To examine the effect of increased gluconeogenesis [by increasing free fatty acids (FFA)] on hepatic glucose output (HGO) and on the first substrate (glucose) cycle, a primed continuous infusion of [2-3H]- and [6-14C]glucose was infused to isotopic steady state in 12 normal male volunteers after an overnight fast. Blood samples for the determination of glucose specific activity were obtained before and after an infusion of saline (n = 6) or 10% Intralipid and heparin (90 mU.kg-1.min-1, n = 6). Plasma FFA (593.3 +/- 74.5 to 971.1 +/- 127.1 mumol/l, P = 0.007) and glycerol (68.0 +/- 5.9 vs. 222.4 +/- 32.0 mumol/l, P = 0.002) increased during the lipid infusion, and beta-hydroxybutyrate levels rose from 0.24 +/- 0.12 to 0.50 +/- 0.17 mmol/l (P = 0.01). No change in plasma glucose, insulin, or glucagon levels was observed during the study, and levels of the gluconeogenic substrates alanine and lactate were also unchanged. Baseline rates of glucose cycling (rate of appearance of [2-3H]glucose minus rate of appearance of [6-14C]glucose) were similar in the two groups [1.44 +/- 0.33 vs. 1.33 +/- 0.44 mumol.kg-1.min-1, not significant (NS)] and did not change during either saline or lipid infusion, respectively. However, Cori cycle activity (the conversion of [6-14C]- to [1-14C]glucose) increased significantly from 0.59 +/- 0.19 to 1.28 +/- 0.19 mumol.kg-1.min-1 (P = 0.002) after FFA and glycerol levels had been increased, in marked contrast to the saline control (0.51 +/- 0.18 to 0.39 +/- 0.18 mumol.kg-1.min-1, NS).(ABSTRACT TRUNCATED AT 250 WORDS)
We previously reported a fall in hepatic glucose output (HGO) during sleep accompanied by reductions in glucose utilization (Rd) and free fatty acids (FFAs). This study was undertaken to determine the potential role of changes in Rd and FFA on HGO in nondiabetic men. To determine if the fall in HGO during sleep could be reversed by FFA elevation, seven nondiabetic men underwent [3-3H]glucose infusions from 2200 to 0800, with heparin (90 mU.kg-1.min-1) added at 0200. Glucose appearance (Ra) fell from 11.7 +/- 1.1 at 2430 to 8.9 +/- 0.8 mumol.kg-1.min-1 (P less than 0.05) at 0200. The fall in Ra was associated with decreases in FFA (0.57 +/- 0.10 to 0.48 +/- 0.07 mM) and glycerol (0.08 +/- 0.01 to 0.06 +/- 0.01 mM). Infusion of heparin significantly increased FFA and glycerol (1.09 +/- 0.21 and 0.11 +/- 0.01 mM, respectively, P less than 0.01) and resulted in a significant fall in plasma alanine, suggesting that gluconeogenesis had been increased. However, rates of glucose turnover were indistinguishable from overnight studies without heparin. In additional studies (n = 6), intralipid and heparin-induced FFA elevation (from 0.61 +/- 0.07 to 0.95 +/- 0.05 mM, P less than 0.01) stimulated gluconeogenesis ([U-14C]alanine to glucose) twofold (188 +/- 22% increase compared to 114 +/- 6% in saline control studies, P less than 0.01). However, despite increasing gluconeogenesis, overall HGO did not change (10.6 +/- 0.5 vs. 10.7 +/- 0.6 mumol.kg-1.min-1) during lipid infusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Numerous physiological agents and conditions modulate cellular insulin sensitivity by downregulating or upregulating total cellular insulin receptors. In this study, we examined the effects of replacing complete medium in the absence or presence of insulin on the regulation of insulin-receptor gene expression in cultured human hepatoma cells (HepG2). Failure to replace complete medium resulted in growth arrest of HepG2 cells and a six- to sevenfold increase in insulin-receptor mRNA due to the prolongation of insulin-receptor mRNA half-life. Northern analysis revealed multiple insulin-receptor mRNA species; the largest species (11 kilobases) was disproportionately increased in growth-arrested cells. High concentrations of insulin (500 ng/ml) induced a 33.8% decrease in the abundance of insulin-receptor mRNA (n = 14). At lower concentrations, a trend of inhibition was observed but was not statistically significant. Insulin (500 ng/ml) did not affect insulin-receptor mRNA stability. The effect of conditioned media, insulin, and dexamethasone on insulin-receptor promoter activity was also examined. Various constructs of the 5'-flanking region of the insulin-receptor gene were attached immediately upstream to a chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into HepG2 cells via a pBR322-derived plasmid (pCAT). In cells replaced with complete medium, 12 and 118% of the promoter activity was contained within 578 and 877 base pairs, respectively, from the major translational initiation site. Conditioned media from growth-arrested cells in culture for 7 days increased promoter activity approximately twofold in 48 h. However, this increase failed to localize to any specific region on the insulin-receptor promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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