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The aim of this study was to examine the effects of insulin and phorbol 12-myristate 13-acetate (PMA), an activator of classic and novel PKCs, on the translocation of PKC from cytosol to membrane in H4IIE (H4) rat hepatoma cells. Six PKC isoforms were expressed, including PKC-mu and PKC-lambda, identified for the first time in this hepatoma-cell line. Insulin induced translocation of PKC-delta from the cytosol to the membrane fraction as early as 15 min and maximally at 60 min with levels returning to that of controls by 180 min. Insulin also decreased levels of PKC-zeta in membranes at 5, 10, 15, and 30 min, but had no effect on cytosol levels. Ten minutes of PMA treatment translocated PKC-delta completely, and 24 h of PMA treatment downregulated PKC-delta. Neither acute nor chronic PMA had any effect on PKC-zeta. These studies establish the ability of both insulin and PMA to activate PKC-delta in H4 cells, and coupled with our previous work demonstrating a diminution of the effect of insulin on gene transcription in PKC downregulated cells, suggest that insulin may exert specific effects, in part, through a PKC-dependent pathway.

Samples of breast tissue obtained at biopsy or mastectomy from women with benign breast disease and infiltrating duct or anaplastic carcinoma were maintained for 2 weeks in organ culture synthetic medium 199 without additional serum or hormones. Media were changed every 48 hours. Media withdrawn from the tissues were assayed for insulin, prolactin (Prl), and parathyroid hormone (PTH). In addition, tissue explants were extracted in acid-alcohol and assayed for insulin by standard radioimmunoassay (RIA) procedures. At day 0 portions of breast tissue from patients with malignant or benign disease were fixed in Bouin solution; they were then embedded in paraffin; and serial sections were obtained for histologic and immunocytochemical examination. The dissection media assayed for insulin and PTH by RIA showed that the hormones were present in media from patients with benign as well as malignant disease. However, there was no significant difference between the two groups of women. Only traces of Prl were detected in media. The amount of insulin present in certain tissue explants appeared to increase with time in culture. Immunocytochemical studies showed that insulin-like or PTH-like immunostaining appeared most often in malignant tumor tissue and was observed infrequently or not at all in patients with benign disease. Prl-positive cells were rare. These data suggest that breast tissues contain and may synthesize significant amounts of certain hormones that may influence the growth and proliferation of breast cells.

Acid-ethanol extracts of homogenates from the parotid and submaxillary salivary glands of normal and streptozocin-induced diabetic adult male mice were investigated for insulin-like material. Extracts of both the parotid and submaxillary glands contained insulin-like immunoreactivity. The values were 156 +/- 72 ng/g wet tissue in the parotid and 104 +/- 36 ng/g wet tissue in the submaxillary gland. Fractionation of this material on Sephadex G-50 (superfine) columns revealed a single peak corresponding to the elution volume of isotopically labeled insulin. Isolated fat cells were stimulated by these extracts to convert [14C]glucose to 14CO2. This effect was blocked by preincubation with anti-insulin serum. It was observed with the avidin-biotin immunocytochemical technique that both the parotid and submaxillary glands of adult male mice possess a population of cells containing an insulin-like material. After intraperitoneal injection of streptozocin there was a marked decrease of insulin-like material extractable from both the parotid and submaxillary glands. However, this beta-cell cytotoxic agent did not completely destroy the salivary cells containing the insulin-like material. These data suggest that both the parotid and submaxillary salivary glands may be extrapancreatic sources of insulin in mice.

Four types of human breast lesions and C3H mouse mammary adenocarcinomas (type A) were examined for the immunocytochemical localization of cells containing hormone-like substances. Insulin- or somatostatin-like immunoreactive material was observed in scattered single cells and nests of tumor cells in seven of eight infiltrating duct carcinomas, and in the majority of tumor cells from an anaplastic carcinoma. A few somatostatin-immunoreactive cells were observed in only one of seven fibroadenomas studied. No immunoreactive cells were observed in mouse adenocarcinomas or in human breast dysplasias. These results suggest that cells with hormone-like immunoreactivity may be a common feature in two types of malignant human breast tumors.

A complete system for neural regulation of insulin and glucagon secretion from the ventral hypothalamus to the autonomic cholinergic and adrenergic nerves of the endocrine pancreas is described. Both physiologic and pathophysiologic states of altered metabolism can be partly explained by activation of this neural system. Direct hypothalamic humoral control of the endocrine pancreas has been evaluated by studies of the hypothalamic hormone somatostatin, or somatotrophin release inhibiting factor (SRIF). Somatostatin has been shown to inhibit pancreatic glucagon and insulin secretion when infused into the pancreatic artery of intact dogs in vivo in the absence of a change of systemic glucose concentration. Although less inhibition is observed when the same amount of hormone is infused intraportally, the potency of somatostatin when given directly into the liver suggests extrapancreatic effects as well as direct pancreatic effects on insulin and glucagon secretion.

To test the hypothesis that somatostatin is a proportional inhibitor of glucose-induced insulin release, we examined the effect of somatostatin on the release of insulin stimulated either by endogenous signals (basal), by glucose infusion, or by glucose injection. Somatostatin infusions (1.7 micrograms/min x 30 min) produced a decrement of basal insulin output from the right lobe of the canine pancreas that was proportional to the initial rate of basal insulin secretion (r = -0.87, p < 0.001, n = 16). Glucose infusions of 1-6 mg/kg/min produced much higher rates of insulin output; again, the decrement of insulin output produced by somatostatin correlated with the initial rate of insulin secretion (r = -0.68, p < 0.01, n = 16). Rapid intravenous injection of either 2 or 20 g of glucose produced a wide range of acute insulin responses (AIR). Somatostatin produced a decrement that was proportional to the original magnitude of the AIR (r = -0.70, p < 0.005, n = 16). Thus, the absolute amount of inhibition produced by somatostatin increases, not decreases, with the magnitude of the stimulus. These data suggest that the inhibitory effect of somatostatin cannot be overcome by increasing the size of glucose stimulus. Thus, exogenous somatostatin, and presumably endogenous somatostatin as well, will produce an inhibitory effect at any physiologic level of glucose stimulation than the beta-cell receives.

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.

Through the use of radioimmunoassay and immunocytochemical techniques, it was found that the parotid glands of male rats possess a population of cells that contain an insulin-like substance. These cells were situated mainly in groups along the intercalated ducts of the gland, or less frequently as isolated cells dispersed throughout the acini. No cells displaying insulin-like immunoreactivity were observed in the striated or main excretory ducts of the parotid. After intravenous (i.v.) injections of streptozotocin (STZ) there was a marked depletion of insulin from the pancreatic islets of rats having diabetes for a 3-27-day interval. Although this cytotoxin also reduced the amount of insulin extractable from the parotid, it did not destroy the insulin-like immunoreactive cells found in this gland. The results of this study suggest that the parotid may be an important source of extrapancreatic insulin. Moreover, these findings indicate that insulin-immunoreactive cells of this salivary gland are spared from the cytotoxic action of STZ.

The effects of substantia nigra lesions on the volume densities of islet cells and on the content of insulin and glucagon in the pancreas were examined using five groups of age-matched, Sprague-Dawley rats. Two groups received bilateral substantia nigra lesions using intrathecal injections of either a low (6 micrograms/hemisphere) or a high (12 micrograms/hemisphere) dose of 6-hydroxydopamine. Rats given sham lesions served as controls for the effects of the neurotoxic drug. These three groups, plus a fourth consisting of unoperated controls, were provided with a high-fat diet to minimize lesion-induced alterations of food intake and body weight. Eleven weeks after lesion placement, tissue was collected from all animals for the assessment of islet cell volume densities and the pancreatic content of insulin and glucagon. Plasma samples also were obtained to determine the levels of glucose, insulin, and glucagon. Data from those animals were compared with that obtained from a fifth group, termed "pre-lesion controls", sacrificed at the beginning of the experiment. Linear-scan morphometry documented an increase of B-cell volume density in the pancreas of non-lesioned rats over the 11-week period (p less than 0.05). However, the volume density of B cells in the pancreas of lesioned animals did not increase compared with that of pre-lesion controls. In terms of A or D cells, no significant differences of volume density were found between the five groups. Compared with that of the pre-lesion controls, pancreatic insulin and glucagon content increased in the lesioned and neurally-intact animals.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was undertaken to determine the influence of alpha-adrenergic blockade upon the somatostatin-mediated inhibition of insulin secretion. Somatostatin inhibited both the basal and glucose-stimulated pancreaticoduodenal vein insulin output of dogs. This effect was not observed during a simultaneous infusion of phentolamine, an alpha-adrenergic blocker. It is proposed that alpha-adrenergic mechanisms play a role in the inhibition of insulin secretion by somatostatin.

We examined the effect of somatostatin (SS) and its analogues on basal insulin and glucagon output from the right lobe of the in situ canine pancreas. Somatostatin (0.17 and 1.7 microgram/min, iv) significantly inhibited insulin (delta = -48 +/- 9% and delta = -88 +/- 3%) and glucagon (delta = -13 +/- 16%, P = NS and delta = -55 +/- 8%). Des-Asn5-SS Significantly inhibited insulin (delta = -40 +/- 9% and delta = -92 +/- 3%) but not glucagon (delta = +35 +/- 18% and delta = +4 +/- 12%). Likewise, [D-Ser13]-SS significantly inhibited insulin (delta = -40 +/- 14% and delta = -71 +/- 8%) with only slight inhibition of glucagon (delta = +26 +/- 15%, P = NS and delta = -16 +/- 6%, P less than 0.05). In contrast, [D-Cys14]-SS significantly inhibited both inhibited both insulin and glucagon. We conclude that structural changes of cyclic somatostatin can dissociate its ability to inhibit pancreatic insulin and glucagon secretion. Because the putative receptors on the pancreatic alpha- and beta-cell appear to recognize different configurations of the somatostatin molecule, it is suggested that the receptors themselves are different.

Immunocytochemical and radioimmunoassay studies were performed on pancreatic and parotid tissues from diabetic BB and control Wistar rats. Compared with those of normoglycemic controls, the pancreata of diabetic BB rats generally lacked insulin-containing B-cells. Extracts from the parotid glands of diabetic rats contained less immunoassayable insulin-like material than was present in parotid extracts of controls. However, the parotid glands of both groups of animals contained numerous cells displaying insulin-like immunoreactivity. These insulin-immunoreactive cells, located mainly in the intercalated portion of the duct system, were comparable to those we reported recently in the parotid glands of normal and streptozocin-diabetic Sprague-Dawley rats. The presence of an insulin-like material in the parotid salivary gland of two types of diabetic animals suggests that such cells may be spared, in part, from the effects of both chemical and hereditary diabetogenic factors.

These studies assessed the ability of glucose infusions to potentiate the acute insulin response (AIR) to iv isoproterenol (12 micrograms), arginine (750 mg), or glucose (5 g) that was previously inhibited by an infusion of somatostatin (SRIF). SRIF (1.7 micrograms/min) markedly inhibited the AIR to isoproterenol (AIR before SRIF, 28 +/- 1 microU/ml; AIR during SRIF, 8 +/- microU/ml; P less than 0.025), arginine (AIR before SRIF, 6 +/- 2 microU/ml; AIR during SRIF, 1 +/- 1 microU/ml; P less than 0.01), and glucose (AIR before SRIF, 19 +/- 7 microU/ml; AIR during SRIF, 1 +/- microU/ml; P less than 0.05). The administration of a glucose infusion of 105 mg/min partially restored the AIR to isoproterenol and arginine. Glucose infused at 440 mg/min fully restored the AIR to both isoproterenol (AIR during SRIF plus glucose, 31 +/- 4 microU/ml) and arginine (AIR during SRIF plus glucose, 9 +/- 2 microU/ml). In contrast, the AIR to glucose was not affected by infusion of glucose (AIR during SRIF plus glucose, 0 +/- 1 microU/ml). In the absence of SRIF, glucose infusion potentiates the AIR to isoproterenol and arginine but not to glucose. Therefore, during SRIF infusion, glucose retains the ability to potentiate the AIR to nonglucose stimuli despite the loss of the ability to stimulate insulin release directly. These data suggest that the potentiating effects of glucose and the inhibiting effects of SRIF may be mediated by a common mechanism affecting insulin release.

The role of insulin in the regulation of human adipose tissue lipoprotein lipase was evaluated. Adipose tissue heparin-releasable lipoprotein lipase (thought to be related to peripheral clearance of plasma triglycerides) was low in insulin-deficient, untreated hyperglycemic diabetic subjects (P less than 0.001) and treatment of hyperglycemia returned the activity to normal. In chronic hyperinsulinism, represented by obesity, heparin-releasable activity among control subjects was correlated to percent of ideal body weight (r=0.53, P less than 0.05) and to fat cell size (r=0.61, P less than 0.02). Acetone-ether powder lipoprotein lipase activity (presumed to reflect total tissue enzyme) was also related to percent of ideal body weight (r=0.76, P less than 0.001 for controls; r=0.67, P less than 0.05 for diabetics) and to fat cell size (r=0.71, P less than 0.01 for controls; r=0.85, P less than 0.01 for diabetics. Postprandial-stimulated insulin secretion was related to diet-induced changes in lipoprotein lipase in control subjects; both were dependent upon the amount of dietary carbohydrate. In contrast, the diabetic patients with low insulin responses, failed to increase lipoprotein lipase activity with feeding. The changes in heparin-releasable (r=0.66, P less than 0.01) and acetone-ether powder (r=0.69, P less than 0.01) activity during feeding were related to the percent increase in plasma insulin. Thus, insulin appears to be important in the regulation of human adipose tissue lipoprotein lipase activity. Elevated insulin levels in obesity and increased insulin secretion after eating were associated with increased lipoprotein lipase activity. Defects in insulin secretion, both in postabsorptive and postprandial states, are associated with low adipose tissue lipoprotein lipase and may lead to hypertriglyceridemia in diabetic man.