DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.

Loss of immunity-related GTPase GM4951 leads to nonalcoholic fatty liver disease without obesity.

Obesity and diabetes are well known risk factors for nonalcoholic fatty liver disease (NAFLD), but the genetic factors contributing to the development of NAFLD remain poorly understood. Here we describe two semi-dominant allelic missense mutations (Oily and Carboniferous) of Predicted gene 4951 (Gm4951) identified from a forward genetic screen in mice. GM4951 deficient mice developed NAFLD on high fat diet (HFD) with no changes in body weight or glucose metabolism. Moreover, HFD caused a reduction in the level of Gm4951, which in turn promoted the development of NAFLD. Predominantly expressed in hepatocytes, GM4951 was verified as an interferon inducible GTPase. The NAFLD in Gm4951 knockout mice was associated with decreased lipid oxidation in the liver and no defect in hepatic lipid secretion. After lipid loading, hepatocyte GM4951 translocated to lipid droplets (LDs), bringing with it hydroxysteroid 17ß-dehydrogenase 13 (HSD17B13), which in the absence of GM4951 did not undergo this translocation. We identified a rare non-obese mouse model of NAFLD caused by GM4951 deficiency and define a critical role for GTPase-mediated translocation in hepatic lipid metabolism.

Hepatic deletion of Mboat7 (LPIAT1) causes activation of SREBP-1c and fatty liver.

Genetic variants that increase the risk of fatty liver disease and cirrhosis have recently been identified in the proximity of membrane-bound O-acyltransferase domain-containing 7 (MBOAT7). To elucidate the link between these variants and fatty liver disease, we characterized Mboat7 liver-specific KO mice (Mboat7 LSKO). Chow-fed Mboat7 LSKO mice developed fatty livers and associated liver injury. Lipidomic analysis of liver using MS revealed a pronounced reduction in 20-carbon PUFA content in phosphatidylinositols (PIs) but not in other phospholipids. The change in fatty acid composition of PIs in these mice was associated with a marked increase in de novo lipogenesis because of activation of SREBP-1c, a transcription factor that coordinates the activation of genes encoding enzymes in the fatty acid biosynthesis pathway. Hepatic removal of both SREBP cleavage-activating protein (Scap) and Mboat7 normalized hepatic triglycerides relative to Scap-only hepatic KO, showing that increased SREBP-1c processing is required for Mboat7-induced steatosis. This study reveals a clear relationship between PI fatty acid composition and regulation of hepatic fat synthesis and delineates the mechanism by which mutations in MBOAT7 cause hepatic steatosis.

Osteocalcin and Non-Alcoholic Fatty Liver Disease: Lessons From Two Population-Based Cohorts and Animal Models.

Osteocalcin regulates energy metabolism in an active undercarboxylated/uncarboxylated form. However, its role on the development of non-alcoholic fatty liver disease (NAFLD) is still controversial. In the current study, we investigated the causal relationship of circulating osteocalcin with NAFLD in two human cohorts and studied the effect of uncarboxylated osteocalcin on liver lipid metabolism through animal models. We analyzed the correlations of serum total/uncarboxylated osteocalcin with liver steatosis/fibrosis in a liver biopsy cohort of 196 participants, and the causal relationship between serum osteocalcin and the incidence/remission of NAFLD in a prospective community cohort of 2055 subjects from Shanghai Changfeng Study. Serum total osteocalcin was positively correlated with uncarboxylated osteocalcin (r = 0.528, p < .001). Total and uncarboxylated osteocalcin quartiles were inversely associated with liver steatosis, inflammation, ballooning, and fibrosis grades in both male and female participants (all p for trend <.05). After adjustment for confounding glucose, lipid, and bone metabolism parameters, the male and female participants with lowest quartile of osteocalcin still had more severe liver steatosis, with multivariate-adjusted odds ratios (ORs) of 7.25 (1.07-49.30) and 4.44 (1.01-19.41), respectively. In the prospective community cohort, after a median of 4.2-year follow-up, the female but not male participants with lowest quartile of osteocalcin at baseline had higher risk to develop NAFLD (hazard ratio [HR] = 1.90; 95% confidence interval [CI] 1.14-3.16) and lower chance to achieve NAFLD remission (HR = 0.56; 95% CI 0.31-1.00). In wild-type mice fed a Western diet, osteocalcin treatment alleviated hepatic steatosis and reduced hepatic SREBP-1 and its downstream proteins expression. In mice treated with osteocalcin for a short term, hepatic SREBP-1 expression was decreased without changes of glucose level or insulin sensitivity. When SREBP-1c was stably expressed in a human SREBP-1c transgenic rat model, the reduction of lipogenesis induced by osteocalcin treatment was abolished. In conclusion, circulating osteocalcin was inversely associated with NAFLD. Osteocalcin reduces liver lipogenesis via decreasing SREBP-1c expression. © 2020 American Society for Bone and Mineral Research (ASBMR).

Cholesterol auxotrophy and intolerance to ezetimibe in mice with SREBP-2 deficiency in the intestine.

SREBP-2 activates transcription of all genes needed for cholesterol biosynthesis. To study SREBP-2 function in the intestine, we generated a mouse model (Vil-BP2-/- ) in which Cre recombinase ablates SREBP-2 in intestinal epithelia. Intestines of Vil-BP2-/- mice had reduced expression of genes required for sterol synthesis, in vivo sterol synthesis rates, and epithelial cholesterol contents. On a cholesterol-free diet, the mice displayed chronic enteropathy with histological abnormalities of both villi and crypts, growth restriction, and reduced survival that was prevented by supplementation of cholesterol in the diet. Likewise, SREBP-2-deficient enteroids required exogenous cholesterol for growth. Blockade of luminal cholesterol uptake into enterocytes with ezetimibe precipitated acutely lethal intestinal damage in Vil-BP2-/- mice, highlighting the critical interplay in the small intestine of sterol absorption via NPC1L1 and sterol synthesis via SREBP-2 in sustaining the intestinal mucosa. These data show that the small intestine requires SREBP-2 to drive cholesterol synthesis that sustains the intestinal epithelia when uptake of cholesterol from the gut lumen is not available, and provide a unique example of cholesterol auxotrophy expressed in an intact, adult mammal.

Expression of SREBP-1c Requires SREBP-2-mediated Generation of a Sterol Ligand for LXR in Livers of Mice.

The synthesis of cholesterol and fatty acids (FA) in the liver is independently regulated by SREBP-2 and SREBP-1c, respectively. Here, we genetically deleted Srebf-2 from hepatocytes and confirmed that SREBP-2 regulates all genes involved in cholesterol biosynthesis, the LDL receptor, and PCSK9; a secreted protein that degrades LDL receptors in the liver. Surprisingly, we found that elimination of Srebf-2 in hepatocytes of mice also markedly reduced SREBP-1c and the expression of all genes involved in FA and triglyceride synthesis that are normally regulated by SREBP-1c. The nuclear receptor LXR is necessary for Srebf-1c transcription. The deletion of Srebf-2 and subsequent lower sterol synthesis in hepatocytes eliminated the production of an endogenous sterol ligand required for LXR activity and SREBP-1c expression. These studies demonstrate that cholesterol and FA synthesis in hepatocytes are coupled and that flux through the cholesterol biosynthetic pathway is required for the maximal SREBP-1c expression and high rates of FA synthesis.

CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking.

Histone deacetylase 9 represses cholesterol efflux and alternatively activated macrophages in atherosclerosis development.

OBJECTIVE: Recent genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke. HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries. The molecular mechanisms how HDAC9 might increase atherosclerosis is not clear. APPROACH AND RESULTS: In this study, we show that systemic and bone marrow cell deletion of HDAC9 decreased atherosclerosis in LDLr(-/-) (low density lipoprotein receptor) mice with minimal effect on plasma lipid concentrations. HDAC9 deletion resulted upregulation of lipid homeostatic genes, downregulation of inflammatory genes, and polarization toward an M2 phenotype via increased accumulation of total acetylated H3 and H3K9 at the promoters of ABCA1 (ATP-binding cassette transporter), ABCG1, and PPAR-γ (peroxisome proliferator-activated receptor) in macrophages. CONCLUSIONS: We conclude that macrophage HDAC9 upregulation is atherogenic via suppression of cholesterol efflux and generation of alternatively activated macrophages in atherosclerosis.

Leptin ameliorates insulin resistance and hepatic steatosis in Agpat2-/- lipodystrophic mice independent of hepatocyte leptin receptors.

Leptin is essential for energy homeostasis and regulation of food intake. Patients with congenital generalized lipodystrophy (CGL) due to mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) and the CGL murine model (Agpat2(-/-) mice) both have severe insulin resistance, diabetes mellitus, hepatic steatosis, and low plasma leptin levels. In this study, we show that continuous leptin treatment of Agpat2(-/-) mice for 28 days reduced plasma insulin and glucose levels and normalized hepatic steatosis and hypertriglyceridemia. Leptin also partially, but significantly, reversed the low plasma thyroxine and high corticosterone levels found in Agpat2(-/-) mice. Levels of carbohydrate response element binding protein (ChREBP) were reduced, whereas lipogenic gene expression were increased in the livers of Agpat2(-/-) mice, suggesting that deregulated ChREBP contributed to the development of fatty livers in these mice and that this transcription factor is a target of leptin's beneficial metabolic action. Leptin administration did not change hepatic fatty acid oxidation enzymes mRNA levels in Agpat2(-/-) mice. The selective deletion of leptin receptors only in hepatocytes did not prevent the positive metabolic actions of leptin in Agpat2(-/-) mice, supporting the notion that the majority of metabolic actions of leptin are dependent on its action in nonhepatocyte cells and/or the central nervous system.

Omega-3 fatty acids ameliorate atherosclerosis by favorably altering monocyte subsets and limiting monocyte recruitment to aortic lesions.

OBJECTIVE: Fish oil, containing omega-3 fatty acids, attenuates atherosclerosis. We hypothesized that omega-3 fatty acid-enriched oils are atheroprotective through alteration of monocyte subsets and their trafficking into atherosclerotic lesions. METHODS AND RESULTS: Low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice were fed diets containing 10% (calories) palm oil and 0.2% cholesterol, supplemented with an additional 10% palm oil, echium oil (containing 18:4 n-3), or fish oil. Compared with palm oil-fed low-density lipoprotein receptor knockout mice, echium oil and fish oil significantly reduced plasma cholesterol, splenic Ly6C(hi) monocytosis by ≈50%, atherosclerosis by 40% to 70%, monocyte trafficking into the aortic root by ≈50%, and atherosclerotic lesion macrophage content by 30% to 44%. In contrast, atherosclerosis and monocyte trafficking into the artery wall was not altered by omega-3 fatty acids in apolipoprotein E(-/-) mice; however, Ly6C(hi) splenic monocytes positively correlated with aortic root intimal area across all diet groups. In apolipoprotein E(-/-) mice, fish oil reduced the percentage of blood Ly6C(hi) monocytes, despite an average 2-fold higher plasma cholesterol relative to palm oil. CONCLUSIONS: The presence of splenic Ly6C(hi) monocytes parallels the appearance of atherosclerotic disease in both low-density lipoprotein receptor knockout and apolipoprotein E(-/-) mice. Furthermore, omega-3 fatty acids favorably alter monocyte subsets independently from effects on plasma cholesterol and reduce monocyte recruitment into atherosclerotic lesions.

Macrophage 12/15 lipoxygenase expression increases plasma and hepatic lipid levels and exacerbates atherosclerosis.

12/15 lipoxygenase (12/15LO) oxidizes polyunsaturated fatty acids (PUFAs) to form bioactive lipid mediators. The role of 12/15LO in atherosclerosis development remains controversial. We evaluated atherosclerosis development and lipid metabolism in 12/15LO-LDL receptor (LDLr) double knockout (DK) vs. LDLr knockout (SK) mice fed a PUFA-enriched diet to enhance production of 12/15LO products. Compared with SK controls, DK mice fed a PUFA-enriched diet had decreased plasma and liver lipid levels, hepatic lipogenic gene expression, VLDL secretion, and aortic atherosclerosis and increased VLDL turnover. Bone marrow transplantation and Kupffer cell ablation studies suggested both circulating leukocytes and Kupffer cells contributed to the lipid phenotype in 12/15LO-deficient mice. Conditioned medium from in vitro incubation of DK vs. SK macrophages reduced triglyceride secretion in McArdle 7777 hepatoma cells. Our results suggest that, in the context of dietary PUFA enrichment, macrophage 12/15LO expression adversely affects plasma and hepatic lipid metabolism, resulting in exacerbated atherosclerosis.

Niemann-Pick C1-Like 1 deletion in mice prevents high-fat diet-induced fatty liver by reducing lipogenesis.

Niemann-Pick C1-Like 1 (NPC1L1) mediates intestinal absorption of dietary and biliary cholesterol. Ezetimibe, by inhibiting NPC1L1 function, is widely used to treat hypercholesterolemia in humans. Interestingly, ezetimibe treatment appears to attenuate hepatic steatosis in rodents and humans without a defined mechanism. Over-consumption of a high-fat diet (HFD) represents a major cause of metabolic disorders including fatty liver. To determine whether and how NPC1L1 deficiency prevents HFD-induced hepatic steatosis, in this study, we fed NPC1L1 knockout (L1-KO) mice and their wild-type (WT) controls an HFD, and found that 24 weeks of HFD feeding causes no fatty liver in L1-KO mice. Hepatic fatty acid synthesis and levels of mRNAs for lipogenic genes are substantially reduced but hepatic lipoprotein-triglyceride production, fatty acid oxidation, and triglyceride hydrolysis remain unaltered in L1-KO versus WT mice. Strikingly, L1-KO mice are completely protected against HFD-induced hyperinsulinemia under both fed and fasted states and during glucose challenge. Despite similar glucose tolerance, L1-KO relative WT mice are more insulin sensitive and in the overnight-fasted state display significantly lower plasma glucose concentrations. In conclusion, NPC1L1 deficiency in mice prevents HFD-induced fatty liver by reducing hepatic lipogenesis, at least in part, through attenuating HFD-induced insulin resistance, a state known to drive hepatic lipogenesis through elevated circulating insulin levels.

Targeted deletion of hepatocyte ABCA1 leads to very low density lipoprotein triglyceride overproduction and low density lipoprotein hypercatabolism.

Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11.

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5' proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3' hepatic control region, derived from a region approximately 18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3' splice acceptor sites causing deletion of cloned 5' untranslated mRNA sequences and, in some cases, deletion of the 5' end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3' splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1-exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.

[Occupational exposure profiles of polycyclic aromatic hydrocarbons in coke oven workers].

OBJECTIVE: To study the characteristics of occupational exposure of polycyclic aromatic hydrocarbons (PAHs) in coke oven workers. METHODS: Samples were collected individually and PAHs concentration in the ambient air were assayed by high performance liquid chromatography (HPLC) and toxic equivalency factors (TEFs) were introduced to assess the carcinogenic potency. RESULTS: The levels of PAHs occupational exposure in oven workers at topside was higher than at side oven and bottom oven (P < 0.05). Non-carcinogenic PAHs were more than 70% of total PAHs and benzo[a]pyrene accounted for 65.5% approximately 72.4% of total benzo[a]pyrene equivalents. Total occupational exposure level of PAHs in coke oven workers was positively related to the content of benzo[a]pyrene and pyrene, respectively (r(2) = 0.84, r(2) = 0.94, P < 0.05). CONCLUSION: Coke oven workers were exposed to a high level of PAHs which possessed some extent of carcinogenic potency, and benzo[a]pyrene is the chief carcinogenic substance.