Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity

Abstract

The endoplasmic reticulum (ER) is the main site of protein and lipid synthesis, membrane biogenesis, xenobiotic detoxification and cellular calcium storage, and perturbation of ER homeostasis leads to stress and the activation of the unfolded protein response. Chronic activation of ER stress has been shown to have an important role in the development of insulin resistance and diabetes in obesity. However, the mechanisms that lead to chronic ER stress in a metabolic context in general, and in obesity in particular, are not understood. Here we comparatively examined the proteomic and lipidomic landscape of hepatic ER purified from lean and obese mice to explore the mechanisms of chronic ER stress in obesity. We found suppression of protein but stimulation of lipid synthesis in the obese ER without significant alterations in chaperone content. Alterations in ER fatty acid and lipid composition result in the inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and ER stress. Correcting the obesity-induced alteration of ER phospholipid composition or hepatic Serca overexpression in vivo both reduced chronic ER stress and improved glucose homeostasis. Hence, we established that abnormal lipid and calcium metabolism are important contributors to hepatic ER stress in obesity.

Figure 1. Proteomic and lipidomic landscape of the lean and obese ER

a, Biological pathways associated with significantly regulated proteins in the obese ER proteome. Bar colors indicate the fold enrichment with significance values (negative log of p-values) superimposed. b,c, Transcript levels of genes involved in lipid metabolism in the lean and obese mouse liver. d, Alterations of liver ER lipidome. Heatmap display of all significant (p<0.05) alterations present between lean and obese ER lipidomes. The color corresponds to differences in the relative abundance (nmol%) of each fatty acid among individual lipid groups detected in the lean and obese liver ER. e, The relative abundance of PC and PE in lean and obese liver ER samples. Values are mean±SEM (n=6 for each group). “*” denotes p<0.05, Student's t-test.

Figure 2. Elevated PC/PE ratio impairs SERCA activity and ER homeostasis

a, Calcium transport activity of microsomes loaded with PC and PE in vitro. Transcript levels of Pemt (b) and corresponding microsomal calcium transport activities (c) of Hepa1-6 cells expressing control (Gfp) or mouse Pemt ORF. d, Calcium transport activity (top) and SERCA protein levels (bottom) of microsomes prepared from lean and obese mouse liver. Liver Serca2b transcript levels (e) and microsomal calcium transport activities (f), immunoblot (g) and quantitative RT-PCR (h) measurement of ER stress markers in the livers of lean mice expressing either LacZ (control) or Serca2b shRNAs. “*” in g denotes the phosphorylated IRE1α and “*” in other panels denotes significant difference (p<0.05, n=4) by student's t-test. Values are mean±SEM.

Figure 3. Suppression of liver Pemt expression corrects ER PC/PE ratio, relieves ER stress, and improves systemic glucose homeostasis in obesity

a, PC/PE ratio, and b, calcium transport activity of liver ER from ob/ob mice expressing LacZ (control) or Pemt shRNAs. Immunoblot (c) and quantitative PCR (d) measurement of ER stress markers in the liver. Expression of hepatic lipogenesis and gluconeogenesis genes (e), triglyceride content (f), and Hematoxylin & Eosin staining (g and h) of liver samples. Plasma glucose (i) and insulin (j) levels in control and Pemt shRNA-treated ob/ob mice after 6-hour food withdrawal. k-l, Plasma glucose levels of control and Pemt shRNA-treated ob/ob mice after intraperitoneal administration of either 1g/kg of glucose (k) or 1IU/kg of insulin (l). All data are mean±SEM (n=4 for a-e, n=6 for f-l), “*” denotes p<0.05 (one-way ANOVA for data presented in k and l, and Student's t-test for others).

Figure 4. Exogenous Serca expression alleviates ER stress and improves systemic glucose homeostasis

Liver Serca2b transcript levels (a) and microsomal calcium transport activities (b) of control or Serca2b overexpressing obese mice. Plasma glucose (c) Plasma insulin levels (d), tissue weights (e) of ob/ob mice as in panel a. Triglyceride content (f), H&E staining (g, h) and immunoblot analyses (i) of ER stress markers (IRE1α and eIF2α phosphorylation, and CHOP) and secretory proteins (ASGR and HP) in the obese liver expressing Serca2b compared to controls. All values are mean±SEM (n=4 for a-b, n=6 for c-h), “*” denotes p<0.05 (Student's t-test).