Modulation by nutrients and drugs of liver acyl-CoAs analyzed by mass spectrometry.

The profile of liver acyl-CoAs induced by dietary fats of variable compositions or by xenobiotic hypolipidemic amphipathic carboxylates was evaluated in vivo using a novel electrospray ionization tandem mass spectrometry methodology of high resolution, sensitivity, and reliability. The composition of liver fatty acyl-CoAs was found to reflect the composition of dietary fat. Treatment with hypolipidemic carboxylates resulted in liver dominant abundance of their respective acyl-CoAs accompanied by an increase in liver fatty acyl-CoAs. Cellular effects exerted by dietary fatty acids and/or xenobiotic carboxylic drugs may be transduced in vivo by their respective acyl-CoAs.

Suppression of hepatocyte nuclear factor-4alpha by acyl-CoA thioesters of hypolipidemic peroxisome proliferators.

Hepatocyte nuclear factor-4alpha (HNF-4alpha) modulates the expression of liver-specific genes that control the production (e.g. apolipoprotein [apo] A-I and apo B) and clearance (e.g. apo C-III) of plasma lipoproteins. We reported that the CoA thioesters of amphipathic carboxylic hypolipidemic drugs (e.g. clofibric acid analogues currently used for treating hyperlipidemia in humans and substituted long-chain dicarboxylic acids) were formed in vivo, bound to HNF-4alpha, inhibited its transcriptional activity, and suppressed the expression of HNF-4alpha-responsive genes. Hypolipidemic PPARalpha (peroxisome proliferator-activated receptor alpha) activators that were not endogenously thioesterified into their respective acyl-CoAs were shown to be effective in rats but not in humans, implying that the hypolipidemic activity transduced by PPARalpha in rats was PPARalpha-independent in humans. The suppressed acyl-CoA synthase of PPARalpha knockout mice left unresolved the contribution made by the acyl-CoA/HNF-4alpha pathway to the hypolipidemic effect of PPARalpha agonists in rodents. Hence, suppression of HNF-4alpha activity by the CoA thioesters of hypolipidemic "peroxisome proliferators" may account for their hypolipidemic activity independently of PPARalpha activation by their respective free carboxylates. The hypolipidemic activity of peroxisome proliferators is mediated in rats and humans by the PPARalpha and HNF-4alpha pathways, respectively.

Fatty acid cycling in the fasting rat.

Adipose tissue lipolysis and fatty acid reesterification by liver and adipose tissue were investigated in rats fasted for 15 h under basal and calorigenic conditions. The fatty acid flux initiated by adipose fat lipolysis in the fasted rat is mostly futile and is characterized by reesterification of 57% of lipolyzed free fatty acid (FFA) back into adipose triglycerides (TG). About two-thirds of FFA reesterification are carried out before FFA release into plasma, whereas the rest consists of plasma FFA extracted by adipose tissue. Thirty-six percent of the fasting lipolytic flux is accounted for by oxidation of plasma FFA, whereas only a minor fraction is channeled into hepatic very low density lipoprotein-triglycerides (VLDL-TG). Total body calorigenesis induced by thyroid hormone treatment and liver-specific calorigenesis induced by treatment with beta, beta'-tetramethylhexadecanedioic acid (Medica 16) are characterized by a 1.7- and 1.3-fold increase in FFA oxidation, respectively, maintained by a 1.5-fold increase in adipose fat lipolysis. Hepatic reesterification of plasma FFA into VLDL-TG is negligible under both calorigenic conditions. Hence, total body fatty acid metabolism is regulated by adipose tissue as both source and sink. The futile nature of fatty acid cycling allows for its fine tuning in response to metabolic demands.

Evaluation of brain long-chain acylcarnitines during cerebral ischemia.

Concentration and distribution of long-chain acylcarnitines in control microwaved and ischemic (decapitated) rat brain were measured by electrospray ionization tandem mass spectrometry. The total acylcarnitine concentration from control rat brains equaled 7-8 nmol/g wet weight brain, about one-fourth the total concentration of long-chain acyl-CoA, indicating a small role in buffering the total acyl-CoA pool concentration. Furthermore, acylcarnitine did not differ between ischemic and control rat brain with regard to total concentration or concentrations of molecular species of acylcarnitine. Therefore, the size of the acylcarnitine pool in brain is not affected by the dramatic increase in unesterified fatty acids (approximately 4x) that occurs in ischemia.

Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue.

Calcium-dependent uncoupling of liver mitochondrial oxidative phosphorylation by a non-metabolizable long chain fatty acyl analogue was compared with uncoupling induced by in vivo thyroid hormone treatment. beta,beta'-Methyl-substituted hexadecane alpha, omega-dioic acid (Medica 16) is reported here to induce a saturable 20-30% decrease in liver mitochondrial DeltaPsi, DeltapH and protonmotive force which proceeds in the presence of added Ca(2+) to cyclosporin A-sensitive mitochondrial permeabilization. Ca(2+)-dependent uncoupling by Medica 16 was accompanied by atractylate-enhanced, bongkrekic-inhibited activation of mitochondrial Ca(2+) efflux. The direct mitochondrial effect exerted in vitro by Medica 16 is similar to that induced by in vivo thyroid hormone treatment. Hence, the thyromimetic protonophoric activity of Medica 16 and the uncoupling activity of TH converge onto components of the mitochondrial permeabilization transition pore.

Mitochondria uncoupling by a long chain fatty acyl analogue.

Mitochondria uncoupling by fatty acids in vivo is still questionable, being confounded by their dual role as substrates for oxidation and as putative genuine uncouplers of oxidative phosphorylation. To dissociate between substrate and the uncoupling activity of fatty acids in oxidative phosphorylation, the uncoupling effect was studied here using a nonmetabolizable long chain fatty acyl analogue. beta,beta'-Methyl-substituted hexadecane alpha,omega-dioic acid (MEDICA 16) is reported here to induce in freshly isolated liver cells a saturable oligomycin-insensitive decrease in mitochondrial proton motive force with a concomitant increase in cellular respiration. Similarly, MEDICA 16 induced a saturable decrease in membrane potential, proton gradient, and proton motive force in isolated liver and heart mitochondria accompanied by an increase in mitochondrial respiration. Uncoupling by MEDICA 16 in isolated mitochondria was partially suppressed by added atractyloside. Hence, fatty acids may act as genuine uncouplers of cellular oxidative phosphorylation by interacting with specific mitochondrial proteins, including the adenine nucleotide translocase.

Sensitization to insulin induced by beta,beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) in obese Zucker rats in vivo.

Beta,beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) consists of a nonmetabolizable long-chain fatty acid designed to probe the effect exerted by fatty acids on insulin sensitivity. The effect of MEDICA 16 was evaluated in insulin-resistant Zucker (fa/fa) rats in terms of liver, muscle, and adipose tissue response to clamped euglycemic hyperinsulinemia in vivo. Nontreated Zucker rats were insulin resistant, maintaining basal rates of total-body glucose disposal, glucose production in liver, free fatty acid (FFA) flux into plasma, and FFA reesterification in adipose tissue, irrespective of the insulin levels induced. MEDICA 16 treatment resulted in an insulin-induced decrease in hepatic glucose production, together with an insulin-induced increase in total-body glucose disposal. Intracellular reesterification of lipolysed FFA in adipose tissue was specifically activated by MEDICA 16, resulting in a pronounced decrease in FFA release, with a concomitant decrease in plasma FFA. In conclusion, MEDICA 16 treatment results in the sensitization of liver, muscle, and adipose tissue to insulin in an animal model for obesity-induced insulin resistance.

Mitochondrial permeability transition is induced by in vivo thyroid hormone treatment.

Thyroid hormone treatment in vivo results in activation of mitochondrial Ca2+ efflux and temperature-dependent enhanced swelling of Ca(2+)-loaded rat liver mitochondria. Thyroid hormone-induced swelling was effectively prevented in the presence of excess EGTA or cyclosporin A. Thyroid hormone treatment similarly resulted in a dramatic decrease in mitochondrial membrane potential (80%), proton gradient (45%), and proton motive force (69%) measured in Ca(2+)-loaded mitochondria. All three parameters were essentially restored to euthyroid values in the presence of excess EGTA or cyclosporin A. Mitochondrial energy-linked transhydrogenase activity measured in the presence of Ca2+ was 33% increased and 38% decreased in hypothyroid and L-T3-treated hypothyroid rats, respectively, compared to that in euthyroid rats. Hence, in vivo thyroid hormone treatment may induce mitochondrial permeability transition mediated by the cyclosporin A-sensitive permeability transition pore.

Thyromimetic effect of peroxisome proliferators.

Xenobiotic amphipathic carboxylates of varying hydrophobic backbones, known collectively as 'peroxisome proliferators' (PP), affect lipoprotein metabolism, calorigenesis, liver redox and phosphate potentials and adipose conversion. Some biological effects exerted by PP are strikingly similar to those exerted by thyroid hormones (TH). Furthermore, similarly to TH, these compounds have been recently found to induce in euthyroid as well as thyroidectomized rats or in rat hepatocytes cultured in TH-free media, liver activities classically considered as TH-dependent, eg malic enzyme (ME) and S14. The thyromimetic effect of PP could be accounted for by transcriptional activation of TH-dependent genes as verified by run-on transcription assays. The thyromimetic effect of PP was found not to be mediated by the TH nuclear receptor. Moreover, in contrast to TH, PP were ineffective as thyromimetic agents in the rat heart or pituitary cells, suggesting a tissue specificity different from that of TH. The overall thyromimetic effect of PP appears to involve transcriptional activation of TH-dependent genes, yet being mediated by a novel transduction pathway.

Tissue selective modulation of redox and phosphate potentials by beta,beta'-methyl-substituted hexadecanedioic acid.

beta,beta'-Methyl-substituted hexadecanedioic acid (MEDICA 16) shares some of the calorigenic-hypolipidemic characteristics of thyroid hormones. In light of this similarity, MEDICA 16 was further evaluated here as a modulator of rat liver redox and phosphate potentials as well as an effector of cardiac high energy intermediates level in comparison to thyroid hormone. 1) Similarly to thyroid hormone, MEDICA 16 treatment resulted in 3.5-fold decrease in cytosolic redox potential. With both treatment modes, the induced decrease in cytosolic redox potential resulted in a concomitant increase in the liver capacity of handling an ethanol or xylitol load. 2) The apparent liver cytosolic phosphate potential calculated from the glycerol-3-phosphate 3-phosphoglyceric acid ratio evaluated under conditions of rapid equilibrium within the glycerol-3 phosphate/3-phosphoglyceric acid metabolic section was found to remain unaffected by either MEDICA 16 or T3 treatment. However, the apparent cellular phosphate potential was substantially decreased by both treatment modes, thus reflecting a decrease in the apparent liver mitochondrial phosphate potential. 3) Similarly to thyroid hormone, the effect of MEDICA 16 on liver redox and phosphate potentials could be accounted for by induction of mitochondrial glycerol-3-phosphate dehydrogenase. 4) In contrast to liver, heart high energy intermediates were affected by thyroid hormone but not by MEDICA 16 treatment. 5) The effect of T3 and MEDICA 16 with respect to liver redox and phosphate potentials may partially account for the calorigenic-hypolipidemic effect of both. MEDICA 16 may however serve as a selective liver thyromimetic agent lacking the cardiac affect induced by thyroid hormone.

Effect of thyroid hormone treatment on redox and phosphate potentials in rat liver.

In the present study rat liver cytosolic/mitochondrial redox and phosphate potentials were evaluated as a function of thyroid hormone status. T3 treatment resulted in a dose-dependent 2-fold decrease in the cytosolic redox potential, as reflected by the liver lactate/pyruvate ratio, with a concomitant increase in the liver capacity of handling an ethanol load. The effect of T3 on liver cytosolic redox potential was correlated with a T3-induced increase in mitochondrial glycerol-3-phosphate dehydrogenase activity. The apparent liver cytosolic phosphate potential was calculated from the glycerol-3-phosphate/3-phosphoglyceric acid ratio, using 31P-nmr under conditions of rapid equilibrium within the glycerol-3-phosphate/3- phosphoglyceric acid metabolic section and was found to be essentially unaffected by T3 treatment. The apparent total cellular phosphate potential measured by 31P-nmr, however, was decreased in T3-treated animals, reflecting a decrease in the apparent liver mitochondrial phosphate potential induced by T3 treatment. Also, while the apparent cellular phosphate potential of euthyroid rats was independent of ethanol administration, the reduced cellular phosphate potential of T3-treated rats was normalized by ethanol treatment. In conclusion, thyroid hormone treatment induces in vivo a dramatic decrease in the liver cytosolic redox potential, with a concomitant increase in the liver oxidizing capacity. The decrease in cytosolic redox potential induced by thyroid hormone treatment is accompanied by a decrease in mitochondrial phosphate potential. Liver mitochondrial ATP production in thyroid hormone-treated animals appears to be rate limited by the availability of cytosolic reducing equivalents.

A study of galactose intolerance in human and rat liver in vivo by 31P magnetic resonance spectroscopy.

An oral load of 20 mg/kg galactose produces significant changes in the 31P magnetic resonance spectrum of the liver of a galactosemic patient. The peak at 5.2 ppm (which includes inorganic phosphate and galactose-1-phosphate) increased on two occasions to about twice its original size 60 min after galactose administration. An oral load of 10 mg/kg galactose given to a second patient produced no discernible changes at 30 min. We have also used an animal model of galactose intolerance, in which galactose metabolism in rats was blocked by the acute administration of ethanol. Studies in vivo and in vitro showed that the increase in the peak at 5.2 ppm was largely due to galactose-1-phosphate. We have shown in this preliminary study that small amounts of galactose can produce significant elevation of hepatic galactose-1-phosphate, which can be detected by 31P magnetic resonance spectroscopy.

Glucose recycling and production in glycogenosis type I and III: stable isotope technique study.

Glucose carbon recycling, endogenous glucose production, and glucose turnover rates were measured, by stable isotope methodology, in five patients with glycogen storage disease type I (GSD-I), two patients with glycogen storage disease type III (GSD-III), and three control children. A primed-constant infusion of D-[U-13C]glucose was administered nasogastrically to fasted subjects. The isotopic enrichments and 13C isotopomer distribution of plasma glucose were measured by chemical ionization gas chromatography mass spectroscopy. In response to increasing rates of glucose infusion, endogenous glucose production decreased, whereas the rate of glucose appearance or total glucose flux increased. Recycling of infused D-[U-13C]-glucose, calculated from changes in the isotopomer distribution of plasma [13C]glucose, was not detectable in GSD-I but reached 50% in GSD-III. In GSD-I the gluconeogenic pool was found to be highly labeled and recycled, whereas plasma glucose was diluted but not recycled. It is suggested that in GSD-I dilution of plasma glucose is due to release of glucose from branch points in glycogen. We propose that studies of the extent of glucose recycling and of isotopic enrichment of gluconeogenic precursors can be used as a noninvasive test for diagnosis of GSD-I and other defects in glucose production.

Estimation of glucose carbon recycling in children with glycogen storage disease: A 13C NMR study using [U-13C]glucose.

A stable isotope procedure to estimate hepatic glucose carbon recycling and thereby elucidate the mechanism by which glucose is produced in patients lacking glucose 6-phosphatase is described. A total of 10 studies was performed in children with glycogen storage disease type I (GSD-I) and type III (GSD-III) and control subjects. A primed dose-constant nasogastric infusion of D-[U-13C]glucose (greater than 99% 13C-enriched) or an infusion diluted with nonlabeled glucose solution was administered following different periods of fasting. Hepatic glucose carbon recycling was estimated from 13C NMR spectra. The recycling parameters were derived from plasma beta-glucose C-1 splitting pattern, doublet/singlet values of plasma glucose C-1 in comparison to doublet/singlet values of known mixtures of [U-13C]glucose and unlabeled glucose as a function of 13C enrichment of glucose C-1. The fractional glucose C-1 enrichment of plasma glucose samples was analyzed by 1H NMR spectroscopy and confirmed by gas chromatography/mass spectroscopy. The values obtained for GSD-I patients coincided with the standard [U-13C]glucose dilution curve. These results indicate that the plasma glucose of GSD-I subjects comprises only a mixture of 99% 13C-enriched D-[U-13C]glucose and unlabeled glucose but lacks any recycled glucose. Significantly different glucose carbon recycling values were obtained for two GSD-III patients in comparison to GSD-I patients. Our results eliminate a mechanism for glucose production in GSD-I children involving gluconeogenesis. However, glucose release by amylo-1,6-glucosidase activity would result in endogenous glucose production of non-13C-labeled and nonrecycled glucose carbon, as was found in this study. In GSD-III patients gluconeogenesis is suggested as the major route for endogenous glucose synthesis. The contribution of the triose-phosphate pathway in these patients has been determined. The significant difference of the glucose C-1 splitting pattern in plasma GSD-III and control subjects, in comparison to GSD-I plasma, can be used as a parameter for estimating glucose recycling. This approach can be developed as a noninvasive diagnostic test for inborn enzymatic defects involving gluconeogenesis.

Glucose recycling and production in children with glycogen storage disease type I, studied by gas chromatography/mass spectrometry and (U-13C)glucose.

Glucose recycling and production were determined in plasma of three children with glycogen storage disease type I (GSD-I) in comparison to normal children. A primed-constant infusion of (U-13C)glucose was introduced nasogastrically at different rates. Endogenous glucose production rates were found to be correlated with rates of glucose infusion. A range of glucose production of 3.5-1.8 mg kg-1 min-1 was found when glucose infusion rates increased from 0.13 to 6 mg kg-1 min-1. The isotopomer distribution of the infused (U-13C)glucose and the plasma glucose in GSD-I children, measured by chemical ionization gas chromatography/mass spectrometry, were identical, indicating absence of glucose recycling. However, a significant change in the isotopomer distribution of plasma glucose was observed in normal subjects. It is suggested that the origin of endogenous glucose production in GSD-I children, during (U-13C)glucose infusion, is from non-labelled sources. The absence of glucose recycling is indicative of a deficiency of glucose 6-phosphatase activity in the liver of GSD-I patients.

Effects of streptozotocin-induced diabetes on drug metabolism in rats.

The metabolism of salicylic acid was investigated in male normal and diabetic rats. The results showed a decrease in urinary excretion of salicylic-glucuronic acid in diabetic animals and this decrease is statistically significant (P less than or equal to 0.01), while the urinary excretion of salicyluric acid in the diabetic group was higher than in controls and statistically significant (P less than or equal to 0.001). There was no marked difference between normal and diabetic rats in total urinary excretion of salicylic acid and metabolites.

A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C-labeled substrates.

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2-13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC-MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl-CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by 'metabolic exchange' with the oxaloacetate pool, via the Krebs cycle, is less than 7%.

Metabolic pathways leading to liver glycogen repletion in vivo, studied by GC-MS and NMR.

A quantitative analysis of the pathways leading to glycogen repletion in rats was conducted. [U-13C]Glucose was administered intra-intestinally into awake fasted animals. The distribution of glucose isotopomers derived from liver glycogen, liver extracts and plasma was performed by GC-MS and 13C NMR. The potential gluconeogenic precursors for liver glycogen, lactate, alanine, glutamate and glutamine, were also analyzed. The amount of glycogen that is synthesized by the direct pathway was found to be 35%. The 13C enrichment of liver lactate, alanine and glucose is similar, indicating that they are the major precursors for liver glycogen synthesis via the indirect pathway. Our results demonstrate that after 24 h fasting, when glucose is supplied, gluconeogenesis from endogenous sources is not shut off.

Characterization of stages in development of obesity-diabetes syndrome in sand rat (Psammomys obesus).

Sand rats (Psammomys obesus) maintained on a diet providing a free choice between laboratory chow and salt bush (Atriplex halimus) were classified into four groups differing in extent of the diabetic syndrome: A, normoglycemic-normoinsulinemic; B, normoglycemic-hyperinsulinemic; C, hyperglycemic-hyperinsulinemic; or D, hyperglycemic with reduced insulin levels. The metabolic pattern of these groups was characterized by measuring the uptake of fatty acid-labeled, very-low-density lipoprotein-borne triglycerides (VLDL-TG) and [3H]-2-deoxyglucose (2-DOG) into muscle and adipose tissues; incorporation of [14C]alanine into glycogen in vivo; gluconeogenesis from lactate, pyruvate, and alanine in hepatocytes; the effect of insulin on glycogen synthesis from glucose; the oxidation of albumin-bound [1-14C]palmitate and [14C]glucose in strips of soleus muscle; activities of muscle and adipose tissue lipoprotein lipase; and activities of rate-limiting enzymes of glycolysis, gluconeogenesis, and fatty acid synthesis in liver. In group A, uptake of VLDL-TG and activity of lipoprotein lipase were higher in adipose tissue and lower in muscle than in albino rats. In the liver, gluconeogenesis and the activity of phosphoenolpyruvate carboxykinase, as well as lipid synthesis and the activity of NADP-malate dehydrogenase, were higher than in albino rats, whereas activity of pyruvate kinase was lower. In group B, uptake of VLDL-TG by adipose tissue and muscle and lipoprotein lipase activity were similar or higher than in group A. Uptake of 2-DOG by muscle and adipose tissue and activity of liver phosphoenolpyruvate carboxykinase were lower than in group A. In groups C and D, uptake of VLDL-TG and lipoprotein lipase activity in muscle were further increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipogenesis in the sand rat (Psammomys obesus).

Synthesis of fatty acids was measured in the liver and in epididymal adipose tissue of sand rats and albino rats. In chow-fed sand rats the rate of hepatic lipogenesis, as measured by the incorporation of 3H2O into fatty acids, was four- to sevenfold higher than in albino rats and in sand rats on a low-calorie saltbush diet. The contribution of [14C]glucose to lipogenesis in sand rat liver was lower than in albino rats. In fed sand rats lipogenesis incorporating 3H2O was stimulated by casein but not by glucose. In adipose tissue, lipogenesis measured 1 h after administration of 3H2O was much lower in sand rats than in albino rats. In vitro incorporation of [14C]glucose or acetate into adipose tissue fatty acids was negligible. In adipose tissue, uptake of very-low-density lipoproteins (VLDL) and lipoprotein lipase activity were sevenfold higher than in albino rats. Activities of NADP-malate dehydrogenase, acetyl CoA carboxylase, and fatty acid synthetase were considerably higher in the liver of chow-fed sand rats than in albino rats. It was concluded that obesity in sand rats originates from hepatic lipogenesis without a significant contribution of local fatty acid synthesis in adipose tissue.