Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy.

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 apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents.

ER stress can cause hepatic insulin resistance and steatosis. Increased VLDL secretion could protect the liver from ER stress-induced steatosis, but the effect of lipid-induced ER stress on the secretion of VLDL is unknown. To determine the effect of lipids on hepatic ER stress and VLDL secretion, we treated McA-RH7777 liver cells with free fatty acids. Prolonged exposure increased cell triglycerides, induced steatosis, and increased ER stress. Effects on apoB100 secretion, which is required for VLDL assembly, were parabolic, with moderate free fatty acid exposure increasing apoB100 secretion, while greater lipid loading inhibited apoB100 secretion. This decreased secretion at higher lipid levels was due to increased protein degradation through both proteasomal and nonproteasomal pathways and was dependent on the induction of ER stress. These findings were supported in vivo, where intravenous infusion of oleic acid (OA) in mice increased ER stress in a duration-dependent manner. apoB secretion was again parabolic, stimulated by moderate, but not prolonged, OA infusion. Inhibition of ER stress was able to restore OA-stimulated apoB secretion after prolonged OA infusion. These results suggest that excessive ER stress in response to increased hepatic lipids may decrease the ability of the liver to secrete triglycerides by limiting apoB secretion, potentially worsening steatosis.

PCSK9 dominant negative mutant results in increased LDL catabolic rate and familial hypobetalipoproteinemia.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central player in the regulation of cholesterol homeostasis, increasing the low-density lipoprotein (LDL) receptor degradation. Our study aimed at exploring the pathogenic consequences in vivo and in vitro of a PCSK9 prodomain mutation found in a family with hypobetalipoproteinemia (FHBL). METHODS AND RESULTS: A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, approximately 50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9. CONCLUSIONS: These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL.

C-C chemokine receptor 2 (CCR2) regulates the hepatic recruitment of myeloid cells that promote obesity-induced hepatic steatosis.

OBJECTIVE: Obesity induces a program of systemic inflammation that is implicated in the development of many of its clinical sequelae. Hepatic inflammation is a feature of obesity-induced liver disease, and our previous studies demonstrated reduced hepatic steatosis in obese mice deficient in the C-C chemokine receptor 2 (CCR2) that regulates myeloid cell recruitment. This suggests that a myeloid cell population is recruited to the liver in obesity and contributes to nonalcoholic fatty liver disease. RESEARCH DESIGN AND METHODS: We used fluorescence-activated cell sorting to measure hepatic leukocyte populations in genetic and diet forms of murine obesity. We characterized in vivo models that increase and decrease an obesity-regulated CCR2-expressing population of hepatic leukocytes. Finally, using an in vitro co-culture system, we measured the ability of these cells to modulate a hepatocyte program of lipid metabolism. RESULTS: We demonstrate that obesity activates hepatocyte expression of C-C chemokine ligand 2 (CCL2/MCP-1) leading to hepatic recruitment of CCR2(+) myeloid cells that promote hepatosteatosis. The quantity of these cells correlates with body mass and in obese mice represents the second largest immune cell population in the liver. Hepatic expression of CCL2 increases their recruitment and in the presence of dietary fat induces hepatosteatosis. These cells activate hepatic transcription of genes responsible for fatty acid esterification and steatosis. CONCLUSIONS: Obesity induces hepatic recruitment of a myeloid cell population that promotes hepatocyte lipid storage. These findings demonstrate that recruitment of myeloid cells to metabolic tissues is a common feature of obesity, not limited to adipose tissue.

The effects of obesity-associated insulin resistance on mRNA expression of peroxisome proliferator-activated receptor-gamma target genes, in dogs.

Visceral adipose tissue and skeletal muscle have central roles in determining whole-body insulin sensitivity. The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a potential mediator of insulin sensitivity. It can directly modulate the expression of genes that are involved in glucose and lipid metabolism, including GLUT4, lipoprotein lipase (LPL) and adipocytokines (leptin and adiponectin). In this study, we aimed to determine the effects of obesity-associated insulin resistance on mRNA expression of PPARgamma and its target genes. Dogs were studied when they were lean and at the end of an overfeeding period when they had reached a steady obese state. The use of a sensitive, real-time PCR assay allowed a relative quantification of mRNA expression for PPARgamma, LPL, GLUT4, leptin and adiponectin, in adipose tissue and skeletal muscle. In visceral adipose tissue and/or skeletal muscle, mRNA expression of PPARgamma, LPL and GLUT4 were at least 2-fold less in obese and insulin-resistant dogs compared with the same animals when they were lean and insulin-sensitive. The mRNA expression and plasma concentration of leptin was increased, whereas the plasma level and mRNA expression of adiponectin was decreased, by obesity. In adipose tissue, PPARgamma expression was correlated with leptin and adiponectin. These findings, in an original model of obesity induced by a prolonged period of overfeeding, showed that insulin resistance is associated with a decrease in PPARgamma mRNA expression that could dysregulate expression of several genes involved in glucose and lipid metabolism.