Hepatic Fat Oxidation Research Articles

Progressive adaptation of hepatic ketogenesis in mice fed a high-fat diet

Hepatic ketogenesis provides a vital systemic fuel during fasting because ketone bodies are oxidized by most peripheral tissues and, unlike glucose, can be synthesized from fatty acids via mitochondrial beta-oxidation. Since dysfunctional mitochondrial fat oxidation may be a cofactor in insulin-resistant tissue, the objective of this study was to determine whether diet-induced insulin resistance in mice results in impaired in vivo hepatic fat oxidation secondary to defects in ketogenesis. Ketone turnover (micromol/min) in the conscious and unrestrained mouse was responsive to induction and diminution of hepatic fat oxidation, as indicated by an eightfold rise during the fed (0.50+/-0.1)-to-fasted (3.8+/-0.2) transition and a dramatic blunting of fasting ketone turnover in PPARalpha(-/-) mice (1.0+/-0.1). C57BL/6 mice made obese and insulin resistant by high-fat feeding for 8 wk had normal expression of genes that regulate hepatic fat oxidation, whereas 16 wk on the diet induced expression of these genes and stimulated the function of hepatic mitochondrial fat oxidation, as indicated by a 40% induction of fasting ketogenesis and a twofold rise in short-chain acylcarnitines. Together, these findings indicate a progressive adaptation of hepatic ketogenesis during high-fat feeding, resulting in increased hepatic fat oxidation after 16 wk of a high-fat diet. We conclude that mitochondrial fat oxidation is stimulated rather than impaired during the initiation of hepatic insulin resistance in mice.

Testosterone stimulates extra‐hepatic but not hepatic fat oxidation (Fox): comparison of oral and transdermal testosterone administration in hypopituitary men

Fat mass is increased in hypogonadal men and the changes are reversed by testosterone replacement. Testosterone administration enhances whole body fat oxidation (Fox). Fat is oxidized in the liver and in extra-hepatic tissues. To determine whether the stimulation of Fox by testosterone arises primarily from the liver or from extra-hepatic tissues. This was an open-label cross-over study. Thirteen men with hypopituitarism (age 53.1 +/- 4.1 years) with both growth hormone (GH) and testosterone deficiency were studied sequentially after 2 weeks of treatment with transdermal testosterone (5 mg), no treatment, and stepwise incremental doses of oral crystalline testosterone (10, 20, 40 and 80 mg) in the absence of GH replacement. Serum testosterone, IGF-I, metabolic effects [resting energy expenditure (REE) and Fox], SHBG, and thyroid binding globulin (TBG) as markers of excessive hepatic androgen exposure, were measured at the end of each treatment period. When compared to the no-treatment phase, mean blood testosterone levels rose into the physiological range after transdermal testosterone delivery but did not significantly change after 10, 20, 40 or 80 mg oral testosterone treatment. Blood SHBG and TBG fell significantly with 80 mg oral testosterone dose but were unaffected by any other testosterone treatment. Fox increased significantly with transdermal but not with any dose of oral testosterone. Mean plasma IGF-I and REE were unaffected by testosterone, regardless of the route or dose. Short-term testosterone administration does not stimulate hepatic fat oxidation but enhances whole body fat oxidation by acting on extra-hepatic tissues.

Preliminary report: pharmacologic 11 β-hydroxysteroid dehydrogenase type 1 inhibition increases hepatic fat oxidation in vivo and expression of related genes in rats fed an obesogenic diet

This study aimed to explore in a model of diet-induced steatosis the impact of pharmacologic 11 β-hydroxysteroid dehydrogenase type 1 (11 β-HSD1) inhibition, under conditions of unchanged ingestive behavior, on liver fat oxidation. Male Sprague-Dawley rats were fed an obesogenic diet and were continuously treated or not with an 11 β-HSD1 inhibitor (Compound A, 3 mg/[kg d]; Merck Research Laboratories, Rahway, NJ), after which liver expression of oxidative genes and in vivo hepatic fat oxidation were quantified. Treatment with Compound A reduced liver triglyceride concentration (−28%), increased hepatic expression of several genes coding for enzymes of mitochondrial and peroxisomal β-oxidation, and concomitantly enhanced in vivo liver fat oxidation (+38%). The study demonstrates, under conditions that avoided changes in food intake seen in gene knockout or higher-dose pharmacologic models, the efficacy of 11 β-HSD1 inhibition to up-regulate hepatic fat oxidation gene expression, which functionally translates into enhanced hepatic lipid oxidation in vivo.

Effects of Insulin on the Metabolic Control of Hepatic Gluconeogenesis In Vivo

OBJECTIVEInsulin represses the expression of gluconeogenic genes at the mRNA level, but the hormone appears to have only weak inhibitory effects in vivo. The aims of this study were 1) to determine the maximal physiologic effect of insulin, 2) to determine the relative importance of its effects on gluconeogenic regulatory sites, and 3) to correlate those changes with alterations at the cellular level.RESEARCH DESIGN AND METHODSConscious 60-h fasted canines were studied at three insulin levels (near basal, 4×, or 16×) during a 5-h euglycemic clamp. Pancreatic hormones were controlled using somatostatin with portal insulin and glucagon infusions. Glucose metabolism was assessed using the arteriovenous difference technique, and molecular signals were assessed.RESULTSInsulin reduced gluconeogenic flux to glucose-6-phosphate (G6P) but only at the near-maximal physiological level (16× basal). The effect was modest compared with its inhibitory effect on net hepatic glycogenolysis, occurred within 30 min, and was associated with a marked decrease in hepatic fat oxidation, increased liver fructose 2,6-bisphosphate level, and reductions in lactate, glycerol, and amino acid extraction. No further diminution in gluconeogenic flux to G6P occurred over the remaining 4.5 h of the study, despite a marked decrease in PEPCK content, suggesting poor control strength for this enzyme in gluconeogenic regulation in canines.CONCLUSIONSGluconeogenic flux can be rapidly inhibited by high insulin levels in canines. Initially decreased hepatic lactate extraction is important, and later reduced gluconeogenic precursor availability plays a role. Changes in PEPCK appear to have little or no acute effect on gluconeogenic flux.

Alterations in hepatic macronutrient metabolism in response to short term high fat feeding in rats

Insulin resistance (IR) is coupled to obesity or high fat feeding by ectopic lipid metabolism and accumulation of intermediates that disrupt insulin signaling. We recently reported that hepatic mitochondrial fat metabolism is dysfunctional in Zucker Diabetic Fatty (ZDF) rats. The objective of this study was to test whether similar effects on hepatic mitochondrial metabolism occur in lean (ZDF+/−) rats following a short term (7d) high fat (60% fat calories) challenge. Conscious, unrestrained rats were infused with stable isotope tracers followed by NMR or LC‐MS isotopomer analysis of plasma metabolites to determine hepatic fluxes. Fasting hepatic ketogenesis and fat oxidation is impaired in obese diabetic rats. In contrast, a short term high fat diet increased fasting ketone concentrations and did not affect TCA cycle flux or GNG. Ureagenesis, an energy intensive pathway that spans both cytosolic and mitochondrial compartments, was also lower in fat fed lean ZDF+/− rats compared to obese ZDF−/− rats. We conclude that short‐term high fat feeding elicits a physiological response in several hepatic mitochondrial and cytosolic pathways that facilitates whole body metabolism of lipid rich macronutrients, but that this adaptation is fundamentally different from the patho‐physiological features of hepatic fat metabolism in obese diabetic rats.

Postprandial metabolism with 1,3-diacylglycerol oil versus equivalent intakes of long-chain and medium-chain triacylglycerol oils

Objective We assessed the ability of novel lipid structures including medium-chain triacylglycerols (MCTs) and 1,3-diacylglycerol (DG) oil to lower postprandial triacylglycerol (TG) elevation and increase hepatic fat oxidation when substituted for dietary TG, which may be useful in the prevention and treatment of obesity and other related metabolic conditions, such as dyslipidemias. Methods This double-blind, randomized, crossover trial evaluated the effects of an oral fat load containing DG or MCTs compared with equivalent intakes of long-chain triacylglycerols (LCTs) on the postprandial metabolic responses of insulin-resistant men and women ( n = 36). Each subject consumed a single oral fat load on each test day. The fat loads were delivered in milkshakes that contained 30 g of one of the three test oils. Results The postprandial TG incremental area under the curve after MCT was 73% lower, and that for DG was 22% lower, compared with the response after LCT oil. The incremental area under the curve values for chylomicron TG were reduced versus LCT by 89% and 28%, respectively, in the MCT and DG conditions. Compared with the LCT treatment, β-hydroxybutyrate concentration was increased after MCT oil, but not after DG. Conclusion These results indicate that dietary DG decreased postprandial triglyceridemia compared with LCT, but to a lesser extent than MCT.

Adipose tissue fatty acid metabolism in insulin-resistant men

Increased NEFA production and concentrations may underlie insulin resistance. We examined systemic and adipose tissue NEFA metabolism in insulin-resistant overweight men (BMI 25-35 kg/m2). In a cohort study we examined NEFA concentrations in men in the upper quartile of fasting insulin (n = 124) and in men with fasting insulin below the median (n = 159). In a metabolic study we examined NEFA metabolism in the fasting and postprandial states, in ten insulin-resistant men and ten controls. In the cohort study, fasting NEFA concentrations were not significantly different between the two groups (median values: insulin-resistant men, 410 micromol/l; controls, 445 micromol/l). However, triacylglycerol concentrations differed markedly (1.84 vs 1.18 mmol/l respectively, p < 0.001). In the metabolic study, arterial NEFA concentrations again did not differ between groups, whereas triacylglycerol concentrations were significantly higher in insulin-resistant men. Systemic NEFA production and the release of NEFA from subcutaneous adipose tissue, expressed per unit of fat mass, were both reduced in insulin-resistant men compared with controls (fasting values by 32%, p = 0.02, and 44%, p = 0.04 respectively). 3-Hydroxybutyrate concentrations, an index of hepatic fat oxidation and ketogenesis, were lower (p = 0.03). Adipose tissue NEFA output is not increased (per unit weight of tissue) in insulin resistance. On the contrary, it appears to be suppressed by high fasting insulin concentrations. Alterations in triacylglycerol metabolism are more marked than those in NEFA metabolism and are indicative of altered metabolic partitioning of fatty acids (decreased oxidation, increased esterification) in the liver.

Partial resistance to peroxisome proliferator-activated receptor-alpha agonists in ZDF rats is associated with defective hepatic mitochondrial metabolism.

OBJECTIVE—Fluxes through mitochondrial pathways are defective in insulin-resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The purpose of this study is to determine whether abnormal mitochondrial metabolism plays a role in the dysregulation of both hepatic fat and glucose metabolism during diabetes.RESEARCH DESIGN AND METHODS—Mitochondrial fluxes were measured using 2H/13C tracers and nuclear magnetic resonance spectroscopy in ZDF rats during early and advanced diabetes. To determine whether defects in hepatic fat oxidation can be corrected by peroxisome proliferator–activated receptor (PPAR-)-α activation, rats were treated with WY14,643 for 3 weeks before tracer administration.RESULTS—Hepatic mitochondrial fat oxidation in the diabetic liver was impaired twofold secondary to decreased ketogenesis, but tricarboxylic acid (TCA) cycle activity and pyruvate carboxylase flux were normal in newly diabetic rats and elevated in older rats. Treatment of diabetic rats with a PPAR–α agonist induced hepatic fat oxidation via ketogenesis and hepatic TCA cycle activity but failed to lower fasting glycemia or endogenous glucose production. In fact, PPAR-α agonism overstimulated mitochondrial TCA cycle flux and induced pyruvate carboxylase flux and gluconeogenesis in lean rats.CONCLUSIONS—The impairment of certain mitochondrial fluxes, but preservation or induction of others, suggests a complex defect in mitochondrial metabolism in the diabetic liver. These data indicate an important codependence between hepatic fat oxidation and gluconeogenesis in the normal and diabetic state and potentially explain the sometimes equivocal effect of PPAR-α agonists on glycemia.

Activation of glucagon receptor signaling stimulates regulators of hepatic fat oxidation in vivo

Evidence suggests that glucagon (GN) has persistent effects on the liver that we hypothesize are related to AMPK, PPARα, and FGF‐21. These proteins are known to promote hepatic oxidative processes. We aimed to define signaling pathways downstream of the hepatic GN receptor in vivo. The role of GN stimulation was tested using wild type (WT) and glucagon receptor null (KO) mice catheterized in a carotid artery and jugular vein 5d prior to study. In 5h fasted mice, GN (10ng/kg/min) or saline (SAL) was infused for 2h and the interaction of GN with circulating fat levels was tested in separate studies in which intralipid (IL) was also infused. Pfloridzin was infused in all studies to prevent high glucose and insulin levels. Euglycemia (150mg/dL) was maintained using a variable glucose infusion. Elevated GN (∼5‐fold) in WT increased glucose production and caused increases in AMP:ATP (7.7±0.5 fold), p‐AMPK (2.1±0.3 fold), and PPARα and FGF‐21 mRNA (2.0±0.3 and 2.0±0.4 fold) in liver compared to SAL. IL alone had no effects. However, IL led to marked potentiation of GN‐induced stimulation of FGF‐21 mRNA (12.3±4.2‐fold) in WT mice. Effects of GN with and without IL were eliminated in KO mice. We show in vivo that glucagon drops the liver to a low energy state which corresponds to increased activation of AMPK and induction of PPARα and FGF‐21 mRNA. Effects of GN on FGF‐21 are potentiated by IL. In conclusion, GN not only acutely regulates glucose homeostasis, but also causes adaptations in processes that improve the ability of the liver to oxidize fat. Supported by DK 50277.

Regulation of fat metabolism in the liver: link to non-alcoholic hepatic steatosis and impact of physical exercise.

Hepatic steatosis may develop as a consequence of several dysfunctions. An increased circulating non-esterified fatty acid (NEFA) pool seems to be a major determinant in the pathogenesis of non-alcoholic fatty liver disease. Increased activation of the transcription factor sterol-regulatory-element-binding protein-1c, which promotes fatty acid synthesis, also contributes to hepatic fat accumulation. Increased hepatic fat oxidation with hepatic steatosis may be triggered by increased hepatic fat concentrations through the action of hepatic peroxisomes mediated by peroxisome proliferator-activated receptor alpha. Finally, inhibition in very low density lipoprotein secretion may also result in hepatic steatosis. This appears to be mainly controlled by the esterification of NEFAs into triacylglycerols by diacyglycerol acyltransferase-1 and -2 and the microsomal transfer protein. Physical exercise would interfere with the development of hepatic steatosis by stimulating lipid oxidation and inhibiting lipid synthesis in liver through the activation of the AMP-activated protein kinase pathway.

Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2.

Hepatic steatosis is a core feature of the metabolic syndrome and type 2 diabetes and leads to hepatic insulin resistance. Malonyl-CoA, generated by acetyl-CoA carboxylases 1 and 2 (Acc1 and Acc2), is a key regulator of both mitochondrial fatty acid oxidation and fat synthesis. We used a diet-induced rat model of nonalcoholic fatty liver disease (NAFLD) and hepatic insulin resistance to explore the impact of suppressing Acc1, Acc2, or both Acc1 and Acc2 on hepatic lipid levels and insulin sensitivity. While suppression of Acc1 or Acc2 expression with antisense oligonucleotides (ASOs) increased fat oxidation in rat hepatocytes, suppression of both enzymes with a single ASO was significantly more effective in promoting fat oxidation. Suppression of Acc1 also inhibited lipogenesis whereas Acc2 reduction had no effect on lipogenesis. In rats with NAFLD, suppression of both enzymes with a single ASO was required to significantly reduce hepatic malonyl-CoA levels in vivo, lower hepatic lipids (long-chain acyl-CoAs, diacylglycerol, and triglycerides), and improve hepatic insulin sensitivity. Plasma ketones were significantly elevated compared with controls in the fed state but not in the fasting state, indicating that lowering Acc1 and -2 expression increases hepatic fat oxidation specifically in the fed state. These studies suggest that pharmacological inhibition of Acc1 and -2 may be a novel approach in the treatment of NAFLD and hepatic insulin resistance.

Effects of Zidovudine, Stavudine and β-Aminoisobutyric Acid on Lipid Homeostasis in Mice: Possible Role in Human Fat Wasting

ObjectiveAlthough mitochondrial DNA (mtDNA) depletion could play a role in nucleoside reverse transcriptase inhibitor-induced lipoatrophy, poor correlations between fat mtDNA levels and lipoatrophy suggest additional mechanism(s). Stavudine (d4T), zidovudine (AZT) and the thymine catabolite, β-aminoisobutyric acid (BAIBA), but not zalcitabine (ddC) or didanosine (ddI), can increase fatty acid oxidation in liver mitochondria and plasma ketone bodies in mice. Since fat oxidation in non-adipose tissue can influence body adiposity, we sought to determine whether d4T, AZT and BAIBA can cause lipoatrophy in mice by this catabolic mechanism.MethodsLean or obese ob/ob mice were treated for 6 weeks with d4T, AZT or BAIBA, and lean mice with ddC or ddI. Body fat mass was assessed by dual energy X-ray absorptiometry, and mtDNA by Slot blot hybridization in epididymal fat.ResultsWhereas ddC or ddI did not change plasma β-hydroxybutyrate and body fat mass, d4T, AZT and BAIBA increased plasma β-hydroxybutyrate in lean mice suggesting increased hepatic fatty acid oxidation and ketogenesis. Despite unchanged food consumption, a supra-pharmacological dose of d4T tended to decrease, whilst AZT and BAIBA decreased body fat mass. Fat mtDNA and plasma triglycerides, cholesterol, glucose, insulin, leptin and adiponectin levels were unchanged. In obese mice, d4T, AZT and BAIBA did not increase plasma β-hydroxybutyrate, and only AZT decreased body fat mass without reducing fat mtDNA.Conclusionsd4T and AZT can enhance hepatic fat oxidation and cause fat wasting, without decreasing adipose tissue mtDNA and without causing insulin resistance in mice. BAIBA, a thymine catabolite, reproduces these effects. These catabolic effects could play a role in the lipoatrophy, which can occur in AZT- or d4T-treated patients.

ENHANCED LONG-CHAIN FATTY ACID METABOLISM BY INTESTINE IN THE SUCKLING RAT.▴ 1838

The newborn infant depends on breast milk lipids as a major source of energy. Increased lipid utilization is reflected by enhanced ketogenesis in the developing liver. We hypothesize that the intestine provides an additional locus for the newborn to metabolize milk-borne lipids and thus augment hepatic fat oxidation. To evaluate long-chain fatty acid oxidation by the small intestine during development, we assayed the in vitro production of CO2, and, as an index of ketogenesis, acid-soluble aqueous products from oleic acid by intestinal slices of suckling (12-14 day old) and weanling(30 day old) rats. Animals were fasted overnight, killed by decapitation and samples of proximal and distal intestine were removed, cut into slices, and incubated in Krebs-Ringer phosphate buffer with 8 mM taurocholate and 0.2 mM(carboxyl-14C) oleic acid for 7.5, 15, 22, 30 and 45 min. Evolved CO2 was trapped using hyamine hydroxide and reactions were terminated with 15% perchloric acid. Aqueous and lipid phases were separated in chloroform:methanol and radioactivity in each phase and in trapped CO2 was quantitated by liquid scintillation spectrophotometry. Results, expressed as nmol oleic acid oxidized per gram wet tissue at 15 min, are shown below.Table

Low-Dose Oral Glyburide Reduces Fasting Blood Glucose by Decreasing Hepatic Glucose Production in Healthy Volunteers Without Increasing Carbohydrate Oxidation

Glyburide is an effective hypoglycemic agent in patients with type II diabetes even after the loss of its ability to increase insulin secretion. The exact mechanism is unknown. In an attempt to describe the direct effect of glyburide on glucose metabolism, a very low dose of glyburide (20 micrograms/kg body weight) was given orally to 12 healthy volunteers in an attempt to increase blood concentrations of the drug without causing a marked increase in insulin secretion. Fasting hepatic glucose production (HGP), carbohydrate oxidation (CO), leucine appearance, leucine oxidation, and fat oxidation were determined between hours 3 and 4 and hours 7 and 8. The changes seen in the glyburide-treated volunteers were compared with the changes seen in 5 non-treated, healthy volunteers during the same 8-hour period. Mean blood glucose decreased greater in the glyburide-treated volunteers (20 +/- 2% vs 5 +/- 2%, P < 0.01). Insulin and C-peptide concentrations after glyburide administration (hour 7 to 8) did not differ significantly from baseline (hour 3 to 4) values (insulin: 53 +/- 9 pmol/L vs 52 +/- 9 pmol/L; C-peptide: 0.34 +/- 0.06 ng/mL vs 0.39 +/- 0.07 ng/mL). This low dose of glyburide resulted in a significantly greater decrease in HGP (16 +/- 2%; P < 0.001) than seen with fasting alone (8 +/- 4%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The Regulation of Mitochondrial Fatty Acid Oxidation and Hepatic Gene Expression by Catecholamine

We hypothesized that dopamine or dobutamine may alter hepatic mitochondrial fatty acid oxidation secondary to an effect on hepatic gene expression. We investigated the effect of dopamine or dobutamine on hepatic fat oxidation and gene transcription by studying the enzyme carnitine palmitoyltransferase (CPT), the rate-limiting step in hepatic mitochondrial long-chain fat oxidation. We incubated either H4IIE rat hepatoma cells or rat hepatocytes in primary cell culture with either dopamine (1,0.1, 0.01 μg/ml), dobutamine (1,0.1, 0.01 μg/ml), or vehicle control for 1, 2, 3, or 4 hr. We investigated the effect on (1) CPT mRNA (Northern or dot blotting) and the possible regulatory mechanism by incubating dopamine (0.1 μg/ml) or dobutamine (0.1 μg/ml) with propranolol or phentolamine, (2) CPT translation (CPT [ 35S]methionine incorporation), and (3) hepatic mitochondrial fatty acid oxidation ([1- 14C]palmitate oxidation to acid-soluble products). We conclude that (1) dopamine or dobutamine increases both hepatic CPT mRNA and CPT protein translation, (2) the effect on CPT mRNA is mediated by the β-receptor, (3) the increase in hepatic mitochondrial fat oxidation induced by dopamine or dobutamine may be, in part, secondary to increased CPT transcription and translation, and (4) the significant difference in hepatic fat oxidation induced by dopamine as compared with that by dobutamine is secondary to factors other than transcriptional or translational mechanisms. We speculate that dopamine treatment in the critically ill may increase hepatic mitochondrial fatty acid oxidation and ketogenesis and that this increase in β-oxidation may be, in part, secondary to increased CPT gene expression.

Hepatic fuel metabolism during muscular work: role and regulation

The increased fuel demands of the working muscle necessitate that metabolic processes within the liver be accelerated accordingly. The sum of changes in hepatic glycogenolysis and gluconeogenesis are closely coupled to the increase in glucose uptake by the working muscle, due to the actions of the pancreatic hormones. The exercise-induced rise in glucagon and fall in insulin interact to stimulate hepatic glycogenolysis, whereas the increase in gluconeogenesis is determined primarily by glucagon action. The increment in gluconeogenesis is caused by increases in hepatic gluconeogenic precursor delivery and fractional extraction as well as in the efficiency of intrahepatic conversion to glucose. Glucagon stimulates the latter two processes. Epinephrine may become important in the regulation of hepatic glucose production during prolonged or heavy exercise when its levels are particularly high. On the other hand, there is no evidence that hepatic innervation is essential for the rise in hepatic glucose production during exercise. Nonesterified fatty acid (NEFA) delivery to, uptake of, and oxidation by the liver are accelerated during prolonged exercise, resulting in an increase in ketogenesis. The rate of the first two of these processes is largely determined by factors that stimulate fat mobilization. The third step is regulated by both NEFA delivery to and glucagon-stimulated fat oxidation within the liver. The increase in hepatic fat oxidation produces energy that fuels gluconeogenesis. The shuttling of amino acids to the liver provides carbon-based compounds that are used for gluconeogenesis, transfers nitrogen to the liver, and supplies substrate for protein synthesis. During exercise, metabolic events within the liver, which are regulated by hormone levels and substrate supply, integrate pathways of carbohydrate, fat, and amino acid metabolism. These processes function to provide substrates for muscular energy metabolism and conserve carbon in glucose and nitrogen in protein.

Effects of choline deficiency and carnitine on palmitic acid oxidation by rat liver homogenates.

The conversion of C14 carboxyl-labeled palmitic acid to CO2 and ketones by liver homogenates prepared from choline deficient rats maintained on a 20% protein diet was within the normal range, indicating that hepatic fat oxidation is not impaired in the absence of choline alone. Carnitine addition in vitro augmented ketogenesis by livers from all groups of rats tested, while choline addition in vitro was without effect. Choline administration in vivo resulted in an enhanced oxidation of palmitic acid to CO2 by liver homogenates obtained from choline-deficient animals. However, liver preparations from animals injected in vivo with choline continued to respond to carnitine addition in vitro with an enhanced ketogenesis. It was concluded that carnitine and choline influence different parameters of fat metabolism.