Selective Acetyl-CoA Carboxylase 1 Inhibitor Improves Hepatic Steatosis and Hepatic Fibrosis in a Preclinical Nonalcoholic Steatohepatitis Model.

Acetyl-CoA carboxylase (ACC) 1 and ACC2 are essential rate-limiting enzymes that synthesize malonyl-CoA (M-CoA) from acetyl-CoA. ACC1 is predominantly expressed in lipogenic tissues and regulates the de novo lipogenesis flux. It is upregulated in the liver of patients with nonalcoholic fatty liver disease (NAFLD), which ultimately leads to the formation of fatty liver. Therefore, selective ACC1 inhibitors may prevent the pathophysiology of NAFLD and nonalcoholic steatohepatitis (NASH) by reducing hepatic fat, inflammation, and fibrosis. Many studies have suggested ACC1/2 dual inhibitors for treating NAFLD/NASH; however, reports on selective ACC1 inhibitors are lacking. In this study, we investigated the effects of compound-1, a selective ACC1 inhibitor for treating NAFLD/NASH, using preclinical in vitro and in vivo models. Compound-1 reduced M-CoA content and inhibited the incorporation of [14C] acetate into fatty acids in HepG2 cells. Additionally, it reduced hepatic M-CoA content and inhibited de novo lipogenesis in C57BL/6J mice after a single dose. Furthermore, compound-1 treatment of 8 weeks in Western diet-fed melanocortin 4 receptor knockout mice-NAFLD/NASH mouse model-improved liver hypertrophy and reduced hepatic triglyceride content. The reduction of hepatic M-CoA by the selective ACC1 inhibitor was highly correlated with the reduction in hepatic steatosis and fibrosis. These findings support further investigations of the use of this ACC1 inhibitor as a new treatment of NFLD/NASH. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a novel selective inhibitor of acetyl-CoA carboxylase (ACC) 1 has anti-nonalcoholic fatty liver disease (NAFLD) and anti-nonalcoholic steatohepatitis (NASH) effects in preclinical models. Treatment with this compound significantly improved hepatic steatosis and fibrosis in a mouse model. These findings support the use of this ACC1 inhibitor as a new treatment for NAFLD/NASH.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

CTRP3 ameliorates fructose-induced metabolic associated fatty liver disease via inhibition of xanthine oxidase-associated oxidative stress.

OBJECTIVES: The incidence of metabolic associated fatty liver disease (MAFLD) induced by high fructose consumption is dramatically increasing in the world while lacking specifically therapeutic drugs. The present study aimed to investigate the effect of complement C1q/tumor necrosis factor-related protein-3 (CTRP3) on fructose-induced MAFLD and its potential mechanisms. METHOD: The animal models with MAFLD were built with Sprague-Dawley (SD) rats drinking 10 % fructose solution for 12 weeks. Then, specific hepatic CTRP3 overexpression was conducted by a single caudal-vein injection of CTRP3-expressing adenoviruses. Rats were sacrificed two weeks later. RESULTS: Drinking 10 % fructose solution for 12 weeks successfully built the rats models with MAFLD. Fructose feeding markedly decreased hepatic CTRP3 expression in rats. However, CTRP3 overexpression in liver alleviated hyperuricemia, dyslipidemia, liver function injury, intrahepatic triglyceride (TG) accumulation and histological changes of hepatic steatosis in rats fed with fructose. CTRP3 overexpression also inhibited hepatic XO activity in liver and improved subsequent oxidative stress, accompanied with downregulation of gene expression of sterol-regulatory element binding protein 1c (SERBP-1c) and fatty acid synthase (FAS). CONCLUSION: CTRP3 attenuates MAFLD induced by fructose, which maybe partially attribute to rescued oxidative stress related with xanthine oxidase overactivity.

Dietary Betaine Mitigates Hepatic Steatosis and Inflammation Induced by a High-Fat-Diet by Modulating the Sirt1/Srebp-1/Pparɑ Pathway in Juvenile Black Seabream (Acanthopagrus schlegelii).

The present study aimed to elucidate the mechanism of dietary betaine, as a lipid-lowering substance, on the regulation of lipid metabolism and inflammation in juvenile black seabream (Acanthopagrus schlegelii) fed a high fat diet. An 8-week feeding trial was conducted in black seabream with an initial weight of 8.39 ± 0.01g fed four isonitrogenous diets including Control, medium-fat diet (11%); HFD, high-fat diet (17%); and HFD supplemented with two levels (10 and 20 g/kg) of betaine, HFD+B1 and HFD+B2, respectively. SGR and FE in fish fed HFD+B2 were significantly higher than in fish fed HFD. Liver histology revealed that vacuolar fat droplets were smaller and fewer in bream fed HFD supplemented with betaine compared to fish fed HFD. Betaine promoted the mRNA and protein expression levels of silent information regulator 1 (Sirt1), up-regulated mRNA expression and protein content of lipid peroxisome proliferator-activated receptor alpha (pparα), and down-regulated mRNA expression and protein content of sterol regulatory element-binding protein-1(srebp-1). Furthermore, the mRNA expression levels of anti-inflammatory cytokines in liver and intestine were up-regulated, while nuclear factor kB (nf-kb) and pro-inflammatory cytokines were down-regulated by dietary betaine supplementation. Likewise, in fish that received lipopolysaccharide (LPS) to stimulate inflammatory responses, the expression levels of mRNAs of anti-inflammatory cytokines in liver, intestine and kidney were up-regulated in fish fed HFD supplemented with betaine compared with fish fed HFD, while nf-kb and pro-inflammatory cytokines were down-regulated. This is the first report to suggest that dietary betaine could be an effective feed additive to alleviate hepatic steatosis and attenuate inflammatory responses in black seabream fed a high fat diet by modulating the Sirt1/Srebp-1/Pparɑ pathway.

Colesevelam Reduces Ethanol-Induced Liver Steatosis in Humanized Gnotobiotic Mice.

Alcohol-related liver disease is associated with intestinal dysbiosis. Functional changes in the microbiota affect bile acid metabolism and result in elevated serum bile acids in patients with alcohol-related liver disease. The aim of this study was to identify the potential role of the bile acid sequestrant colesevelam in a humanized mouse model of ethanol-induced liver disease. We colonized germ-free (GF) C57BL/6 mice with feces from patients with alcoholic hepatitis and subjected humanized mice to the chronic-binge ethanol feeding model. Ethanol-fed gnotobiotic mice treated with colesevelam showed reduced hepatic levels of triglycerides and cholesterol, but liver injury and inflammation were not decreased as compared with non-treated mice. Colesevelam reduced hepatic cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1) protein expression, although serum bile acids were not lowered. In conclusion, our findings indicate that colesevelam treatment mitigates ethanol-induced liver steatosis in mice.

Fatty Acid Desaturase 1 Influences Hepatic Lipid Homeostasis by Modulating the PPARα-FGF21 Axis.

The fatty acid desaturase 1 (FADS1), also known as delta-5 desaturase (D5D), is one of the rate-limiting enzymes involved in the desaturation and elongation cascade of polyunsaturated fatty acids (PUFAs) to generate long-chain PUFAs (LC-PUFAs). Reduced function of D5D and decreased hepatic FADS1 expression, as well as low levels of LC-PUFAs, were associated with nonalcoholic fatty liver disease. However, the causal role of D5D in hepatic lipid homeostasis remains unclear. In this study, we hypothesized that down-regulation of FADS1 increases susceptibility to hepatic lipid accumulation. We used in vitro and in vivo models to test this hypothesis and to delineate the molecular mechanisms mediating the effect of reduced FADS1 function. Our study demonstrated that FADS1 knockdown significantly reduced cellular levels of LC-PUFAs and increased lipid accumulation and lipid droplet formation in HepG2 cells. The lipid accumulation was associated with significant alterations in multiple pathways involved in lipid homeostasis, especially fatty acid oxidation. These effects were demonstrated to be mediated by the reduced function of the peroxisome proliferator-activated receptor alpha (PPARα)-fibroblast growth factor 21 (FGF21) axis, which can be reversed by treatment with docosahexaenoic acid, PPARα agonist, or FGF21. In vivo, FADS1-knockout mice fed with high-fat diet developed increased hepatic steatosis as compared with their wild-type littermates. Molecular analyses of the mouse liver tissue largely corroborated the observations in vitro, especially along with reduced protein expression of PPARα and FGF21. Conclusion: Collectively, these results suggest that dysregulation in FADS1 alters liver lipid homeostasis in the liver by down-regulating the PPARα-FGF21 signaling axis.

Tpl2 kinase regulates inflammation but not tumorigenesis in mice.

Tumor progression locus 2 (Tpl2, gene name MAP3K8), a mitogen-activated protein kinase, is widely expressed in immune and non-immune cells to integrate tumor necrosis factor (TNF), toll-like receptors (TLRs), and interleukin-1 (IL1) receptor signaling to regulate inflammatory response. Given its central role in inflammatory response, Tpl2 is an attractive small molecule drug target. However, the role of Tpl2 as an oncogene or tumor suppressor gene remains controversial, and its function outside immune cells is not understood. We therefore utilized a Tpl2 kinase dead (Tpl2-KD) mouse model in an 18-month aging study to further elucidate Tpl2 effects on lifespan and chronic disease. Histopathological studies revealed the incidence and severity of spontaneous tumors and non-neoplastic lesions were comparable between wild type and Tpl2-KD mice. The only finding was that male Tpl2-KD mice had higher bodyweight and an increased incidence of liver steatosis, suggesting a sex-specific role for Tpl2 in hepatic lipid metabolism. In conclusion, loss of Tpl2 kinase activity did not lead to increased tumorigenesis over aging in mice but affected likely alterations in lipid metabolism in male animals.

Hepatic nNOS impaired hepatic insulin sensitivity through the activation of p38 MAPK.

Neuronal nitric oxide synthase (nNOS) interacts with its adaptor protein NOS1AP through its PZD domain in the neurons. Previously, we had reported that NOS1AP enhanced hepatic insulin sensitivity through its PZD-binding domain, which suggested that nNOS might mediate the effect of NOS1AP. This study aimed to examine the role and underlying mechanisms of nNOS in regulating hepatic insulin sensitivity. nNOS co-localized with NOS1AP in mouse liver. The overexpression of NOS1AP in mouse liver decreased the level of phosphorylated nNOS (p-nNOS (Ser1417)), the active form of nNOS. Conversely, the liver-specific deletion of NOS1AP increased the level of p-nNOS (Ser1417). The overexpression of nNOS in the liver of high-fat diet-induced obese mice exacerbated glucose intolerance, enhanced intrahepatic lipid accumulation, decreased glycogen storage, and blunted insulin-induced phosphorylation of IRbeta and Akt in the liver. Similarly, nNOS overexpression increased triglyceride production, decreased glucose utilization, and downregulated insulin-induced expression of p-IRbeta, p-Akt, and p-GSK3beta in the HepG2 cells. In contrast, treatment with Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, improved glucose tolerance and upregulated insulin-induced phosphorylation of IRbeta and Akt in the liver of ob/ob mice. Furthermore, overexpression of nNOS increased p38MAPK phosphorylation in the HepG2 cells. In contrast, inhibition of p38MAPK with SB203580 significantly reversed the nNOS-induced inhibition of insulin-signaling activity (all P < 0.05). This indicated that hepatic nNOS inhibited the insulin-signaling pathway through the activation of p38MAPK. These findings suggest that nNOS is involved in the development of hepatic insulin resistance and that nNOS might be a potential therapeutic target for diabetes.

Kanglexin, a new anthraquinone compound, attenuates lipid accumulation by activating the AMPK/SREBP-2/PCSK9/LDLR signalling pathway.

Hyperlipidaemia is one of the major risk factors for atherosclerosis, coronary heart disease, stroke and diabetes. In the present study, we synthesized a new anthraquinone compound, 1,8-dihydroxy-3-succinic acid monoethyl ester-6-methylanthraquinone, and named it Kanglexin (KLX). The aim of this study was to evaluate whether KLX has a lipid-lowering effect and to explore the potential molecular mechanism. In this study, Sprague-Dawley rats were fed a high fat diet (HFD) for 5 weeks to establish a hyperlipidaemia model; then, the rats were orally administered KLX (20, 40, and 80 mg kg-1·d-1) or atorvastatin calcium (AT, 10 mg kg-1·d-1) once a day for 2 weeks. KLX had prominent effects on reducing blood lipids, hepatic lipid accumulation, body weight and the ratio of liver weight/body weight. Furthermore, KLXdramatically reduced the total cholesterol (TC) and triglyceride (TG) levels and lipid accumulation in a HepG2 cell model of dyslipidaemia induced by 1 mmol/L oleic acid (OA). KLX may decrease lipid levels by phosphorylating adenosine monophosphate-activated protein kinase (AMPK) and the downstream sterol regulatory element binding protein 2 (SREBP-2)/proprotein convertase subtilisin/kexin type 9 (PCSK9)/low-density lipoprotein receptor (LDLR) signalling pathway in the HFD rats and OA-treated HepG2 cells. The effects of KLX on the AMPK/SREBP-2/PCSK9/LDLR signalling pathway were abolished when AMPK was inhibited by compound C (a specific AMPK inhibitor) in HepG2 cells. In summary, KLX has an efficient lipid-lowering effect mediated by activation of the AMPK/SREBP-2/PCSK9/LDLR signalling pathway. Our findings may provide new insight into and evidence for the discovery of a new lipid-lowering drug for the prevention and treatment of hyperlipidaemia, fatty liver, and cardiovascular disease in the clinic.

Downregulation of glucose-6-phosphatase expression contributes to fluoxetine-induced hepatic steatosis.

Fluoxetine is a first-line selective serotonin reuptake inhibitor widely applied for the treatment of depression; however, it induces abnormal hepatic lipid metabolism. Considering decreased expression or function of glucose-6-phosphatase (G6Pase), a key enzyme in gluconeogenesis, or the upregulation of fatty acid uptake, causes hepatic lipid accumulation. The aim of this study was to elucidate whether G6Pase regulation and fatty acid uptake alteration contribute to fluoxetine-induced abnormal hepatic lipid metabolism. Our study revealed that 8-week oral administration of fluoxetine dose-dependently increased hepatic triglyceride, causing hepatic steatosis. Concomitantly, the expression of G6Pase in mouse livers and primary mouse hepatocytes (PMHs) was downregulated in a concentration-dependent manner. Furthermore, fluoxetine increased the concentrations of glucose-6-phosphate (G6Pase substrate) and acetyl CoA (the substrate for de novo lipogenesis) in mouse livers. Additionally, fluoxetine also induced lipid accumulation and downregulated G6Pase expression in HepG2 cells. However, the uptake of green fluorescent fatty acid (BODIPY™ FL C16) in PMHs was not changed after fluoxetine treatment, indicating that fluoxetine-induced hepatic steatosis was not associated with fatty acid uptake alteration. In conclusion, fluoxetine downregulated hepatic G6Pase expression, subsequently enhanced the transformation of glucose to lipid, and ultimately resulted in hepatic steatosis, but with no impact on fatty acid uptake.

IRE1α-JNK pathway-mediated autophagy promotes cell survival in response to endoplasmic reticulum stress during the initial phase of hepatic steatosis.

AIMS: It has been widely reported that autophagy and inositol-requiring enzyme-1α (IRE1α)-c-Jun N-terminal kinase (JNK) pathway was involved in cell survival under endoplasmic reticulum (ER) stress, but their specific roles in hepatic steatosis remain unclear. This study aimed to determine the interaction between autophagy and IRE1α-JNK pathway on cell survival in response to ER stress during the initial phase of hepatic steatosis. METHODS: Hepatic steatosis was induced in HepG2 cells by supplementing oleic acid (OA). Lipid accumulation was evaluated by BODIPY493/503 staining. ER stress and IRE1α-JNK signaling were investigated by western blot. Autophagy was monitored by western blot, GFP-LC3 plasmid and immunofluorescence staining, while apoptosis was determined by western blotting, Annexin-V-FITC/PI staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. KEY FINDINGS: Aggravated lipid accumulation was found under increased ER stress during the initial phase of hepatic steatosis. Meanwhile, an increase of autophagy and no alteration of apoptosis were observed under increased ER stress. Interestingly, autophagy was induced by ER stress, while autophagy suppression led to an increase of apoptosis in response to ER stress Moreover, further study showed that IRE1α-JNK pathway was activated after ER stress and consequently induced autophagy, which promoted cell survival in the initial phase of hepatic steatosis. SIGNIFICANCE: We conclude that IRE1α-JNK pathway was activated during ER stress in the initial phase of hepatic steatosis and promoted cell survival by enhancing autophagy. Targeting IRE1α-JNK-autophagy signaling may provide new insight into preventive strategies for hepatic steatosis.

Proteomic exploration of cystathionine ß-synthase deficiency: implications for the clinic.

Introduction: Homocystinuria due to cystathionine ß-synthase (CBS) deficiency, the most frequent inborn error of sulfur amino acid metabolism, is characterized biochemically by severely elevated homocysteine (Hcy) and related metabolites, such as Hcy-thiolactone and N-Hcy-protein. CBS deficiency reduces life span and causes pathological abnormalities affecting most organ systems in the human body, including the cardiovascular (thrombosis, stroke), skeletal/connective tissue (osteoporosis, thin/non-elastic skin, thin hair), and central nervous systems (mental retardation, seizures), as well as the liver (fatty changes), and the eye (ectopia lentis, myopia). Molecular basis of these abnormalities were largely unknown and available treatments remain ineffective. Areas covered: Proteomic and transcriptomic studies over the past decade or so, have significantly contributed to our understanding of mechanisms by which the CBS enzyme deficiency leads to clinical manifestations associated with it. Expert opinion: Recent findings, discussed in this review, highlight the involvement of dysregulated proteostasis in pathologies associated with CBS deficiency, including thromboembolism, stroke, neurologic impairment, connective tissue/collagen abnormalities, hair defects, and hepatic toxicity. To ameliorate these pathologies, pharmacological, enzyme replacement, and gene transfer therapies are being developed.

The Inhibition of Aldose Reductase Accelerates Liver Regeneration through Regulating Energy Metabolism.

OBJECTIVES: Our previous study showed that aldose reductase (AR) played key roles in fatty liver ischemia-reperfusion (IR) injury by regulating inflammatory response and energy metabolism. Here, we aim to investigate the role and mechanism of AR in the regeneration of normal and fatty livers after liver surgery. METHODS: The association of AR expression with liver regeneration was studied in the rat small-for-size liver transplantation model and the mice major hepatectomy and hepatic IR injury model with or without fatty change. The direct role and mechanism of AR in liver regeneration was explored in the AR knockout mouse model. RESULTS: Delayed regeneration was detected in fatty liver after liver surgery in both rat and mouse models. Furthermore, the expression of AR was increased in liver after liver surgery, especially in fatty liver. In a functional study, the knockout of AR promoted liver regeneration at day 2 after major hepatectomy and IR injury. Compared to wild-type groups, the expressions of cyclins were increased in normal and fatty livers of AR knockout mice. AR inhibition increased the expressions of PPAR-α and PPAR-γ in both normal liver and fatty liver groups after major hepatectomy and IR injury. In addition, the knockout of AR promoted the expressions of SDHB, AMPK, SIRT1, and PGC1-α and PPAR. CONCLUSIONS: The knockout of AR promoted the regeneration of normal and fatty livers through regulating energy metabolism. AR may be a new potential therapeutic target to accelerate liver regeneration after surgery.

Sesamol Alleviates Obesity-Related Hepatic Steatosis via Activating Hepatic PKA Pathway.

This study aimed to investigate the effect of sesamol (SEM) on the protein kinase A (PKA) pathway in obesity-related hepatic steatosis treatment by using high-fat diet (HFD)-induced obese mice and a palmitic acid (PA)-treated HepG2 cell line. SEM reduced the body weight gain of obese mice and alleviated related metabolic disorders such as insulin resistance, hyperlipidemia, and systemic inflammation. Furthermore, lipid accumulation in the liver and HepG2 cells was reduced by SEM. SEM downregulated the gene and protein levels of lipogenic regulator factors, and upregulated the gene and protein levels of the regulator factors responsible for lipolysis and fatty acid ß-oxidation. Meanwhile, SEM activated AMP-activated protein kinase (AMPK), which might explain the regulatory effect of SEM on fatty acid ß-oxidation and lipogenesis. Additionally, the PKA-C and phospho-PKA substrate levels were higher after SEM treatment. Further research found that after pretreatment with the PKA inhibitor, H89, lipid accumulation was increased even with SEM administration in HepG2 cells, and the effect of SEM on lipid metabolism-related regulator factors was abolished by H89. In conclusion, SEM has a positive therapeutic effect on obesity and obesity-related hepatic steatosis by regulating the hepatic lipid metabolism mediated by the PKA pathway.

Heterogeneity in liver histopathology is associated with GSK-3ß activity and mitochondrial dysfunction in end-stage diabetic rats on differential diets.

While liver histopathology is heterogeneous in diabetes, the underlying mechanisms remain unclear. We investigated whether glycemic variation resulting from differential diets can induce heterogeneity in diabetic liver and the underlying molecular mechanisms. We generated end-stage non-obese diabetic model rats by subtotal-pancreatectomy in male Sprague- Dawley rats and ad libitum diet for 7 weeks (n = 33). The rats were then divided into three groups, and fed a standard- or a low-protein diet (18 or 6 kcal%, respectively), for another 7 weeks: to maintain hyperglycemia, 11 rats were fed ad libitum (18AL group); to achieve euglycemia, 11 were calorierestricted (18R group), and 11 were both calorie- and proteinrestricted with the low-protein diet (6R group). Overnightfasted liver samples were collected after the differential diets together with sham-control (18S group), and histology and molecular changes were compared. Hyperglycemic-18AL showed glycogenic hepatopathy (GH) without steatosis, with the highest GSK-3ß inactivation because of Akt activation during hyperglycemia; mitochondrial function was not impaired, compared to the 18S group. Euglycemic-18R showed neither GH nor steatosis, with intermediate GSK-3ß activation and mitochondrial dysfunction. However, euglycemic-6R showed both GH and steatosis despite the highest GSK-3ß activity and no molecular evidence of increased lipogenesis or decreased ApoB expression, where mitochondrial dysfunction was highest among the groups. In conclusion, heterogeneous liver histopathology developed in end-stage non-obese diabetic rats as the glycemic levels varied with differential diets, in which protein content in the diets as well as glycemic levels differentially influenced GSK-3ß activity and mitochondrial function in insulin-deficient state. [BMB Reports 2020; 53(2): 100-105].

Loss of transglutaminase 2 sensitizes for diet-induced obesity-related inflammation and insulin resistance due to enhanced macrophage c-Src signaling.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes clearance of apoptotic cells (efferocytosis) acting as integrin ß3 coreceptor. Accumulating evidence indicates that defective efferocytosis contributes to the development of chronic inflammatory diseases. Obesity is characterized by the accumulation of dead adipocytes and inflammatory macrophages in the adipose tissue leading to obesity-related metabolic syndrome. Here, we report that loss of TG2 from bone marrow-derived cells sensitizes for high fat diet (HFD)-induced pathologies. We find that metabolically activated TG2 null macrophages express more phospho-Src and integrin ß3, unexpectedly clear dying adipocytes more efficiently via lysosomal exocytosis, but produce more pro-inflammatory cytokines than the wild type ones. Anti-inflammatory treatment with an LXR agonist reverts the HFD-induced phenotype in mice lacking TG2 in bone marrow-derived cells with less hepatic steatosis than in wild type mice proving enhanced lipid clearance. Thus it is interesting to speculate whether LXR agonist treatment together with enhancing lysosomal exocytosis could be a beneficial therapeutic strategy in obesity.

Low abundance of mitofusin 2 in dairy cows with moderate fatty liver is associated with alterations in hepatic lipid metabolism.

High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows.

Pancreastatin inhibitor activates AMPK pathway via GRP78 and ameliorates dexamethasone induced fatty liver disease in C57BL/6 mice.

AIMS: To investigate the role of pancreastatin inhibitor (PSTi8) in lipid homeostasis and insulin sensitivity in dexamethasone induced fatty liver disease associated type 2 diabetes. MAIN METHODS: Glucose releases assay, lipid O staining and ATP/AMP ratio were performed in HepG2 cells. Twenty four mice were randomly divided into 4 groups: Control group (saline), DEX (1 mg/kg, im) for 17 days, DEX+PSTi8 (acute 5 mg/kg and chronic 2 mg/kg, ip) for 10 days. The glucose, insulin and pyruvate tolerance tests (GTT, ITT and PTT), biochemical parameters and Oxymax-CLAMS were performed. Further to elucidate the action mechanisms of PSTi8, we performed genes expression and western blotting of biological samples. KEY FINDINGS: We found that PSTi8 suppresses hepatic glucose release, lipid deposition, oxidative stress induced by DEX, stimulates the cellular energy level in hepatocytes and enhances GRP78 activity. It reduces lipogensis and enhances fatty acid oxidation to improve insulin sensitivity and glucose tolerance in DEX induced diabetic mice. The above cellular effects are the result of activated AMPK signalling pathway in liver, which increases Srebp1c and ACC phosphorylation. The increased ACC phosphorylation suppresses protein kinase C activity and enhances insulin sensitivity. The increased expression of UCP3 in liver elicits fatty acid oxidation and energy expenditure, which suppress oxidative stress. SIGNIFICANCE: Thus the activation of AMPK signalling through GRP78, improves lipid homeostasis, enhances insulin sensitivity via inhibition of PKC activity. PSTi8 suppresses inflammation associated with incomplete fatty acid oxidation. Hence, PSTi8 may be a potential therapeutic agent to treat glucocorticoid-induced fatty liver associated type 2 diabetes.

Metformin delays AKT/c-Met-driven hepatocarcinogenesis by regulating signaling pathways for de novo lipogenesis and ATP generation.

Hepatocellular carcinoma (HCC) is a lethal malignancy with few effective options for therapeutic treatment in its advanced stages. Metformin, a first-line oral agent used in the treatment of type 2 diabetes, exhibits efficacy in metabolic reprogramming fueling changes in cell growth and proliferation for multiple cancer types, including HCC. However, the molecular mechanism by which metformin delays hepatocarcinogenesis in individuals with hepatic steatosis remains rare. Here, we investigate the preventive efficacy of metformin in a rapid AKT/c-Met-triggered HCC mouse model featuring excessive levels of steatosis. Hematoxylin and eosin staining, Oil Red O staining and immunoblotting were applied for mechanistic investigations. Pharmacological and biochemical strategies were employed to illuminate molecular evidence for HCC cell lines. The results show that metformin obstructs the malignant transformation of hepatocytes in AKT/c-Met mice. Mechanistically, metformin reduces the expression of phospho-ERK (Thr202/Tyr204) and two forms of proto-oncogenes, Cyclin D1 and c-Myc, in AKT/c-Met mice. Moreover, metformin ameliorates FASN-mediated aberrant lipogenesis and HK2/PKM2-driven ATP generation in vivo. Furthermore, metformin represses the expression of FASN and HK-2 by targeting c-Myc in an AMPK-dependent manner in vitro. In addition, metformin is effective at inhibiting PKM2 expression in the presence of an AMPK inhibitor compound C, suggesting that its functioning in PKM2 is AMPK-independent. Our study experimentally validates a novel molecular mechanism by which metformin alleviates enhanced lipogenesis and high energy metabolism during hepatocarcinogenesis, indicating that metformin may serve as an agent for the prevention of HCC in patients with nonalcoholic fatty liver diseases.

Discovery of potent liver-selective stearoyl-CoA desaturase-1 (SCD1) inhibitors, thiazole-4-acetic acid derivatives, for the treatment of diabetes, hepatic steatosis, and obesity.

SCD1 is a rate-limiting enzyme in the conversion of saturated fatty acids to monounsaturated fatty acids. SCD1 inhibitors have potential effects on obesity, diabetes, acne, and cancer, but the adverse effects associated with SCD1 inhibition in the skin and eyelids are impediments to clinical development. To avoid mechanism-based adverse effects, we explored the compounds that selectively inhibit SCD1 in the liver in an ex vivo assay. Starting from a systemically active lead compound, we focused on the physicochemical properties tPSA and cLogP to minimize exposure in the off-target tissues. This effort led to the discovery of thiazole-4-acetic acid analog 48 as a potent and liver-selective SCD1 inhibitor. Compound 48 exhibited significant effects in rodent models of diabetes, hepatic steatosis, and obesity, with sufficient safety margins in a rat toxicology study with repeated dosing.