Non-obese diabetic mice were treated daily with a mixture of ganliosides from day 30 until day 250 of life or until the mice became diabetic. Ganglioside treatment reduced diabetes incidence from 80–90% to 47% and from 20–30% to zero in female and male mice respectively. Gangliosides did not affect the frequency of perivasculitis. It is concluded that gangliosides can reduce diabetes incidence in non-obese diabetic mice.

The purpose of this work was to investigate the molecular mechanism responsible for the induction of hepatic glucokinase in diabetic rats acutely treated with insulin. Experimental diabetes was provoked by injection of streptozotocin 8-10 days before the experiments. Regular insulin was given by three intraperitoneal injections at 8-h intervals, and the time course of glucokinase induction was followed over a time period of 24 h. The amount of glucokinase in liver was estimated by Western blotting of total cytosol protein with affinity-purified antibodies, as well as by conventional enzyme activity assay. Both measurements showed that glucokinase was reduced by more than 90% in the livers of diabetic rats as compared to normal controls. Following insulin administration, the amount (and activity) of glucokinase increased in a time-dependent fashion, after an initial lag of 4 h, to reach 65% of the nondiabetic control level 24 h after the initial dose of insulin. Northern blot analysis with a cloned cDNA probe was used to quantitate glucokinase mRNA. In contrast with the slow onset of enzyme accumulation, the amount of glucokinase mRNA was shown to be increased dramatically as early as 1 h after insulin administration. The abundance of specific mRNA increased until 8 h after the initial dose of insulin. Subsequently, the level of the mRNA decayed rapidly so that little message was left after 16 h and virtually none after 24 h. Run-on transcription experiments with isolated nuclei showed that the rate of transcription of the glucokinase gene was increased about 20-fold within 45 min of insulin administration and returned to the prestimulation level after 8 h. From these data, it was concluded that the induction of glucokinase resulted primarily from a burst in the transcriptional activity of the gene, leading to a short-term accumulation of glucokinase mRNA. The more sustained elevation of the enzyme level can be accounted for by the long half-life of the enzyme (greater than 30 h). The virtually immediate activation of glucokinase gene transcription suggests a direct effect of insulin on the liver cell.

The exocrine secretory function of the ventral and dorsal areas of the rat pancreas was investigated separately. In the isolated perfused rat pancreas, caerulein (10(-12), 10(-11), and 10(-10) M) and carbachol (10(-7) and 10(-6) M) caused a greaterfold increase in pancreatic secretion from the dorsal than the ventral area. Caerulein (10(-9) M) and carbachol (10(-5) M) were supramaximal concentrations with respect to secretion from the dorsal area, but this was not the case in the ventral area. Similar findings were also observed when ventral and dorsal secretions were studied in vivo in anesthetized animals. By contrast, in dispersed acini the sensitivity and responsiveness of tissue from the dorsal and ventral areas were similar to both secretagogues. The possible influence of the endocrine pancreas via the insuloacinar portal system was studied in the perfused pancreas of diabetic rats treated with insulin in vivo. Carbachol (10(-6) M)-stimulated secretion from the ventral area was similar (except for amylase) to that of controls, while a significantly reduced (P less than 0.001) output was measured from the dorsal pancreas. These results demonstrate that in the intact pancreas differences of exocrine secretion exist between the ventral and dorsal areas and suggest that insulin may contribute, at least in part, to the expression of such differences.

Isolated rat islets of Langerhans were pulse-labeled (5 min, [3H]leucine) and then exposed to 10 or 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 during an 85-min chase period. There was a dose-related inhibition of the conversion of labeled proinsulin to insulin by Tris. At 50 mM, Tris also inhibited the release of newly synthesized (labeled) proinsulin and insulin. These inhibitory effects of Tris were almost absent if the islets were exposed to 50 mM Tris during only the last 60 min of the 85-min chase period. Both proinsulin and total islet protein synthesis (as indexed by incorporation of [3H]leucine) were inhibited acutely by 50 mM Tris (5-min exposure); after 85 min of exposure to 50 mM Tris, the inhibition of proinsulin biosynthesis was more marked than that of total islet protein. In contrast to its effects on newly synthesized products, 50 mM Tris failed to inhibit the release of immunoreactive insulin during an 85-min incubation. However, when islets were exposed to 50 mM Tris for a longer period, a partial inhibition of immunoreactive insulin release was observed as from 120 min. Insulin released from islets consists of a mixture of older stored material and of newly synthesized products, the latter being released preferentially. These results are consistent with a selective effect of 50 mM Tris on the production of newly synthesized insulin. During the first 120 min of exposure to Tris, islet reserves of newly synthesized products will be depleted thereby leading to a new, reduced, rate of release of immunoreactive material consisting only of older insulin stores. To confirm that Tris neither inhibited the release of stored insulin nor exerted any nonspecific toxic effects on B-cells during prolonged exposure, islets were labeled with [3H]leucine in the absence of Tris for 3 days. The fate of the prelabeled insulin stores was then followed during a 24-h chase period in the presence of 50 mM Tris. The release of the labeled, stored insulin was not affected by Tris, whereas the release of immunoreactive insulin (consisting of both new and old material) was partially inhibited as expected. We conclude that Tris inhibits proinsulin synthesis and processing. The inhibition of newly synthesized proinsulin release reflects perturbation of intracellular events before exocytosis, which, itself, is not affected by TriS.

The object of the present investigation was to determine whether insulin secreted by the endocrine pancreas and carried in the insulo-acinar portal system has a direct effect on pancreatic enzyme secretion. For this purpose, the isolated rat pancreas was perfused in a nonrecirculating system. The perfusate contained 3 mM glucose, and either caerulein or vaso-active intestinal polypeptide was used to stimulate exocrine secretion. The amount of insulin reaching the exocrine pancreas was reduced by two different experimental procedures. In the first, use was made of streptozotocin-diabetic rats treated with insulin in vivo. Treatment was such that the contents of amylase and lipase, vastly altered in the untreated diabetic state, were normalized before the perfusion studies. In the second procedure, insulin reaching the exocrine pancreas was reduced by antiinsulin serum in the perfusate. In these procedures, the reduced insulin bioavailability was associated with a reduction in caerulein- and vasoactive intestinal polypeptide-stimulated enzyme release, which was shown as a reduction of maximum responsiveness to caerulein without alteration of sensitivity. By contrast, in dispersed pancreatic acini where the insulo-acinar axis was completely disrupted, amylase secretion from diabetic and nondiabetic tissue was identical over a wide range of caerulein concentrations, showing that the secretory defect seen in the perfusion studies was not inherent to the exocrine tissue. The results show that basal insulin secretion has a direct effect on pancreatic enzyme output and that the insulo-acinar axis may play an important role in the regulation of acinar cell function.

The involvement of the Ca2+ binding protein, calmodulin, in the regulation of insulin release was studied. Calmodulin was measured in isolated rat islets, rat insulinoma cells, the insulin secreting cell line (RINmSF) and in islets isolated from normal and diabetic Chinese hamsters. Total content of calmodulin was determined by a radioimmunoassay using a rabbit anti-calmodulin serum and was found to lie in the range of 4 to 7 μg/ml protein. When rat islets were maintained in tissue culture for 6 days at 2.8 or 8.3 mM glucose, the content of calmodulin of the two groups was similar. Likewise there was no difference in calmodulin content between islets from normal and diabetic hamsters. This study suggests that a variation of the total cellular calmodulin does not play a role in the process of insulin secretion.

The contents of three major digestive enzymes (amylase, lipase and chymotrypsinogen) were measured in the obese Zucker rat. Only minimal changes were found in 7-week-old rats, but in adult obese rats (14-16 weeks) the amylase content was decreased by 50%, whereas the lipase and chymotrypsinogen contents were increased by 45% and 20%, respectively, compared with lean controls. Abnormalities of enzyme secretion were also found. Since the changes observed in enzyme proportions in adult obese Zucker rats are qualitatively similar to those observed in insulinopenic diabetes and other states associated with decreased glucose metabolism, it is speculated that the abnormalities found in the obese Zucker rat may be due to decreased glucose metabolism in the exocrine tissue consequent to insulin resistance.

The effects of glucose and insulin on pancreatic enzyme release have been investigated using the isolated perfused rat pancreas. Basal and caerulein-stimulated secretion was significantly less in the presence of 15 mM glucose than with 5 mM glucose, except at a supramaximal concentration of caerulein (10(-9) M) where secretion was similar in both groups. Addition of exogenous insulin also caused a reduction in enzyme secretion, but the time of onset of the inhibitory action was delayed compared to that observed with glucose. Furthermore, it was found that the effects of 15 mM glucose and exogenous insulin were not additive at the concentration used in these experiments, and that the inhibitory action of insulin was glucose-dependent. Such glucose-insulin interactions must play an important role in the modulation of pancreatic enzyme secretion.

Modified cytosolic proteins are known to be degraded more rapidly than their native counterparts. In order to determine whether the same applies to a modified protein within the potentially protective environment of secretory granules, rat islets were labelled [(3H]leucine) in the presence or absence (controls) of 3 mM-canavanine and 3 mM-thialysine (analogues of arginine and lysine respectively), followed by a 24h ‘chase’ period without analogues. The results showed the following. (1) Incorporation of the analogues into newly synthesized labelled proinsulin inhibited its conversion into insulin during the chase period. (2) Despite this block in conversion, the modified proinsulin was released from islets at the same rate as native proinsulin and insulin from control islets. (3) Morphometric analysis of high-resolution autoradiographs showed that products labelled in the presence of analogues were sequestered into secretory granules at the same rate as native products in control B-cells. (4) Only 7% of prelabelled proinsulin had been degraded within islet cells during the chase period in control islets, compared with 36% for proinsulin prelabelled in the presence of analogues. (5) Control experiments showed that the analogues had no effect on the release or intracellular degradation of unmodified stored insulin (present in islets before exposure to the analogues). (6) Despite sequestration into secretory granules, modified proinsulin, if not released from B-cells, is thus degraded more rapidly than native products.

The effect of constant, controlled hyperinsulinemia on in vivo and in vitro insulin responsiveness has been investigated in rats that have received insulin infusions through chronically implanted jugular vein catheters. Constant rates of insulin infusion during days 1-4 resulted in stable plasma insulin concentrations. The plasma glucose initially fell from 122 +/- 3 to 53 +/- 4 mg/dl. While the infusion rate was maintained constant, the plasma glucose continued to fall over the subsequent days so that on day 4 the plasma glucose, 40 +/- 2 mg/dl, was significantly lower than that in the same animals on day 1 (P less than 0.02). Subsequently, the rate of insulin infusion was decreased to maintain the plasma glucose level in the 35-40 mg/dl range. Plasma catecholamine levels were high in insulin-infused rats. On the eighth day an in vivo insulin tolerance test (0.5 U/kg) was performed. Insulin-infused rats responded with a hypoglycemia that was both more pronounced and longer sustained than in saline-infused controls. Insulin responsiveness in vitro has been measured in isolated adipocytes. Adipocytes from epididymal fat pads were of similar size in the two groups of animals. Glucose uptake by adipocytes from insulin-infused rats was similar to that of controls under basal (zero insulin) conditions, but showed an increase in the maximum response to insulin. Glucose incorporation into total lipid and fatty acid was greater in adipocytes from insulin-infused rats than in controls under both basal (zero insulin) and insulin-stimulated conditions. Activities of the lipogenic enzymes acetyl CoA carboxylase and fatty acid synthetase were markedly increased in epididymal fat pads of insulin-infused rats.(ABSTRACT TRUNCATED AT 250 WORDS)

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C-peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.

Sensitivity to insulin in vivo was studied in six Type 1 diabetic patients without residual insulin secretion and without clinical insulin resistance, and in eight non-diabetic subjects, using the euglycaemic insulin clamp technique. Insulin was infused for four periods of 2 h sequentially at 0.5, 1.0, 2.0 and 5.0 mU · kg-1 · min-1; for each insulin infusion period the steady-state plasma free insulin levels were comparable in the diabetic and non-diabetic subjects. The mean ±SEM plasma glucose concentration was 4.9±0.03 mmol/l in the diabetic subjects (coefficient of variation of plasma glucose values: 5.7±0.7%) and 4.6±0.01 mmol/l in the control subjects (coefficient of variation: 5.1±0.6%). Insulin-mediated glucose disposal was lower in the diabetic than in the non-diabetic subjects at the two lower insulin infusion rates (mean±SEM = 2.03±0.27 versus 4.8±0.64 mg · kg-1 · min-1 at the first insulin infusion rate, p<0.01, and 5.59±0.59 versus 8.36±0.61 mg · kg-1 · min-1 at the second insulin infusion rate, p<0.01). However, insulin-induced glucose uptake did not differ significantly between the two groups at the third and fourth rates of insulin infusion. These results show that impaired insulin sensitivity in Type 1 diabetes is dependent on insulin concentration.

The effect of previous exposure to glucose on subsequent glucose-stimulated insulin and somatostatin secretion has been investigated using the isolated perfused rat pancreas. As expected, when the pancreases of non-diabetic rats were exposed to 16.7 mM glucose on two occasions, 20 min apart, insulin secretion during the second period of exposure to high glucose was greater than that during the first period. By contrast, there was no potentiation of somatostatin secretion during the second glucose stimulation with respect to that of the first. Indeed, when the basal glucose concentration was low (1.4 or 2.8 m m ) somatostatin secretion during the second glucose stimulation was lower than that during the first. Since exogenous insulin is known to inhibit glucoseinduced somatostatin secretion, it seemed possible that lack of visible potentiation of glucose-induced somatostatin secretion by glucose could have been due to partial D cell inhibition by simultaneously augmented insulin secretion during the second glucose stimulation. In an attempt to exclude such an interaction between B and D cells, somatostatin secretion was also studied in the pancreases of spontaneously diabetic, Wistar (BB) rats (these animals are insulin deficient and are maintained by daily injections of insulin). However, even though insulin secretion was not detectable from these pancreases, glucose potentiation of glucose-induced somatostatin secretion did not occur. Although the pancreatic B and D cells are known to respond in a similar manner to many secretagogues the present results show that glucose potentiation of glucosestimulated somatostatin secretion is not found under circumstances where potentiation of insulin secretion does occur. In addition, the absence of potentiated somatostatin secretion could not be attributed to partial inhibition of the D cell by insulin.

The availability of suitably characterized dispersed islet cell preparations may assist in studies of islet function. Since freshly dispersed adult rat islet cells failed to respond appropiately to secretagogues (no alteration in insulin, glucagon, or somatostatin release after glucose change; modest response to IBMX), these cells were established in primary monolayer culture. We then tested the hypothesis that islet function is at least partially determined by islet structure. B cells which had attached to Petri dishes during a culture period of four days were well preserved at the ultrastructural level, with mitochondria clustered at the cell face attached to the Petri dish and secretory granules concentrated towards the portion of the cell facing the medium. Since it was not possible to estimate cellular hormone content or hormone release as a function of the number of specific types of cells, fractional rates of release and hormone content ratios were compared with those for intact islets maintained in culture in parallel. Whereas the ratio of somatostatin:insulin content was similar for islets and cells (∼0.7:100), the dispersed cell population appeared depleted in glucagon (glucagon:insulin ratios being 17:100 for islets and 4:100 for cells) reflecting either degranulation or relative loss of A cells. In contrast to the lack of responsiveness seen with freshly dispersed islet cells, the cultured cells released insulin in response to glucose and glucose plus IBMX in a fashion comparable to that seen with cultured islets. Proinsulin biosynthesis (incorporation of [3H] leucine) was higher in cultured cells than islets. Somatostatin release was lower from dispersed cells than from islets while the opposite was true for glucagon. Dispersed islet cells in monolayer thus appear to be a valuable model system for the study of islet structure-function relationships, and these results reveal several ways in which the function of dispersed cells differs from that of intact islets.

Rat islets were maintained for 3 days in tissue culture at 2.8, 8.3, or 16.7 mM glucose, in the presence of [3H]leucine to label insulin stores. The immunoreactive insulin content of islets after 3 days at either 2.8 or 16.7 mM glucose was lower than that of islets exposed to 8.3 mM glucose. The specific radioactivity of stored labeled insulin, however, increased with increasing glucose. Islet insulin turnover was thus highest at 16.7 mM glucose. During a subsequent 1-day chase period at 2.8, 8.3, or 16.7 mM glucose for each of the three groups of islets prelabeled at these same three concentrations, all islets responded to glucose with increased insulin release. Release of immunoreactive insulin was highest at all three chase glucose concentrations for those islets preexposed for 3 days to 16.7 mM glucose. After 3-day labeling of islets at 8.3 mM glucose, there was an inverse relationship between the rate of release of labeled insulin and its intracellular degradation in response to the three glucose concentrations during the chase period. Those islets prelabeled at either 2.8 or 16.7 mM glucose showed much lower rates of intracellular degradation of labeled insulin stores during the chase period regardless of the ambient glucose or of the rate of release of the labeled hormone. Adaptive changes in islet insulin handling thus occurred during the 3-day maintenance period at different glucose concentrations. Whereas glucose concentration is, of course, thought to regulate insulin biosynthesis and release directly, regulation of degradation of stored insulin within B-cells appears to reflect both the relative activity of biosynthesis and release of the hormone and the insulin content of the islets. This last and striking relation is best interpreted as reflecting two labile, exchangeable, granule pools, one preferentially available for release and the other for intracellular degradation.

Rat islet cell tumours induced by injection of streptozotocin and nicotinamide have been studied in vivo and after the establishment of monolayer cultures of tumour cells. During an intravenous glucose tolerance test, tumour-bearing rats had increased release of immunoreactive insulin, with a high proportion of proinsulin, as well as accelerated glucose disposal relative to control rats. The tumours were rich in immunoreactive insulin and somatostatin, poor in glucagon. Non-tumour pancreatic tissue or isolated islets contained 10% or less of the corresponding normal amounts of insulin whereas the islet content of somatostatin was unchanged and that of glucagon increased. This is best interpreted as a selective suppression of non-tumour B cells, further supported by the observation that the initially reduced insulin release and content of non-tumour islets were partially restored after 2 days in tissue culture. In monolayer culture, tumour cells maintained insulin production and acute responsiveness to glucose for prolonged periods. There was no sign of cell proliferation. It is concluded that primary, chemically-induced insulin-producing pancreatic islet cell tumours retain several features characteristic to normal B cells and continue to influence glucose homeostasis in vivo.