Effect of physical training on glucose transporters in fat cell fractions

Physical training increases maximally insulin-stimulated glucose assimilation and 3-O-methylglucose transport in epididymal fat cells. In the present report, glucose-inhibitable cytochalasin B binding in subcellular fractions of epididymal adipocytes was measured to assess changes in number of glucose transporters induced by training. Groups of rats trained by swimming were compared to control groups of the same age, matched with respect to body weight by restricted feeding. It was found that in trained rats the number of glucose transporters in the low density microsome fractions from non-insulin-stimulated fat cells was larger than in untrained rats. In both groups of rats, insulin stimulation of adipocytes decreased the number of glucose transporters in low-density microsomes by about 60% and increased the number of glucose transporters in the plasma membrane fractions. The number of glucose transporters in the plasma membrane fractions from maximally insulin-stimulated fat cells was larger in trained rats than in control rats. [U-¹⁴C]Glucose incorporation into lipids varied in proportion to plasma membrane cytochalasin B binding per cell under all conditions tested. The results explain the enhancing effect of training on insulin responsiveness transport of hexose in fat cells.     

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver

The cleavage of insulin by the partially purified insulin protease was studied using the four [¹²⁵I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 > A-14 > A-19 > B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the ‘insulin’ peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the ‘insulin’ peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g. containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).     

Diversity of prolactin systems in the insect Leucophaea maderae: Use of antiserum polyclonality for immunocytochemical detection of neuropeptide heterogeneity

Summary The presence of prolactin-like neuropeptides was demonstrated immunocytochemically in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Use of the unlabelled peroxidase-antiperoxidase method of Sternberger revealed a rather widespread and differential distribution of reaction products resembling human (hPRL) and ovine (oPRL) prolactin. Tests with antirat PRL antibody were negative. The specificity of the antibodies used was established by liquid-phase absorptions and confirmed in tissue control systems. In L. maderae, anti-oPRL identifies part of an oPRL-like molecule different from human and rat PRL. Anti-hPRL reveals part of a human and ovine PRL-like molecule different from rat prolactin. These results indicate the occurrence, in the nervous tissue of one insect species, of at least two types of prolactin-like molecules.Supported in part by SNF grants 11-5082 and 11-6652 (G.N.H.) and NIH Grant NS 22344-02 (B.S.). The authors are indebted to Mrs. Bente Hershøj for skillful technical assistance     

SUMOylation of Pancreatic Glucokinase Regulates Its Cellular Stability and Activity

Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 β-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic β-cells.