brain abnormalities in db/db mice in specific areas that are involved in processing of neural signals that can affect the islets of Langerhans, gonadotrophin secretory patterns, and many other visceral functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental patterns for pancreatic opioids in the rat.

Developmental patterns for rat pancreatic opioid peptides and islet hormones were studied from gestational day 20 through adulthood. Fetal tissue was obtained as well as pancreas at birth (day 0), and postnatal days 3,7, 14, and 21, and 7 weeks. The hormones measured included insulin, glucagon, and somatostatin. The opioids measured were β-endorphin, Met- and Leuenkephalins, and the high molecular weight enkephalin precursors. Pancreata were pooled as necessary and extracted (acid alcohol, or hot acetic acid), and opioids were further purified on reversed-phase C-18 (Sep-pak) cartridges. In all instances measurements were made by radioimmunoassays. Precursor pep- tides were first digested (with trypsin and carboxypeptidase B) prior to immunoassay. All opioids and hormones except the precursors for enkephalins showed a well-defined surge in pancreatic concentration during the first post- natal week. In contrast, the precursors had the highest concentration in the fetus, and by the seventh day of life had decreased by >50%. This progressive decrease may represent maturation of the enkephalin convertase and trypsin like enzymes in the islets. The opioid and hormonal surges that we have described are similar to the surge in islet concentration of thyroid-releasing hormone (TRH) previously described in neonatal rat islets. It is suggested that these postnatal alterations in opioid and hormone concentration relate to a specific function in the development of the endocrine pancreas.