Effect of Continuous Feeding of Ayu-Narezushi on Lipid Metabolism in a Mouse Model of Metabolic Syndrome.

Ayu-narezushi, a traditional Japanese fermented food, comprises abundant levels of lactic acid bacteria (LAB) and free amino acids. This study aimed to examine the potential beneficial effects of ayu-narezushi and investigated whether ayu-narezushi led to improvements in the Tsumura Suzuki obese diabetes (TSOD) mice model of spontaneous metabolic syndrome because useful LAB are known as probiotics that regulate intestinal function. In the present study, the increased body weight of the TSOD mice was attenuated in those fed the ayu-narezushi-comprised chow (ayu-narezushi group) compared with those fed the normal rodent chow (control group). Serum triglyceride and cholesterol levels were significantly lower in the Ayu-narezushi group than in the control group at 24 weeks of age. Furthermore, hepatic mRNA levels of carnitine-palmitoyl transferase 1 and acyl-CoA oxidase, which related to fatty acid oxidation, were significantly increased in the ayu-narezushi group than in the control group at 24 weeks of age. In conclusion, these results suggested that continuous feeding with ayu-narezushi improved obesity and dyslipidemia in the TSOD mice and that the activation of fatty acid oxidation in the liver might contribute to these improvements.

Renal tubule Cpt1a overexpression protects from kidney fibrosis by restoring mitochondrial homeostasis.

Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of short- and middle-chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD.

Adipocytes fuel gastric cancer omental metastasis via PITPNC1-mediated fatty acid metabolic reprogramming.

Omental metastasis occurs frequently in gastric cancer (GC) and is considered one of the major causes of gastric cancer-related mortality. Recent research indicated that omental adipocytes might mediate this metastatic predilection. Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) was identified to have a crucial role in metastasis. However, whether PITPNC1 participates in the interaction between adipocytes and GC omental metastasis is unclear. Methods: We profiled and analyzed the expression of PITPNC1 through analysis of the TCGA database as well as immunohistochemistry staining using matched GC tissues, adjacent normal gastric mucosa tissues (ANTs), and omental metastatic tissues. The regulation of PITPNC1 by adipocytes was explored by co-culture systems. By using both PITPNC1 overexpression and silencing methods, the role of PITPNC1 in anoikis resistance and metastasis was determined through in vitro and in vivo experiments. Results: PITPNC1 was expressed at higher rates in GC tissues than in ANTs; notably, it was higher in omental metastatic lesions. Elevated expression of PITPNC1 predicted higher rates of omental metastasis and a poor prognosis. PITPNC1 promoted anoikis resistance through fatty acid metabolism by upregulating CD36 and CPT1B expression. Further, PITPNC1 was elevated by adipocytes and facilitated GC omental metastasis. Lastly, in vivo studies showed that PITPNC1 was a therapeutic indicator of fatty acid oxidation (FAO) inhibition. Conclusion: Elevated expression of PITPNC1 in GC is correlated with an advanced clinical stage and a poor prognosis. PITPNC1 promotes anoikis resistance through enhanced FAO, which is regulated by omental adipocytes and consequently facilitates GC omental metastasis. Targeting PITPNC1 might present a promising strategy to treat omental metastasis.

Alogliptin alleviates hepatic steatosis in a mouse model of nonalcoholic fatty liver disease by promoting CPT1a expression via Thr172 phosphorylation of AMPKα in the liver.

Pioglitazone (PIO) has been reported to be effective for nonalcoholic fatty liver disease (NAFLD) and alogliptin (ALO) may have efficacy against NAFLD progression in patients with type 2 diabetes mellitus (T2DM). The present study examined the effectiveness of ALO in a rodent model of NAFLD and diabetes mellitus. KK­Ay mice were used to produce an NAFLD model via administration of a choline­deficient (CD) diet. To examine the effects of alogliptin, KK­Ay mice were provided with a CD diet with 0.03% ALO and/or 0.02% PIO orally for 8 weeks. Biochemical parameters, pathological alterations and hepatic mRNA levels associated with fatty acid metabolism were assessed. Severe hepatic steatosis was observed in KK­Ay mice fed with a CD diet, which was alleviated by the administration of ALO and/or PIO. ALO administration increased the hepatic carnitine palmitoyltransferase 1a (CPT1a) mRNA expression level and enhanced the Thr172 phosphorylation of AMP­activated protein kinase α (AMPKα) in the liver. PIO administration tended to decrease the hepatic fatty acid synthase mRNA expression level and increase the serum adiponectin level. Homeostasis model of assessment­insulin resistance values tended to improve with ALO and PIO administration. ALO and PIO alleviated hepatic steatosis in KK­Ay mice fed with a CD diet. ALO increased hepatic mRNA expression levels associated with fatty acid oxidation. In addition, the results of the present study suggested that ALO promotes CPT1a expression via Thr172 phosphorylation of AMPKα.

p38α MAPK antagonizing JNK to control the hepatic fat accumulation in pediatric patients onset intestinal failure.

The p38α mitogen-activated protein kinase (MAPK) has been related to gluconeogenesis and lipid metabolism. However, the roles and related mechanisms of p38α MAPK in intestinal failure (IF)-associated liver steatosis remained poor understood. Here, our experimental evidence suggested that p38α MAPK significantly suppressed the fat accumulation in livers of IF patients mainly through two mechanisms. On the one hand, p38α MAPK increased hepatic bile acid (BA) synthesis by upregulating the expression of the rate-limiting enzyme cholesterol 7-α-hydroxylase (CYP7A1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which in turn activated the transcription of the CYP7A1. On the other hand, p38α MAPK promoted fatty acid (FA) ß-oxidation via upregulating peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes carnitine palmitoyltransferase 1A (CPT1A) and peroxisomal acyl-coenzyme aoxidase 1 (ACOX1). Dual luciferase assays indicated that p38α MAPK increased the transcription of PPARα, PGC-1α and CYP7A1 by upregulating their promoters' activities. In addition, in vitro and in vivo assays indicated p38α MAPK negatively regulates the hepatic steatosis by controlling JNK activation. In conculsion, our findings demonstrate that hepatic p38α MAPK functions as a negative regulator of liver steatosis in maintaining BA synthesis and FAO by antagonizing the c-Jun N-terminal kinase (JNK).

Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

Impaired skeletal muscle mitochondrial fatty acid oxidation (mFAO) has been implicated in the etiology of insulin resistance. Carnitine palmitoyltransferase-1 (CPT1) is a key regulatory enzyme of mFAO whose activity is inhibited by malonyl-CoA, a lipogenic intermediate. Whereas increasing CPT1 activity in vitro has been shown to exert a protective effect against lipid-induced insulin resistance in skeletal muscle cells, only a few studies have addressed this issue in vivo. We thus examined whether a direct modulation of muscle CPT1/malonyl-CoA partnership is detrimental or beneficial for insulin sensitivity in the context of diet-induced obesity. By using a Cre-LoxP recombination approach, we generated mice with skeletal muscle-specific and inducible expression of a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA inhibition. When fed control chow, homozygous CPT1mt transgenic (dbTg) mice exhibited decreased CPT1 sensitivity to malonyl-CoA inhibition in isolated muscle mitochondria, which was sufficient to substantially increase ex vivo muscle mFAO capacity and whole body fatty acid utilization in vivo. Moreover, dbTg mice were less prone to high-fat/high-sucrose (HFHS) diet-induced insulin resistance and muscle lipotoxicity despite similar body weight gain, adiposity, and muscle malonyl-CoA content. Interestingly, these CPT1mt-protective effects in dbTg-HFHS mice were associated with preserved muscle insulin signaling, increased muscle glycogen content, and upregulation of key genes involved in muscle glucose metabolism. These beneficial effects of muscle CPT1mt expression suggest that a direct modulation of the malonyl-CoA/CPT1 partnership in skeletal muscle could represent a potential strategy to prevent obesity-induced insulin resistance.

Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes.

The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders.

Narrow time window of metabolic changes associated with transition to overt heart failure in Tgaq*44 mice.

BACKGROUND: The timing and consequences of alternations in substrate utilization in heart failure (HF) and their relationship with structural changes remain unclear. This study aimed to analyze metabolic changes associated with transition to overt heart failure in transgenic mouse model of HF resulting from cardiac-specific overexpression of constitutively active Gαq*. METHODS: Structural changes quantified by morphometry, relative cardiac mRNA and protein expression of PPARα, FAT/CD36, CPT-1, GLUT-4 and glycolytic efficiency following administration of 1-(13)C glucose were investigated in 4-14-month-old Tgαq*44 mice (TG), compared with age-matched FVB wild type mice (WT). RESULTS: Initial hypertrophy in TG (4-10-month of age) was featured by an accelerated glycolytic pathway that was not accompanied by structural changes in cardiomyocytes. In 10-month-old TG, cardiomyocyte elongation and hypertrophic remodeling and increased glycolytic flux was accompanied by relatively low expression of FAT/CD36, CPT-1 and PPARα. During the transition phase (12-month-old TG), a pronounced increase in PPARα with an increase in relative fatty acid (FA) flux was associated with anomalies of cardiomyocytes with accumulation of lipid droplets and glycogen as well as cell death. At the stage of overt heart failure (14-month-old TG), an accelerated glycolytic pathway with a decline in FA oxidation was accompanied by further structural changes. CONCLUSION: Tgαq*44 mice display three distinct phases of metabolic/structural changes during hypertrophy and progression to HF, with relatively short period of increase in FA metabolism, highlighting a narrow metabolic changes associated with transition to overt heart failure in Tgaq*44 mice that have therapeutic significance.

Forms of n-3 (ALA, C18:3n-3 or DHA, C22:6n-3) Fatty Acids Affect Carcass Yield, Blood Lipids, Muscle n-3 Fatty Acids and Liver Gene Expression in Lambs.

The effects of supplementing diets with n-3 alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA) on plasma metabolites, carcass yield, muscle n-3 fatty acids and liver messenger RNA (mRNA) in lambs were investigated. Lambs (n = 120) were stratified to 12 groups based on body weight (35 ± 3.1 kg), and within groups randomly allocated to four dietary treatments: basal diet (BAS), BAS with 10.7 % flaxseed supplement (Flax), BAS with 1.8 % algae supplement (DHA), BAS with Flax and DHA (FlaxDHA). Lambs were fed for 56 days. Blood samples were collected on day 0 and day 56, and plasma analysed for insulin and lipids. Lambs were slaughtered, and carcass traits measured. At 30 min and 24 h, liver and muscle samples, respectively, were collected for determination of mRNA (FADS1, FADS2, CPT1A, ACOX1) and fatty acid composition. Lambs fed Flax had higher plasma triacylglycerol, body weight, body fat and carcass yield compared with the BAS group (P < 0.001). DHA supplementation increased carcass yield and muscle DHA while lowering plasma insulin compared with the BAS diet (P < 0.01). Flax treatment increased (P < 0.001) muscle ALA concentration, while DHA treatment increased (P < 0.001) muscle DHA concentration. Liver mRNA FADS2 was higher and CPT1A lower in the DHA group (P < 0.05). The FlaxDHA diet had additive effects, including higher FADS1 and ACOX1 mRNA than for the Flax or DHA diet. In summary, supplementation with ALA or DHA modulated plasma metabolites, muscle DHA, body fat and liver gene expression differently.

Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer.

PURPOSE: The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof. METHODS: Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52). RESULTS: The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054-22.59, p = 0.043). CONCLUSION: Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.

Impaired adipogenesis in adipose tissue associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet.

AIMS: Chronic inflammation might be associated with hepatic lipid deposition independent of overnutrition. However, the mechanism is not fully understood. In this study, we investigate if impaired adipogenesis in adipose tissue is associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet. MAIN METHODS: Casein injection in C57BL/6J mice was given every other day to induce chronic inflammation. All mice were sacrificed after 18weeks of injections. The serum, liver and adipose tissue were collected for analysis. Real-time polymerase chain reaction and western blotting were used to examine the gene and protein expressions of molecules involved in hepatic lipid metabolism and adipose adipogenesis. KEY FINDINGS: Casein injection elevated serum levels of insulin, free fatty acid (FFA) and proinflammatory factors. The gene expression of proinflammatory factors of adipose tissue and the liver also increased in the casein group as compared with the control group. Chronic inflammation up-regulated the hepatic expression of fatty acid translocase (CD36) and down-regulated microsomal triacylglycerol transfer protein (MTP), carnitine palmitoyltransferase 1a (CPT1a) and acyl-coenzyme a oxidase 1 (ACOX1). Meanwhile, chronic inflammation not only diminished the size of adipocytes, but also down-regulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding proteinα (C/EBPα), both indicating an impaired adipogenesis. SIGNIFICANCE: Besides disturbed lipid metabolism in the liver per se, impaired adipogenesis in the adipose tissue might also be associated with hepatic lipid deposition induced by chronic inflammation in mice with chew diet.

Impact of fasting time on hepatic lipid metabolism in nutritional animal studies.

Many animal studies on improvement of lipid metabolism, using dietary components, fast the animals on the final day of the feeding. Although fasting has a significant impact on lipid metabolism, its time-dependent influence is not fully understood. We examined the effects of several fasting times on lipid metabolism. Rats fed with a semisynthetic diet for 2 wk were killed after 0 (9:00 am), 6 (7:00 am-1:00 pm), 9 (0:00 am-9:00 am), and 13 h (8:00 pm-9:00 am) of fasting. Compared to the 0 h group, marked reduction of liver weight and hepatic triacylglycerol content was observed in the 9 and 13 h groups. Activities of hepatic enzymes involved in fatty acid synthesis gradually decreased during fasting. In contrast, drastic time-dependent reduction of gene expression, of the enzymes, was observed. Expression of carnitine palmitoyltransferase mRNA was higher in the fasting groups than in the 0 h group. Our study showed that fasting has a significant impact on several parameters related to lipid metabolism in rat liver.

Comparison of the catalytic activities of three isozymes of carnitine palmitoyltransferase 1 expressed in COS7 cells.

The enzyme carnitine palmitoyltransferase 1 (CPT1) catalyzes the transfer of an acyl group from acyl-CoA to carnitine to form acylcarnitine, and three isozymes of it, 1a, 1b, and 1c, have been identified. Interestingly, the 1c isozyme was reported to show no enzymatic activity, but it was not clearly demonstrated whether this inactivity was due to its dysfunction or due to its poor expression. In the present study, we (a) expressed individual CPT1 isozymes in COS7 cells, (b) evaluated quantitatively their expression levels by Western blotting using the three bacterially expressed CPT1 isozymes as standards, and (c) evaluated their catalytic activities. With these experiments, we successfully demonstrated that the absence of the enzymatic activity of the 1c isozyme was due to its dysfunction. In addition, experiments on the preparation of standard CPT1 isozymes revealed that the 1c isozyme did not show the standard relationship between migration in an SDS-PAGE gel and molecular size. We further tried to determine why the 1c isozyme was inert by preparing chimeric CPT1 between 1a and 1c, but no clear conclusion could be drawn because one of the chimeric CPT1s was not sufficiently expressed.

Whey protein isolate counteracts the effects of a high-fat diet on energy intake and hypothalamic and adipose tissue expression of energy balance-related genes.

The intake of whey protein isolate (WPI) is known to reduce high-fat diet (HFD)-induced body-weight gain and adiposity. However, the molecular mechanisms are not fully understood. To this end, we fed C57BL/6J mice for 8 weeks with diets containing 10 % energy as fat (low-fat diet, LFD) or 45 % energy as fat (HFD) enriched with either 20 % energy as casein (LFD and HFD) or WPI (high-fat WPI). Metabolic parameters and the hypothalamic and epididymal adipose tissue expression of energy balance-related genes were investigated. The HFD increased fat mass and plasma leptin levels and decreased the dark-phase energy intake, meal number, RER, and metabolic (VO2 and heat) and locomotor activities compared with the LFD. The HFD increased the hypothalamic tissue mRNA expression of the leptin receptor, insulin receptor (INSR) and carnitine palmitoyltransferase 1b (CPT1b). The HFD also reduced the adipose tissue mRNA expression of GLUT4 and INSR. In contrast, WPI reduced fat mass, normalised dark-phase energy intake and increased meal size in HFD-fed mice. The dietary protein did not have an impact on plasma leptin, insulin, glucose or glucagon-like peptide 1 levels, but increased plasma TAG levels in HFD-fed mice. At a cellular level, WPI significantly reduced the HFD-associated increase in the hypothalamic tissue mRNA expression of the leptin receptor, INSR and CPT1b. Also, WPI prevented the HFD-induced reduction in the adipose tissue mRNA expression of INSR and GLUT4. In comparison with casein, the effects of WPI on energy intake and hypothalamic and adipose tissue gene expression may thus represent a state of reduced susceptibility to weight gain on a HFD.

Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for ß-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and ß-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

Induction of carnitine palmitoyl transferase 1 and fatty acid oxidation by retinoic acid in HepG2 cells.

The vitamin A derivative retinoic acid (RA) is an important regulator of mammalian adiposity and lipid metabolism, primarily acting at the gene expression level through nuclear receptors of the RA receptor (RAR) and retinoid X receptor (RXR) subfamilies. Here, we studied cell-autonomous effects of RA on fatty acid metabolism, particularly fatty acid oxidation, in human hepatoma HepG2 cells. Exposure to all-trans RA (ATRA) up-regulated the expression of carnitine palmitoyl transferase-1 (CPT1-L) in HepG2 cells in a dose- and time-dependent manner, and increased cellular oxidation rate of exogenously added radiolabeled palmitate. The effect of ATRA on gene expression of CPT1-L was: dependent on ongoing transcription, reproduced by both 9-cis RA and a pan-RXR agonist (but not a pan-RAR agonist) and abolished following RXRα partial siRNA-mediated silencing. CPT1-L gene expression was synergistically induced in HepG2 cells simultaneously exposed to ATRA and a selective peroxisome proliferator-activated receptor α agonist. We conclude that ATRA treatment enhances fatty acid catabolism in hepatocytes through RXR-mediated mechanisms that likely involve the transactivation of the PPARα:RXR heterodimer. Knowledge of agents and nutrient-derivatives capable of enhancing substrate oxidation systemically and specifically in liver, and their mechanisms of action, may contribute to new avenues of prevention and treatment of fatty liver, obesity and other metabolic syndrome-related disorders.

Ceramide levels regulated by carnitine palmitoyltransferase 1C control dendritic spine maturation and cognition.

The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.

Dysregulated phosphorylation and nuclear translocation of cyclic AMP response element binding protein (CREB) in rat liver after chronic ethanol binge.

Binge ethanol during chronic ethanol abuse augments liver injury but the underlying mechanism remains unknown. CREB (cyclic AMP response element binding protein) is implicated as a key transcription factor in liver regeneration and hepatic glucose and lipid metabolism. We examined the effects of ethanol on the phosphorylation of CREB in hepatocytes, and in vivo in rat liver after chronic ethanol binge. For in vivo studies, rats were fed ethanol in liquid diet for 4 weeks followed by single binge administration of ethanol (intragastric, 5 g/kg body weight). Four hours after binge administration, liver samples were collected and analyzed. Treatment of hepatocytes with ethanol caused increased phosphorylation of p38 MAPK (mitogen activated protein kinase), MSK-1 (mitogen and stress activated kinase) and CREB in the nuclear compartment without activation of ERK1/2 (extracellular regulated kinase); whereas angiotensin II induced activation of CREB was accompanied by activation of ERK1/2. In chronic ethanol-binge studies, analysis of the whole cell extracts showed increased phosphorylation of CREB, with no effect on CREB protein levels; increased phospho-ERK1/2, and decreased phospho-p38 MAPK. In contrast, the nuclear levels of phospho-CREB and CREB protein were reduced. Reduction in phospho-CREB and CREB proteins in the nuclear extracts was accompanied by suppression of mRNA levels for CPT-1 (carnitine palmitoyl transferase-1) and increase in hepatic steatosis after binge. It is concluded that binge ethanol causes defect in the nuclear accumulation of CREB protein, phospho-CREB, and an exaggerated hepatic steatosis. These in vivo effects are distinct from the effects of ethanol on hepatocytes in vitro.

Myocardial and metabolic dysfunction in type 2 diabetic rats: impact of ghrelin.

Diabetes mellitus (DM) is commonly associated with metabolic and cardiac dysfunctions. The aim of this study was to examine the effect of ghrelin on metabolic and cardiac dysfunctions in a type-2 diabetes mellitus (T2DM) rat model. For this, 48 male adult Sprague-Dawley rats were divided equally into 4 groups: Group I, fed normal chow, served as normal control group; Groups II-IV, were fed a high-fat diet for 2 weeks followed by injection of streptozotocin (STZ) (35 mg/kg body mass) to create a model of T2DM; Group II, were not treated; Group III, were treated with the vehicle (saline); Group IV, were treated with ghrelin (40 µg/kg body mass) twice daily for 10 days. The untreated diabetic rats showed a significant increase in serum fasting blood glucose, insulin homeostasis model assessment (HOMA) index, triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), total serum cholesterol (TC), and body mass, with a decrease in high-density lipoprotein cholesterol (HDL-C) (p < 0.05). Hearts isolated from diabetic rats showed a significant increase in myocardial fat content, a significant decrease in GLUT4, and an increase in acyl-CoA oxidase enzyme mRNA (p < 0.05). Ghrelin administration for 10 days caused a significant improvement in lipid profile, HOMA index, and body mass, and significantly corrected the myocardial mass, significantly reduced the fat content of the myocardium, significantly increased GLUT4, and decreased acyl CoA oxidase mRNA (p < 0.05). Thus, ghrelin improves both the metabolic functions and the disturbed energy metabolism in the cardiac muscle of obese diabetic rats.

Oleate-enriched diet improves insulin sensitivity and restores muscle protein synthesis in old rats.

BACKGROUND & AIMS: Age-related inflammation and insulin resistance (IR) have been implicated in the inability of old muscles to properly respond to anabolic stimuli such as amino acids (AA) or insulin. Since fatty acids can modulate inflammation and IR in muscle cells, we investigated the effect of palmitate-enriched diet and oleate-enriched diet on inflammation, IR and muscle protein synthesis (MPS) rate in old rats. METHODS: Twenty-four 25-month-old rats were fed either a control diet (OC), an oleate-enriched diet (HFO) or a palmitate-enriched diet (HFP) for 16 weeks. MPS using labeled amino acids and mTOR activation were assessed after AA and insulin anabolic stimulation to mimic postprandial state. RESULTS: IR and systemic and adipose tissue inflammation (TNFα and IL1ß) were improved in the HFO group. Muscle genes controlling mitochondrial ß-oxidation (PPARs, MCAD and CPT-1b) were up-regulated in the HFO group. AA and insulin-stimulated MPS in the HFO group only, and this stimulation was related to activation of the Akt/mTOR pathway. CONCLUSIONS: The age-related MPS response to anabolic signals was improved in rats fed an oleate-enriched diet. This effect was related to activation of muscle oxidative pathways, lower IR, and a decrease in inflammation.