A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties.

The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.

GH administration rescues fatty liver regeneration impairment by restoring GH/EGFR pathway deficiency.

GH pathway has been shown to play a major role in liver regeneration through the control of epidermal growth factor receptor (EGFR) activation. This pathway is down-regulated in nonalcoholic fatty liver disease. Because regeneration is known to be impaired in fatty livers, we wondered whether a deregulation of the GH/EGFR pathway could explain this deficiency. Hepatic EGFR expression and triglyceride levels were quantified in liver biopsies of 32 obese patients with different degrees of steatosis. We showed a significant inverse correlation between liver EGFR expression and the level of hepatic steatosis. GH/EGFR down-regulation was also demonstrated in 2 steatosis mouse models, a genetic (ob/ob) and a methionine and choline-deficient diet mouse model, in correlation with liver regeneration defect. ob/ob mice exhibited a more severe liver regeneration defect after partial hepatectomy (PH) than methionine and choline-deficient diet-fed mice, a difference that could be explained by a decrease in signal transducer and activator of transcription 3 phosphorylation 32 hours after PH. Having checked that GH deficiency accounted for the GH signaling pathway down-regulation in the liver of ob/ob mice, we showed that GH administration in these mice led to a partial rescue in hepatocyte proliferation after PH associated with a concomitant restoration of liver EGFR expression and signal transducer and activator of trnascription 3 activation. In conclusion, we propose that the GH/EGFR pathway down-regulation is a general mechanism responsible for liver regeneration deficiency associated with steatosis, which could be partially rescued by GH administration.

Hepatic carnitine palmitoyl transferase 1 (CPT1 A) deficiency in North American Hutterites (Canadian and American): evidence for a founder effect and results of a pilot study on a DNA-based newborn screening program.

We describe six patients with hepatic carnitine palmitoyl transferase (CPT1 A) deficiency who are members of a large extended Hutterite kindred living in widely scattered communities in the United States and Canadian Prairies. Two patients have significant neurological impairment due to severe recurrent hypoglycemic crises. The remaining four patients with earlier detection and treatment have near normal outcomes. The Canadian and American Hutterite families share two common ancestors who married in 1812, about 60 years before the Hutterites arrived in North America and prior to their subdivision into the three groups (Schmiedeleut, Dariusleut, and the Lehrerleut). These patients share a common haplotype on chromosome 11q13 and are all homozygous for a common CPT1 A G710E mutation, suggesting a founder effect. The clustering of such a rare disorder of fatty acid oxidation prompted us to initiate a pilot DNA-based neonatal screening program to determine the carrier frequency of this mutation in Hutterite newborns with the participation and support of the community. To date our carrier frequency is 1/16, close to the predicted frequency based on diagnosed patients and number of births. We believe our newborn screening program for CPT1 A deficiency in the Hutterite community will serve as a prototype model for delivery of targeted genetic services to other similar unique genetic isolates.

Molecular and enzymatic characterization of a unique carnitine palmitoyltransferase 1A mutation in the Hutterite community.

Hepatic carnitine palmitoyltransferase 1 (CPT1A) deficiency is a rare disorder of mitochondrial fatty acid oxidation inherited as an autosomal recessive trait. Symptomatology comprises attacks of hypoketotic hypoglycemia with risk of sudden death or neurological sequelae. Only one CPT1A mutation has been reported so far. Identification of the disease-causing mutations allows both insights into the structure-function relationships of CPT1A and management of the patients and their relatives. The molecular analysis of CPT1A deficiency in a large Hutterite kindred illustrates this point. Both cDNA and genomic DNA analysis demonstrate that the affected patients are homozygous for a 2129G>A mutation predicting a G710E substitution. Studies in fibroblasts from one patient as well as heterologous expression of the mutagenized CPT1A in yeast show that the G710E mutation alters neither mitochondrial targeting nor stability of the CPT1A protein. By contrast, kinetic studies conclusively establish that the mutant CPT1A is totally inactive, indicating that the G710E mutation dramatically impairs the catalytic function of CPT1A. Finally, due to a strongly suspected founder effect for the origin of CPT1A deficiency in this Hutterite kindred, identification of this disease-causing mutation allows the setup of a targeted DNA-based newborn screening in this at-risk population.

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients.

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.

The N-terminal domain of rat liver carnitine palmitoyltransferase 1 contains an internal mitochondrial import signal and residues essential for folding of its C-terminal catalytic domain.

We have previously shown that the first 147 N-terminal residues of the rat liver carnitine palmitoyltransferase 1 (CPT1), encompassing its two transmembrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM). In the present study, we have identified the precise import sequence in this polytopic OMM protein. In vitro import studies with fusion and deletion CPT1 proteins demonstrated that none of its TM segments behave as a signal anchor sequence. Analysis of the regions flanking the TM segments revealed that residues 123-147, located immediately downstream of TM2, function as a noncleavable, matrix-targeting signal. They specify mitochondrial targeting, whereas the hydrophobic TM segment(s) acts as a stop-transfer sequence that stops and anchors the translocating CPT1 into the OMM. Heterologous expression in Saccharomyces cerevisiae of several deleted CPT1 proteins not only confirms the validity of the "stop-transfer" import model but also indicates that residues 1-82 of CPT1 contain a putative microsomal targeting signal whose cellular significance awaits further investigation. Finally, we identified a highly folded core within the C-terminal domain of CPT1 that is hidden in the entire protein by its cytosolic N-terminal residues. Functional analysis of the deleted CPT1 proteins indicates that this folded C-terminal core, which may belong to the catalytic domain of CPT1, requires TM2 for its correct folding achievement and is in close proximity to residues 1-47.

Carnitine palmitoyltransferase deficiencies.

Carnitine palmitoyltransferase (CPT) deficiencies are common disorders of mitochondrial fatty acid oxidation. The CPT system is made up of two separate proteins located in the outer- (CPT1) and inner- (CPT2) mitochondrial membranes. While CPT2 is a ubiquitous protein, two tissue-specific CPT1 isoforms-the so-called "liver" (L) and "muscle" (M) CPT1s-have been shown to exist. Amino acid and cDNA nucleotide sequences have been identified for all of these proteins. L-CPT1 deficiency (13 families reported) presents as recurrent attacks of fasting hypoketotic hypoglycemia. Two L-CPT1 mutations have been reported to date. M-CPT1 deficiency has not been hitherto identified. CPT2 deficiency has several clinical presentations. The "benign" adult form (more than 150 families reported) is characterized by episodes of rhabdomyolysis triggered by prolonged exercise. The prevalent S113L mutation is found in about 50% of mutant alleles. The infantile-type CPT2 deficiency (10 families reported) presents as severe attacks of hypoketotic hypoglycemia, occasionally associated with cardiac damage commonly responsible for sudden death before 1 year of age. In addition to these symptoms, features of brain and kidney dysorganogenesis are frequently seen in the neonatal-onset CPT2 deficiency (13 families reported), almost always lethal during the first month of life. More than 25 CPT2 mutations (private missense or truncating mutations) have hitherto been detected. Treatment is based upon avoidance of fasting and/or exercise, a low-fat diet enriched with medium chain triglycerides and carnitine ("severe" CPT2 deficiency). Prenatal diagnosis may be offered for pregnancies at a 1/4 risk of infantile/severe-type CPT2 deficiency.

The N-terminal domain of rat liver carnitine palmitoyltransferase 1 mediates import into the outer mitochondrial membrane and is essential for activity and malonyl-CoA sensitivity.

The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1-150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonyl-CoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain of L-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity.

Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae.

The rat liver carnitine palmitoyltransferase 1 (L-CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria. Comparison of the biochemical properties of the yeast-expressed L-CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl-CoA inhibition and affinity for carnitine. Normal Michaelis-Menten kinetics towards palmitoyl-CoA were observed when careful experimental conditions were used for the CPT assay. Thus, the expression in S. cerevisiae is a valid model to study the structure-function relationships of L-CPT 1.

Role of the mitochondrial DnaJ homologue, Mdj1p, in the prevention of heat-induced protein aggregation.

The role of the mitochondrial Hsp70 system in the prevention of heat-induced protein aggregation was studied in isolated mitochondria from Saccharomyces cerevisiae. Firefly luciferase was employed as a thermolabile tester protein. After shift to 40 degrees Celsius transient increase of mt-Hsp70/luciferase complex was observed, which required functional Mdj1p and Mge1p, the mitochondrial homologues of DnaJ and GrpE. The kinetics of luciferase aggregation, however, were not influenced by mutations in either mt-Hsp70 or Mge1p. Only the absence of Mdj1p led to enhanced protein aggregation. Thus, a central role in the transient protection against heat stress is attributed to this mitochondrial DnaJ homologue.

The role of the GrpE homologue, Mge1p, in mediating protein import and protein folding in mitochondria.

Mge1p, a mitochondrial GrpE homologue, has recently been identified in the yeast Saccharomyces cerevisiae and a role for this protein in precursor import has been reported. To dissect the molecular mechanism of Mge1p function, conditional mge1 mutants were constructed. Cells harbouring mutant mge1 accumulated precursor proteins at restrictive temperature. Both kinetics and efficiency of import were reduced in mitochondria isolated from strains possessing mutant mge1. Binding of mitochondrial-Hsp70 (mt-Hsp70) to incoming precursor proteins was abolished at restrictive temperature. Nucleotide-dependent dissociation of mt-Hsp70 from the import component MIM44 was reduced in mitochondria from mutant mge1 strains. Furthermore, at restrictive temperature an increase of incompletely folded, newly imported protein and enhanced protein aggregation was observed in mitochondria isolated from the mutant strains. We conclude that Mge1p exerts an essential function in import and folding of proteins by controlling the nucleotide-dependent binding of mt-Hsp70 to substrate proteins and the association of mt-Hsp70 with MIM44.

Induction of fatty-acid-synthase gene expression by glucose in primary culture of rat hepatocytes. Dependency upon glucokinase activity.

Fatty acid synthase (FAS) expression is low in liver and adipose tissue of suckling rats and increases markedly after weaning on to a high-carbohydrate low-fat diet. It has been shown previously that glucose alone, via an increase in intracellular glucose-6-phosphate level, stimulated the accumulation of FAS mRNA in cultured white adipose tissue of suckling rats. The regulation of FAS expression by glucose and hormones (insulin, dexamethasone and triiodothyronine) was studied in cultured hepatocytes from suckling rats. In hepatocytes cultured for 48 h in the absence of hormones and glucose, FAS mRNA, as well as glucokinase mRNA, levels remained undetectable. Glucose alone was unable to stimulate FAS expression. The combination of hormones, in the absence of glucose, has a marginal effect on FAS mRNA levels. However, FAS mRNA levels were increased in the presence of both glucose and the combination of hormones. This demonstrated that the hormonal induction of FAS mRNA was dependent on the presence of glucose in the culture medium. We have then investigated if glucokinase expression could be a prerequisite for the stimulation of FAS expression in response to glucose. Hepatocytes were cultured for 48 h in the absence of glucose but in the presence of insulin, dexamethasone and triiodothyronine. In these conditions, glucokinase mRNA and activity were markedly increased but there was no accumulation of FAS mRNA. When these hepatocytes were then exposed to various levels of glucose, FAS mRNA rapidly accumulated. Glucose stimulation of FAS expression was observed only in hepatocytes which expressed glucokinase activity. The importance of glucokinase expression for the induction of FAS mRNA by glucose is supported by the striking correlation between glucose-6-phosphate concentrations and the levels of FAS mRNA. This study clearly demonstrates that: (a) glucose metabolism is directly involved in the regulation of FAS gene expression; (b) the effect of hormones is partly due to their capacity to induce in the hepatocytes the capacity for glucose phosphorylation.

[Regulation of expression of genes coding for lipogenesis enzymes during development in the rat]. / Régulation de l'expression des gènes codant les enzymes de la lipogénèse au cours du développement chez le rat.

In vivo studies in the rat during the weaning period and in vitro studies on cultured cells have shown that nutriments and pancreatic hormones play a major role in lipogenic enzyme gene expression in liver and adipose tissue. Activation of lipogenic enzyme gene expression in liver and adipose tissue is dependent upon the increased supply of glucose and the concomitant hyperinsulinemia that occur after weaning. In contrast, an elevated supply of polyunsaturated or an increased plasma glucagon level prevent lipogenic enzyme gene expression in liver and adipose tissue. Glucose-6-phosphate seems to be the intracellular metabolite that mediates the transcriptional activation of lipogenic enzyme genes in response to glucose.

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding.

Mdj1p, a novel member of the DnaJ family, is a heat shock protein that is associated with the inner membrane of mitochondria of Saccharomyces cerevisiae. Disruption of the MDJ1 gene resulted in a petite phenotype, loss of mitochondrial DNA, and inviability at 37 degrees C. Import of precursor proteins was not affected by a lack of Mdj1p, but folding of newly imported proteins was markedly impaired. The efficiency of refolding of a tester protein, dihydrofolate reductase, was significantly reduced in mitochondria lacking Mdj1p after incubation at elevated temperature. We conclude that Mdj1p is an important mitochondrial chaperone that participates in the folding of newly imported proteins and in the protection of proteins against heat denaturation and aggregation.

Regulation of lipogenic enzyme gene expression by nutrients and hormones.

In vivo and in vitro experiments strongly support the view that marked increases in the levels of mRNA and in the activities of lipogenic enzymes that occur in liver and white adipose tissue of the rat after weaning to a high-carbohydrate diet are dependent on an increase in plasma glucose and insulin concentrations. An increased glucose metabolism is necessary for the expression of insulin effects on fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) mRNA accumulation in white adipose tissue, as insulin is ineffective in vitro in the absence of glucose. It is suggested that intracellular glucose-6-phosphate could play an important role in the effect of insulin on lipogenic enzyme gene expression in white adipose tissue. Other hormones and substrates could also play a role in the surge of lipogenesis after weaning. The fall in plasma glucagon after weaning to a high-carbohydrate diet could reinforce the insulin-induced accumulation of FAS and ACC mRNA, as this hormone inhibits the accumulation of lipogenic enzyme mRNA in liver and white adipose tissue. The decrease in the dietary supply of fat after weaning to a high-carbohydrate diet could also potentiate the accumulation of FAS and ACC mRNA in liver because long-chain poly-unsaturated fatty acids are potent inhibitors of the expression of the genes encoding liver lipogenic enzymes. A direct effect of fatty acids on a cis-acting element of the lipogenic enzyme genes could be involved, as the regulatory region of FAS gene contains a polyunsaturated fatty acid response element that shares some similarity with the peroxisome proliferator-activated receptor recently described.

Evidence for an impaired long-chain fatty acid oxidation and ketogenesis in Fao hepatoma cells.

Fatty acid metabolism has been studied in Fao rat hepatoma cells. In basal conditions of culture, [1-14C]oleate is mainly esterified (85% of oleate uptake) in Fao cells, phospholipids being the most important esterified products (60% of oleate esterified). Addition of N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (0.1 mM) in Fao cells does not change the metabolic fate of oleate whereas it induces gluconeogenesis and phosphoenolpyruvate carboxykinase mRNA accumulation. It is shown that the limitation of oleate oxidation is located at the level of the entry into mitochondria since octanoate is actively oxidized in Fao cells. Neither the activities of carnitine palmitoyltransferase (CPT) I and II nor the CPT II protein amount are affected by cAMP addition. The limitation of oleate oxidation in Fao cells results from (a) a high rate of lipogenesis and a high malonyl-CoA concentration, (b) a CPT I very sensitive to malonyl-CoA inhibition. The presence of an active oleate oxidation in mitochondria isolated from Fao cells confirms that CPT I is the limiting step of oleate oxidation. Moreover, Fao cells are unable to perform ketogenesis. This particular feature results from a specific deficiency in mitochondrial hydroxymethylglutaryl-CoA synthase protein, activity and gene expression. The metabolic characteristics observed in Fao cells could be a common feature in hepatoma cell lines with regard to the low capacity for long-chain fatty acid oxidation and ketone body production observed in the rat H4IIE and the human HepG2 cells.

Characterization of mitochondrial imidazoline-guanidinium receptive sites (IGRS) in liver.

Some imidazoline and guanidinium antihypertensive drugs display high affinity for a nonadrenergic membrane protein, the imidazoline-guanidinium receptive site (IGRS), which is insensitive to catecholamine and physically distinct from alpha 2-adrenoceptor. In the present report, we characterized IGRS in human and rabbit liver using [3H]idazoxan as radioligand. By performing subcellular fractionation, we showed a significant increase in [3H]idazoxan binding sites on membrane fractions enriched in cytochrome oxidase activity, a mitochondrial marker. A further enrichment in [3H]idazoxan binding (53-fold with respect to the homogenate) was found in a purified preparation of mitochondrial outer membranes. This localization of IGRS will facilitate the characterization of its functional activity in liver.

Hormonal control of fatty acid oxidation during the neonatal period.

The development of long-chain fatty acid (LCFA) oxidation, either in the liver for ketone body and energy productions or in peripheral tissues as oxidative fuels, is essential for the newborn mammals. At least in the liver, the postnatal development of LCFA oxidation and ketogenesis seems regulated by pancreatic hormones which plasmatic concentrations are markedly changed at birth (fall in insulin and rise in glucagon levels). In cultured hepatocytes from rabbit fetuses (no LCFA oxidation), the addition of glucagon or cyclic AMP induces LCFA oxidation at a level similar to that found in 24-h-old newborns (high LCFA oxidation). The presence of insulin inhibits totally the effects of glucagon. It seems that carnitine palmitoyltransferase I (CPT I), a key enzyme of LCFA oxidation, represents the main site for hormonal control of LCFA oxidation. This regulation is not due to changes in the hepatic malonyl-CoA concentration (a metabolic intermediate in lipogenesis and a potent inhibitor of CPT I) but to modifications in the sensitivity of CPT I to malonyl-CoA inhibition. The molecular mechanisms responsible for the changes in the sensitivity of CPT I are discussed.