Joanne K. Kelleher

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.

Regulation of mouse hepatic genes in response to diet induced obesity, insulin resistance and fasting induced weight reduction

BackgroundObesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrys and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO)-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions.ResultsTranscriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/ weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced, hepatic glucose output via phosphoenolpyruvate carboxykinase (PCK1), whose mRNA levels were also elevated.ConclusionThese findings demonstrate that hepatocytes respond to DIO and weight reduction by controlling gene transcription in a variety of important molecular pathways. Future studies that characterize the physiological significance of the identified genes in modulating energy homeostasis could provide a better understanding of the mechanisms linking DIO with insulin resistance.

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.

Calcium sensitive isocitrate and 2-oxoglutarate dehydrogenase activities in rat liver and AS-30D hepatoma mitochondria.

NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase in extracts of mitochondria from the highly malignant AS-30D rat hepatoma cell line demonstrate Ca2+ sensitivities and affinities for substrates similar to those of normal liver mitochondria. However, the maximal activities of NAD+- and NADP+-dependent isocitrate dehydrogenase were found to be 8 and 3.5 fold higher in hepatoma mitochondrial extracts than those of liver mitochondria, whereas maximal activities of succinate and 2-oxoglutarate dehydrogenases were similar in the two tissues. At pyridine nucleotide concentrations giving the lowest physiological NADH/NAD+ ratio, NAD+-isocitrate dehydrogenase activity in hepatoma mitochondrial extracts was completely inhibited at subsaturating concentrations of Ca2+, substrate, and NAD+, in contrast to rat liver mitochondrial extracts which retained significant activity.

Monte Carlo spreadsheet modeling of stable isotope biosynthesis

Metabolic physiologists often introduce stable isotopes, atoms containing additional neutrons, into molecules during biosynthesis. This tags the newly synthesized material by altering its mass. Monte Carlo analysis is implemented on a popular spreadsheet to analyze this process. An example is provided where acetoacetate is synthesized by condensation of two acetate moieties. The precursor acetate is present as a mixture of natural, and 13C enriched, acetate. Monte Carlo spreadsheet modeling captures the complexity of the multi-species isotope biosynthesis by repetitively performing multiple simultaneous Boolean calculations. The effects of increasing the number of molecules synthesized on the goodness of fit of between model and an exact analytical solution is illustrated.

Isotope Labeling Ratios: A Tool for the Exploration of Metabolic Compartments

Reports of metabolic compartmentation often stem from data viewed as unfortunate by the reporters. Namely, an investigation of a metabolic pathway by isotopic tracer techniques yields results incompatible with the starting assumption that a single pool of each metabolite is present in the system. Thus compartmentation is added so that the data are consistent. A reader offered a specific compartmentalized arrangement in the closing paragraphs of a paper may wonder what other possible compartmental arrangements might have been uncovered if, from the onset of the project, multiple compartments of each metabolite had been assumed. Our goal is to describe techniques for determining the information available from steady state isotopic tracer studies if we assume that multiple pools of each metabolite exist which mix when the system is analyzed. Essentially we assume that the specific radioactivity (SA) of metabolite pools cannot be determined by experiment. We are particularly interested in the application of these techniques to investigations of oxidative energy metabolism and pathways involving metabolic cycles. Our previous studies have focused on the TCA cycle (Kelleher, 1984; Mallet et al., 1984). In this area of metabolism, the presence of enzyme aggregates and a variety of compartments including mitochondrial and cytoplasmic spaces render the estimation rates from isotopic tracer studies especially perilous.