Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Identification of dicarboxylate carrier Slc25a10 as malate transporter in de novo fatty acid synthesis

Mitochondrial solute carrier family 25 member 10 (Slc25a10) transports dicarboxylates such as malate or succinate across the mitochondrial inner membrane. Although fatty acid synthesis in adipose tissue or the liver is initiated by citrate transport in exchange for malate across the mitochondrial membrane, the transporter responsible for supplying malate during citrate transport has not been identified. In the present study, we clarified the role of Slc25a10 in supplying malate for citrate transport and examined the effect of Slc25a10 suppression on the lipogenic pathway and lipid accumulation. We have reported an Slc25a10 increase in white adipose tissue in obese mouse models and a decrease in a fasted mouse model using expression profiles. Next, we examined the effect of Slc25a10 suppression by small interfering RNA on citrate transport in the lipogenic cell lines HepG2 and 3T3-L1. We observed that inhibition of malate transport by Slc25a10 suppression significantly reduced the citrate transport from the mitochondria to the cytosol. We also found that suppression of Slc25a10 down-regulated the lipogenic pathway, indicated by decreases in ACC1 expression and malonyl-CoA level. Furthermore, suppression of Slc25a10 decreased triglyceride lipid accumulation in adipose-differentiated 3T3-L1 cells. These results suggested that Slc25a10 plays an important role in supplying malate for citrate transport required for fatty acid synthesis and indicated that inhibition of Slc25a10 might effectively reduce lipid accumulation in adipose tissues.     

Diurnal variations of lipogenic enzymes, their substrate and effector levels, and lipogenesis from tritiated water in rat liver

The activities of glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthetase and acetyl-CoA carboxylase (extracted with or without phosphatase inhibitor) in rat liver did not vary significantly during 24 h. The hepatic levels of glucose 6-phosphate and malate increased coordinately 3-6 h after the beginning (1900 h) of food intake and were high until morning, whereas the levels of acetyl-CoA and citrate peaked at 1900 h and then decreased. However, it is remarkable that the in vivo incorporation of 3H from tritiated water into fatty acids in liver increased with the level of malonyl-CoA after food intake. Comparing the substrate and effector levels with the Km and Ka values for the enzymes, the levels of acetyl-CoA, malonyl-CoA and citrate appear to limit the enzyme activities. It is suggested that, after food intake, the physiological activity of acetyl-CoA carboxylase was increased with the substrate increase and/or with the catalytic activation with citrate, and consequently, the fatty acid synthetase activity was also increased, whereas the enzyme activities measured under optimum conditions were not.     

Citrate carrier and lipogenic enzyme activities in lead nitrate-induced proliferative and apoptotic phase in rat liver.

After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in rat liver. The rate of citrate transport was greatly reduced in rats during both the proliferative phase (3 days after the lead nitrate administration) and the involutive phase (5 days after the metal injection), which follows hepatic hyperplasia and corresponds to the peak of hepatocyte apoptosis. In both phases, a decrease of the lipogenic enzyme activities has been detected. In treated animals, an alteration of mitochondrial lipid composition has also been found. The modified lipid microenvironment could be responsible for the decreased carrier activity which, in turn, may account for the reduced activities of the lipogenic enzymes.     

Altered interactions between lipogenesis and fatty acid oxidation in regenerating rat liver.

The concentrations of malonyl-CoA, citrate, ketone bodies and long-chain acylcarnitine were measured in freeze-clamped liver samples from fed or starved normal, partially hepatectomized or sham-operated rats. These parameters were used in conjunction with measurements of the concentration of plasma non-esterified fatty acids and the rates of hepatic lipogenesis to obtain correlations between rates of fatty acid delivery to the liver, lipogenesis and fatty acid oxidation to ketone bodies and CO2. These correlations indicated that the development of fatty liver after partial hepatectomy is due to an increased partitioning of long-chain acyl-CoA towards acylglycerol synthesis and away from acylcarnitine formation. However, this did not appear to be due to an altered relationship between hepatic malonyl-CoA concentration and acylcarnitine formation. For any concentration of long-chain acylcarnitine, the concentrations of both hepatic and blood ketone bodies were significantly lower in partially hepatectomized rats than in normal or sham-operated animals. This indicated that a lower proportion of the product of beta-oxidation was used for ketone-body formation and more for citrate synthesis in the regenerating liver, especially during the first 24 h after resection. This inference was supported by the changes in hepatic citrate concentrations observed. The high rates of lipogenesis that occurred in the liver remnant were accompanied by an altered relationship between lipogenic rate and hepatic malonyl-CoA concentration, such that much lower concentrations of malonyl-CoA were associated with any given rate of lipogenesis. These adaptations are discussed in relation to the requirements by the remnant for high rates of energy formation through the tricarboxylic acid cycle during the first 24 h after resection, and the possibility that cycling between fatty acid oxidation and synthesis may occur to a greater degree in regenerating liver.     

Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco

ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi. To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco. This was sufficient to provide import of the heterologous protein into the plastids. In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA. Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile. Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.     

Effect of nitrogen sources on the activities of lipogenic enzymes in oleaginous fungus Cunninghamella echinulata

Various inorganic and organic nitrogen sources were used to compare their effects on the lipogenesis and the activities of lipogenic enzymes (providing acetyl-CoA and donating NADPH) in gamma-linolenic acid-producing fungus Cunninghamella echinulata. Lipid accumulation was enhanced by organic nitrogen, among them the presence of corn-steep led to almost 40% oil in the biomass. While organic nitrogen increased activities of acetyl-CoA carboxylase (ACC) and malic enzyme (ME), ATP:citrate lyase (ACL) was rapidly enhanced by ammonium ion. The use of NaNO(3) resulted in high activities of glucose 6-phosphate dehydrogenase (GPD) and 6-phosphogluconate dehydrogenase (PGD). NADP-isocitrate dehydrogenase (NADP-ICD) was more active when the fungus utilized all inorganic N-compounds. The rise of nitrogen concentration in medium was accompanied with reduced lipid accumulation and a fall of ACL, ACC, and ME. In contrast, N-sufficient conditions favored biomass growth and elevated activities of GPD and PGD. Kinetic experiments also suggest that a significant portion of the required acetyl-CoA was being provided via ACL and ACC, and ME (probably coupled with GPD) channeled the NADPH into the fatty acid biosynthesis. The contribution of the lipogenic enzymes to metabolic pathways other than lipogenesis is also discussed.     

IL-1 Signaling in Obesity-Induced Hepatic Lipogenesis and Steatosis

Non-alcoholic fatty liver disease is prevalent in human obesity and type 2 diabetes, and is characterized by increases in both hepatic triglyceride accumulation (denoted as steatosis) and expression of pro-inflammatory cytokines such as IL-1β. We report here that the development of hepatic steatosis requires IL-1 signaling, which upregulates Fatty acid synthase to promote hepatic lipogenesis. Using clodronate liposomes to selectively deplete liver Kupffer cells in ob/ob mice, we observed remarkable amelioration of obesity-induced hepatic steatosis and reductions in liver weight, triglyceride content and lipogenic enzyme expressions. Similar results were obtained with diet-induced obese mice, although visceral adipose tissue macrophage depletion also occurred in response to clodronate liposomes in this model. There were no differences in the food intake, whole body metabolic parameters, serum β-hydroxybutyrate levels or lipid profiles due to clodronate-treatment, but hepatic cytokine gene expressions including IL-1β were decreased. Conversely, treatment of primary mouse hepatocytes with IL-1β significantly increased triglyceride accumulation and Fatty acid synthase expression. Furthermore, the administration of IL-1 receptor antagonist to obese mice markedly reduced obesity-induced steatosis and hepatic lipogenic gene expression. Collectively, our findings suggest that IL-1β signaling upregulates hepatic lipogenesis in obesity, and is essential for the induction of pathogenic hepatic steatosis in obese mice.     

Leptin Contributes to the Adaptive Responses of Mice to High-Fat Diet Intake through Suppressing the Lipogenic Pathway

Background

Leptin is an adipocyte-derived hormone that plays a critical role in energy homeostasis and lipid metabolism. Overnutrition-associated obesity is known to be accompanied by hyperleptinemia. However, the physiological actions of leptin in the metabolic responses to high-fat diet (HFD) intake remain to be completely elucidated. Here we characterized the metabolic features of mice fed high-fat diets and investigated the impact of leptin upon the lipogenic program which was found to be suppressed by HFD feeding through a proteomics approach.

Results

When maintained on two types of high-fat diets for up to 16 weeks, mice with a higher fat intake exhibited increased body fat accumulation at a greater pace, developing more severely impaired glucose tolerance. Notably, HFD feeding at 4 weeks elicited the onset of marked hyperleptinemia, prior to the occurrence of apparent insulin resistance and hyperinsulinemia. Proteomic analysis revealed dramatically decreased expression of lipogenic enzymes in the white adipose tissue (WAT) from HFD-fed mice, including ATP-citrate lyase (ACL) and fatty acid synthase (FAS). The expression of ACL and FAS in the liver was similarly suppressed in response to HFD feeding. By contrast, HFD-induced downregulation of hepatic ACL and FAS was significantly attenuated in leptin receptor-deficient db/db mice. Furthermore, in the liver and WAT of wild type animals, intraperitoneal leptin administration was able to directly suppress the expression of these two lipogenic enzymes, accompanied by reduced triglyceride levels both in the liver and serum.

Conclusions

These results suggest that leptin contributes to the metabolic responses in adaptation to overnutrition through suppressing the expression of lipogenic enzymes, and that the lipogenic pathway represents a key targeted peripheral component in exerting leptin''s liporegulatory actions.     

Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.     

Compensatory increase in lipogenic gene expression in adipose tissue of transgenic mice expressing constitutively active AMP-activated protein kinase-alpha1 in liver

We previously described a line of transgenic mice selectively expressing constitutively active AMPK-α1 under the control of liver-specific human apoE promoter with the hepatic control region sequence. In the short-term activation, the CA-AMPK-α1 transgenic mice at age 10–12 weeks exhibited normal hepatic triglyceride content as compared to wild-type mice due to compensatory increase in mRNA expression of genes in the cholesterol and fatty acid synthesis pathways. But it was not known whether the lipogenic gene expression in white adipose tissue also changed. Here we characterized mRNA expression profile of main lipogenic genes in the cholesterol and fatty acid biosynthesis pathway in white adipose tissue. The data show that short-term chronic activation of AMPK in liver caused marked compensatory increase in lipogenic gene expression both in liver due to induction of Srebp-2 and in white adipose tissue due to upregulation of Srebp-1c. These results support the notion that in addition to its well-recognized function for fat storage adipose tissue can play an adaptive role in fatty acid synthesis when fatty acid synthesis is severely reduced in liver, the main lipogenic organ in mammals.     

Decreased hepatic fatty acid oxidation at weaning in the rat is not linked to a variation of malonyl-CoA concentration

In rats weaned on a high-carbohydrate diet, hepatic fatty acid oxidation capacity is decreased when compared to suckling rats. Previous studies (Benito et al., 1979) suggested that a malonyl-CoA-dependent mechanism could be at the origin of this decrease. Studies on isolated hepatocytes show that despite, respectively, a low and a high lipogenic rate in suckling and weaned rats, malonyl-CoA concentrations are similar in the two groups. This might be due to the lower ratio fatty acid synthetase/acetyl-CoA carboxylase (EC 6.4.1.2) activities during suckling than after weaning. Different rates of hepatic fatty acid oxidation despite similar malonyl-CoA concentrations can be explained by the 2.5-fold higher carnitine palmitoyltransferase I (EC 2.3.1.21) activity in suckling rats together with a 7-fold higher Ki for malonyl-CoA. This precludes a tight control of fatty acid oxidation by [malonyl-CoA] in suckling rats. Weaning on a high-fat carbohydrate-free diet abolishes the changes previously described for the kinetic characteristics of carnitine palmitoyltransferase I suggesting that nutritional modifications rather than a developmental stage are involved. Thus, during the suckling-weaning transition, a variation of [malonyl-CoA] is not responsible for the decrease in hepatic fatty acid oxidation. It involves, in addition, a decrease in carnitine palmitoyltransferase I activity and an increase of the sensitivity of this enzyme to malonyl-CoA.     

Determination of malonyl-coenzyme A in rat heart,kidney, and liver: A comparison between acetyl-coenzyme A and butyryl-coenzyme A as fatty acid synthase primers in the assay procedure

The malonyl-CoA assay was nonlinear at low malonyl-CoA concentrations when labeled acetyl-CoA was used as fatty acid synthase primer. Linearity was obtained with low concentrations of both fatty acid synthase and labeled acetyl-CoA, but then the assay was disturbed by the diluting effect of endogenous acetyl-CoA. The problems of nonlinearity and dilution of radioactivity by endogenous compounds were absent when labeled butyryl-CoA was used as primer. The levels of malonyl-CoA in rat heart, kidney, and liver were determined. The use of butyryl-CoA gave higher values of malonyl-CoA.     

Pharmacological doses of triiodothyronine upregulate lipogenic enzyme gene expression in rat white adipose tissue.

Triiodothyronine (T (3)) is known to increase liver lipogenic enzyme gene expression both in vivo and in tissue culture. Conflicting results have been reported on the effect of T (3) on lipogenic enzyme gene expression in white adipose tissue. The results presented in this paper indicate that administration of pharmacological doses of T (3) in rats leads to increased fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACL) and malic enzyme (ME) activity in white adipose tissue. The increase in lipogenic enzyme activity was associated with increased FAS, ACC, ACL and ME mRNA levels. The response was dose-dependent. Activity of lipogenic enzyme and the lipogenic enzyme mRNA levels were positively correlated to serum T (3) concentration. The in vivo effect of T (3) on lipogenic enzyme gene expression could be reproduced in primary white rat adipocyte culture. In conclusion, the results presented in this paper indicate that T (3) exerts a stimulatory effect on lipogenic enzyme gene expression in white adipose tissue both in vivo and in tissue culture. Significant effects of T (3) on lipogenic enzyme gene expression were only observed in the presence of relatively high (pharmacological) concentrations of the hormone.