A. L. Holleran

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of14C-glutamine to14CO2 and of14C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle α-ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label α-ketoglutarate carbon 5, [2-14C]glucose, [1-14C]acetate and [5-14C]glutamine. The analysis indicated that the probability of flux of TCA cycle α-ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of α-ketoglutarate through α-ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [14C]glutamine to14CO2, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through α-ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of α-ketoglutarate was demonstrated by measuring incorporation of [5-14C]glutamine into14C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.

Progestins block cholesterol synthesis to produce meiosis-activating sterols

The resumption of meiosis is regulated by meiosis‐preventing and meiosis‐activating substances in testes and ovaries. Certain C29 precursors of cholesterol are present at elevated levels in gonadal tissue, but the mechanism by which these meiosis‐activating sterols (MAS) accumulate has remained an unresolved question. Here we report that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF‐MAS and T‐MAS). These C29 sterols accumulated as a result of inhibition of Δ24‐reduction and 4α‐demethylation. Progesterone, pregnenolone, and 17α‐OH‐pregnenolone were potent inhibitors of Δ24‐reduction in an in vitro cell assay and led to the accumulation of desmosterol, a Δ5,24 sterol precursor of cholesterol. A markedly different effect was observed for 17α‐OH‐progesterone, which caused the accumulation of sterols associated with inhibition of 4α‐demethylation. The flux of 13C‐acetate into lathosterol and cholesterol was decreased by progestins as measured by isotopomer spectral analysis, whereas newly synthesized MAS accumulated. The combined evidence that MAS concentrations can be regulated by physiological levels of progestins and their specific combination provides a plausible explanation for the elevated concentration of MAS in gonads and suggests a new role for progestins in fertility.—Lindenthal, B., Holleran, A. L., Aldaghlas, T. A., Ruan, B., Schroepfer, G. J., Jr., Wilson, W. K., and Kelleher, J. K. Progestins block cholesterol synthesis to produce meiosis‐activating sterols. FASEB J. 15, 775‐784 (2001)

Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes

The objective of this study was to investigate the mechanisms by which tamoxifen modifies cholesterol metabolism in cellular models of liver metabolism, HepG2 cells and rat hepatocytes. The effect of tamoxifen on cholesterol and triglyceride-palmitate synthesis was measured using isotopomer spectral analysis (ISA) and gas chromatography-mass spectrometry (GC-MS) and compared with the effects of progesterone, estradiol, the antiestrogen ICI 182,780, and an oxysterol, 25-hydroxycholesterol (25OHC). Cholesterol synthesis in cells incubated in the presence of either [1-(13)C]acetate, [U-13C]glucose, or [4,5-(13)C]mevalonate for 48 hours was reduced in the presence of 10 micromol/L tamoxifen and 12.4 micromol/L 25OHC in both HepG2 cells and rat hepatocytes. The ISA methodology allowed a clear distinction between effects on synthesis and effects on precursor enrichment, and indicated that these compounds did not affect enrichment of the precursors of squalene. Progesterone was effective in both cell types at 30 micromol/L and only in HepG2 cells at 10 micromol/L. Estradiol and ICI 182,780 at 10 micromol/L did not inhibit cholesterol synthesis. None of the compounds altered the synthesis of triglyceride-palmitate in either cell type. Treatment of cells with tamoxifen produced accumulation of three sterol precursors of cholesterol, zymosterol, desmosterol, and delta8 cholesterol. This pattern of precursors indicates inhibition of delta24,25 reduction in addition to the previously described inhibition of delta8 isomerase. We conclude that tamoxifen is an effective inhibitor of the conversion of lanosterol to cholesterol in cellular models at concentrations comparable to those present in the plasma of tamoxifen-treated individuals. Our findings indicate that this mechanism may contribute to the effect of tamoxifen in reducing plasma cholesterol in humans.

Acetoacetate metabolism in AS-30D hepatoma cells

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetly CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells.The major findings were:1.Acetoacetate was utilized by AS-30D cells, with14C-lipid and14CO2 as major products of [3-14C] acetoacetate.2.Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium.3.Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate.4.Succinly CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria.5.Addition of acetoacetate, but not β-hydroxybutyrate decreased conversion of [1-14C] acetate to14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer.6.In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis. These data demonstrate for the first time the flux of acetoacetate carbon to lipid and CO2 in hepatoma cells and suggest that increases in the ambient concentration of acetoacetate, occurring in fasting or malignant cachexia, could produce increases in the utilization of this ketone body by hepatoma cells containing 3-oxyacid CoA transferase activity.