Disruption of retinoid homeostasis induces RBP4 overproduction in diabetes: O-GlcNAcylation involved.

BACKGROUND: Retinol-binding protein 4 (RBP4) is elevated and associated with inflammation in metabolic diseases. Disruption of the retinol cascade and O-GlcNAcylation of the RBP4 receptor (STRA6) are found in diabetic kidneys. OBJECTIVES: We investigated whether the disruption of the retinol cascade induces RBP4 overproduction and if O-linked GlcNAc modification targets RBPR2 and contributes to the disruption of retinol cascades in diabetic livers. METHODS: Western blot or immunohistochemistry for RBPR2, CRBP1, LRAT, RALDH, RARα, RARγ, RXRα, RBP4, GFAT, OGT, OGA and inflammatory markers, as well as ELISA for RBP4, were performed in livers of db/db and ob/ob mice and high glucose-cultured hepatocytes. Immunoprecipitation and dual fluorescence staining were used to explore O-GlcNAc-modified RBPR2 and RBP4 binding activity on RBPR2. Transfection of the CRBP1 gene was done to verify whether a disrupted retinol cascade induces RBP4 overproduction. OGT silencing was done to investigate the association of O-GlcNAcylation with the disruption of retinol cascade. RESULTS: Disruption of retinol cascade, RBP4 overproduction, O-GlcNAcylation of RBPR2, decreased RBP4 binding activity on RBPR2 and inflammation were found in livers of db/db and ob/ob mice and high glucose-cultured hepatocytes. CRBP1 gene transfection reversed the suppression of the cellular retinol cascade and simultaneously attenuated the RBP4 overproduction and inflammation in high glucose-treated hepatocytes. The silencing of OGT reversed the disruption of the cellular retinol cascade, RBP4 overproduction and inflammation induced by high glucose in hepatocytes. CONCLUSIONS: This study indicates that the disruption of cellular retinol cascade is strongly associated with RBP4 overproduction and inflammation in diabetic livers. RBPR2 is one target for high glucose-mediated O-linked GlcNAc modification, which causes liver retinol dyshomeostasis.

O-GlcNAcylation disrupts STRA6-retinol signals in kidneys of diabetes.

BACKGROUND: O-GlcNAcylation is an important mechanism of diabetic complication. Retinoid homeostasis regulates cell-physiological functions through STRA6-retinol signaling. Therefore, we investigated whether O-GlcNAcylation disrupted STRA6-retinol signals in diabetes. METHODS: Immunoprecipitation and proximity ligation assay were used to investigate O-GlcNAcylation of STRA6-retinol signals in kidneys of db/db and ob/ob mice. Western blot and immunohistochemistry were done for STRA6/CRBP1/LRAT/RALDH1/RARs pathway, GFAT, OGT, TGFß1 and collagen 1 level. HPLC and ELISA for retinol, retinal, and retinoic acid concentrations were performed in vivo and vitro. RBP4 binding with STRA6 was measured in vitro. To verify whether O-GlcNAcylation disrupted STRA6-retinol signals, treatment of TMG and OSMI-1, transfection of OGA and OGT, and OGT siRNA were performed in HK-2 cells. RESULTS: STRA6 and RALDH1 were highly O-GlcNAc-modified in glomeruli and tubules of db/db and ob/ob mice. RBP4, p-Try, p-JAK2, and p-STAT5 on STRA6 immunoprecipitate were reduced. Cellular retinol signals (CRBP1, LRAT, ADH, retinol, retinal, RA, RARα, RARγ and RXRα) remarkably decreased in kidneys of db/db, ob/ob mice and HG-cultured cells. TMG and OGT transfection induced O-GlcNAcylation of STRA6 and RALDH1, repressed RBP4-bound STRA6, and retinol signals in NG-cultured cells. OSMI-1, OGA transfection, and OGT silence reversed O-GlcNAc-modification of STRA6 and RALDH1, and rescued the decrease of retinol signals, and reversed the increase of TGFß1 and collagen 1 in HG-treated cells. CONCLUSIONS: O-GlcNAcylation significantly modified STRA6 and RALDH1, suppressed RBP4 binding activity, and disrupted retinol signals in the kidney of diabetes. GENERAL SIGNIFICANCE: This study first indicates that STRA6-retinol signals were directly disrupted by O-GlcNAcylation in diabetic kidney.

Prevention of 4-hydroxynonenal-induced lipolytic activation by carnosic acid is related to the induction of glutathione S-transferase in 3T3-L1 adipocytes.

Induction of 4-hydroxynonenal (4-HNE), a major lipid peroxidation aldehyde, is observed in patients with obesity and type 2 diabetes mellitus. The lipolytic response by 4-HNE has been linked to insulin resistance. In this study, we investigated the effects of carnosic acid (CA) on 4-HNE-induced lipolysis and the inhibition of ß-oxidation in 3T3-L1 adipocytes. The results indicated that cells pretreated with CA reduced 4-HNE-mediated free fatty acid (FFA) release. Furthermore, CA reversed the inhibition of phosphorylation of Tyr632 of insulin receptor substrate-1 (IRS-1) and Akt and the phosphorylation of Ser307 of IRS-1. CA inhibited 4-HNE-induced phosphorylation of protein kinase A (PKA) and hormone-sensitive lipase (HSL), and reversed the suppression by 4-HNE of phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (p < 0.05). Pretreatment of cells with forskolin (a cAMP agonist) and compound C (an AMPK inhibitor) reversed these effects, respectively (p < 0.05). In human subcutaneous adipocytes, CA also attenuated 4-HNE-induced FFA release and the phosphorylation of PKA and HSL (p < 0.05). Moreover, CA increased the protein expression of glutathione S-transferase (GST) A and M. Pretreatment with ethacrynic acid, a GST inhibitor, prevented the 4-HNE-conjugated proteins suppression, the PKA and HSL phosphorylation reduction, and the FFA release inhibition by CA (p < 0.05). CONCLUSION: The attenuation by CA of the lipolytic response by 4-HNE is likely related to the induction of GST, which in turn reduced 4-HNE-conjugated proteins and decreased the activation of the PKA/HSL pathway. The observed effects may explain how CA improves 4-HNE-induced insulin resistance.