Insulin-resistant apoB/BATless mice have hypertriglyceridemia because of increased assembly and secretion of very low density apolipoprotein B (apoB) and triglycerides compared with mice expressing only apoB (Siri, P., Candela, N., Ko, C., Zhang, Y., Eusufzai, S., Ginsberg, H. N., and Huang, L. S. (2001) J. Biol. Chem. 276, 46064-46072). Despite increased very low density lipoprotein secretion, apoB/BATless mice have fatty livers. We found that hepatic mRNA levels of key lipogenic enzymes, acetyl-CoA carboxylase, fatty-acid synthase, and stearoyl-CoA desaturase-1 were increased in apoB/BATless mice compared with levels in apoB mice, suggesting increased lipogenesis in apoB/BATless mice. This was confirmed by determining incorporation of tritiated water into fatty acids. Neither the hepatic mRNA of the lipogenic transcription factor, SREBP-1c (sterol-response element-binding protein 1c), nor the nuclear levels of the mature form of SREBP-1 protein were elevated in apoB/BATless mice. By contrast, hepatic levels of peroxisomal proliferator-activated receptor 2 (PPARγ2) mRNA and protein were specifically increased in apoB/BATless mice, as were hepatic mRNA levels of two targets of PPARγ, CD36 and aP2. Treatment of apoB/BATless mice for 4 weeks with intraperitoneal injections of a PPARγ antisense oligonucleotide resulted in dramatic reductions of both PPARγ1 and PPARγ2 mRNA, PPARγ2 protein, and mRNA levels of fatty-acid synthase and acetyl-CoA carboxylase. These changes were associated with decreased hepatic de novo lipogenesis and hepatic triglyceride concentrations. We conclude that hepatic steatosis in apoB/BATless mice is associated with elevated rates of hepatic lipogenesis that are linked directly to increased hepatic expression of PPARγ2. The mechanism whereby hepatic Pparγ2 gene expression is increased and how PPARγ2 stimulates lipogenesis is under investigation.

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.

Inhibition of VLDL secretion reduces plasma levels of atherogenic apolipoprotein B (apoB) lipoproteins but can also cause hepatic steatosis. Approaches targeting apoB synthesis, which lies upstream of VLDL secretion, have potential to effectively reduce dyslipidemia but can also lead to hepatic accumulation of unsecreted triglycerides (TG). Here, we found that treating mice with apoB antisense oligonucleotides (ASOs) for 6 weeks decreased VLDL secretion and plasma cholesterol without causing steatosis. The absence of steatosis was linked to an increase in ER stress in the first 3 weeks of ASO treatment, followed by development of ER autophagy at the end of 6 weeks of treatment. The latter resulted in increased fatty acid (FA) oxidation that was inhibited by both chloroquine and 3-methyl adenine, consistent with trafficking of ER TG through the autophagic pathway before oxidation. These findings support the concept that inhibition of apoB synthesis traps lipids that have been transferred to the ER by microsomal TG transfer protein (MTP), inducing ER stress. ER stress then triggers ER autophagy and subsequent lysosomal lipolysis of TG, followed by mitochondrial oxidation of released FA, leading to prevention of steatosis. The identification of this pathway indicates that inhibition of VLDL secretion remains a viable target for therapies aiming to reduce circulating levels of atherogenic apoB lipoproteins.

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.

Abetalipoproteinemia (ABL) is an inherited disorder of lipoprotein metabolism resulting from mutations in microsomal triglyceride transfer protein (MTTP). In addition to expression in the liver and intestine, MTTP is expressed in cardiomyocytes, and cardiomyopathy has been reported in several ABL cases. Using induced pluripotent stem cells (iPSCs) generated from an ABL patient homozygous for a missense mutation (MTTPR46G), we show that human hepatocytes and cardiomyocytes exhibit defects associated with ABL disease, including loss of apolipoprotein B (apoB) secretion and intracellular accumulation of lipids. MTTPR46G iPSC-derived cardiomyocytes failed to secrete apoB, accumulated intracellular lipids, and displayed increased cell death, suggesting intrinsic defects in lipid metabolism due to loss of MTTP function. Importantly, these phenotypes were reversed after the correction of the MTTPR46G mutation by CRISPR/Cas9 gene editing. Together, these data reveal clear cellular defects in iPSC-derived hepatocytes and cardiomyocytes lacking MTTP activity, including a cardiomyocyte-specific regulated stress response to elevated lipids.