Interaction of the tyrosine phosphatase inhibitor ortho-vanadate on stimulus--secretion coupling in pancreatic acinar cells.

BACKGROUND: The effect of the tyrosine phosphatase inhibitor ortho-vanadate on stimulus-secretion coupling was investigated in isolated rat pancreatic acini. METHODS AND RESULTS: Ortho-vanadate (10(3)M) reduced cholecystokinin (CCK)-8 (10(10) M)-stimulated amylase release by 40% (IC50 = 5 x 10(4) M). In contrast, preincubation with 10(3) M ortho-vanadate increased secretin (5 x 10(9) M) and vasoactive intestinal peptide (VIP) (10(7) M)-induced amylase release by 65% and 80% (IC50= 3 x 10(-4) M), respectively. 8-Bromo-cyclic adenosine-5-monophosphate (cAMP) (10(-4) M) and phorbol ester (10(-5) M)-induced secretion was increased by 60% and 50%, respectively, whereas thapsigargin-induced amylase release was not affected. Ortho-vanadate did not affect CCK-8 binding or VIP-induced cAMP synthesis in isolated acini. In contrast, preincubation with 10(-4) M ortho-vanadate resulted in a significant reduction of CCK-8-induced intracellular calcium release. In streptolysin-O-permeabilized acini, ortho-vanadate reduced calcium-induced amlyase secretion by 50%. CONCLUSIONS: The present data provide indirect evidence of a differential involvement of protein tyrosine dephosphorylation in both cAMP- and IP3/Ca(2+)-mediated pancreatic secretion. The differential effects of ortho-vanadate on cAMP- versus calcium-mediated secretion correspond to the results obtained with receptor-independent intracellularly acting secretagogues. Further experiments must define the tyrosine phosphatases involved in both signal-transduction pathways.

Differential inhibitory effects of serine/threonine phosphatase inhibitors and a calmodulin antagonist on phosphoinositol/calcium- and cyclic adenosine monophosphate-mediated pancreatic amylase secretion.

BACKGROUND: Protein phosphorylation and dephosphorylation events are considered to be key steps in the control of agonist-induced pancreatic enzyme release. This study was designed to characterize the role of serine/threonine phosphatases in phosphoinositol/calcium- and cyclic adenosine monophosphate (cAMP)-mediated stimulus-secretion coupling in rat pancreatic acini. METHODS: Isolated rat pancreatic acini were incubated with either the serine/threonine phosphatase inhibitors okadaic acid, calyculin A, and cyclosporin A or the calmodulin antagonist W-7. Amylase secretion was stimulated with cholecystokinin (CCK)-8, secretin, vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP), and the intracellular second messengers calcium and cAMP were determined. RESULTS: Okadaic acid or calyculin A reduced secretagogue-stimulated amylase release to near-basal levels. Inhibition of cAMP-mediated secretion (by VIP, secretin, or PACAP) occurred at lower concentrations than with inositol triphosphate (IP3)/Ca(2+)-dependent enzyme release (via CCK). Cyclosporin A diminished CCK-8-stimulated secretion by 35%, whereas secretion in response to cAMP-mediated secretagogues was not affected. W-7 completely inhibited acinar secretion in response to cAMP-or IP3/Ca(2+)-mediated secretagogues. Binding of 125I-CCK-8- or 125I-PACAP-(1-27) to acini was not influenced by the phosphatase inhibitors or W-7. Okadaic acid and calyculin A affected neither CCK-8-stimulated intracellular Ca2+ release nor PACAP-(1-27)-stimulated cAMP synthesis, whereas W-7 inhibited by 50% and 40%, respectively. CONCLUSIONS: The inhibitory profiles of okadaic acid, calyculin A, cyclosporin A, and W-7 indicate that phosphatases 1 and 2A play a relevant role in cAMP-mediated enzyme release, whereas phosphatases 1 and 2B are predominantly involved in IP3/Ca(2+)-dependent stimulus-secretion coupling. The calmodulin antagonist W-7 interferes at multiple steps of intracellular signal-transduction pathways.

Calyculin A, okadaic acid and W-7 interfere with a distal step in pancreatic acinar signal transduction.

The effects of the serine/threonine phosphatase inhibitors calyculin A, okadaic acid and the calmodulin antagonist W-7 on amylase secretion were studied in pancreatic acini. Calyculin A and okadaic acid dose-dependently inhibited amylase secretion to basal levels when stimulated with the intracellularly acting secretagogues thapsigargin, 8-br-cAMP or PMA. W-7 dose-dependently inhibited thapsigargin- or 8-br-cAMP-induced amylase secretion. In combination, thapsigargin, 8-br-cAMP and PMA induced amylase secretion comparable to the stimulation by cholecystokinin. Their effect was significantly inhibited by calyculin A, okadaic acid or W-7. These data imply that type 1- and 2b-phosphatases and calmodulin play a key role in the stimulation of exocrine pancreatic secretion at a distal step of both the Ca2+/IP3- and cAMP-mediated signal-transduction pathways.

Serine/threonine phosphatases play a role in stimulus-secretion coupling in pancreatic acinar cells.

The role of serine/threonine phosphatases in Ca2+/IP3- and cAMP- mediated stimulus-secretion coupling was investigated in isolated pancreatic acinar cells. Cyclosporine A, an inhibitor of type 2b serine/threonine phosphatases, maximally reduced CCK-8-stimulated amylase secretion by 33%. In contrast, the secretory response to secretin or PACAP-(1-27) was not significantly altered by cyclosporine A independent of the secretagogue-concentration okadaic acid significantly reduced amylase release, induced by Ca2+/IP3-mediated- (CCK-8) or cAMP-mediated agonists (secretin, PACAP-(1-27), VIP) at concentrations that primarily inactivate type 1 and 2b phosphatases. Calyculin A, another type 1 and 2a phosphatase inhibitor, had a similar inhibitory effect on CCK-8-, secretin- or PACAP-(1-27)-induced secretion. In permeabilized acini, cyclosporine A reduced calcium-induced amylase release by 20%, whereas okadaic acid and calyculin A had an inhibitory effect by 55% and 52%, respectively. The ultrastructure of CsA-incubated acinar cells was not different from vehicle-incubated control lobules. In contrast, incubation with okadaic acid for 60 min resulted in morphological alterations of the Golgi apparatus, leading to a fragmentation of Golgi cisternae into small vesicles. Our data suggest a role of type 1 and 2b phospatases in stimulus-secretion coupling of both signal-transduction pathways in pancreatic acinar cells. These phosphatases might also be important for the maintenance of pancreatic cellular ultrastructure.

Energy expenditure and substrate metabolism after oral fructose in patients with cirrhosis.

There is little information on the metabolic response to ingested fructose in patients with cirrhosis. Glucose kinetics, plasma lipid and blood lactate levels, whole body substrate oxidation rates and energy expenditure were measured following ingestion of 75 g fructose, in 8 cirrhotic patients and 6 controls. Fasting plasma glucose levels and rates of glucose appearance (Ra) and disappearance (Rd) were similar. The basal rate of lipolysis was higher in cirrhotic patients (P < 0.05), but whole body lipid and carbohydrate oxidation rates and energy expenditure were similar. After fructose ingestion, plasma fructose levels were much higher in cirrhotic patients (P < 0.001) and the incremental area under the plasma glucose curve was twice that of controls (P < 0.05). The increase in glucose in patients with cirrhosis was due to an increase in glucose Ra and an initial reduction in glucose Rd. Plasma non-esterified fatty acid levels fell to similar low levels in both groups. Glycerol levels fell in controls (P < 0.05) but not in cirrhotic patients. Blood lactate levels, fasting and after oral fructose, were similar in cirrhotics and controls. The time course of suppression of lipid oxidation and stimulation of carbohydrate oxidation was more closely related to fructose levels than to serum fatty acid levels in both groups. The percent suppression and total quantity of lipid oxidized in 4 h after fructose were not significantly different, but the suppressed lipid oxidation rates and elevated carbohydrate oxidation rates were sustained for longer in the cirrhotics. The data suggest that fructose uptake and metabolism inhibits oxidation of intracellular lipid. There was a smaller increase in energy expenditure after fructose in cirrhotics (P < 0.001), but normal overall storage of fructose; the likely explanation is reduced first pass hepatic fructose uptake in cirrhotics making more fructose available to the periphery for incorporation into muscle glycogen. The energy cost of storing fructose as muscle glycogen is less than that of storing it as liver glycogen. Preferential incorporation of fructose carbon into muscle glycogen, with lower rates of hepatic glycogen and triglyceride synthesis, would therefore result in less energy expenditure after a fructose load in cirrhotics.

Metabolic handling of orally administered glucose in cirrhosis.

We used a dual-isotope method (oral [1-14C]glucose and intravenous [6-3H]glucose) to examine whether the oral glucose intolerance of cirrhosis is due to (a) a greater input of glucose into the systemic circulation (owing to a lower first-pass hepatic uptake of ingested glucose, or to impaired inhibition of hepatic glucose output), (b) a lower rate of glucose removal, or (c) a combination of these mechanisms. Indirect calorimetry was used to measure oxidative and nonoxidative metabolism. Basal plasma glucose levels (cirrhotics, 5.6 +/- 0.4[SE], controls, 5.1 +/- 0.2 mmol/liter), and rates of glucose appearance (Ra) and disappearance (Rd) were similar in the two groups. After 75 g of oral glucose, plasma glucose levels were higher in cirrhotics than controls, the curves diverging for 80 min despite markedly higher insulin levels in cirrhotics. During the first 20 min, there was very little change in glucose Rd and the greater initial increase in plasma glucose in cirrhotics resulted from a higher Ra of ingested [1-14C]glucose into the systemic circulation, suggesting a reduced first-pass hepatic uptake of portal venous glucose. The continuing divergence of the plasma glucose curves was due to a lower glucose Rd between 30 and 80 min (cirrhotics 236 +/- 17 mg/kg in 50 min, controls 280 +/- 17 mg/kg in 50 min, P < 0.05, one-tailed test). Glucose metabolic clearance rate rose more slowly in cirrhotics and was significantly lower than in controls during the first 2 h after glucose ingestion (2.24 +/- 0.17 vs 3.30 +/- 0.23 ml/kg per min, P < 0.005), in keeping with their known insulin insensitivity. Despite the higher initial glucose Ra in cirrhotics, during the entire 4-h period the quantity of total glucose and of ingested glucose (cirrhotics 54 +/- 2 g [72% of oral load], controls 54 +/- 3 g) appearing in the systemic circulation were similar. Overall glucose Rd (cirrhotics 72.5 +/- 3.8 g/4 h, controls 77.2 +/- 2.2 g/4h) and percent suppression of hepatic glucose output over 4 h (cirrhotics, 53 +/- 10%, controls 49 +/- 8%) were also similar. After glucose ingestion much of the extra glucose utilized was oxidized to provide energy that in the basal state was derived from lipid fuels. Glucose oxidation after glucose ingestion was similar in both groups and accounted for approximately two-thirds of glucose Rd. The reduction in overall nonoxidative glucose disposal did not reach significance (21 +/- 5 vs. 29 +/- 3 g/4 h, 0.05 < P < 0.1). Although our data would be compatible with an impairment of tissue glycogen deposition after oral glucose, glucose storage as glycogen probably plays a small part part in overall glucose disposal. Our results suggest that the higher glucose levels seen in cirrhotics after oral glucose are due initially to an increase in the amount of ingested glucose appearing in the systemic circulation, and subsequently to an impairment in glucose uptake by tissues due to insulin insensitivity. Impaired suppression of hepatic glucose output does not contribute to oral glucose intolerance.

Metabolic actions of insulin-like growth factor II in cultured adult rat hepatocytes are not mediated through the insulin-like growth factor II receptor.

Short- and long-term regulation of hepatic carbohydrate metabolism by insulin-like growth factor II was studied in primary cultures of adult rat hepatocytes and compared to the metabolic potency of insulin. Insulin-like growth factor II stimulated glycogen synthesis from [14C]glucose, uptake of [3H]aminoisobutyric acid and [14C]lactate formation from [14C]glucose up to three-fold. Basal glycogenolysis was inhibited to about 10%, and glucagon-activated glycogenolysis was blocked completely. The enzymatic activity of glucokinase and pyruvate kinase was induced two-fold, the glucagon-dependent induction of phosphoenolpyruvate carboxykinase was antagonized. Compared to insulin, half-maximal responses required up to 50 times higher insulin-like growth factor II concentrations ranging from 10-20 nmol/l. A similar difference was observed for binding affinity of insulin-like growth factor II to the insulin receptor. The interaction with the insulin-like growth factor II/mannose 6-phosphate (IGF-II/Man-6-P) receptor was examined by studying 125I-insulin-like growth factor II binding and uptake of lysosomal enzymes. The affinity of insulin-like growth factor II to the IGF-II/Man-6-P receptor was considerably higher than for the insulin receptor. Antibodies against the IGF-II/Man-6-P receptor did not affect metabolic responses to insulin-like growth factor II, while binding to its receptor and the receptor-mediated endocytosis of arylsulphatase A were strongly inhibited. Thus, in adult rat liver insulin-like growth factor II appeared to exert metabolic actions not via interaction with its own receptor but through low affinity binding to hepatic insulin receptors.

Mechanism of insulin resistance in CCl4-induced cirrhosis of rats.

Insulin action was studied in rats with CCl4/phenobarbital-induced cirrhosis of the liver using the euglycemic hyperinsulinemic clamp technique coupled with isotopic measurement of individual tissue glucose uptake, glycogen formation, and lipogenesis. In cirrhotic rats, dose response curves showed a reduction of insulin-stimulated total body glucose disposal of about 30%. Insulin action on tissue glucose uptake and initial phosphorylation (assessed with [3H]2-deoxyglucose) were unchanged; however, incorporation of [14C]glucose into lipids and particularly into glycogen was reduced substantially (being most pronounced in skeletal muscle and diaphragm) at maximally as well as half-maximally effective serum insulin concentrations during euglycemic clamping. At identical IV insulin infusion rates, steady-state serum insulin concentrations were elevated up to fourfold in cirrhotic animals. Antilipolytic action of insulin was unaltered. These data suggest that the principal metabolic pathway affected in insulin resistance of rats with experimental cirrhosis appeared to be insulin-stimulated glycogen formation in muscle tissues.