Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor.

Two highly sensitive, nonradiolabeled assays for protein phosphotyrosine phosphatase (PTPase) have been developed. The first assay is based on the use of chemically synthesised phosphotyrosine-containing peptides that can be separated from the dephosphorylated peptide products by HPLC. In this assay, partially purified placental PTPase 1B dephosphorylated three dodecaphosphopeptides (corresponding to insulin receptor autophosphorylation sites at positions PY1146, PY1150, and PY1151) with approximately equal affinity (Km 1.3-2.5 microM), indicating that PTPase 1B shows no distinct preference for the site of dephosphorylation in these peptides. The second assay employs either a phosphopeptide or an autophosphorylated tyrosine kinase domain immobolized on microtiter plate wells. After reaction with PTPase, the remaining unconverted phosphosubstrate is detected in an ELISA using anti-phosphotyrosine antibodies. The latter assay was used to monitor PTPase activity during purification procedures and for characterizing PTPases. Modulation of PTPase activity by orthovanadate, heparin, Zn2+, and EDTA gave similar results in both assays. The immobilized autophosphorylated IR tyrosine kinase domain was a poor substrate for bovine liver alkaline phosphatase and seminal fluid acid phosphatase. The second assay also offers the potential for comparing PTPase activity toward several autophosphorylated tyrosine kinase domains, including those of the insulin, epidermal growth factor, and platelet-derived growth factor receptors.

Ketone-body metabolism after partial hepatectomy in the rat.

Fed or 24 h-starved rats were subjected to two-thirds partial hepatectomy or sham-operation and subsequently starved for 4, 14 or 24 h. Despite high plasma fatty acid concentrations, the partially hepatectomized rats failed to respond to post-operative starvation with increased blood and liver ketone-body concentrations or to maintain the high ketone-body concentrations associated with pre-operative starvation. Hypoglycaemia and hyperlactaemia were observed within 30 min of functional hepatectomy, but not partial hepatectomy, of 24 h-starved rats, and, even after a further 24 h starvation of partially hepatectomized rats, blood glucose concentrations were only slightly decreased. The results are discussed with reference to fat oxidation and gluconeogenesis in the liver remaining after partial hepatectomy.

The development of tolerance to antilipolytic agents in rats.

The development of tolerance to the action of certain antilipolytic agents has been investigated in vivo in rats. Tolerance to oral nicotinic acid developed during twice daily dosing for 4 days at 100 and 250 mg/kg but not at 10, 25 or 50 mg/kg. Tolerance induced by high doses of nicotinic acid was no longer detectable after a further week without treatment. Tolerance developed to a dose of 10 mg/kg nicotinic acid when dosing was repeated at hourly intervals for up to 6 hr. Rats made tolerant to nicotinic acid also became tolerant to both 5-methylpyrazole-3-carboxylic acid and to pyridyl-3-tetrazole and rats made tolerant to these antilipolytic agents were also tolerant to nicotinic acid. Rats made tolerant to nicotinic acid still responded to the antilipolytic activity of the prostaglandin analogue, sulprostone. These results suggest that nicotinic acid, pyridyl-3-tetrazole and 5-methylpyrazole-3-carboxylic acid act through a common mechanism or receptor and that the development of tolerance is associated with this receptor or the mechanism by which it is linked to adenylate cyclase.

The development of tolerance to antilipolytic agents by isolated rat adipocytes.

Using an isolated rat epididymal adipocyte system we have studied the development of tolerance to and cross-tolerance between nicotinic acid, 5-methylpyrazole-3-carboxylic acid and pyridyl-3-tetrazole. Preincubating isolated adipocytes with any one of these compounds results in a reduction of the antilipolytic activity of that compound when the cells are exposed to a subsequent challenge dose. Furthermore, preincubation with nicotinic acid, 5-methylpyrazole-3-carboxylic acid or pyridyl-3-tetrazole results in a reduction of the antilipolytic response to challenge with either of the other two compounds. Preincubation of isolated adipocytes with nicotinic acid does not affect the subsequent antilipolytic activity of the PGE2 analogue, sulprostone. Preincubation with sulprostone does not lead to the development of tolerance to its own antilipolytic actions. The results obtained from these studies suggest that nicotinic acid, 5-methylpyrazole-3-carboxylic acid and pyridyl-3-tetrazole exert their antilipolytic activity via a common biochemical pathway which is distinct from that mediating the antilipolytic activity of prostaglandins. These findings also indicate that the development of tolerance occurs prior to the involvement of adenylate cyclase in lipolysis.

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate.

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of the hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extracellular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by 14CO2 evolution from [1-14C]pyruvate and [3-14C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of 1 mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate, and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.

Direction of carbon flux in starvation and after refeeding: in vitro and in vivo effects of 3-mercaptopicolinate.

3-Mercaptopicolinate (3-MPA) is a specific inhibitor of phosphoenolpyruvate carboxykinase (PEP CK). In vivo the hypoglycaemic action of 3-MPA in 24 h-starved rats was abolished on intragastric glucose refeeding. Nonetheless, 3-MPA decreased hepatic glycogen content and rate of synthesis in starved animals re-fed glucose. The inference is that on re-feeding after starvation hepatic glycogen is synthesised mainly de novo via glyconeogenesis involving PEP CK. 3-MPA increased hepatic lipogenesis in water- and glucose-fed normal and diabetic rats. This increase is presumed to result from inhibition of PEP CK and consequent diversion of pyruvate from gluconeogenesis to lipogenesis. In contrast, 3-MPA inhibited brown-fat lipogenesis in water- and glucose-fed rats.