Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

VIP (Vasoactive Intestinal Polypeptide)-immunoreactive neurons in the retina of the rat

Summary Immunoreactive vasoactive intestinal polypeptide (VIP) was detected in a population of amacrine cells in the retina of the rat. Processes of these cells reach both the inner and outer half of the inner plexiform layer where they form sublayers. The VIP neurons are different from previously known amacrine cell types.     

Somatostatin and VIP neurons in the retina of different species

Summary Neurons displaying somatostatin or vasoactive intestinal polypeptide (VIP) immunoreactivity were detected among the amacrine cells in the retina of baboon, cynomolgus monkey, squirrel monkey, cow, pig, cat, rabbit, guinea-pig, rat, mouse, frog and goldfish. Generally, immunoreactive cell bodies were located in the inner nuclear layer with processes ramifying in three more or less well-defined sublayers in the inner plexiform layer. The density of the sublayers and their location varied with the peptide and species investigated. In most cases there was a sublayer in the outermost part (Ramon y Cajal's sublamina 1) of the inner plexiform layer and this sublayer was usually the best developed. In some species a few somatostatin fibres were also detected in the outer plexiform layer, suggesting that some interplexiform cells contain somatostatin. In the baboon VIP was found exclusively in interstitial amacrine cells which have their cell bodies and processes entirely within the inner plexiform layer.     

Removal of nonionic detergent from proteins fractionated by electrofocusing.

The nonionic detergent Nonipol TD 12 (an alkyl polyoxyethylene alcohol) has been removed from solubilised proteins after their fractionation by electrofocusing. Following electrofocusing in nonionic detergent an anionic or cationic detergent was added to the focussing medium and the focusing was allowed to continue. The ionic detergent formed mixed micelles with the nonionic detergent. Thus charged, the mixed micelles migrated to either electrode, removing nonionic detergent from the fractionated proteins. Applying this technique to an adipose tissue preparation, detergent-inhibited activity of a lipolytic enzyme was restored and the binding of adenosine 3':5'-cyclic monophosphate (cyclic AMP) to a protein kinase was increased.     

Praealbonemertes whangateaunienses n. gen. and n. sp., a new heteronemertean from New Zealand

Abstract

A new genus and species of heteronemertean, Praealbonemertes whangateaunienses n. gen. and n. sp., is described and illustrated. The species is characterised by inter alia a cephalic lacuna with strands of longitudinal muscle fibres, a proboscis with three muscle layers, and a well-developed muscle plate dorsal to the foregut and anterior intestine. The material was collected in New Zealand.     

5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside

Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5'-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling.     

Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol

The respective roles of monoacylglycerol lipase and hormone-sensitive lipase in the sequential hydrolysis of adipose tissue triacylglycerols have been examined. An adipose tissue preparation, containing both lipases in approximately the same proportion as in the intact tissue, hydrolyzed emulsified tri- or dioleoylglycerol to fatty acids and glycerol, with little accumulation of di- or monooleoylglycerol. Selective removal of the monoacylglycerol lipase by immunoprecipitation markedly reduced the glycerol release. Isolated hormone-sensitive lipase hydrolyzed acylglycerols with a marked accumulation of monoacylglycerol in accordance with the positional specificity of this enzyme (Fredrikson, G. and Belfrage, P. (1983) J. Biol. Chem. 258, 14253-14256). Addition of increasing amounts of isolated monoacylglycerol lipase led to a corresponding increase in glycerol release, due to hydrolysis of the monoacylglycerols formed. The reaction proceeded to completion when the relative proportion of the two lipases was similar to that in the intact tissue. These findings indicate that hormone-sensitive lipase catalyzes the hydrolysis of triacylglycerol in the rate-limiting step of adipose tissues lipolysis, and of the resulting diacylglycerol, whereas the action of monoacylglycerol lipase is required in the final hydrolysis of the 2-monoacylglycerols produced.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

IRS-4 mediated mitogenic signalling by insulin and growth hormone in LB cells,a murine T-cell lymphoma devoid of IGF-I receptors

Insulin and growth hormone (GH) induce mitogenic and metabolic signals in cells, GH either directly or indirectly via IGF-I production. We have studied a spontaneous murine T-cell lymphoma (LB cells) devoid of IGF-1 receptors in which proliferation is maintained by insulin [Int. J. Cancer 50 (1992) 80], and show that GH is more potent than insulin, with both GH and insulin dose-response curves for thymidine incorporation being bell-shaped. Binding showed somatogenic rather than lactogenic GH receptors. Insulin stimulated phosphorylation of the insulin receptor and of a 160-kDa protein, identified as the IRS-4 protein. This phosphorylated IRS-4 associated with PI3-kinase, which was activated along with the downstream p70(S6) kinase, whereas the Ras-MAPK pathway was not. Using selective inhibitors, the PI3-kinase, but not p70(S6) kinase or MEK, was found to be involved in insulin-stimulated DNA synthesis. GH induced tyrosine phosphorylation of IRS-4 and nuclear translocation of STAT5. The LB cells constitute a new model for studying GH and insulin signalling without interference of IGF-1 receptors.