Streptozotocin diabetes increases mRNA expression of ketogenic enzymes in the rat heart.

BACKGROUND: Diabetic cardiomyopathy develops in insulin-dependent diabetic patients who have no hypertension, cardiac hypertrophy or vascular disease. Diabetes increases cardiac fatty acid oxidation, but cardiac hypertrophy limits fatty acid oxidation. Here we examined effects of diabetes on gene expression in rat hearts. METHODS: We used oligonucleotide microarrays to examine effects of insulindependent diabetes in the rat heart. RTQ PCR confirmed results of microarrays. Specific antibodies were used to examine changes in the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). RESULTS: A surprising result of diabetes was increased mRNA encoding all enzymes of the ketone body synthesis pathway. Increased mRNA expression for these enzymes was confirmed by RTQ PCR. The mRNA encoding HMGCS2, the rate-controlling enzyme, was 27 times greater in diabetic hearts. Total HMGCS2 protein increased 8-fold in diabetic hearts, but no difference was found in HMGCS2 protein in control vs. diabetic liver. CONCLUSIONS: Insulin-dependent diabetes induced the enzymes of ketone body synthesis in the heart, including HMGCS2, as well as increasing enzymes of fatty acid oxidation. GENERAL SIGNIFICANCE: The mammalian heart does not export ketone bodies to other tissues, but rather is a major consumer of ketone bodies. Induction of HMGCS2, which is normally expressed only in the fetal and newborn heart, may indicate an adaptation by the heart to combat "metabolic inflexibility" by shifting the flux of excess intramitochondrial acetyl-CoA derived from elevated fatty acid oxidation into ketone bodies, liberating free CoA to balance the acetyl-CoA/CoA ratio in favor of increased glucose oxidation through the pyruvate dehydrogenase complex.

Membrane microenvironment regulation of carnitine palmitoyltranferases I and II.

CPT (carnitine palmitoyltransferase) 1 and CPT2 regulate fatty acid oxidation. Recombinant rat CPT2 was isolated from the soluble fractions of bacterial extracts and expressed in Escherichia coli. The acyl-CoA chain-length-specificity of the recombinant CPT2 was identical with that of the purified enzyme from rat liver mitochondrial inner membranes. The Km for carnitine for both the mitochondrial preparation and the recombinant enzyme was identical. In isolated mitochondrial outer membranes, cardiolipin (diphosphatidylglycerol) increased CPT1 activity 4-fold and the Km for carnitine 6-fold. It decreased the Ki for malonyl-CoA inhibition 60-fold, but had no effect on the apparent Km for myristoyl-CoA. Cardiolipin also activated recombinant CPT2 almost 4-fold, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylcholine activated the enzyme 3-, 2- and 2-fold respectively. Most of the recombinant CPT2 was found to have substantial interaction with cardiolipin. A model is proposed whereby cardiolipin may hold the fatty-acid-oxidizing enzymes in the active functional conformation between the mitochondrial inner and outer membranes in conjunction with the translocase and the acyl-CoA synthetase, thus combining all four enzymes into a functional unit.

Regulation of pyruvate dehydrogenase kinase 4 (PDK4) by CCAAT/enhancer-binding protein beta (C/EBPbeta).

The conversion of pyruvate to acetyl-CoA in mitochondria is catalyzed by the pyruvate dehydrogenase complex (PDC). Activity of PDC is inhibited by phosphorylation via the pyruvate dehydrogenase kinases (PDKs). Here, we examined the regulation of Pdk4 gene expression by the CCAAT/enhancer-binding protein ß (C/EBPß). C/EBPß modulates the expression of multiple hepatic genes including those involved in metabolism, development, and inflammation. We found that C/EBPß induced Pdk4 gene expression and decreased PDC activity. This transcriptional induction was mediated through two C/EBPß binding sites in the Pdk4 promoter. C/EBPß participates in the hormonal regulation of gluconeogenic genes. Previously, we reported that Pdk4 was induced by thyroid hormone (T(3)). Therefore, we investigated the role of C/EBPß in the T(3) regulation of Pdk4. T(3) increased C/EBPß abundance in primary rat hepatocytes. Knockdown of C/EBPß with siRNA diminished the T(3) induction of the Pdk4 and carnitine palmitoyltransferase (Cpt1a) genes. CPT1a is an initiating step in the mitochondrial oxidation of long chain fatty acids. Our results indicate that C/EBPß stimulates Pdk4 expression and participates in the T(3) induction of the Cpt1a and Pdk4 genes.

Peroxisome proliferator activated receptor alpha (PPARalpha) and PPAR gamma coactivator (PGC-1alpha) induce carnitine palmitoyltransferase IA (CPT-1A) via independent gene elements.

Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARalpha) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARalpha ligands. Here, we characterized a binding site for PPARalpha in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARalpha binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1alpha did not enhance CPT-1A transcription through the PPARalpha binding site in the second intron. Following knockdown of PGC-1alpha with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARalpha and PGC-1alpha stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.

Regulation of pyruvate dehydrogenase kinase 4 (PDK4) by thyroid hormone: role of the peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha).

PDK4 (pyruvate dehydrogenase kinase 4) regulates pyruvate oxidation through the phosphorylation and inhibition of the pyruvate dehydrogenase complex (PDC). PDC catalyzes the conversion of pyruvate to acetyl-CoA and is an important control point in glucose and pyruvate metabolism. PDK4 gene expression is stimulated by thyroid hormone (T(3)), glucocorticoids, and long chain fatty acids. The effects of T(3) on gene expression in the liver are mediated via the thyroid hormone receptor. Here, we have identified two binding sites for thyroid hormone receptor beta in the promoter of the rat PDK4 (rPDK4) gene. In addition, we have investigated the role of transcriptional coactivators and found that the PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator) enhances the T(3) induction of rPDK4. Following T(3) administration, there is an increase in the association of PGC-1 alpha with the rPDK4 promoter. Interestingly, this increased association is with the proximal rPDK4 promoter rather than the distal region of the gene that contains the T(3) response elements. Administration of T(3) to hypothyroid rats elevated the abundance of PGC-1 alpha mRNA and protein in the liver. In addition, we observed greater association of PGC-1 alpha not only with the rPDK4 gene but also with phosphoenolpyruvate carboxykinase and CPT-1a (carnitine palmitoyltransferase 1a) genes. Knockdown of PGC-1 alpha in rat hepatocytes reduced the T(3) induction of PDK4, PEPCK, and CPT-1a genes. Our results indicate that T(3) regulates PGC-1 alpha abundance and association with hepatic genes, and in turn PGC-1 alpha is an important participant in the T(3) induction of selected genes.

Dysregulation of sterol regulatory element binding protein-1c in livers of morbidly obese women is associated with altered suppressor of cytokine signaling-3 and signal transducer and activator of transcription-1 signaling.

We compared hepatic expression of genes that regulate lipid biosynthesis and metabolic signaling in liver biopsy specimens from women who were undergoing gastric bypass surgery (GBP) for morbid obesity with that in women undergoing ventral hernia repair who had experienced massive weight loss (MWL) after prior GBP. Comprehensive metabolic profiles of morbidly obese (MO) (22 subjects) and MWL (9 subjects) were also compared. Analyses of gene expression in liver biopsies from MO and MWL were accomplished by Affymetrix microarray, real-time polymerase chain reaction, and Western blotting techniques. After GBP, MWL subjects had lost on average 102 lb as compared with MO subjects. This was accompanied by effective reversal of the dyslipidemia and insulin resistance that were present in MO. As compared with MWL, livers of MO subjects exhibited increased expression of sterol regulatory element binding protein (SREBP)-1c and its downstream lipogenic targets, fatty acid synthase and acetyl-coenzyme A-carboxylase-1. Livers of MO subjects also exhibited enhanced expression of suppressor of cytokine signaling-3 protein and attenuated Janus kinase signal transducer and activator of transcription (JAK/STAT) signaling. Consistent with these findings, we found that the human SREBP-1c promoter was positively regulated by insulin and negatively regulated by STAT3. These data support the hypothesis that suppressor of cytokine signaling-3-mediated attenuation of the STAT signaling pathway and resulting enhanced expression of SREBP-1c, a key regulator of de novo lipid biosynthesis, are mechanistically related to the development of hepatic insulin resistance and dyslipidemia in MO women.

Regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) gene expression by glucocorticoids and insulin.

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRalpha) stimulates the expression of PDK4. Here, we determined that one of the ERRalpha binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRalpha binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.

Hepatic gene expression in morbidly obese women: implications for disease susceptibility.

The objective of this study was to determine the molecular bases of disordered hepatic function and disease susceptibility in obesity. We compared global gene expression in liver biopsies from morbidly obese (MO) women undergoing gastric bypass (GBP) surgery with that of women undergoing ventral hernia repair who had experienced massive weight loss (MWL) following prior GBP. Metabolic and hormonal profiles were examined in MO vs. MWL groups. Additionally, we analyzed individual profiles of hepatic gene expression in liver biopsy specimens obtained from MO and MWL subjects. All patients underwent preoperative metabolic profiling. RNAs were extracted from wedge biopsies of livers from MO and MWL subjects, and analysis of mRNA expression was carried out using Affymetrix HG-U133A microarray gene chips. Genes exhibiting greater than twofold differential expression between MO and MWL subjects were organized according to gene ontology and hierarchical clustering, and expression of key genes exhibiting differential regulation was quantified by real-time-polymerase chain reaction (RT-PCR). We discovered 154 genes to be differentially expressed in livers of MWL and MO subjects. A total of 28 candidate disease susceptibility genes were identified that encoded proteins regulating lipid and energy homeostasis (PLIN, ENO3, ELOVL2, APOF, LEPR, IGFBP1, DDIT4), signal transduction (MAP2K6, SOCS-2), postinflammatory tissue repair (HLA-DQB1, SPP1, P4HA1, LUM), bile acid transport (SULT2A, ABCB11), and metabolism of xenobiotics (GSTT2, CYP1A1). Using gene expression profiling, we have identified novel candidate disease susceptibility genes whose expression is altered in livers of MO subjects. The significance of altered expression of these genes to obesity-related disease is discussed.

Control of aquaporin 2 expression in collecting ducts of quail kidneys.

Birds and mammals are the only vertebrates that can concentrate urine. Avian kidneys contain structurally primitive loopless nephrons and also more advanced looped nephrons, in the cortical and medullary regions, respectively. We have identified the gene sequence of an aquaporin 2 (AQP2)-homologue water channel in collecting ducts of kidneys from adult quail, Coturnix japonica. Although immunoreactive quail AQP2 (qAQP2) was found in both types of nephrons, the expression is enhanced more clearly in the medullary regions after water deprivation. We therefore hypothesized that regulation of qAQP2 expression in quail kidneys via antidiuretic hormone (ADH) may require more advanced nephron structure. In this study, we determined the expression of qAQP2 mRNA in tissues isolated from the cortical and medullary regions before and after water deprivation, by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In both normally hydrated and water-deprived groups, qAQP2 mRNA levels in the medullary regions were significantly higher (P<0.01) than in the cortical regions. In medullary areas, qAQP2 mRNA levels (real-time PCR normalized with 18S) were significantly higher (P<0.01, ANOVA) after water deprivation (1.09+/-0.10) than in normally hydrated controls (0.46+/-0.08). In cortical areas, qAQP2 mRNA levels were also higher after water deprivation (0.37+/-0.05) than in controls (0.11+/-0.02). qAQP2 mRNA signals determined by in situ hybridization of digoxigenin-labeled riboprobe were also enhanced after water deprivation in both cortical and medullary collecting ducts. The results suggest that, contrary to our hypothesis, the endogenous production of ADH by water deprivation stimulates qAQP2 mRNA in both loopless and looped nephrons.

Two distinct aquaporin-4 cDNAs isolated from medullary cone of quail kidney.

Water deprivation or arginine vasotocin upregulates aquaporin-2 (AQP2) expression in apical and subapical regions of medullary collecting duct (CD) cells of Coturnix coturnix quail (q) kidneys. We therefore aimed to determine whether the CD has AQPs mediating water exit from the intracellular to the extracellular (interstitial) space. Using a homologue cloning technique, we isolated two distinct qAQP4 cDNAs from quail medullary cones; long (L, open reading frames) and short (S) cDNA encoded 335 (qAQP4-L) and 301 (qAQP4-S) amino acids with, respectively, 80% and 87% identity to human long- and short-form AQP4. qAQP4-S is identical to qAQP4-L from the second initiation site. Both isoforms have two NPA motifs, but lack cysteine at the known mercury-sensitive site. qAQP4-L and qAQP4-S are expressed in membranes of Xenopus laevis oocytes, but both failed to increase the water permeability (P(f)) of oocytes exposed to a hypotonic solution. Glutamate (Q242) replacement with histidine did not increase P(f). With conventional RT-PCR and real-time PCR, qAQP4-L/S mRNA signals were detected in the brain, lung, heart, intestine, adrenal gland, skeletal muscle, liver, and kidney (higher in medulla than in cortical region). qAQP4-L mRNA was detected only in the brain and adrenal gland. Orthogonal arrays of intramembranous particles were not detected in quail CDs. The results suggest that although qAQP4-L and qAQP4-S have high homology to mammalian AQP4, their physiological function may be different.

Regulation of carnitine palmitoyltransferase I (CPT-Ialpha) gene expression by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) isoforms.

The peroxisome proliferator activated receptor gamma coactivators (PGC-1) have important roles in mitochondrial biogenesis and metabolic control in a variety of tissues. There are multiple isoforms of PGC-1 including PGC-1alpha and PGC-1beta. Both the PGC-1alpha and beta isoforms promote mitochondrial biogenesis and fatty acid oxidation, but only PGC-1alpha stimulates gluconeogenesis in the liver. Carnitine palmitoyltransferase I (CPT-I) is a key enzyme regulating mitochondrial fatty acid oxidation. In these studies, we determined that PGC-1beta stimulated expression of the "liver" isoform of CPT-I (CPT-Ialpha) but that PGC-1beta did not induce pyruvate dehydrogenase kinase 4 (PDK4) which is a regulator of pyruvate metabolism. The CPT-Ialpha gene is induced by thyroid hormone. We found that T3 increased the expression of PGC-1beta and that PGC-1beta enhanced the T3 induction of CPT-Ialpha. The thyroid hormone receptor interacts with PGC-1beta in a ligand dependent manner. Unlike PGC-1alpha, the interaction of PGC-1beta and the T3 receptor does not occur exclusively through the leucine-X-X-leucine-leucine motif in PGC-1beta. We have found that PGC-1beta is associated with the CPT-Ialpha gene in vivo. Overall, our results demonstrate that PGC-1beta is a coactivator in the T3 induction of CPT-Ialpha and that PGC-1beta has similarities and differences with the PGC-1alpha isoform.

Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway.

Tetraspanin CD82 attenuates cellular morphogenesis through down-regulating integrin alpha6-mediated cell adhesion.

Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.

Peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) enhances the thyroid hormone induction of carnitine palmitoyltransferase I (CPT-I alpha).

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-controlling step in the pathway of mitochondrial fatty acid oxidation. Thyroid hormone will stimulate the expression of the liver isoform of CPT-I (CPT-I alpha). This induction of CPT-I alpha gene expression requires the thyroid hormone response element in the promoter and sequences within the first intron. The peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) is a coactivator that promotes mitochondrial biogenesis, mitochondrial fatty acid oxidation, and hepatic gluconeogenesis. In addition, PGC-1 alpha will stimulate the expression of CPT-I alpha in primary rat hepatocytes. Here we report that thyroid hormone will increase PGC-1 alpha mRNA and protein levels in rat hepatocytes. In addition, overexpression of PGC-1 alpha will enhance the thyroid hormone induction of CPT-I alpha indicating that PGC-1 alpha is a coactivator for thyroid hormone. By using chromatin immunoprecipitation assays, we show that PGC-1 alpha is associated with both the thyroid hormone response element in the CPT-I alpha gene promoter and the first intron of the CPT-I alpha gene. Our data demonstrate that PGC-1 alpha participates in the stimulation of CPT-I alpha gene expression by thyroid hormone and suggest that PGC-1 alpha is a coactivator for thyroid hormone.

Peroxisomal proliferator activated receptor gamma coactivator (PGC-1alpha) stimulates carnitine palmitoyltransferase I (CPT-Ialpha) through the first intron.

Peroxisomal proliferator activated receptor gamma coactivator-1 (PGC-1alpha) is a transcriptional coactivator that promotes mitochondrial biogenesis and energy metabolism in brown fat, skeletal muscle and heart. Previous studies demonstrated that PGC-1alpha is present at low levels in the liver but that the hepatic abundance of PGC-1alpha is elevated in diabetic and fasted animals. Elevated PGC-1alpha expression is associated with increased fatty acid oxidation and hepatic glucose production. Carnitine palmitoyltransferase-I (CPT-I) is a rate controlling step in the mitochondrial oxidation of long chain fatty acids. CPT-I transfers the acyl moiety from fatty acyl-CoA to carnitine for the translocation of long chain fatty acids across the mitochondrial membrane. There are two isoforms of CPT-I including a liver isoform CPT-Ialpha and a muscle isoform CPT-Ibeta. Here, we characterized the regulation of CPT-Ialpha isoform by PGC-1alpha. PGC-1alpha stimulates CPT-Ialpha primarily through multiple sites in the first intron. We found that PGC-1alpha can induce CPT-Ialpha gene expression in cardiac myocytes and primary hepatocytes. Our results indicate that PGC-1alpha elevates the expression of CPT-Ialpha via a unique mechanism that utilizes elements within the intron.

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts.

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.

Expression of three carnitine palmitoyltransferase-I isoforms in 10 regions of the rat brain during feeding, fasting, and diabetes.

Inhibition of carnitine palmitoyltransferase-I (CPT-I) activity in the brain has been shown to decrease food intake in rats. We examined the expression of mRNA encoding all three known CPT-I isoforms (alpha, beta, and gamma in 10 different major regions of the rat brain in normal, chow-fed rats, in fasting rats, and in insulin-dependent diabetic rats. Compared with the effects of fasting and diabetes on CPT-I mRNA in the liver and heart, there was either less effect or no effect depending on the particular brain region examined. These results suggest that the regulation of CPT-I mRNA levels is different in the brain than in other tissues. A surprising result of this study was the discovery of very high, unique expression of CPT-Ibeta (the muscle isoform) in the cerebellum.

Inhibition of carnitine palmitoyltransferase in the rat small intestine reduces export of triacylglycerol into the lymph.

Following digestion of dietary triacylglycerol (TAG), intestinal epithelial cells absorb fatty acids and monoacylglycerols that are resynthesized into TAG by enzymes located on the endoplasmic reticulum (ER). A study in rat liver (Abo-Hashema, K. A., M. H. Cake, G. W. Power, and D. J. Clarke. 1999. Evidence for TAG synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J. Biol. Chem. 274: 35577-35582) showed that there is a carnitine-dependent ER lumenal synthesis of TAG. We wanted to test the hypothesis that a similar pathway was present in rat intestine by utilizing etomoxir, a specific inhibitor of carnitine palmitoyltransferase (CPT). Intraduodenal infusion of etomoxir inhibited CPT activity in the ER by 69%. Etomoxir did not affect either the uptake of intraduodenally infused [3H]glyceryltrioleate by the intestinal mucosa or the production of mucosal [3H]TAG, excluding the possibility that etomoxir interfered with TAG absorption or synthesis. Etomoxir did not inhibit protein synthesis, glucose, cholesterol or palmitate absorption or metabolism, or ATP concentrations. Etomoxir substantially (74%) diminished lymph TAG output from intralumenally infused glyceryltrioleate. In conclusion, these data strongly support the hypothesis that an ER CPT system exists and is necessary for processing dietary TAG into chylomicrons. The significant reduction in lymphatic output of chylomicron TAG on etomoxir treatment suggests that the major source of chylomicron TAG is a diacylglyceroltransferase on the lumenal surface of the ER.

Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity, leading to PKC-dependent insulin exocytosis.

Hypoglycemic sulfonylureas such as glibenclamide have been widely used to treat type 2 diabetic patients for 40 yr, but controversy remains about their mode of action. The widely held view is that they promote rapid insulin exocytosis by binding to and blocking pancreatic beta-cell ATP-dependent K+ (KATP) channels in the plasma membrane. This event stimulates Ca2+ influx and sets in motion the exocytotic release of insulin. However, recent reports show that >90% of glibenclamide-binding sites are localized intracellularly and that the drug can stimulate insulin release independently of changes in KATP channels and cytoplasmic free Ca2+. Also, glibenclamide specifically and progressively accumulates in islets in association with secretory granules and mitochondria and causes long-lasting insulin secretion. It has been proposed that nutrient insulin secretagogues stimulate insulin release by increasing formation of malonyl-CoA, which, by blocking carnitine palmitoyltransferase 1 (CPT-1), switches fatty acid (FA) catabolism to synthesis of PKC-activating lipids. We show that glibenclamide dose-dependently inhibits beta-cell CPT-1 activity, consequently suppressing FA oxidation to the same extent as glucose in cultured fetal rat islets. This is associated with enhanced diacylglycerol (DAG) formation, PKC activation, and KATP-independent glibenclamide-stimulated insulin exocytosis. The fat oxidation inhibitor etomoxir stimulated KATP-independent insulin secretion to the same extent as glibenclamide, and the action of both drugs was not additive. We propose a mechanism in which inhibition of CPT-1 activity by glibenclamide switches beta-cell FA metabolism to DAG synthesis and subsequent PKC-dependent and KATP-independent insulin exocytosis. We suggest that chronic CPT inhibition, through the progressive islet accumulation of glibenclamide, may explain the prolonged stimulation of insulin secretion in some diabetic patients even after drug removal that contributes to the sustained hypoglycemia of the sulfonylurea.

A thyroid hormone response unit formed between the promoter and first intron of the carnitine palmitoyltransferase-Ialpha gene mediates the liver-specific induction by thyroid hormone.

Carnitine palmitoyltransferase-I (CPT-I) catalyzes the rate-controlling step of fatty acid oxidation. CPT-I converts long-chain fatty acyl-CoAs to acylcarnitines for translocation across the mitochondrial membrane. The mRNA levels and enzyme activity of the liver isoform, CPT-Ialpha, are greatly increased in the liver of hyperthyroid animals. Thyroid hormone (T3) stimulates CPT-Ialpha transcription far more robustly in the liver than in non-hepatic tissues. We have shown that the thyroid hormone receptor (TR) binds to a thyroid hormone response element (TRE) located in the CPT-Ialpha promoter. In addition, elements in the first intron participate in the T3 induction of CPT-Ialpha gene expression, but the CPT-Ialpha intron alone cannot confer a T3 response. We found that deletion of sequences in the first intron between +653 and +744 decreased the T3 induction of CPT-Ialpha. Upstream stimulatory factor (USF) and CCAAT enhancer binding proteins (C/EBPs) bind to elements within this region, and these factors are required for the T3 response. The binding of TR and C/EBP to the CPT-Ialpha gene in vivo was shown by the chromatin immunoprecipitation assay. We determined that TR can physically interact with USF-1, USF-2, and C/EBPalpha. Transgenic mice were created that carry CPT-Ialpha-luciferase transgenes with or without the first intron of the CPT-Ialpha gene. In these mouse lines, the first intron is required for T3 induction as well as high levels of hepatic expression. Our data indicate that the T3 stimulates CPT-Ialpha gene expression in the liver through a T3 response unit consisting of the TRE in the promoter and additional factors, C/EBP and USF, bound in the first intron.