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Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the I[C.sub.50] for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecularweight effectors of phosphorylase a activity. However, at higher concentrations (10-30 [micro]M), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme. glucose

Facilitative glucose transporters exhibit variable hexose affinity and tissue-specific expression. These characteristics contribute to specialized metabolic properties of cells. Here we describe the characterization of a novel glucose transporter-like molecule, GLUT-12. GLUT-12 was identified in MCF-7 breast cancer cells by homology to the insulin-regulatable glucose transporter GLUT-4. The GLUT-12 cDNA encodes 617 amino acids, which possess features essential for sugar transport. Di-leucine motifs are present in [NH.sup.2] and COOH termini at positions similar to the GLUT-4 FQQI and LL targeting motifs. GLUT-12 exhibits 29% amino acid identity with GLUT-4 and 40% to the recently described GLUT-10. Like GLUT-10, a large extracellular domain is predicted between transmembrane domains 9 and 10. Genomic organization of GLUT-12 is highly conserved with GLUT-10 but distinct from GLUTs 1-5. Immunofluorescence showed that, in the absence of insulin, GLUT-12 is localized to the perinuclear region in MCF-7 cells. Immunoblotting demonstrated GLUT-12 expression in skeletal muscle, adipose tissue, and small intestine. Thus GLUT-12 is potentially part of a second insulin-responsive glucose transport system. facilitative glucose transporter; insulin-responsive tissues; breast cancer

Genetically diabetic (db/db) mice were analyzed to determine the effects of diabetes on cerebral glucose utilization (CGU), cerebral glucose homeostasis and GLUT-1 or GLUT-3 expression. The genetically diabetic db/db mice exhibited reduced cerebral metabolism, CGU and altered brain growth. Diabetes also increased the expression of GLUT-1 and reduced GLUT-3 expression. Furthermore, altered brain metabolism in diabetic mice indicated the insulin sensitivity of normal brain growth.

The intense activity of phosphatidylinositol 3'-kinase (PtdIns 3'-kinase) results from the association of two phosphorylated tyrosines carrying YMXM sequences on insulin receptor substrate 1 with two carboxy SH2 domains of PtdIns 3'-kinase. Phosphotyrosine peptides carrying phosphorylated tyrosine YZPZ motifs in 3T3-L1 adipocytes exhibit enormous affinity for binding SH2 domains of PtdIns-3'kinase compared to those containing singular YZPZ sequence.

Transgenic mice expressing high levels of GLUT4/muscle-fat specific facilitative glucose transporter were produced in a suitable tissue-specific fashion to study their physiological function in mediating glucose homeostrains. A drastic fall in fasting glucose levels resulted from high-level expression of GLUT4 protein. A constitutively high level of cell surface GLUT4 protein expression and better metabolic regulation of fluctuations in plasma glucose concentrations results from enhanced expression of human GLUT4 gene in vivo.

Belke, Darrell D., Terje S. Larsen, E. Michael Gibbs, and David L. Severson. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab 279: E1104-E1113, 2000.--Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-[lepr.sup.db]/[lepr.sup.db] (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-sub.3]H]glucose, [U-sup.14]C]glucose, and [9,[10-sup.3]H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts. diabetic cardiomyopathy