The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism.

Cancer cells integrate multiple biosynthetic demands to drive unrestricted proliferation. How these cellular processes crosstalk to fuel cancer cell growth is still not fully understood. Here, we uncover the mechanisms by which the transcription factor Carbohydrate responsive element binding protein (ChREBP) functions as an oncogene during hepatocellular carcinoma (HCC) development. Mechanistically, ChREBP triggers the expression of the PI3K regulatory subunit p85α, to sustain the activity of the pro-oncogenic PI3K/AKT signaling pathway in HCC. In parallel, increased ChREBP activity reroutes glucose and glutamine metabolic fluxes into fatty acid and nucleic acid synthesis to support PI3K/AKT-mediated HCC growth. Thus, HCC cells have a ChREBP-driven circuitry that ensures balanced coordination between PI3K/AKT signaling and appropriate cell anabolism to support HCC development. Finally, pharmacological inhibition of ChREBP by SBI-993 significantly suppresses in vivo HCC tumor growth. Overall, we show that targeting ChREBP with specific inhibitors provides an attractive therapeutic window for HCC treatment.

miR-21-5p promotes NASH-related hepatocarcinogenesis.

BACKGROUND AND AIMS: The mechanisms governing the progression of non-alcoholic fatty liver disease (NAFLD) towards steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain elusive. Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis. METHODS: Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n = 199) or HCC (n = 366 HCC and n = 11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks to induce NASH and NASH-HCC, respectively. RESULTS: In obese individuals, hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice exhibited peroxisome-proliferator-activated receptor α (PPARα) activation, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis, with the pro-inflammatory/fibrogenic milieu reversing to baseline levels. In parallel, Mir21 KO mice displayed reduced number of pre-neoplastic nodules, hepatocyte proliferation and activation of oncogenic signalling, being protected from NASH-associated carcinogenesis. The hsa-miRNA-21-5p/PPARα pathway was similarly deregulated in patients with HCC- or NASH-related HCC, correlating with HCC markers and worse prognosis. CONCLUSIONS: Hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. The inhibition of hsa-miR-21-5p, leading to a pro-metabolic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC.

Plasma Metabolomic and Lipidomic Profiling of Metabolic Dysfunction-Associated Fatty Liver Disease in Humans Using an Untargeted Multiplatform Approach.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a complex disorder that is implicated in dysregulations in multiple biological pathways, orchestrated by interactions between genetic predisposition, metabolic syndromes and environmental factors. The limited knowledge of its pathogenesis is one of the bottlenecks in the development of prognostic and therapeutic options for MAFLD. Moreover, the extent to which metabolic pathways are altered due to ongoing hepatic steatosis, inflammation and fibrosis and subsequent liver damage remains unclear. To uncover potential MAFLD pathogenesis in humans, we employed an untargeted nuclear magnetic resonance (NMR) spectroscopy- and high-resolution mass spectrometry (HRMS)-based multiplatform approach combined with a computational multiblock omics framework to characterize the plasma metabolomes and lipidomes of obese patients without (n = 19) or with liver biopsy confirmed MAFLD (n = 63). Metabolite features associated with MAFLD were identified using a metabolome-wide association study pipeline that tested for the relationships between feature responses and MAFLD. A metabolic pathway enrichment analysis revealed 16 pathways associated with MAFLD and highlighted pathway changes, including amino acid metabolism, bile acid metabolism, carnitine shuttle, fatty acid metabolism, glycerophospholipid metabolism, arachidonic acid metabolism and steroid metabolism. These results suggested that there were alterations in energy metabolism, specifically amino acid and lipid metabolism, and pointed to the pathways being implicated in alerted liver function, mitochondrial dysfunctions and immune system disorders, which have previously been linked to MAFLD in human and animal studies. Together, this study revealed specific metabolic alterations associated with MAFLD and supported the idea that MAFLD is fundamentally a metabolism-related disorder, thereby providing new perspectives for diagnostic and therapeutic strategies.

UCP2 silencing restrains leukemia cell proliferation through glutamine metabolic remodeling.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy derived from early T cell progenitors. Since relapsed T-ALL is associated with a poor prognosis improving initial treatment of patients is essential to avoid resistant selection of T-ALL. During initiation, development, metastasis and even in response to chemotherapy, tumor cells face strong metabolic challenges. In this study, we identify mitochondrial UnCoupling Protein 2 (UCP2) as a tricarboxylic acid (TCA) cycle metabolite transporter controlling glutamine metabolism associated with T-ALL cell proliferation. In T-ALL cell lines, we show that UCP2 expression is controlled by glutamine metabolism and is essential for their proliferation. Our data show that T-ALL cell lines differ in their substrate dependency and their energetic metabolism (glycolysis and oxidative). Thus, while UCP2 silencing decreases cell proliferation in all leukemia cells, it also alters mitochondrial respiration of T-ALL cells relying on glutamine-dependent oxidative metabolism by rewiring their cellular metabolism to glycolysis. In this context, the function of UCP2 in the metabolite export of malate enables appropriate TCA cycle to provide building blocks such as lipids for cell growth and mitochondrial respiration. Therefore, interfering with UCP2 function can be considered as an interesting strategy to decrease metabolic efficiency and proliferation rate of leukemia cells.

Loss and gain of function of Grp75 or mitofusin 2 distinctly alter cholesterol metabolism, but all promote triglyceride accumulation in hepatocytes.

In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells. Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. Further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell. In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. METHODS: C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. RESULTS: When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlkl in fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in ß-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. CONCLUSION: The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. LAY SUMMARY: There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

UCP2 Deficiency Increases Colon Tumorigenesis by Promoting Lipid Synthesis and Depleting NADPH for Antioxidant Defenses.

Colorectal cancer (CRC) is associated with metabolic and redox perturbation. The mitochondrial transporter uncoupling protein 2 (UCP2) controls cell proliferation in vitro through the modulation of cellular metabolism, but the underlying mechanism in tumors in vivo remains unexplored. Using murine intestinal cancer models and CRC patient samples, we find higher UCP2 protein levels in tumors compared to their non-tumoral counterparts. We reveal the tumor-suppressive role of UCP2 as its deletion enhances colon and small intestinal tumorigenesis in AOM/DSS-treated and ApcMin/+ mice, respectively, and correlates with poor survival in the latter model. Mechanistically, UCP2 loss increases levels of oxidized glutathione and proteins in tumors. UCP2 deficiency alters glycolytic pathways while promoting phospholipid synthesis, thereby limiting the availability of NADPH for buffering oxidative stress. We show that UCP2 loss renders colon cells more prone to malignant transformation through metabolic reprogramming and perturbation of redox homeostasis and could favor worse outcomes in CRC.

The histone demethylase Phf2 acts as a molecular checkpoint to prevent NAFLD progression during obesity.

Aberrant histone methylation profile is reported to correlate with the development and progression of NAFLD during obesity. However, the identification of specific epigenetic modifiers involved in this process remains poorly understood. Here, we identify the histone demethylase Plant Homeodomain Finger 2 (Phf2) as a new transcriptional co-activator of the transcription factor Carbohydrate Responsive Element Binding Protein (ChREBP). By specifically erasing H3K9me2 methyl-marks on the promoter of ChREBP-regulated genes, Phf2 facilitates incorporation of metabolic precursors into mono-unsaturated fatty acids, leading to hepatosteatosis development in the absence of inflammation and insulin resistance. Moreover, the Phf2-mediated activation of the transcription factor NF-E2-related factor 2 (Nrf2) further reroutes glucose fluxes toward the pentose phosphate pathway and glutathione biosynthesis, protecting the liver from oxidative stress and fibrogenesis in response to diet-induced obesity. Overall, our findings establish a downstream epigenetic checkpoint, whereby Phf2, through facilitating H3K9me2 demethylation at specific gene promoters, protects liver from the pathogenesis progression of NAFLD.

Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets.

AIMS/HYPOTHESIS: Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells. METHODS: We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling. RESULTS: We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid ß-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated. CONCLUSIONS/INTERPRETATION: Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

Oleate dose-dependently regulates palmitate metabolism and insulin signaling in C2C12 myotubes.

Because the protective effect of oleate against palmitate-induced insulin resistance may be lessened in skeletal muscle once cell metabolism is overloaded by fatty acids (FAs), we examined the impact of varying amounts of oleate on palmitate metabolic channeling and insulin signaling in C2C12 myotubes. Cells were exposed to 0.5mM of palmitate and to increasing doses of oleate (0.05, 0.25 and 0.5mM). Impacts of FA treatments on radio-labelled FA fluxes, on cellular content in diacylglycerols (DAG), triacylglycerols (TAG), ceramides, acylcarnitines, on PKCθ, MAPKs (ERK1/2, p38) and NF-ΚB activation, and on insulin-dependent Akt phosphorylation were examined. Low dose of oleate (0.05mM) was sufficient to improve palmitate complete oxidation to CO2 (+29%, P<0.05) and to alter the cellular acylcarnitine profile. Insulin-induced Akt phosphorylation was 48% higher in that condition vs. palmitate alone (p<0.01). Although DAG and ceramide contents were significantly decreased with 0.05mM of oleate vs. palmitate alone (-47 and -28%, respectively, p<0.01), 0.25mM of oleate was required to decrease p38 MAPK and PKCθ phosphorylation, thus further improving the insulin signaling (+32%, p<0.05). By contrast, increasing oleate concentration from 0.25 to 0.5mM, thus increasing total amount of FA from 0.75 to 1mM, deteriorated the insulin signaling pathway (-30%, p<0.01). This was observed despite low contents in DAG and ceramides, and enhanced palmitate incorporation into TAG (+27%, p<0.05). This was associated with increased incomplete FA ß-oxidation and impairment of acylcarnitine profile. In conclusion, these combined data place mitochondrial ß-oxidation at the center of the regulation of muscle insulin sensitivity, besides p38 MAPK and PKCθ.

Novel Grb14-Mediated Cross Talk between Insulin and p62/Nrf2 Pathways Regulates Liver Lipogenesis and Selective Insulin Resistance.

A long-standing paradox in the pathophysiology of metabolic diseases is the selective insulin resistance of the liver. It is characterized by a blunted action of insulin to reduce glucose production, contributing to hyperglycemia, while de novo lipogenesis remains insulin sensitive, participating in turn to hepatic steatosis onset. The underlying molecular bases of this conundrum are not yet fully understood. Here, we established a model of selective insulin resistance in mice by silencing an inhibitor of insulin receptor catalytic activity, the growth factor receptor binding protein 14 (Grb14) in liver. Indeed, Grb14 knockdown enhanced hepatic insulin signaling but also dramatically inhibited de novo fatty acid synthesis. In the liver of obese and insulin-resistant mice, downregulation of Grb14 markedly decreased blood glucose and improved liver steatosis. Mechanistic analyses showed that upon Grb14 knockdown, the release of p62/sqstm1, a partner of Grb14, activated the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), which in turn repressed the lipogenic nuclear liver X receptor (LXR). Our study reveals that Grb14 acts as a new signaling node that regulates lipogenesis and modulates insulin sensitivity in the liver by acting at a crossroad between the insulin receptor and the p62-Nrf2-LXR signaling pathways.

Enhancing hepatic mitochondrial fatty acid oxidation stimulates eating in food-deprived mice.

Hepatic fatty acid oxidation (FAO) has long been implicated in the control of eating. Nevertheless, direct evidence for a causal relationship between changes in hepatic FAO and changes in food intake is still missing. Here we tested whether increasing hepatic FAO via adenovirus-mediated expression of a mutated form of the key regulatory enzyme of mitochondrial FAO carnitine palmitoyltransferase 1A (CPT1mt), which is active but insensitive to inhibition by malonyl-CoA, affects eating and metabolism in mice. CPT1mt expression increased hepatocellular CPT1 protein levels. This resulted in an increase in circulating ketone body levels in fasted CPT1mt-expressing mice, suggesting an increase in hepatic FAO. These mice did not show any significant changes in cumulative food intake, energy expenditure, or respiratory quotient after 4-h food deprivation. After 24-h food deprivation, however, the CPT1mt-expressing mice displayed increased food intake. Thus expression of CPT1mt in the liver increases hepatic FAO capacity, but does not inhibit eating. Rather, it may even stimulate eating after prolonged food deprivation. These data do not support the hypothesis that an increase in hepatic FAO decreases food intake.

Mitochondrial retrograde signaling mediated by UCP2 inhibits cancer cell proliferation and tumorigenesis.

Cancer cells tilt their energy production away from oxidative phosphorylation (OXPHOS) toward glycolysis during malignant progression, even when aerobic metabolism is available. Reversing this phenomenon, known as the Warburg effect, may offer a generalized anticancer strategy. In this study, we show that overexpression of the mitochondrial membrane transport protein UCP2 in cancer cells is sufficient to restore a balance toward oxidative phosphorylation and to repress malignant phenotypes. Altered expression of glycolytic and oxidative enzymes mediated the effects of this metabolic shift. Notably, UCP2 overexpression increased signaling from the master energy-regulating kinase, adenosine monophosphate-activated protein kinase, while downregulating expression of hypoxia-induced factor. In support of recent new evidence about UCP2 function, we found that UCP2 did not function in this setting as a membrane potential uncoupling protein, but instead acted to control routing of mitochondria substrates. Taken together, our results define a strategy to reorient mitochondrial function in cancer cells toward OXPHOS that restricts their malignant phenotype.

Enhancing liver mitochondrial fatty acid oxidation capacity in obese mice improves insulin sensitivity independently of hepatic steatosis.

BACKGROUND & AIMS: Despite major public health concern, therapy for non-alcoholic fatty liver, the liver manifestation of the metabolic syndrome often associated with insulin resistance (IR), remains elusive. Strategies aiming to decrease liver lipogenesis effectively corrected hepatic steatosis and IR in obese animals. However, they also indirectly increased mitochondrial long-chain fatty acid oxidation (mFAO) by decreasing malonyl-CoA, a lipogenic intermediate, which is the allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT1A), the key enzyme of mFAO. We thus addressed whether enhancing hepatic mFAO capacity, through a direct modulation of liver CPT1A/malonyl-CoA partnership, can reverse an already established hepatic steatosis and IR in obese mice. METHODS: Adenovirus-mediated liver expression of a malonyl-CoA-insensitive CPT1A (CPT1mt) in high-fat/high-sucrose (HF/HS) diet-induced or genetically (ob/ob) obese mice was followed by metabolic and physiological investigations. RESULTS: In association with increased hepatic mFAO capacity, liver CPT1mt expression improved glucose tolerance and insulin response to a glucose load in HF/HS and ob/ob mice, showing increased insulin sensitivity, and corrected IR in ob/ob mice. Surprisingly, hepatic steatosis was not affected in CPT1mt-expressing obese mice, indicating a clear dissociation between hepatic steatosis and IR. Moreover, liver CPT1mt expression rescued HF/HS-induced impaired hepatic insulin signaling at the level of IRS-1, IRS-2, Akt, and GSK-3ß, most likely through the observed decrease in the HF/HS-induced accumulation of lipotoxic lipids, oxidative stress, and JNK activation. CONCLUSIONS: Enhancing hepatic mFAO capacity is sufficient to reverse a state of IR and glucose intolerance in obese mice independently of hepatic steatosis.

Modulation of the hepatic malonyl-CoA-carnitine palmitoyltransferase 1A partnership creates a metabolic switch allowing oxidation of de novo fatty acids.

Liver mitochondrial beta-oxidation of LCFAs (long-chain fatty acids) is tightly regulated through inhibition of CPT1A (carnitine palmitoyltransferase 1A) by malonyl-CoA, an intermediate of lipogenesis stimulated by glucose and insulin. Moreover, CPT1A sensitivity to malonyl-CoA inhibition varies markedly depending on the physiopathological state of the animal. In the present study, we asked whether an increase in CPT1A activity solely or in association with a decreased malonyl-CoA sensitivity could, even in the presence of high glucose and insulin concentrations, maintain a sustained LCFA beta-oxidation and/or protect from triacylglycerol (triglyceride) accumulation in hepatocytes. We have shown that adenovirus-mediated expression of rat CPT1wt (wild-type CPT1A) and malonyl-CoA-insensitive CPT1mt (CPT1AM593S mutant) in cultured fed rat hepatocytes counteracted the inhibition of oleate beta-oxidation induced by 20 mM glucose/10 nM insulin. Interestingly, the glucose/insulin-induced cellular triacylglycerol accumulation was prevented, both in the presence and absence of exogenous oleate. This resulted from the generation of a metabolic switch allowing beta-oxidation of de novo synthesized LCFAs, which occurred without alteration in glucose oxidation and glycogen synthesis. Moreover, CPT1mt expression was more effective than CPT1wt overexpression to counteract glucose/insulin effects, demonstrating that control of CPT1A activity by malonyl-CoA is an essential driving force for hepatic LCFA metabolic fate. In conclusion, the present study highlights that CPT1A is a prime target to increase hepatic LCFA beta-oxidation and that acting directly on the degree of its malonyl-CoA sensitivity may be a relevant strategy to prevent and/or correct hepatic steatosis.

The transcription factor COUP-TFII is negatively regulated by insulin and glucose via Foxo1- and ChREBP-controlled pathways.

COUP-TFII has an important role in regulating metabolism in vivo. We showed this previously by deleting COUP-TFII from pancreatic beta cells in heterozygous mutant mice, which led to abnormal insulin secretion. Here, we report that COUP-TFII expression is reduced in the pancreas and liver of mice refed with a carbohydrate-rich diet and in the pancreas and liver of hyperinsulinemic and hyperglycemic mice. In pancreatic beta cells, COUP-TFII gene expression is repressed by secreted insulin in response to glucose through Foxo1 signaling. Ex vivo COUP-TFII reduces insulin production and secretion. Our results suggest that beta cell insulin secretion is under the control of an autocrine positive feedback loop by alleviating COUP-TFII repression. In hepatocytes, both insulin, through Foxo1, and high glucose concentrations repress COUP-TFII expression. We demonstrate that this negative glucose effect involves ChREBP expression. We propose that COUP-TFII acts in a coordinate fashion to control insulin secretion and glucose metabolism.

Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity.

We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N-C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N-C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N-C interactions. This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N-C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24-->Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40-47) as being involved in the chemical N-C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.

The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria.

A genetic dimorphism encodes for either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of human manganese superoxide dismutase (MnSOD) and has been reported to modulate the risk of some cancers, neurodegenerative diseases and severe alcoholic liver disease. Although functional consequences of this dimorphism on MnSOD activity have not been assessed, computer models predict a partial alpha-helix structure for the Ala-MnSOD/MTS, but a beta-sheet structure for the Val-variant, which could hamper mitochondrial import. To investigate this hypothesis, we studied the in-vitro import of chimaeric proteins composed of either one of the MnSOD/MTS fused to the mouse dihydrofolate reductase (DHFR) protein, and the import of the two human MnSOD precursor variants into rat liver mitochondria. Compared to Ala-proteins, the Val-MnSOD/MTS-DHFR precursor and Val-MnSOD precursor were both partly arrested within the inner mitochondrial membrane. The Ala-MnSOD precursor generated 30-40% more of the active, matricial, processed MnSOD homotetramer than the Val-MnSOD precursor. These results show that the Ala-MnSOD/MTS allows efficient MnSOD import into the mitochondrial matrix, while the Val-variant causes partial arrest of the precursor within the inner membrane and decreased formation of the active MnSOD tetramer in the mitochondrial matrix.

Organization of the human liver carnitine palmitoyltransferase 1 gene ( CPT1A) and identification of novel mutations in hypoketotic hypoglycaemia.

Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation. CPT1 controls the import of long-chain fatty acids into the mitochondria, where they are oxidized. Two CPT1 isoforms, the so-called "liver" and "muscle" CPT1s encoded by the CPT1Aand CPT1Bgenes, respectively, have been identified so far. While the cDNA sequences of both isoforms are known, only CPT1Bgene organization has yet been described. We took advantage of the working draft data to characterize the organization of the human CPT1A gene. We have shown the existence of 20 exons, spanning 60 kb of DNA. Two alternate promoters and numerous transcription factor-binding sites were identified within the 5' upstream region of the gene. In the 3' untranslated region, the major polyA signal was suggested to lie about 2 kb downstream of the stop codon. These data enabled us to characterize six novel mutations in four CPT1A-deficient patients; namely Q100X (exon 4), A414 V (exon 11), Y498C (exon 13), 1876-1G>A (intron 15), a 113-bp intronic insertion in the mature CPT1A mRNA (exon 13-14 junction), and a large 8-kb deletion encompassing intron 14 to exon 17. Thus, identification of the CPT1A gene organization contributes to improve the molecular screening in patients and provides tools for the study of the human CPT1A gene expression.