Esterification Of Exogenous Fatty Acids Research Articles

Specific role for acyl CoA

Nonalcoholic fatty liver disease, characterized by the accumulation of triacylglycerols (TGs) and other lipids in the liver, often accompanies obesity and is a risk factor for nonalcoholic steatohepatitis and fibrosis. To treat or prevent fatty liver, a thorough understanding of hepatic fatty acid and TG metabolism is crucial. To investigate the role of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of TG synthesis, in fatty liver development, we studied mice with global and liver-specific knockout of Dgat1. DGAT1 was required for hepatic steatosis induced by a high-fat diet and prolonged fasting, which are both characterized by delivery of exogenous fatty acids to the liver. Studies in primary hepatocytes showed that DGAT1 deficiency protected against hepatic steatosis by reducing synthesis and increasing the oxidation of fatty acids. In contrast, lipodystrophy (aP2-SREBP-1c436) and liver X receptor activation (T0901317), which increase de novo fatty acid synthesis in liver, caused steatosis independently of DGAT1. Pharmacologic inhibition of Dgat1 with antisense oligonucleotides protected against fatty liver induced by a high-fat diet. Our findings identify a specific role for hepatic DGAT1 in esterification of exogenous fatty acids and indicate that DGAT1 contributes to hepatic steatosis induced by this mechanism.

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.

The mammalian target of rapamycin regulates lipid metabolism in primary cultures of rat hepatocytes

The mammalian target of rapamycin (mTOR) is a conserved serine-threonine kinase that regulates cell growth and metabolism in response to nutrient signals. However, the specific involvement of mTOR in regulation of energy metabolism is poorly understood. To determine if signaling via mTOR might be directly involved in regulation of fatty acid metabolism in hepatocytes, we performed studies with rapamycin, a specific inhibitor of mTOR. Rapamycin-mediated inhibition of mTOR (18-48 hours) increased oxidation of exogenous fatty acids (46%-100%, respectively). In addition, esterification of exogenous fatty acids and de novo lipid synthesis were reduced (40%-60%, respectively). Consistent with inhibition of lipogenic pathways, rapamycin decreased expression of genes encoding acetyl–coenzyme A carboxylase I and mitochondrial glycerol phosphate acyltransferase. Non–insulin-dependent glucose transport and glycogen synthesis were decreased by 20% to 30%, whereas glucose utilization was unaffected by rapamycin. The data suggest that the hyperlipidemia observed with the drug in vivo is likely not the result of enhanced hepatic synthesis, but rather of delayed peripheral clearance. However, these results are consistent with the idea that mTOR may play a significant role, not only in “energy sensing,” but also in regulation of energy production through profound effects on hepatic fatty acid metabolism.

Stearoyl CoA desaturase 1 is elevated in obesity but protects against fatty acid-induced skeletal muscle insulin resistance in vitro

Stearoyl CoA desaturase 1 (SCD1) is implicated in mediating obesity and insulin resistance. Paradoxically, SCD1 converts saturated fatty acids, the lipid species implicated in mediating insulin resistance, to monounsaturated fatty acids. The aim of the present study was to assess the molecular mechanisms that implicate SCD1 in the aetiology of fatty acid-induced insulin resistance. SCD1 protein was transiently decreased or increased in rat L6 skeletal muscle myotubes using SCD1 short interfering RNA (siRNA) or liposome-mediated transfection of pcDNA3.1/Hygro-mSCD1, respectively. Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol (DAG) and ceramide. Insulin-stimulated Akt activity and phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and DAG accumulation and protected myotubes from fatty acid-induced insulin resistance. SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites.

Regulatory factors in the development of fatty infiltration of the liver during gram-negative sepsis

To further understand the development of fatty liver during gram-negative sepsis, we measured fatty acid uptake in addition to esterification and secretion of lipids by freshly isolated hepatocytes from fasted and fed control and Escherichia coli-treated rats. Rats were made septic by intravenous (IV) injection of 8 × 10 7 live E coli colonies per 100 g body weight. For the fasted groups, food was removed after E coli injection. Fed rats received a nutritionally adequate diet intragastrically for 5 days before and 24 hours after inducing sepsis. Twenty-four hours after E coli injection, the esterification of newly synthesized fatty acids, as measured by 3H 2O incorporation, and the esterification of exogenous fatty acids, measured from 14C-palmitate incorporation, into triglyceride (TG), total cholesterol, and total phospholipid phosphorus were significantly greater in hepatocytes from fasted septic rats compared with their control rats. In fed septic rats, esterification of 14C-palmitate into TG was fourfold greater than in the fed control rats. The increased rates of esterification in hepatocytes from fasted and fed septic rats were not accompanied by an increase in the labeled TG in the medium. This inability to secrete the additional TG that the hepatocytes produce resulted in a higher concentration of cellular TG in fasted and fed septic rats than in their controls. The enzymes glycerol-3-phosphate acyltransferase (GPAT) and phosphatidate phosphohydrolase (PPH) do not appear to be factors contributing to the increased TG synthesis, since the increase in enzyme activity was not accompanied by a similar increase in TG synthesis. When sepsis was induced in the fasted but not in the fed state, the significant increase in free fatty acid (FFA) uptake and the content of glycerol-3-phosphate (G3P) contributed to the increase in TG synthesis. These results demonstrate that lipids accumulate in hepatocytes from septic rats because of the elevated rate of TG synthesis without a similar increase in secretion rates. Increased intracellular lipolysis/re-esterification and altered hormonal levels may be more important factors than enzyme regulation in determining why the liver retains TG during sepsis.

Zonation of fatty acid metabolism in rat liver.

Fatty acid metabolism was studied in periportal and perivenous hepatocytes isolated by the method of Chen & Katz [Biochem. J. (1988) 255, 99-104]. The rate of fatty acid synthesis and the activity of acetyl-CoA carboxylase were markedly enhanced in perivenous hepatocytes as compared with periportal cells. However, the response of these two parameters to short-term modulation by cellular effectors such as the hormones insulin and glucagon, the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and the xenobiotics ethanol and acetaldehyde was similar in the two zones of the liver. In addition, perivenous hepatocytes showed a higher capacity of esterification of exogenous fatty acids into both cellular and very-low-density-lipoprotein lipids. Nevertheless, no difference between the two cell sub-populations seemed to exist in relation to the secretion of very-low-density lipoproteins. On the other hand, the rate of fatty acid oxidation was increased in periportal cells. This could be accounted for by a higher activity of carnitine palmitoyltransferase I and a lower sensitivity of this enzyme to inhibition by malonyl-CoA in the periportal zone. No differences were observed between periportal and perivenous hepatocytes in relation to the short-term response of fatty acid oxidation and carnitine palmitoyltransferase I activity to the cellular modulators mentioned above. In conclusion, our results show that: (i) lipogenesis is achieved at higher rates in the perivenous zone of the liver, whereas the fatty-acid-oxidative process occurs with a certain preference in the periportal area of this organ; (ii) the short-term response of the different fatty-acid-metabolizing pathways to cellular effectors is quantitatively similar in the two zones of the liver.

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [ 3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [ 3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 μM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.

Esterification of exogenous and endogenous fatty acids by rat adipocytes in vitro

Rat adipocytes were used in vivo to compare the esterification of exogenous fatty acids and fatty acids formed de novo from glucose or acetate. Pure single fatty acids added to the medium were esterified at comparable rates but marked differences were observed when the same acids were supplied as components of a fatty acid mixture of a composition similar to that in the tissue. Fatty acids synthesised de novo from acetate by adipocytes in a medium containing high concentrations of acetate were located predominantly in diacylglycerols. The effect was most marked with adipocytes from older rats and was enhanced by the presence of exogenous long-chain fatty acids. Exogenous oleic acid was esterified predominantly into triacylglycerols at all concentrations of acetate. No such accumulation of endogenously-synthesised fatty acids in diacylglycerols occurred when glucose was the precursor for fatty acid synthesis. The diacylglycerols formed were almost entirely of the sn-1,2-configuration.

The esterification of exogenous fatty acids by adipose tissue of hypertriglyceridaemic subjects with or without diabetes mellitus.

1. The esterification of exogenous palmitate to diglyceride and triglyceride in adipocytes was studied in obese and diabetic patients with and without hypertriglyceridaemia. The rate of esterification correlated significantly with the triglyceride content of adipocytes. 2. In diabetic patients with hypertriglyceridaemia, the rate of esterification to triglyceride was significantly greater than in diabetic patients with normotriglyceridaemia. This difference could not be attributed to differences in glucose tolerance or to the degree of obesity. 3. Fasting plasma insulin levels were greater in the hypertriglyceridaemic group than in the normotriglyceridaemic group. The difference in esterification rates could have been due to differences in adipocyte size. 4. The esterification of fatty acid in adipose tissue of diabetic patients was lower than in non-diabetic subjects and this difference could not be accounted for by differences in adipocyte size or differences in the intracellular pools of fatty acid in adipose tissue. 5. The role of esterification of exogenous fatty acids in adipose tissue as a possible determinant of the uptake of glyceride fatty acids from plasma is discussed.

Utilisation of exogenous fatty acids by rat epididymal fat cells in vitro

When isolated rat fat cells are incubated with radioactively labelled palmitate the extracellular fatty acid pool is diluted by fatty acids of endogenous origin. Using fat cell concentration as an experimental variable, a simple graphical procedure has been devised to correct for the difficulty such precursor dilution causes in measurement of oxidation and esterification of exogenous fatty acids. After application of such corrections it was calculated that extracellular palmitate was utilised for oxidation with a composite K m of approx. 7 μM. The composite K m for palmitate esterification was 29 μM (no other substrates present, albumin included at 10 mg/ml).