Hepatic retinaldehyde deficiency is involved in diabetes deterioration by enhancing PCK1- and G6PC-mediated gluconeogenesis

Abstract

Type 2 diabetes (T2D) is often accompanied with an induction of retinaldehyde dehydrogenase 1 (RALDH1 or ALDH1A1) expression and a consequent decrease in hepatic retinaldehyde (Rald) levels. However, the role of hepatic Rald deficiency in T2D progression remains unclear. In this study, we demonstrated that reversing T2D-mediated hepatic Rald deficiency by Rald or citral treatments, or liver-specific Raldh1 silencing substantially lowered fasting glycemia levels, inhibited hepatic glucogenesis, and downregulated phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6-phosphatase (G6PC) expression in diabetic db/db mice. Fasting glycemia and Pck1/G6pc mRNA expression levels were strongly negatively correlated with hepatic Rald levels, indicating the involvement of hepatic Rald depletion in T2D deterioration. A similar result that liver-specific Raldh1 silencing improved glucose metabolism was also observed in high-fat diet-fed mice. In primary human hepatocytes and oleic acid-treated HepG2 cells, Rald or Rald + RALDH1 silencing resulted in decreased glucose production and downregulated PCK1/G6PC mRNA and protein expression. Mechanistically, Rald downregulated direct repeat 1-mediated PCK1 and G6PC expression by antagonizing retinoid X receptor α, as confirmed by luciferase reporter assays and molecular docking. These results highlight the link between hepatic Rald deficiency, glucose dyshomeostasis, and the progression of T2D, whilst also suggesting RALDH1 as a potential therapeutic target for T2D.

Keywords: Gluconeogenesis; Glucose-6-phosphatase; Oleic acid; Phosphoenolpyruvate carboxykinase 1; Retinaldehyde; Retinaldehyde dehydrogenase 1; Retinoid X receptor; Type 2 diabetes.

Graphical abstract

Figure 1

Effects of retinaldehyde (Rald) or Rald+citral treatment on type 2 diabetes (T2D) progression in db/db mice. Schematic diagram of the experiment (A). Changes in body weight (B), food intake (C), water consumption (D), and fasting blood glucose (FBG) levels (E) of db/m (CON) mice, db/db (DM) mice, Rald-treated db/db (DM-R) mice, and Rald+citral-treated db/db (DM-RC) mice. Blood glucose levels and their area under the curve (AUC) following intraperitoneal 1 g/kg sodium pyruvate (F), 0.75 g/kg glucose (G), or 1 IU/kg insulin (H) injection in CON, DM, DM-R, and DM-RC mice. The expression of RALDH1 protein (I), levels of Rald (J) and RA (K), and mRNA levels of Cyp26a (L) in the livers of CON, DM, DM-R, and DM-RC mice. Correlation analysis of hepatic Rald levels with FBG (M) and the AUC of the intraperitoneal pyruvate (N) or glucose tolerance test (O) in CON, DM, DM-R, and DM-RC mice. Data are mean ± SD, n = 6. ∗P < 0.05, ∗∗P < 0.01 vs. DM mice; ^##P < 0.01 vs. DM-R mice; ^&&P < 0.01 vs. Week 0.

Figure 2

Liver-specific Raldh1 silencing increases hepatic Rald levels to attenuate T2D deterioration. (A) In vivo imaging of CON mice (db/m mice receiving saline injection), DM-ShCON mice (db/db mice infected with AAV9-shEmpty), and DM-ShRaldh1 mice (db/db mice infected with AAV9-shRaldh1) at 3 weeks after AAV9 injection. Levels of hepatic RALDH1 mRNA and protein (B), hepatic Rald (C), and FBG (D) in CON, DM-ShCON, and DM-ShRaldh1 mice. Blood glucose levels and their AUC in each group of mice following intraperitoneal injection of 1 g/kg sodium pyruvate (E) or 0.75 g/kg glucose (F). Data are mean ± SD, n = 6. ∗P < 0.05, ∗∗P < 0.01 vs. DM-ShCON mice; ^&&P < 0.01 vs. Day 0.

Figure 3

Decreases in hepatic Rald upregulates PCK1 and G6PC expression in db/db mice. GCK, PCK1, and G6PC protein levels (A, B) in the livers of CON, DM, DM-R, and DM-RC mice (n = 6). (C) Gck, Pck1, and G6pc mRNA levels in the livers of CON (n = 5), DM (n = 6), DM-R (n = 6), and DM-RC (n = 6) mice. Correlation analysis of hepatic Rald concentrations with the mRNA levels of Pck1 (D), G6pc (E), and Gck (F). GCK, PCK1, and G6PC protein (G, H) and mRNA (I) levels in the livers of CON, DM-ShCON, and DM-ShRaldh1 mice (n = 6). Data are mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. DM or DM-ShCON mice.

Figure 4

Liver-specific Raldh1 silencing increases hepatic Rald to inhibit high-fat diet (HFD)-induced glucose dyshomeostasis. Schematic diagram of the experiment (A). Changes in the hepatic Rald levels (B), body weight (C), FBG levels (D), and food consumption (E) of SCD-ShCON mice (standard chow diet-fed AAV9-shEmpty-infected C57BL/6J mice), HFD-ShCON mice (HFD-fed AAV9-shEmpty-infected C57BL/6J mice), and HFD-ShRaldh1 mice (HFD-fed AAV9-shRaldh1-infected C57BL/6J mice). Blood glucose levels and their AUC in the SCD-ShCON, HFD-ShCON, and HFD-ShRaldh1 mice following intraperitoneal injection of 2 g/kg sodium pyruvate (F) or glucose (G). PCK1 and G6PC protein levels (H) in SCD-ShCON, HFD-ShCON, and HFD-ShRaldh1 mice. Data are mean ± SD, n = 6. ∗P < 0.05, ∗∗P < 0.01 vs. HFD-ShCON mice.

Figure 5

Effects of Rald and RALDH1 silencing (Si) on the expression of enzymes related to glucose metabolism in oleic acid (OA)-treated HepG2 cells and primary human hepatocytes. Effects of OA treatment on the cellular levels of triglyceride (A), OA (B), palmitic acid (C), glucose utilization (D), glucose production (E), and mRNA levels of PCK1, G6PC, and GCK (F) (n = 4) in HepG2 cells. Effects of Rald, Si, and Rald + Si on glucose production (G), the mRNA expression of PCK1 and G6PC (H), glucose utilization (I), and the mRNA expression of GCK (J) in OA-treated HepG2 cells (n = 4). Effects of Rald, Si, and Rald + Si on the expression of PCK1, G6PC, and GCK (K) proteins in OA-treated HepG2 cells (n = 6). Changes in the mRNA levels of RALDH1 under Si treatment (L), changes in the levels of glucose production (M), and PCK1, G6PC, and GCK mRNA expression (N) under treatment with Si, Rald, or their combination in primary hepatocytes from four human donors (n = 3 for each donor). PCK1 and G6PC protein levels in primary hepatocytes from donors 2 and 3 (n = 3 for each donor) under Rald or Rald + Si treatment (O). Data are mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. CON group; ^#P < 0.05, ^##P < 0.01 vs. OA group.

Figure 6

Rald inhibits PCK1 and G6PC expression by antagonizing RXR. The effects of antagonists or agonists of RXR (HX531 or LG100268) (A), RAR (Ro 41–5253 or TTNPB) (B), and PPARγ (GW9662 or rosiglitazone) (C) on PCK1 and G6PC mRNA expression in OA-treated HepG2 cells. The effects of HX531, Ro 41–5253, and Rald on LG100268+TTNPB-induced PCK1 (D), G6PC (E), and CYP26A (K) mRNA expression. The effects of Rald on LG100268- (F and G) or TTNPB- (H and I) induced PCK1 and G6PC mRNA expression. CYP26A gene expression under TTNPB, Ro 41–5253, and Rald treatment (J). In the above experiments, the concentrations of HX531, LG100268, TTNPB, GW9662, and rosiglitazone were 1 μmol/L; the concentration of Ro 41–5253 was 5 μmol/L; and the concentration of Rald was 2 μmol/L. Data are mean ± SD, n = 4. ∗∗P＜0.01 vs. CON group; ^$P＜0.05, ^$$P＜0.01 vs. LG100268, TTNPB, or LG100268+TTNPB group; ^##P＜0.01 vs. Rald group. RAR, retinoid acid receptor; RXR, retinoid X receptor; PPARγ, peroxisome proliferator-activated receptor-gamma.

Figure 7

Effects of Rald on RXRα and RARα activation. The effects of Rald or RA on RARα-ligand-binding domain (LBD) activation (A). The effects of concentration-elevated Rald with 0.5 μmol/L RA (B) and concentration-elevated RA with or without 0.2 μmol/L Rald (C) on RARα-LBD activation. The effects of Rald, RA, OA, or 9-cis-RA (9C-RA) on RXRα-LBD activation (D). The effects of concentration-elevated RA (E) or OA (F) with or without 5 μmol/L Rald on RXRα-LBD activation. The effects of concentration-elevated Rald with 0.4 mmol/L OA (G) or 1 μmol/L RA (H) on RXRα-LBD activation. (I) The docking score of Rald, RA, OA, or 9C-RA calculated by the glide function of Schrödinger Maestro 11.5. Close-up view of the above ligands (J) and their binding modes (K) in the RXRα binding pocket. (L) Superimposition of the RXRα crystal structures with different ligands bound. Data are mean ± SD, n = 3. ∗P < 0.05, ∗∗P < 0.01 vs. concentration 0.

Figure 8

Rald downregulates PCK1 and G6PC expression by inhibiting RXR/DR1 activation. (A) Heatmap showing the changes in the levels of different genes in human primary hepatocytes under Rald treatment, RALDH1 silencing (Si), and combination of them (n = 3 per donor). The relative luciferase activation (R.L.A) under the treatment of 0.2 μmol/L 9C-RA with concentration-elevated Rald or HX531 in HEK293T cells transfected with the RXR:RXR-DR1 (B), RAR:RXR-DR1 (C) and RAR:RXR-DR5 systems (D) (n = 3). Data are mean ± SD. ∗∗P＜0.01 vs. CON group; ^#P＜0.05, ^##P＜0.01 vs. 9C-RA group.