Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways

Abstract

Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine beta-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element-binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia.

Figure 1

Intracellular homocysteine levels in HepG2 cells. HepG2 cells were cultured in the absence or presence of 1 mM (circles) or 5 mM (triangles) homocysteine for the indicated time periods, washed, and lysed by three freeze/thaw cycles. Total intracellular homocysteine was determined and normalized to total protein. Data are presented as the mean ± SD of three separate experiments.

Figure 2

Homocysteine induces the expression of GRP78/BiP and GADD153 in HepG2 cells. (a) Northern blot analysis of the steady-state mRNA levels of GRP78/BiP and GADD153 in HepG2 cells cultured for 4 hours in the absence (control) or presence of either 5 mM homocysteine, 5 mM cysteine, 5 mM methionine, 10 μg/ml tunicamycin, 2.5 mM DTT, 5 mM homoserine, or 5 mM glycine. Total RNA (10 μg/lane) was size fractionated by agarose-gel electrophoresis, transferred to nylon membranes, and subjected to blot hybridization using radiolabeled cDNA probes encoding human GRP78/BiP or GADD153. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe. (b) Immunoblot analysis of GRP78/BiP and GADD153 protein in HepG2 cells cultured in the absence or presence of 5 mM homocysteine for the indicated time periods. HepG2 cells were also treated with 2.5 mM DTT or 10 μg/ml tunicamycin for 8 hours. Total protein lysates (40 μg/lane) were separated on 12% SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes and immunostained with Ab’s against either GRP78/BiP (anti-KDEL) or GADD153.

Figure 3

Homocysteine induces the expression of SREBP-1 in HepG2 cells. (a) Immunoblot analysis of SREBP-1 protein in HepG2 cells cultured in the absence or presence of 5 mM homocysteine for the indicated time periods. Total protein lysates (40 μg/lane) were separated on 10% SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes, and immunostained with an mAb that recognizes both the precursor (P) and mature (M) forms of SREBP-1. (b) Northern blot analysis of the steady-state mRNA levels of SREBP-1 in HepG2 cells cultured in the presence of 5 mM homocysteine for the indicated time periods. Total RNA (10 μg/lane) was size fractionated by agarose-gel electrophoresis, transferred to nylon membranes, and subjected to blot hybridization using a radiolabeled cDNA probe encoding human SREBP-1. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe.

Figure 4

Homocysteine induces the steady-state mRNA levels of genes involved in cholesterol biosynthesis in HepG2 cells. Northern blot analysis of the steady-state mRNA levels of IPP isomerase, HMG-CoA reductase, and FPP synthase in HepG2 cells cultured in the absence or presence of 5 mM homocysteine for the indicated time periods. Total RNA (10 μg/lane) was size fractionated by agarose-gel electrophoresis, transferred to nylon membranes, and subjected to blot hybridization using the appropriate radiolabeled cDNA probe. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe.

Figure 5

Effect of ER stress agents on steady-state mRNA levels of IPP isomerase in HepG2 cells. Equivalent amounts of total RNA (10 μg/lane) isolated from HepG2 cells cultured for 4 hours in the absence (control) or presence of 5 mM homocysteine, 2.5 mM DTT, 5 mM β-mercaptoethanol, 10 μM of the Ca²⁺ ionophore A23187, or 10 μg/ml tunicamycin were examined by Northern blot analysis using a radiolabeled IPP isomerase cDNA probe. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe.

Figure 6

Overexpression of GRP78/BiP prevents the increase in steady-state mRNA levels of SREBP-1 and IPP isomerase by homocysteine. (a) Immunoblot analysis of GRP78/BiP and GRP94 in wild-type (T24/83), vector-transfected (T24/83-pcDNA), or GRP78/BiP–overexpressing (T24/83-GRP78) cells. Total protein lysates (40 μg/lane) were separated on 10% SDS-polyacrylamide gels under reducing conditions and either stained with Coomassie blue (upper panel) or immunostained with an anti-KDEL mAb. (b) Immunolocalization of GRP78/BiP in the ER of wild-type or GRP78/BiP–overexpressing T24/83 cells. Cells grown on glass coverslips were fixed, permeabilized, and immunostained with an anti-GRP78/BiP polyclonal Ab. ×800. (c) Northern blot analysis of the steady-state mRNA levels of SREBP-1 and IPP isomerase in vector-transfected (T24/83-pcDNA) or GRP78/BiP–overexpressing (T24/83-GRP78) cells cultured in the absence or presence of 1 mM homocysteine for the indicated time periods. Total RNA (10 μg/lane) was size fractionated by agarose-gel electrophoresis, transferred to nylon membranes, and subjected to blot hybridization using radiolabeled cDNA probes encoding human SREBP-1 or IPP isomerase. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe.

Figure 7

Homocysteine increases intracellular total cholesterol in cultured human cells. HUVECs, HASMCs, and HepG2 cells were incubated for 48 hours in media containing 0, 0.2, 1, or 5 mM homocysteine. Cells were washed in PBS, harvested in 0.2 M NaOH, and lipids extracted as described in Methods. Total cholesterol was normalized for protein content, and values were expressed as a percentage versus cells treated in the absence of homocysteine. Data are presented as the mean ± SD from three separate experiments. ^AP < 0.05 between control and homocysteine-treated cells.

Figure 8

Effect of homocysteine on LDL uptake in HUVECs, HASMCs, and HepG2. Cells treated in the absence or presence of 5 mM homocysteine for 8 hours were washed with PBS followed by incubation for an additional 2 hours at 37°C in media containing 10 μg/ml BODIPY FL LDL. After washing with PBS, cells were fixed, and LDL binding/uptake was detected by fluorescence microscopy. ×375.

Figure 9

Hepatic morphology of CBS^+/+ and CBS^+/– mice fed control diet or high-methionine/low-folate diet. CBS^+/+ mice fed control (a) or high-methionine/low-folate diet (c) for 20 weeks. CBS^+/– mice fed control (b) or high-methionine/low-folate diet (d) for 16 weeks. The hepatocytes from mice fed the high-methionine/low-folate diet (c and d) are enlarged, multinucleated, and contain extensive microvesicular and macrovesicular lipid with no apparent fibrosis or necrosis. Hematoxylin and eosin staining. ×400.

Figure 10

VLDL-triglyceride secretion rates in CBS^+/+ mice fed control or hyperhomocysteinemic diets. CBS^+/+ mice were fed a control diet, a high-methionine diet (HM), a high-methionine/low-folate diet (HMLF), or a very high-methionine/low-folate diet (SHH) for 2 weeks. Plasma VLDL-triglyceride levels were determined in mice 0, 2, and 4 hours after Triton WR1339 injection. Hepatic VLDL-triglyceride secretion rate was calculated from the slope of the curve. Data are presented as the mean ± SD (n = 4 mice/group). Mean total plasma homocysteine concentration for mice on the SHH diet was 334 ± 100 μM. ^AP < 0.05 between control and mice having hyperhomocysteinemia.

Figure 11

Increased steady-state mRNA levels of GADD153, SREBP-1, and the LDL receptor in the livers of mice having hyperhomocysteinemia. CBS^+/+ mice were fed a control diet (C), a high-methionine diet (HM), or a combination high-methionine/low-folate diet (HMLF). After 2 weeks, the animals were sacrificed and tissues harvested. Total RNA (10 μg/lane) isolated from the livers of two randomly selected animals from each group (n = 5) was examined by Northern blot analysis using radiolabeled cDNA probes encoding human GADD153, SREBP-1, or LDL receptor. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe.