Carnitine Palmitoyltransferase mRNA Research Articles

Aerobic Exercise’s Reversal of Insulin Resistance by Activating AMPKα–ACC–CPT1 Signaling in the Skeletal Muscle of C57BL/6 Mice

Insulin resistance (IR) is a common pathophysiological feature of Type 2 diabetes. Although the mechanisms leading to IR are still elusive, evidence has shown that aerobic exercise can reverse this process. To investigate the effects of aerobic exercise on IR, the authors created an IR animal model by feeding C57BL/6 mice a high-fat diet for 8 wk. They then compared the effect of 6 wk of treadmill training (60 min/d) at 75% VO2max on mice in normal-diet (NE) and high-fat-diet (HE) groups with their sedentary control groups. Levels of skeletal-muscle AMPKα (AMP-activated protein kinase α), ACC (acetyl-CoA carboxylases), and CPT1 (carnitine palmitoyltransferase 1) mRNA and AMPKα, pAMPK-Thr172, ACC, pACC-Ser79, and CPT1 protein expressions were analyzed. In addition, fasting serum levels of insulin, triglyceride, and cholesterol were measured. The results demonstrate that 6 wk of exercise increased AMPKα mRNA expression by 11% and 25 % (p<.01) in the NE and HE groups, respectively, and AMPKα protein expression by 37.9% and 20.1% (p<.01) in NE and HE compared with their sedentary control. In addition, ACC mRNA and protein expressions declined, whereas CPT1 mRNA and protein expressions were elevated in both exercise groups compared with sedentary control groups. In addition, pAMPK-Thr172 and pACC-Ser79 expression increased significantly in the NE and HE groups compared with sedentary control groups. In conclusion, our results demonstrate that 6 wk of aerobic exercise can effectively ameliorate IR by increasing the expression of AMPKα and pAMPK-Thr172, thereby activating the key enzymes that facilitate lipid metabolism.

Effects of Changes of Diet from High‐fat to Low‐fat on Hepatic mRNA Levels of ACS, CPT‐1, and ACC

AimsThe purpose of this study was to investigate the effects of dietary changes from high‐fat diet (HFD, 45% of total calories from fat) to low‐fat diet (LFD, 10% of total calories from fat) on hepatic mRNA levels of acyl coenzyme A synthetase(ACS), carnitine palmitoyltransferase‐1(CPT‐1), and acetyl coenzyme A carboxylase(ACC) as well as on serum and hepatic lipids in mice.MethodsFive weeks‐old male C57BL/6 mice were fed high‐fat diet for 9 weeks then switched to a low‐fat diet or continued the high‐fat diet for 3 weeks. Body weight, serum and hepatic lipid levels were measured. Expressions of ACS, CPT‐1, and ACC mRNA were also analyzed.ResultsThe results showed that after the high‐fat diets were switched to low fat diets, body weight, epididymal and retroperitoneal fats were significantly reduced (p&lt;0.05). The levels of serum and hepatic total cholesterol were not different between the two groups however, serum TG levels of high‐fat diet followed by low‐fat diet group were significantly lower than those of high‐fat diet group (p&lt;0.05). Hepatic mRNA levels of CPT‐1 and ACC were higher significantly in the group of high‐fat diet followed by low‐fat diet (p&lt;0.05) however mRNA levels of ACS were not different between the two groups.ConclusionsChanges in the composition of dietary fat from high to low decreased significantly body fats and the serum TGs. Furthermore, changes of dietary fat composition affected differently the hepatic mRNA levels of ACS, CPT‐1 and ACC.

Aldose Reductase Regulates Hepatic Peroxisome Proliferator-activated Receptor α Phosphorylation and Activity to Impact Lipid Homeostasis

Aldose reductase (AR) is implicated in the development of a number of diabetic complications, but the underlying mechanisms remain to be fully elucidated. We performed this study to determine whether and how AR might influence hepatic peroxisome proliferator-activated receptor alpha (PPARalpha) activity and lipid metabolism. Our results in mouse hepatocyte AML12 cells show that AR overexpression caused strong suppression of PPARalpha/delta activity (74%, p < 0.001) together with significant down-regulation of mRNA expression for acetyl-CoA oxidase and carnitine palmitoyltransferase-1. These suppressive effects were attenuated by the selective AR inhibitor zopolrestat. Furthermore, AR overexpression greatly increased the levels of phosphorylated PPARalpha and ERK1/2. Moreover, AR-induced suppression of PPARalpha activity was attenuated by treatment with an inhibitor for ERK1/2 but not that for phosphoinositide 3-kinase, p38, or JNK. Importantly, similar effects were observed for cells exposed to 25 mm glucose. In streptozotocin-diabetic mice, AR inhibitor treatment or genetic deficiency of AR resulted in significant dephosphorylation of both PPARalpha and ERK1/2. With the dephosphorylation of PPARalpha, hepatic acetyl-CoA oxidase and apolipoprotein C-III mRNA expression was greatly affected and that was associated with substantial reductions in blood triglyceride and nonesterified fatty acid levels. These data indicate that AR plays an important role in the regulation of hepatic PPARalpha phosphorylation and activity and lipid homeostasis. A significant portion of the AR-induced modulation is achieved through ERK1/2 signaling.

Effects of fenofibrate on gene expression of carnitine palmitoyltransferase 1 in liver and skeletal muscle and its influence on insulin sensitivity

To study the effects of fenofibrate (FF), a peroxisome proliferator activated receptor (PPAR) alpha activator, on the expression of carnitine palmitoyltransferase 1 (CPT-1) mRNA in liver and muscle and its influence on insulin sensitivity. Thirty-two normal 8 week-old male SD rats were randomly divided into 3 groups: normal control group, fed with normal food for 3 weeks (NC group, n = 10), high fat diet group, fed with high fat food (HF group, n = 10), and high fat diet supplemented with FF group, fed with high fat food and given with gastric perfusion of FF (50 mg x kg(-1) x d(-1)) (FF group, n = 12). Fast serum triglyceride (TG) level was tested by automatic biochemical analyzer after 8-10 h fasting. Euglycemic-hyperinsulinemic clamp method was used to calculate the glucose infusion rate (GIR) so as to evaluate the insulin sensitivity. By the end of experiment the rats were killed with their internal organs taken out. The triglyceride (TG) contents of liver and skeletal muscle were measured using Folch method. Real-time PCR was used to detect the mRNA expression. of CPT-1 in the liver and skeletal muscles. As compared with the NC group, the TG levels of serum, liver, and skeletal muscle of the HF group were higher than those of the NC group by 0.45-fold, 2.14-fold, and 10.64-fold respectively. The GIR of the HF group was (6.2 +/- 0.8) mg x kg(-1) x min(-1), significantly lower than that of the NC group (15.8 +/- 2.1) mg x kg(-1) x min(-1), (P < 0.01), and that of the P < 0.01. The CPT-1 mRNA expression in liver of the HF group was not significantly different from that of the NC group (P > 0.05); the expression of CPT-1 mRNA in skeletal muscle of the HF group was lower than that of the NC group by 71% (P < 0.01). The CPT-1 mRNA expression in liver and skeletal muscle of the FF group were significantly higher than those of the HF group by 1.00 and 1.05 times respectively (both P < 05). The GIR was negatively correlated with the levels in the liver (r = -0.87, P < 0.01) and in the skeletal muscle (r = -0.78, P < 0.01). Fenofibrate promotes the oxygenation of fatty acids by up-regulating the CPT-1 mRNA expression in the liver and skeletal muscles, thus improving the insulin sensitivity.

Carbacyclin induces carnitine palmitoyltransferase-1 in cardiomyocytes via peroxisome proliferator-activated receptor (PPAR) δ independent of the IP receptor signaling pathway

Prostacyclin (PGI2) and its analogues exert cardioprotective effects via the rhodopsin type membrane PGI2 receptor, IP. Peroxisome proliferator-activated receptor (PPAR) δ is a nuclear receptor abundantly expressed in cardiomyocytes and plays a pivotal role in maintaining constitutive mitochondrial fatty acid β-oxidation (FAO). Recently, a novel signaling pathway of PGI2 via PPARδ has been demonstrated in non-cardiac tissues. We therefore examined whether carbacyclin (cPGI2), a PGI2 analogue, up-regulates transcriptional expression of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzyme in mitochondrial FAO, via PPARδ in cardiomyocytes. Intraperitoneal injection of cPGI2 increased CPT-1 mRNA expression in murine hearts. Transcriptional activity was evaluated by PPAR responsive element (PPRE)-luciferase reporter gene assay in cultured neonatal rat cardiomyocytes. CPT-1 mRNA expression and PPRE promoter activity were significantly increased by cPGI2 in a concentration-dependent manner, where PPRE has been mapped to the promoter region of the CPT-1 gene. Moreover, the elevation of CPT-1 mRNA expression and PPRE promoter activity by cPGI2 was not abolished by H-89, a potent protein kinase A inhibitor, but was significantly inhibited in cardiomyocytes over-expressing a dominant-negative type of PPARδ. Furthermore, electrophoretic mobility shift assays demonstrated that binding of PPARδ to PPRE in the CPT-1 gene promoter is enhanced in response to cPGI2 stimulation. In addition, down-regulation of CPT-1 mRNA expression in cardiomyocytes subjected to hypoxia was attenuated by cPGI2. These results indicate that cPGI2 induces CPT-1 mRNA expression through PPARδ, independent of the IP receptor signaling pathway, suggesting a possibility that cPGI2 modulates cardiac energy metabolism by activating FAO via PPARδ.

Carbacyclin induces CPT-1 in cardiac myocytes via PPARδ, independent of the IP receptor signaling pathway

Prostacyclin (PGI2) has been reported to exert cardioprotective effects via the rhodopsin type PGI2 receptor, IP. Recently, a second signaling pathway of PGI2 via peroxisome proliferator-activated receptor (PPAR) δ, was demonstrated. PPARδ exerts a pivotal role in maintaining constitutive myocardial fatty acid β oxidation (FAO).The objective of this study was to examine whether carbacyclin (cPGI2), a PGI2 analogue, up-regulates transcriptional expression of carnitine palmitoyl-transferase-1 (CPT-1), the rate-limiting enzyme in mitochondrial FAO, via PPARδ in cardiomyocytes. CPT-1 mRNA expression in the cardiac tissue was enhanced in mice injected with 100 μg cPGI2. Transcriptional activity was evaluated by PPAR responsive element (PPRE)-luciferase reporter gene assay in cultured neonatal rat cardiomyocytes. CPT-1 mRNA expression and PPRE promoter activity were significantly increased by cPGI2 in a dose-dependent manner, w here PPRE has been known to be mapped in the CPT-1 promoter region. cPGI2 also induced a dose-dependent phosphorylation of CREB indicating activation of the IP signaling pathway. Moreover, the elevated CPT-1 mRNA expression and PPRE promoter activity by cPGI2 were not abolished by inhibition of the IP receptor signaling pathway, but were significantly attenuated in cardiomyocytes transfected with a dominant negative form of PPARδ utilizing adenovirus vectors, indicating that cPGI2 induces CPT-1 mRNA expression through PPARδ in cardiomyocytes. These results suggest that cPGI2 might modulate cardiac energy metabolism by activating FAO via PPARδ, independent of the IP receptor signaling pathway.

Fatty acid‐induced effect on glucagon secretion is mediated via fatty acid oxidation

While the effect of fatty acids and ectopic triglyceride storage in pancreatic beta cells has been well-defined, only limited information is available on alpha cells. This study evaluates the long-term impact of fatty acids on alpha cell function and proliferation as well as fatty acid oxidation. Clonal alpha cells were cultured with fatty acids in the presence of high glucose for up to 3 days. The influence of fatty acids on glucagon secretion, glucagon content and triglyceride accumulation from 24 to 72 h was investigated. After a - 72 h culture, cell proliferation, carnitine palmitoyl transferase-1 mRNA level and the effect of etomoxir were also elucidated. Fatty acids stimulated glucagon secretion and increased triglyceride accumulation in a time- and dose-dependent manner, but inhibited alpha cell proliferation. Lower concentrations (0.125-0.25 mM) of fatty acids significantly increased glucagon secretion at 48 and 72 h, but did not affect triglyceride content. However, a marked increment in triglyceride accumulation occurred in the presence of 0.5 mM fatty acids. Fatty acids caused an up-regulation of the expression of carnitine palmitoyl transferase-1 gene. Etomoxir (1 microM) reversed fatty acid-induced glucagon hypersecretion, but did not inhibit carnitine palmitoyl transferase-1 mRNA level. Our data indicates that compared with triglyceride accumulation, glucagon secretion is more sensitive to changes in fatty acid concentration. The effect of fatty acids on the glucagon response is mediated through their oxidation. The high carnitine palmitoyl transferase-1 gene expression and the accumulation of triglyceride may initially be a compensatory oxidation reaction to elevated fatty acids.

Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells: role of fatty acid β-oxidation and glucose

Mitochondrial cytopathy has been associated with modifications of lipid metabolism in various situations, such as the acquisition of an abnormal adipocyte phenotype observed in multiple symmetrical lipomatosis or triglyceride (TG) accumulation in muscles associated with the myoclonic epilepsy with ragged red fibers syndrome. However, the molecular signaling leading to fat metabolism dysregulation in cells with impaired mitochondrial activity is still poorly understood. Here, we found that preadipocytes incubated with inhibitors of mitochondrial respiration such as antimycin A (AA) accumulate TG vesicles but do not acquire specific markers of adipocytes. Although the uptake of TG precursors is not stimulated in 3T3-L1 cells with impaired mitochondrial activity, we found a strong stimulation of glucose uptake in AA-treated cells mediated by calcium and phosphatidylinositol 3-kinase/Akt1/glycogen synthase kinase 3beta, a pathway known to trigger the translocation of glucose transporter 4 to the plasma membrane in response to insulin. TG accumulation in AA-treated cells is mediated by a reduced peroxisome proliferator-activated receptor gamma activity that downregulates muscle carnitine palmitoyl transferase-1 expression and fatty acid beta-oxidation, and by a direct conversion of glucose into TGs accompanied by the activation of carbohydrate-responsive element binding protein, a lipogenic transcription factor. Taken together, these results could explain how mitochondrial impairment leads to the multivesicular phenotype found in some mitochondria-originating diseases associated with a dysfunction in fat metabolism.

Dietary trans octadecenoic acids upregulate the liver gene encoding Peroxisome Proliferator-Activated Receptor-α in transition dairy cows

Effects of feeding calcium salts of conjugated linoleic acid (CLA) or trans octadecenoic acids (trans 18:1) on lipid metabolism and hepatic contents of mRNA encoding carnitine palmitoyltransferase 1 (CPT1), microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor alpha (PPARalpha) were examined in 15 early post-partum Holstein cows. Dietary treatments were initiated at approximately 4 weeks prior to expected calving dates and continued for 7 weeks post partum. Treatments prepartum consisted of 1) a basal diet (Control), 2) basal diet+150 g/d of CLA mix (CLA), or 3) basal diet+150 g/d of trans 18:1 mix (TRANS). Intakes of calcium salts of CLA and trans 18:1 mixes were adjusted to 225 g/d during the 7-week postpartum treatment period. Blood samples were collected at weeks 1, 2 and 4 post partum and plasma was harvested immediately for subsequent hormone and metabolite assays. Concentrations of insulin, insulin-like growth factor-I (IGF-I), and leptin in blood did not vary among cows fed the three diets. Plasma nonesterified fatty acid (NEFA) concentrations decreased between weeks 1 and 4 of lactation and were lower in cows fed the diet supplemented with trans 18:1 than in those fed a control diet at week 2 post partum. Periparturient fat supplementation had no detectable effects on CPT1 mRNA content in the liver. Steady-state concentration of MTP mRNA in the liver was greater in the TRANS treatment group than in the control group at week 1 postpartum. Feeding trans 18:1 supplements to transition dairy cows upregulated hepatic PPARalpha mRNA content during the first month of lactation. Under the present experimental conditions, dietary CLA had minimal effects on plasma and hepatic lipid metabolite concentrations in early lactation Holstein cows. Results indicate that dietary trans fatty acids may affect liver lipid metabolism in post-partum dairy cows through alterations in PPARalpha gene expression.

Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats

Polyunsaturated fatty acids (PUFA) and a number of drugs (metformin, thiazolidinediones) and hormones (leptin, adiponectin) that activate AMP-activated protein kinase (AMPK) have been reported to improve insulin sensitivity. To determine whether PUFA activate AMPK, Sprague–Dawley rats were adapted to a 3 h meal-feeding regimen using a fat-free diet (FFD) supplemented with fish oil ( n − 3) or triolein ( n − 9) for 7 days. No differences in hepatic AMPK activity were observed between the groups after 21 h of fasting. On the other hand, hepatic AMPK phosphorylation was decreased in rats refed the FFD, the FFD + triolein, and the FFD + PUFA by 80%, 75%, and 50%, respectively, when assessed 2 h after completion of a meal. In keeping with these changes, decreases in acetyl-CoA carboxylase phosphorylation and carnitine palmitoyl transferase-1 mRNA and increases in fatty acid synthase gene expression were greatest in rats fed the FFD and least in the PUFA-fed rats. The results indicate that dietary PUFA enhance hepatic AMPK activity in vivo, and implicate AMPK as a component of the nutrient-sensing mechanism through which dietary fatty acids and especially PUFA influence the regulation of hepatic lipid metabolism and gene expression.

PPAR alpha is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue.

Adenovirus-induced hyperleptinemia causes rapid disappearance of body fat in normal rats, presumably by up-regulating fatty acid oxidation within white adipocytes. To determine the role of peroxisomal proliferation-activated receptor (PPAR)alpha expression, which was increased during the rapid loss of fat, we infused adenovirus-leptin into PPAR alpha(-/-) and PPAR alpha(+/+) mice. Despite similar degrees of hyperleptinemia and reduction in food intake, epididymal fat pad weight declined 55% in wild-type but only 6% in PPAR alpha(-/-) mice; liver triacylglycerol fell 39% in the wild-type group but was unchanged in PPAR(-/-) mice. Carnitine palmitoyl transferase-1 mRNA rose 52% in the wild-type mice but did not increase in PPAR alpha(-/-) mice. PPAR gamma coactivator-1 alpha rose 3-fold in the fat and 46% in the liver of wild-type mice but was unchanged in PPAR alpha(-/-) mice. Although AMP-activated protein kinase could not be implicated in the lipopenic actions of hyperleptinemia, acetyl CoA carboxylase protein was reduced in the liver of wild-type but not in PPAR alpha(-/-) mice. Thus, in PPAR alpha(-/-) mice, up-regulation of carnitine palmitoyl transferase-1 mRNA in fat, down-regulation of acetyl CoA carboxylase in liver, and up-regulation of PPAR gamma coactivator-1 alpha mRNA in both tissues are abolished, as is the reduction in their triacylglycerol content.

The Effect of Surgical Treatment Following Peritoneal Sepsis on Hepatic Gene Expression

Peritoneal sepsis results in downregulation of the gene that codes for the hepatic mitochondrial enzyme carnitine palmitoyltransferase (CPT). The inhibition of hepatic CPT transcription by sepsis is thought to be mediated, in part, by increased expression of the leucine-zipper DNA transcription factor c-fos. In a cecal ligation and puncture (CLP) model, we examined the temporal effect of surgical treatment (cecal excision) on sepsis-induced inhibition of CPT gene expression. We investigated the hypothesis that Fos protein level will inversely correlate with the regulation of CPT gene expression. Specifically, we studied hepatic Fos nucleoprotein accumulation and CPT gene expression as measured by total mitochondrial CPT activity, CPT protein, and CPT mRNA. We investigated the following groups: (i) CLP followed by cecal excision 6, 12, or 24 hr following initial insult, (ii) concurrent CLP control group, and (iii) concurrent sham CLP reference group. When measured 48 hr following initial surgical insult, we conclude that: (i) in the absence of surgical treatment, peritoneal contamination results in a decrease in hepatic CPT gene expression and an increase in Fos nucleoprotein accumulation; (ii) surgical treatment at 6 or 12 hr following initial insult prevents the downregulation in hepatic CPT gene expression and does not result in Fos nucleoprotein accumulation; and (iii) surgical treatment at 24 hr following insult did not prevent the downregulation of hepatic CPT gene expression and results in an increase in hepatic Fos nucleoprotein accumulation. These data are consistent with the hypothesis that sepsis-induced regulation of hepatic c-fos gene expression, in part, is responsible for the downregulation of CPT gene expression.

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.

Turnover of carnitine palmitoyltransferase mRNA and protein in H4IIE cells. Effect of cyclic AMP and insulin

Regulation of the 68 kDa carnitine palmitoyltransferase (CPT) synthesis by (chlorophenylthio) cyclic AMP (cAMP) and insulin was studied in H4IIE cells in culture. Addition of 0.1 mM- or 1.0 mM-(chlorophenylthio) cAMP induced CPT mRNA and rate of transcription 2-4-fold by 15 min, reaching a plateau at 4-6-fold by 30 min. Addition of 5-15 nM-insulin plus 1.0 mM-cAMP suppressed the increases in transcription rate and mRNA levels occurring with cAMP alone. The t1/2 for CPT mRNA was 70-80 min and was not affected by cAMP. The t1/2 for CPT protein was 70 min, and was increased to 240 min in the presence of cAMP. The rate of CPT synthesis was also increased in the presence of cAMP. The data indicate that CPT synthesis is increased by cAMP via induction of transcription and subsequent increase in the CPT mRNA. Insulin acts to depress transcription and CPT mRNA. In addition, cAMP prolongs the t1/2 of CPT.

Regulation of Carnitine Palmitoyltransferase Synthesis in Spontaneously Diabetic BB Wistar Rats

The long-term regulation of hepatic mitochondrial carnitine palmitoyltransferase (CPT) was studied in control, insulin-treated, and untreated spontaneously diabetic BB Wistar rats. The activity of CPT was elevated approximately twofold in the untreated diabetic rats. This corresponded to an approximately equivalent elevation in the immunoreactive CPT activity. mRNACPT was assayed by reticulocyte lysate translation and by dot blot to a CPT oligonucleotide probe. The level of mRNACPT was approximately proportional to the observed CPT activity. A cDNA probe to CPT was developed, and transcriptional activity for CPT was assessed in isolated hepatic nuclei. Again, transcription of CPT mRNA was approximately proportional to the observed activity. We therefore conclude that at least part of the long-term regulation of hepatic CPT in spontaneously diabetic BB Wistar rats is the product of increased de novo synthesis of CPT protein brought about by regulation at the transcriptional level. Additional control of the amount of CPT may be via the regulation of RNA processing and turnover and enzyme insertion into the mitochondrial membrane.