Insight into mechanism of in vitro insulin secretion increase induced by antipsychotic clozapine: role of FOXA1 and mitochondrial citrate carrier.

The use of clozapine and other antipsychotic drugs is known to be associated with a number of adverse metabolic side effects, including diabetes mellitus. These side effects could be, at least in part, the result of impaired islet cell function and abnormal insulin secretion, although the underlying mechanisms are unknown. The aim of this study is the identification of targets for clozapine related to the abnormal insulin secretion. We identify a specific activation of the transcriptional factor FOXA1, but not FOXA2 and FOXA3, by clozapine in HepG2 cells. Clozapine enhances FOXA1 DNA-binding and its transcriptional activity, increasing mitochondrial citrate carrier gene expression, which contains a FOXA1 site in its promoter. Haloperidol, a conventional antipsychotic drug, does not determine any increase of FOXA1 gene expression. We also demonstrate that clozapine upregulates FOXA1 and CIC gene expression in INS-1 cells only at basal glucose concentration. In addition, we find that abnormal insulin secretion in basal glucose conditions could be completely abolished by FOXA1 silencing in INS-1 cells treated with clozapine. The identification of FOXA1 as a novel target for clozapine may shed more light to understand molecular mechanism of abnormal insulin secretion during clozapine treatment.

New 2-(aryloxy)-3-phenylpropanoic acids as peroxisome proliferator-activated receptor α/γ dual agonists able to upregulate mitochondrial carnitine shuttle system gene expression.

The preparation of a series of 2-(aryloxy)-3-phenylpropanoic acids, resulting from the introduction of different substituents into the biphenyl system of the previously reported peroxisome proliferator-activated receptor α/γ (PPARα/γ) dual agonist 1, allowed the identification of new ligands with higher potency on PPARα and fine-tuned moderate PPARγ activity. For the most promising stereoisomer (S)-16, X-ray and calorimetric studies in PPARγ revealed, at high ligand concentration, the presence of two molecules simultaneously bound to the receptor. On the basis of these results and docking experiments in both receptor subtypes, a molecular explanation was provided for its different behavior as a full and partial agonist of PPARα and PPARγ, respectively. The effects of (S)-16 on mitochondrial acylcarnitine carrier and carnitine-palmitoyl-transferase 1 gene expression, two key components of the carnitine shuttle system, were also investigated, allowing the hypothesis of a more beneficial pharmacological profile of this compound compared to the less potent PPARα agonist fibrates currently used in therapy.

Aberrant mRNA splicing associated with coding region mutations in children with carnitine-acylcarnitine translocase deficiency.

This report describes three infants with genetic defects of carnitine-acylcarnitine translocase (CACT), an inner mitochondrial membrane carrier that is essential for long-chain fatty acid oxidation. Two of the patients were of European and Chinese origin; the third was from consanguineous Turkish parents. CACT activity was totally deficient in cultured skin fibroblasts from all three patients. Patient 1 was heterozygous for a paternal frameshift mutation (120 del T in exon 1) and a maternal lariat branch point mutation (-10 T --> G in intron 2). Patient 2 was heterozygous for the same lariat branch point (-10T --> G intron 2) mutation, derived from the father, and a maternal frameshift mutation (362 del G in exon 3). Patient 3 was homozygous for a frameshift mutation (306 del C in exon 3). All of the three frameshift mutations give rise to the same stop codon at amino acid residue 127 which is predicted to cause premature protein truncation. In addition, cDNA transcript analysis showed that these coding sequence mutations also increase the amount of aberrant mRNA splicing and exon skipping at distances up to 7.7 kb nucleotides from mutation sites. The data suggest that the stability of mRNA transcripts is decreased or the frequency of aberrant splicing is increased in the presence of CACT coding sequence mutations. These results confirm that CACT is the genetic locus of the recessive mutations responsible for the fatal defects of fatty acid metabolism previously associated with deficiency of translocase activity in these three cases.

Genomic organization and mapping of the gene (SLC25A19) encoding the human mitochondrial deoxynucleotide carrier (DNC).

The deoxynucleotide carrier (DNC) transports deoxynucleotides into mitochondria and is therefore essential for mtDNA synthesis. The human DNC gene (SLC25A19) spans about 16.5 kb and consists of nine exons with the translation start site in exon 4. It is located on chromosome 17q25.3. Three transcripts, which differ in their 5' ends and are generated by alternative splicing, have been identified.

Functional analysis of mutant human carnitine acylcarnitine translocases in yeast.

Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (<1%) in the milder patient. Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient approximately 27% residual activity was found. Sequencing of the CACT cDNAs revealed a c.241G>A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast. Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive.

Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae.

The mitochondrial carrier protein for carnitine has been identified in Saccharomyces cerevisiae. It is encoded by the gene CRC1 and is a member of the family of mitochondrial transport proteins. The protein has been over-expressed with a C-terminal His-tag in S. cerevisiae and isolated from mitochondria by nickel affinity chromatography. The purified protein has been reconstituted into proteoliposomes and its transport characteristics established. It transports carnitine, acetylcarnitine, propionylcarnitine and to a much lower extent medium- and long-chain acylcarnitines.

The structure and organization of the human carnitine/acylcarnitine translocase (CACT1) gene2.

The carnitine/acylcarnitine translocase (CACT) transports acylcarnitines into mitochondria in exchange for free carnitine and it is, therefore, essential for the fatty acid beta-oxidation pathway. We have determined the exon-intron structure of the human CACT gene, which is responsible for a genetic disorder of fatty acid oxidation called CACT deficiency. The gene spans about 16.5 kb and consists of nine exons with the translation start site in exon 1. All the splice acceptor and donor sites conform to the AG/GT rules. All the introns except one are located at the level of the sequences coding for the extramembranous loops of CACT. We have designed a series of intronic oligonucleotide primers for amplifying each of the CACT exons together with their flanking intronic sequences, in segments well suited to detect mutations that would affect splicing of mRNA as well as the coding sequence itself.

Bacterial overexpression, purification, and reconstitution of the carnitine/acylcarnitine carrier from rat liver mitochondria.

The carnitine/acylcarnitine carrier from rat liver mitochondria was overexpressed in Escherichia coli. The expressed protein, recovered as inclusion bodies, was solubilized with sarkosyl and purified by Sephadex G-200 and celite chromatography. A yield of 15 mg of purified transport protein per liter of cell culture was obtained. Upon reconstitution into liposomes, the purified carrier catalyzed a [3H]carnitine/carnitine exchange inhibited by maleimides, mercurials, and sulfobetaines. Carnitine esters of various lengths were also transported. The Km for carnitine uptake was 0.47 +/- 0.11 mM, the Vmax of the exchange was 0.78 +/- 0.24 mmol/min per gram of protein, and the Ki for octanoylcarnitine was 13.5 +/- 4.3 microM. The transport properties of the recombinant carrier were virtually identical to those of the native transporter. These studies represent the first overexpression of the functionally active mitochondrial carnitine/acylcarnitine carrier, thus enabling structure/function analysis of this protein by site-directed mutagenesis.

Human mitochondrial transmembrane metabolite carriers: tissue distribution and its implication for mitochondrial disorders.

Mitochondrial transmembrane carrier deficiencies are a recently discovered group of disorders, belonging to the so-called mitochondriocytopathies. We examined the human tissue distribution of carriers which are involved in the process of oxidative phosphorylation (adenine nucleotide translocator, phosphate carrier, and voltage-dependent anion channel) and some mitochondrial substrate carriers (2-oxoglutarate carrier, carnitine-acylcarnitine carrier, and citrate carrier). The tissue distribution on mRNA level of mitochondrial transport proteins appears to be roughly in correlation with the dependence of these tissues on mitochondrial energy production capacity. In general the main mRNA expression of carriers involved in mitochondrial energy metabolism occurs in skeletal muscle and heart. Expression in liver and pancreas differs between carriers. Expression in brain, placenta, lung, and kidney is lower than in the other tissues. Western and Northern blotting experiments show a comparable HVDAC1 protein and mRNA distribution for the tested tissues. Patient's studies showed that cultured skin fibroblasts may not be a reliable alternative for skeletal muscle in screening for human mitochondrial carrier defects.

Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient.

The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC in various human tissues. Furthermore, mutation analysis was performed for a CAC-deficient infant. Direct sequencing of the patient's cDNA revealed a homozygous cytosine nucleotide insertion. This insertion provokes a frameshift and an extension of the open reading frame with 23 novel codons. This is the first report documenting a mutation, in the CAC cDNA, responsible for mitochondrial beta-oxidation impairment.

Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis.

The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.

The mitochondrial carnitine carrier protein: cDNA cloning, primary structure and comparison with other mitochondrial transport proteins.

GENBANK/o acid sequence of the rat carnitine carrier protein, a component of the inner membranes of mitochondria, has been deduced from the sequences of overlapping cDNA clones. These clones were generated in polymerase chain reactions with primers and probes based on amino acid sequence information, obtained from the direct sequencing of internal peptides of the purified carnitine carrier protein from rat. The protein sequence of the carrier, including the initiator methionine, has a length of 301 amino acids. The mature protein has a modified alpha-amino group, although the nature of this modification and the precise position of the N-terminal residue have not been ascertained. Analysis of the carnitine carrier sequence shows that the protein contains a 3-fold repeated sequence about 100 amino acids in length. Dot plot comparisons and sequence alignment demonstrate that these repeated domains are related to each other and also to the repeats of similar length that are present in the other mitochondrial carrier proteins sequenced so far. The hydropathy analysis of the carnitine carrier supports the view that the domains are folded into similar structural motifs, consisting of two transmembrane alpha-helices joined by an extensive extramembranous hydrophilic region. Southern blotting experiments suggest that both the human and the rat genomes contain single genes for the carnitine carrier. These studies provide the primary structure of the mitochondrial carnitine carrier protein and allow us to identify this metabolically important transporter as a member of the mitochondrial carrier family, and the sixth of the members whose biochemical function has already been identified.

Organization and sequence of the human gene for the mitochondrial citrate transport protein.

The citrate (tricarboxylate) carrier transports citrate (or other tricarboxylates) across the inner membranes of mitochondria in an electroneutral exchange for malate (or other dicarboxylic acids). We have determined the sequence of the human citrate transporter gene from overlapping genomic clones generated by polymerase chain reactions by use of primers and probes based on the rat cDNA sequence and on emerging sequences. The gene is spread over 2.8 kb of human DNA and is divided into eight exons. All the introns are located at the level of the sequences coding for the extramembranous loops (and not for the transmembrane segments) of the mature protein. The open reading frame of the human gene encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids to help to target it into mitochondria. 84 identities and 106 highly conservative substitutions are present in CTPs from man to yeast. In addition, we have determined the sequences of two human pseudogenes related to the citrate carrier gene encompassing the coding sequence of the gene between nucleotides 260 and 720.

Cloning and sequencing of the bovine cDNA encoding the mitochondrial tricarboxylate carrier protein.

The tricarboxylate or citrate transporter protein (CTP) catalyzes the transport of citrate across the inner mitochondrial membrane by an exchange for malate or some other anionic metabolite. Using primers based on the rat liver cDNA sequence, overlapping cDNA clones encoding the bovine CTP were isolated from bovine liver poly(A+) cDNA. The entire bovine cDNA is 1151 bp in length with 5' and 3' untranslated regions of 7 and 204 bp, respectively. The open reading frame encodes the mature protein consisting of 298 amino acids, preceded by a presequence of 13 amino acids. The amino acid sequence of the mature bovine CTP is 95.6, 94.9, 32.2% identical to that of the citrate carrier from man, rat and yeast, respectively.

Purification and characterization of the tricarboxylate carrier from eel liver mitochondria.

The tricarboxylate carrier from eel (Anguilla anguilla) liver mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B. On SDS-polyacrylamide gel electrophoresis, the purified fraction showed a single polypeptide band with an apparent molecular mass of 30.4 kDa. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a very active 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. It was purified 641-fold with a recovery of 13.3% and a protein yield of 0.02% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate carrier purified from rat liver mitochondria. These studies provide the first information on the mitochondrial tricarboxylate transport protein of a fish.

The role of sterols in the functional reconstitution of water-soluble mitochondrial porins from plants.

Water-soluble porins were prepared from native mitochondrial porins isolated from different plants (pea and corn). In the water-soluble form the porins have lost their channel-forming properties. The water-soluble porins were investigated for the influence of different sterols on their membrane activity and their channel-forming properties in lipid bilayer membranes. Our experiments demonstrated that the water-soluble porins regained channel forming activity when the protein was preincubated with different sterols in the presence of a detergent. The channels formed in lipid bilayer membranes after this procedure regain in many but not all cases the original properties of the native mitochondrial porins. Preincubation with other sterols led to a change in the single-channel conductance or to a complete loss of the voltage dependence. The sterols had also a strong influence on the channel-forming activity of the porins. Preincubation of water-soluble pea porin with the plant sterol beta-sitosterol resulted in a considerable higher channel-forming activity than with all the other sterols used for preincubation. The role of the sterols in the channel-forming complex is discussed.