A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels
Maja Stefanović-RačićGermán PerdomoBenjamin S. MantellIan SipulaNicholas F. BrownRobert M. O’Doherty
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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.Keywords:
Lipogenesis
Carnitine palmitoyltransferase I
Ketogenesis
Steatosis
DL-2-Rromooctanoate inhibits fatty acid oxidation in perfused rat liver and in mitochondria isolated from rat liver. Perfusion of livers for 12 min with 0.6 mu bromooctanoate causes complete and irreversible inhibition of ketogenesis from octanoate or oleate. Bromooctanoate also inhibits gluconeogenesis from lactate plus pyruvate but not from dihydroxyacetone. In isolated mitochondria, bromooctanoate irreversibly inhibits the oxidation of medium and long chain fatty acids and their L-carnitine esters. The extent of inhibition depends on the concentration of inhibitor and on the concentration of mitochondria. For example, 50% inhibition of palmitoyl L-carnitine oxidation is attained with about 2.5 nmol of bromooctanoate/mg of protein and 100% inhibition is attained with about 6 nmol of bromooctanoate/mg of protein. An energy-dependent activation of bromooctanoate occurs prior to the onset of inhibition. The conversion of bromooctanoate to its inhibitory form is not dependent on carnitine. Direct measurement of the content of coenzyme A and its derivatives in inhibited mitochondria indicates that the inhibitory effects of bromooctanoate are not caused by depletion of mitochondrial free CoA in the form of bromooctanoyl-CoA. Treatment of mitochondria with bromooctanoate does not prevent the subsequent formation of acyl-CoA from added fatty acid substrates. These and other results support the conclusion that an activated derivative of 2-bromooctanoic acid inhibits one or more of the enzymes of p-oxidation.
Ketogenesis
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Dihydroxyacetone
Coenzyme A
Palmitoylcarnitine
Carnitine palmitoyltransferase I
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We examined the potential of overt carnitine palmitoyltransferase (CPT I) to control the hepatic catabolism of palmitoyl-CoA in suckling and adult rats, using a conceptually simplified model of fatty acid oxidation and ketogenesis. By applying top-down control analysis, we quantified the control exerted by CPT I over total carbon flux from palmitoyl-CoA to ketone bodies and carbon dioxide. Our results show that in both suckling and adult rat, CPT I exerts very significant control over the pathways under investigation. However, under the sets of conditions we studied, less control is exerted by CPT I over total carbon flux in mitochondria isolated from suckling rats than in those isolated from adult rats. Furthermore the flux control coefficient of CPT I changes with malonyl-CoA concentration and ATP turnover rate.
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Catabolism
Carnitine palmitoyltransferase I
Carnitine O-palmitoyltransferase
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Fatty acid metabolism has been studied in Fao rat hepatoma cells. In basal conditions of culture, [l‐ 14 C]oleate is mainly esterified (85% of oleate uptake) in Fao cells, phospholipids being the most important esterified products (60% of oleate esterified). Addition of N 6 ,O 2 ′‐dibutyryl‐adenosine 3′,5′‐monophosphate (0.1 mM) in Fao cells does not change the metabolic fate of oleate whereas it induces gluconeogenesis and phospho enol pyruvate carboxykinase mRNA accumulation. It is shown that the limitation of oleate oxidation is located at the level of the entry into mitochondria since octanoate is actively oxidized in Fao cells. Neither the activities of carnitine palmitoyltransferase (CPT) I and II nor the CPT II protein amount are affected by cAMP addition. The limitation of oleate oxidation in Fao cells results from (a) a high rate of lipogenesis and a high malonyl‐CoA concentration, (b) a CPT I very sensitive to malonyl‐CoA inhibition. The presence of an active oleate oxidation in mitochondria isolated from Fao cells confirms that CPT I is the limiting step of oleate oxidation. Moreover, Fao cells are unable to perform ketogenesis. This particular feature results from a specific deficiency in mitochondrial hydroxymethylglutaryl‐CoA synthase protein, activity and gene expression. The metabolic characteristics observed in Fao cells could be a common feature in hepatoma cell lines with regard to the low capacity for long‐chain fatty acid oxidation and ketone body production observed in the rat H4IIE and the human HepG2 cells.
Ketogenesis
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Carnitine palmitoyltransferase I
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Ketogenesis
Malonyl-CoA
Carnitine O-palmitoyltransferase
Acyltransferases
Acyl-CoA
Carnitine palmitoyltransferase I
Acyltransferases
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The development of long-chain fatty acid (LCFA) oxidation, either in the liver for ketone body and energy productions or in peripheral tissues as oxidative fuels, is essential for the newborn mammals. At least in the liver, the postnatal development of LCFA oxidation and ketogenesis seems regulated by pancreatic hormones which plasmatic concentrations are markedly changed at birth (fall in insulin and rise in glucagon levels). In cultured hepatocytes from rabbit fetuses (no LCFA oxidation), the addition of glucagon or cyclic AMP induces LCFA oxidation at a level similar to that found in 24-h-old newborns (high LCFA oxidation). The presence of insulin inhibits totally the effects of glucagon. It seems that carnitine palmitoyltransferase I (CPT I), a key enzyme of LCFA oxidation, represents the main site for hormonal control of LCFA oxidation. This regulation is not due to changes in the hepatic malonyl-CoA concentration (a metabolic intermediate in lipogenesis and a potent inhibitor of CPT I) but to modifications in the sensitivity of CPT I to malonyl-CoA inhibition. The molecular mechanisms responsible for the changes in the sensitivity of CPT I are discussed.
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Carnitine palmitoyltransferase I
Malonyl-CoA
Carnitine O-palmitoyltransferase
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Studied on the oxidation of oleic and octanoic acids to ketone bodies were carried out in homogenates and in mitochondrial fractions of livers taken from fed and fasted rats. Malonyl-CoA inhibited ketogenesis from the former but not from the latter substrate. The site of inhibition appeared to be the carnitine acyltransferase I reaction. The effect was specific and easily reversible. Inhibitory concentrations were in the range of values obtained in livers from fed rats by others. It is proposed that malonyl-CoA functions as both precursor for fatty acid synthesis and suppressor of fatty acid oxidation. As such, it might be an important element in the carbohydrate-induced sparing of fatty acid oxidation.
Ketogenesis
Malonyl-CoA
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Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of long-chain fatty acids (LCFA) for oxidation and for peroxisome proliferator-activated receptor alpha (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced beta-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. During standard diet, mitochondrial HMG CoA synthase mRNA was selectively reduced in L-FABP null liver. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism, whereas L-FABP can activate ketogenic gene expression in fed mice. Thus, the mechanisms whereby L-FABP affects fatty acid oxidation may vary with physiological condition.
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Carnitine O-palmitoyltransferase
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1. An inverse logarithmic relationship was found between lipogenesis and the concentration of serum free fatty acids perfusing the rat liver. 2. Increased concentrations of serum free fatty acids did not alter cholesterol synthesis, triglyceride secretion or increase gluconeogenesis. 3. No direct relationship was found between ketogenesis and either lipogenesis or gluconeogenesis.
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Lipogenesis is increased in hepatocytes from fed lactating rats compared with virgin rats. Inhibition of lipogenesis with 5-(tetradecyloxy)-2-furoic acid resulted in increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased lipogenesis and esterification of [1–14C]oleate and decreased ketogenesis; these changes were reversed by the inhibitor. The reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed rats may be due to alterations in [malonyl-CoA] [McGarry, Mannaerts & Foster (1977) J. Clin. Invest. 60, 265–270; Cook, King & Veech (1978) J. Biol. Chem. 253, 2529–2531], but this mechanism is not considered to be sufficient to explain the increased ketogenesis in starvation completely.
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Dihydroxyacetone
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