Stearoyl-CoA Desaturase-1 Protects Cells against Lipotoxicity-Mediated Apoptosis in Proximal Tubular Cells

Abstract

Saturated fatty acid (SFA)-related lipotoxicity is a pathogenesis of diabetes-related renal proximal tubular epithelial cell (PTEC) damage, closely associated with a progressive decline in renal function. This study was designed to identify a free fatty acid (FFA) metabolism-related enzyme that can protect PTECs from SFA-related lipotoxicity. Among several enzymes involved in FFA metabolism, we identified stearoyl-CoA desaturase-1 (SCD1), whose expression level significantly decreased in the kidneys of high-fat diet (HFD)-induced diabetic mice, compared with non-diabetic mice. SCD1 is an enzyme that desaturates SFAs, converting them to monounsaturated fatty acids (MUFAs), leading to the formation of neutral lipid droplets. In culture, retrovirus-mediated overexpression of SCD1 or MUFA treatment significantly ameliorated SFA-induced apoptosis in PTECs by enhancing intracellular lipid droplet formation. In contrast, siRNA against SCD1 exacerbated the apoptosis. Both overexpression of SCD1 and MUFA treatment reduced SFA-induced apoptosis via reducing endoplasmic reticulum stress in cultured PTECs. Thus, HFD-induced decrease in renal SCD1 expression may play a pathogenic role in lipotoxicity-induced renal injury, and enhancing SCD1-mediated desaturation of SFA and subsequent formation of neutral lipid droplets may become a promising therapeutic target to reduce SFA-induced lipotoxicity. The present study provides a novel insight into lipotoxicity in the pathogenesis of diabetic nephropathy.

Keywords: desaturation; free fatty acid; lipid droplet; lipotoxicity; proximal tubular epithelial cells.

Figure 1

Body weight (A) and blood glucose levels (B) of mice fed with either normal diet (ND, n = 5) or high-fat diet (HFD, n = 5); (C) Expression levels of mRNAs related to lipid metabolism in the kidneys of mice fed an ND or a HFD. Data are expressed as a relative ratio to the ND group; (D) Representative immunoblot of SCD1 and ADRP. β-actin was used as a loading control; (E) Quantitative results of the immunoblots. Data are expressed as a relative ratio to the ND group. p < 0.05 indicates statistical significance; (F) Immunohistological analysis of SCD1 in kidney sections of mice fed an ND or a HFD. Magnification: ×40 (upper), ×200 (lower). Data are shown as mean ± SD. p < 0.05 indicates statistical significance. NS indicates no significance. CPT1, carnitine palmitoyltransferase 1; ACO, acetyl coenzyme A oxidase; MCAD, medium-chain acyl-CoA dehydrogenase; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; SCD, stearoyl-CoA desaturase; DGAT, diacylglycerol transferase; ADRP, adipose differentiation-related protein.

Figure 2

(A) Schema representing the formation of neutral lipid droplets from saturated free fatty acids (FAs) such as palmitate and stearate; (B) Representative immunoblot of cleaved caspase 3 and SCD1 in cultured proximal tubular cells infected with the control retrovirus (pBABE-CONT) or the retrovirus for SCD1 overexpression (pBABE-SCD1), and treated with/without palmitate or oleate. β-actin was used as a loading control; (C) Quantitative result of cleaved caspase 3 (n = 5); (D) Representative pictures of TUNEL staining under the indicated conditions. White arrows indicate TUNEL-positive apoptotic cells; (E) Quantitative results of TUNEL staining; (F) Representative immunoblot of cleaved caspase 3 and SCD1 in cultured proximal tubular cells transfected with the control siRNA or siRNA against SCD1, and treated with/without palmitate or oleate; (G) Quantitative result of cleaved caspase 3 (n = 7). SCD, stearoyl-CoA desaturase. Data are shown as mean ± SD. p < 0.05 indicates statistical significance.

Figure 3

(A) Representative immunoblot of pPERK, PERK, sXBP1 and CHOP in cultured proximal tubular cells infected with the control retrovirus (pBABE-CONT) or the retrovirus for SCD1 overexpression (pBABE-SCD1), and treated with/without palmitate or oleate. β-actin was used as a loading control. Quantitative results of the pPERK/PERK ratio (B), sXBP1 (C) and CHOP (D) (n = 4). Data are shown as mean ± SD. p < 0.05 indicates statistical significance. pPERK, PKR-like ER kinase (PERK) phosphorylated at Thr980; sXBP1, spliced X-box binding protein 1; CHOP, C/EBP homologous protein; SCD, stearoyl-CoA desaturase.

Figure 4

(A) Representative BODIPY staining for visualizing intracellular neutral lipid accumulation in cultured proximal tubular cells infected with the control retrovirus (pBABE-CONT) or the retrovirus for SCD1 overexpression (pBABE-SCD1), and treated with/without palmitate or oleate; (B) mRNA expression levels of DGAT1, DGAT2 and ADRP (n = 6). Data are shown as mean ± SD. NS indicates no significance. DGAT, diacylglycerol transferase; ADRP, adipose differentiation-related protein.

Figure 5

(A) Representative immunoblot of ADRP in cultured proximal tubular cells infected with the control retrovirus (pBABE-CONT) or the retrovirus for ADRP overexpression (pBABE-ADRP); (B) Representative immunoblot of cleaved caspase 3, pPERK, PERK and CHOP in pBABE-CONT or pBABE-ADRP transfected cells, treated with/without palmitate. β-actin was used as a loading control; Quantitative results of cleaved caspase 3 (C), pPERK/PERK ratio (D) and CHOP (E) (n = 4); (F) Representative pictures of TUNEL staining for detecting apoptotic cells in pBABE-CONT or pBABE-ADRP transfected cells, treated with/without palmitate. White arrows indicate TUNEL-positive apoptotic cells; (G) Quantitative results of TUNEL staining. Data are shown as mean ± SD. p < 0.05 indicates statistical significance. ADRP, adipose differentiation-related protein; pPERK, PKR-like ER kinase (PERK) phosphorylated at Thr980; CHOP, C/EBP homologous protein; SCD, stearoyl-CoA desaturase.

Figure 6

Schema showing the renoprotective mechanism of SCD1 against saturated fatty acid (SFA)-bound albumin-induced proximal tubular epithelial cell (PTEC) damage in type 2 diabetic nephropathy. Systemic alteration in FFA metabolism increases urinary SFA-related lipotoxicity. Furthermore, diabetic conditions alter intrarenal FA metabolism, characterized by SCD1 deficiency, enhancing ER stress-related PTEC damage. SCD1 activation may ameliorate SFA-induced PTEC damage via enhancing desaturation and subsequent lipid droplet formation.