RESUMEN
The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."
Asunto(s)
Calcio/metabolismo , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Óvulo/metabolismo , Proteínas de Transporte Vesicular , Animales , Membrana Dobles de Lípidos , Peso Molecular , Proteínas del Tejido Nervioso/metabolismo , Óvulo/citología , Proteínas Qa-SNARE , Proteínas R-SNARE , Proteínas SNARE , Erizos de Mar , Proteína 25 Asociada a Sinaptosomas , Toxina Tetánica/farmacologíaAsunto(s)
Adipocitos/fisiología , Glucosa/metabolismo , Insulina/farmacología , Proteínas Musculares , Proteínas de Transporte Vesicular , Adipocitos/ultraestructura , Animales , Transporte Biológico/efectos de los fármacos , Transportador de Glucosa de Tipo 4 , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas R-SNARE , Ratas , Proteínas SNARE , Fracciones Subcelulares/metabolismo , Proteína 3 de Membrana Asociada a VesículasRESUMEN
The vesicle-associated membrane proteins [VAMPs; vesicle SNAP receptors (v-SNAREs)] present on GLUT4-enriched vesicles prepared from rat adipose cells [Cain, Trimble and Lienhard (1992) J. Biol. Chem. 267, 11681-11684] have been identified as synaptobrevin 2 (VAMP 2) and cellubrevin (VAMP 3) by using isoform-specific antisera. Additional antisera identify syntaxins 2 and 4 as the predominant target membrane SNAP receptors (t-SNAREs) in the plasma membranes (PM), with syntaxin 3 at one-twentieth the level. Syntaxins 2 and 4 are enriched 5-10-fold in PM compared with low-density microsomes (LDM). Insulin treatment results in an 11-fold increase in immunodetectable GLUT4 in PM and smaller (approx. 2-fold) increases in VAMP 2 and VAMP 3, whereas the subcellular distributions of the syntaxins are not altered by insulin treatment. To determine which of the SNAP receptors (SNAREs) in PM might participate in SNARE complexes with proteins from GLUT4 vesicles, complexes were immunoprecipitated with anti-myc antibody from solubilized membranes after the addition of myc-epitope-tagged N-ethylmaleimide-sensitive fusion protein (NSF) and recombinant alpha-soluble NSF attachment protein (alpha-SNAP). These complexes contain VAMPs 2 and 3 and syntaxin 4, but not syntaxins 2 or 3. Complex formation requires ATP and is disrupted by ATP hydrolysis. When all membrane fractions are prepared from basal cells, few or no VAMPs and no syntaxin 4 are immunoprecipitated in SNARE complexes obtained from LDM alone (or from immunoisolated GLUT4 vesicles). The content of syntaxin 4 depends on the presence of PM, and participation of VAMPs 2 and 3 is enhanced 4-6-fold by the addition of solubilized GLUT4 vesicles to PM. The latter increase is greater than can be explained by the 2-fold higher levels of VAMPs added to the reaction mixture. When all membrane fractions are prepared from insulin-stimulated cells, SNARE complexes formed from PM alone contain similar levels of syntaxin 4 but 5-6-fold higher levels of VAMPs 2 and 3 compared with PM alone from basal cells. Addition of GLUT4 vesicle proteins to PM from insulin-treated cells results in a further 2-fold increase in VAMP 2 recovered in SNARE complexes. Therefore the VAMPs in PM of insulin-treated but not basal cells, and in GLUT4-vesicles from cells in either condition, are in a form that readily forms a SNARE complex with PM t-SNAREs and NSF. Insulin seems to activate PM and/or GLUT4 vesicles so as to increase the efficiency of SNARE complex formation.
Asunto(s)
Tejido Adiposo/metabolismo , Etilmaleimida/farmacología , Proteínas de la Membrana/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Tejido Adiposo/efectos de los fármacos , Animales , Encéfalo/metabolismo , Membrana Celular/metabolismo , Epidídimo , Epítopos , Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/efectos de los fármacos , Microsomas/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Miocardio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , Lugares Marcados de Secuencia , Proteína 3 de Membrana Asociada a VesículasRESUMEN
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)
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Glucosa/farmacología , Islotes Pancreáticos/fisiología , Obesidad/genética , Obesidad/fisiopatología , Ratas Endogámicas SHR/genética , Animales , Glucemia/análisis , Western Blotting , Separación Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Hiperglucemia/metabolismo , Hiperglucemia/fisiopatología , Insulina/análisis , Insulina/metabolismo , Islotes Pancreáticos/química , Islotes Pancreáticos/metabolismo , Masculino , Manoheptulosa/farmacología , Proteínas de Transporte de Monosacáridos/análisis , Obesidad/metabolismo , Ratas , Ratas Mutantes , Verapamilo/farmacologíaRESUMEN
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)
Asunto(s)
Amígdala del Cerebelo/metabolismo , Diabetes Mellitus/metabolismo , Eminencia Media/metabolismo , Neuropéptidos/metabolismo , Nervio Vago/metabolismo , Animales , Glucemia/análisis , Encéfalo/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/genética , Disección/métodos , Insulina/sangre , Ratones , Ratones MutantesRESUMEN
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 degrees C (92.4 +/- 7.0 ng.10 islets-1.45 min-1 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 islets-1.45 min-1 at 2.8 mM and 109.8 +/- 8.0 ng.10 islets-1.45 min-1 at 16.8 mM glucose) (P less than 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 less than 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 degrees C, little immunoreactive insulin release occurred at 2.8 mM glucose, despite the persistence of two additional lesions. The glucose-insulin 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Glucosa/farmacología , Insulina/sangre , Islotes Pancreáticos/efectos de los fármacos , Animales , Calcio/farmacocinética , Relación Dosis-Respuesta a Droga , Hipersensibilidad a las Drogas/etiología , Glucagón/análisis , Gliceraldehído/farmacología , Glicéridos/farmacología , Infusiones Intravenosas , Insulina/análisis , Islotes Pancreáticos/química , Islotes Pancreáticos/metabolismo , Cetoácidos/farmacología , Masculino , Manoheptulosa/farmacología , Ratas , Ratas Endogámicas , Somatostatina/análisis , Temperatura , Acetato de Tetradecanoilforbol/farmacología , Tolbutamida/farmacología , Verapamilo/farmacologíaRESUMEN
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)
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Ratones Mutantes/metabolismo , Ratones Obesos/metabolismo , Neuropéptidos/análisis , Páncreas/análisis , Hipófisis/análisis , Animales , Encefalina Leucina/análisis , Encefalina Metionina/análisis , Femenino , Galanina , Técnicas In Vitro , Insulina/sangre , Masculino , Ratones , Obesidad/metabolismo , Péptidos/análisis , Valores de Referencia , Factores Sexuales , Especificidad de la Especie , betaendorfina/análisisRESUMEN
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 microU/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): 1) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus/fisiopatología , Hiperglucemia/fisiopatología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Obesidad/fisiopatología , Ratas Endogámicas SHR/fisiología , Ratas Endogámicas/fisiología , Ratas Mutantes/fisiología , Animales , Glucemia/análisis , Peso Corporal , Técnicas In Vitro , Secreción de Insulina , Perfusión , Ratas , Valores de Referencia , Especificidad de la EspecieRESUMEN
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 beta-endorphin, Met- and Leu-enkephalins, 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 peptides 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 postnatal week. In contrast, the precursors had the highest concentration in the fetus, and by the seventh day of life had decreased by greater than 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.
Asunto(s)
Encefalinas/análisis , Páncreas/análisis , betaendorfina/análisis , Animales , Femenino , Feto/análisis , Glucagón/análisis , Insulina/análisis , Islotes Pancreáticos/análisis , Masculino , Páncreas/embriología , Embarazo , Radioinmunoensayo , Ratas , Ratas Endogámicas WF , Hormona Liberadora de Tirotropina/análisisRESUMEN
Previous reports have provided suggestive evidence for a role of endogenous opiates in modulating feeding behavior as well as insulin secretion from the pancreas. This evidence includes pharmacologic studies as well as studies of endogenous opioid peptides in tissues of genetically obese rodent models. In the present study, content of three opioid peptide immunoreactivities (beta-endorphin, met-enkephalin and leu-enkephalin) were measured in pituitary and pancreas of two recently described, genetically obese rats, the LA/N-cp and the SHR/N-cp. Although both of these inbred strains carry the same cp allele, the SHR-cp rat is obese and diabetic by 8 weeks of age, while the LA/N-cp is obese but remains euglycemic. There were no significant differences in content of immunoreactive beta-endorphin, met-enkephalin or leu-enkephalin in whole pituitary, posterior or anterior lobes of the pituitary, or pancreas which could be ascribed to the effect of the obese phenotype. These data are in contrast to previously reported studies in which significant alterations in opioid peptide immunoreactivities were found in genetically obese Zucker fa/fa rats, C57BL/6 ob/ob mice and C57BL/Ks db/db mice. The present work provides no support for pathological involvement of opioid peptides in the genetically obese cp/cp rat.
Asunto(s)
Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Obesidad/metabolismo , Páncreas/metabolismo , Hipófisis/metabolismo , betaendorfina/metabolismo , Animales , Masculino , Ratas , Ratas Endogámicas SHRRESUMEN
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 (greater than 400 mg/dl), and are hyperinsulinemic, with immunoreactive insulin (IRI) levels 10- to 20-fold greater than 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 greater than 53% decrease in IRI secretion in cp/cp rats (P less than .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.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Glucemia/metabolismo , Modelos Animales de Enfermedad , Genotipo , Insulina/metabolismo , Ratas Endogámicas SHR/sangre , Ratas Endogámicas/sangre , Animales , Arginina/administración & dosificación , Peso Corporal , Ayuno , Femenino , Tamización de Portadores Genéticos , Insulina/sangre , Secreción de Insulina , Masculino , Obesidad/sangre , Páncreas/metabolismo , Perfusión , Ratas , Ratas Endogámicas SHR/genética , Sacarosa/administración & dosificación , Factores de TiempoRESUMEN
Plasma epinephrine (EPI), norepinephrine (NE), beta-endorphin, and corticotropin (ACTH) responses were measured during insulin-induced hypoglycemia in normal subjects and in patients with either multiple system atrophy (MSA) or idiopathic orthostatic hypotension (IOH). In normal subjects, there was a striking rise in EPI, NE, beta-endorphin, and ACTH following the nadir of hypoglycemia. Both beta-endorphin and ACTH responses were significantly lower than normal in patients with MSA, in contrast to normal levels in IOH patients. No correlation was observed between the degree of adrenergic insufficiency and the beta-endorphin and ACTH responses. The normal peptide responses in IOH are consistent with involvement limited to the peripheral sympathetic nervous system, whereas lesions in the central nervous system in MSA interfere with release of beta-endorphin and ACTH in response to hypoglycemia. The strong correlation between beta-endorphin and ACTH levels is consistent with their common origin. Peripheral adrenergic activity is not essential for beta-endorphin and ACTH release in humans.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Enfermedades del Sistema Nervioso Autónomo/fisiopatología , Catecolaminas/sangre , Endorfinas/sangre , Insulina , Glándulas Suprarrenales/fisiopatología , Adulto , Anciano , Enfermedad Crónica , Femenino , Humanos , Hipotensión Ortostática/fisiopatología , Sistema Hipotálamo-Hipofisario/fisiopatología , Masculino , Persona de Mediana Edad , Síndrome de Shy-Drager/fisiopatología , Sistema Nervioso Simpático/fisiopatología , betaendorfinaRESUMEN
Incubation of unfixed, frozen sections of rat pancreas with tritiated guanidinoethylmercaptosuccinic acid (GEMSA) resulted in a localization of [3H]-GEMSA autoradiographic grains over the islets of Langerhans, suggesting that a carboxypeptidase H-like enzyme occurs in the islet.
Asunto(s)
Carboxipeptidasas/metabolismo , Islotes Pancreáticos/metabolismo , Succinatos/metabolismo , Animales , Autorradiografía , Carboxipeptidasa H , Encefalinas/metabolismo , Masculino , RatasRESUMEN
A potential role for somatostatin (SRIF) in the pathogenesis of the hyperinsulinemia of obese rats was considered. SRIF like immunoreactivity (ng/mg protein) was therefore measured in hot 2 N acetic acid extracts of pancreas, stomach, pituitary, and hypothalamus in tissues obtained from three models of genetic obesity in rats. These models included the obese and lean controls of LA/N-cp, SHR/N-cp, and Zucker rats. To assess the effects of diet on SRIF levels, mixed diets were provided ad lib which contained a carbohydrate as either sucrose or starch. Some groups were fed chow diets. No significant dietary effects on tissue levels of SRIF were obtained. However, two of the three models (Zucker and SHR/N-cp) showed phenotypic effects on SRIF levels in pancreas; namely, obese rats showed a significantly greater concentration of SRIF (P less than 0.0005 and less than 0.0002, respectively) than did the lean littermates. These findings were confirmed by measurement of total pancreas SRIF content. Gastric levels were significantly altered only in the obese Zucker rats (P less than 0.005) where obese tissues had lower concentrations than those of lean animals. However similar directional changes in pancreas and stomach were observed in all models. It is concluded that the hyperinsulinemia of the obese animals studied is not due to absolute deficiency in pancreatic SRIF content. It is postulated however that decreased pancreatic SRIF secretion (paracrine or otherwise) relative to pancreatic insulin content could still play a role.
Asunto(s)
Obesidad/genética , Ratas Mutantes/metabolismo , Somatostatina/análisis , Animales , Modelos Animales de Enfermedad , Ayuno , Hipotálamo/análisis , Ratones , Ratones Endogámicos , Obesidad/metabolismo , Páncreas/análisis , Hipófisis/análisis , Ratas , Especificidad de la Especie , Estómago/análisis , Distribución TisularRESUMEN
Free enkephalins (enk) and higher molecular weight enkephalin-containing peptides (enk-c-p) are present in the endocrine pancreas of rats, presumably in B cells. To determine whether these opioid peptides show dynamic alterations as insulin content of pancreas changes, we utilized a copper deficient rat model, in which the exocrine pancreas atrophies and the endocrine pancreas is "intact" and insulin (IRI) content increases. Dietary copper deficiency (-C) was produced in weanling male rats for 4 and 7 weeks. The deficient and copper supplemented (+C) groups were further subdivided to receive all dietary carbohydrate as either 62% fructose (F) or 62% starch (S). -CF rats showed the most severe deficiency. After 7 weeks, total units of pancreatic IRI in -CF were 7.5 +CF 2.1, -CS 7.9 and in +CS 2.8 (p less than 0.001). Pancreatic content of Met5- and Leu5-enk was measured in extracts which were purified on C-18 Seppaks with and without prior treatment with trypsin and carboxypeptidase B. -C animals showed progressive, significant increases in pancreatic content of Leu-enk-c-p, with a decrease in free Leu- and Met-enk (p less than 0.02-0.01). The pancreatic findings are compatible with a co-localization of enkephalins and insulin in the endocrine pancreas and are suggestive of co-regulation.
Asunto(s)
Cobre/deficiencia , Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Hipófisis/metabolismo , Animales , Peso Corporal , Carbohidratos de la Dieta/farmacología , Cinética , Masculino , Tamaño de los Órganos , Proteínas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
We have recently shown that in addition to beta-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 beta-endorphin and Met- and Leu-enkephalins were measured in 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 less than .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 beta-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 beta-endorphin and pituitary Leu-enkephalin; similar elevations in these parameters have been reported previously in C57BL/6J ob/ob mice at approximately 12 wk of age.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Endorfinas/metabolismo , Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Encefalinas/metabolismo , Islotes Pancreáticos/metabolismo , Ratones Obesos/metabolismo , Hipófisis/metabolismo , Envejecimiento , Animales , Peso Corporal , Insulina/sangre , Ratones , betaendorfinaRESUMEN
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 methionine (met)- and leucine (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 leu-enkephalin-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 carboxy-peptidase-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.
Asunto(s)
Encefalina Leucina/análisis , Encefalina Metionina/análogos & derivados , Encefalina Metionina/análisis , Islotes Pancreáticos/análisis , Precursores de Proteínas/análisis , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Masculino , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
Twenty-four male subjects participated in a study in which the effect of feeding diets low in copper (1.03 mg/day) on plasma opiates was determined. The subjects were fed a low-copper diet for 11 wk with either starch or fructose as a major source of carbohydrate. Feeding low-copper diet decreased serum copper level significantly. In addition, plasma leu- and met-enkephalins decreased significantly while beta-endorphin levels rose. On repletion with copper (3 mg/day) for 3 wk, plasma enkephalins increased while beta-endorphin levels decreased to pretest values. These results suggest that feeding low copper decreases plasma enkephalins, which may reflect a copper-dependent process affecting enkephalin biosynthesis and/or release.
Asunto(s)
Cobre/deficiencia , Encefalinas/sangre , Catecolaminas/metabolismo , Ceruloplasmina/sangre , Endorfinas/sangre , Encefalina Leucina/sangre , Encefalina Metionina/sangre , Humanos , Masculino , Superóxido Dismutasa/sangre , betaendorfinaRESUMEN
Seven cytoplasmic enzyme activities were measured in extracts of mononuclear leukocytes (lymphocytes plus monocytes) obtained from 19 type II diabetic humans and 10 healthy control subjects. 6-Phosphofructokinase activity was significantly decreased in cell extracts from diabetics, while other enzyme activities were similar in diabetics and controls. Since the effects of starvation on enzyme activities are sometimes similar to the effects of diabetes, the studies were repeated in 5 control subjects after a 2-day fast. This short period of starvation did not mimic the effect of diabetes on 6-phosphofructokinase activity. The decreased enzyme activity was not correlated with percent specific insulin binding to monocytes in the same cell preparations nor to clinical variables such as obesity or the broad range of fasting plasma glucose values encountered among the diabetics. We conclude that 6-phosphofructokinase activity in mononuclear leukocytes, as in other tissues, may be a marker for a postreceptor lesion associated with the insulin resistance found in type II diabetes mellitus.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Leucocitos/enzimología , Fosfofructoquinasa-1/sangre , Adulto , Anciano , Glucemia/análisis , Diabetes Mellitus Tipo 2/sangre , Femenino , Humanos , Insulina/sangre , Resistencia a la Insulina , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , Monocitos/enzimología , Inanición/enzimologíaRESUMEN
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 microM 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 N-ethylmaleimide, and exhibits apparent competitive inhibition constants (microM) 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 RIN-m 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.