Journal: Experimental and Therapeutic Medicine

Article Title: Sodium sulphate ameliorates hypercholesterolemia via the upregulation of Cyp7a1 in hepatocytes and alleviates hepatic insulin resistance via the downregulation of Trib3 in mice with high cholesterol diets

doi: 10.3892/etm.2024.12650

Figure Lengend Snippet: Effect of sodium sulphate on the insulin signaling pathway in the liver tissues of mice fed an HCD. (A) Kyoto Encyclopedia of Genes and Genomes analysis of the biological pathways of differently expressed mRNAs in the livers of mice from the CON vs. HCD and HCD vs. HCD + MSS groups. (B) RT-qPCR and western blotting results of the Trib3 mRNA and TRB3 protein expression levels in the liver tissues of mice from the CON, HCD, HCD + LSS, HCD + MSS and HCD + HSS groups, respectively. (C) Western blotting results showing the protein expression levels of p-IRS1 (Tyr608), IRS1, p-AKT and AKT in the liver tissues of mice from the CON, HCD, HCD + LSS, HCD + MSS and HCD + HSS groups. The right panel shows the semi-quantitative analysis of the p-AKT/AKT ratio. GAPDH was used as the internal control for western blotting and RT-qPCR. ## P<0.01, ### P<0.001 vs. the CON group; * P<0.05, ** P<0.01, *** P<0.001 vs. the HCD group. HCD, high cholesterol diet; CON, control; LSS, low dose of sodium sulphate; MSS, middle dose of sodium sulphate; HSS, high dose of sodium sulphate; RT-qPCR, reverse transcription-quantitative PCR; Trib3/TRB3, tribbles pseudokinase 3; p-, phosphorylated; IRS1, insulin receptor substrate 1.

Article Snippet: The primary antibodies comprised: Rabbit anti-GAPDH (1:1,000; cat. no. 2118S; CST Biological Reagents Co., Ltd.), mouse anti-CYP7A1 (1:1,000; cat. no. 2683295; MilliporeSigma), rabbit anti-hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 7 (HSD3B7; 1:1,000; cat. no. ab190223; Abcam), rabbit anti-aldo-keto reductase family 1 member D1 (AKR1D1; 1:1,000; cat. no. ab101393; Abcam), rabbit anti-acyl-CoA oxidase 2 (1:1,000; cat. no. ab197808; Abcam), rabbit anti-hydroxysteroid 17-β dehydrogenase 4 (HSD17B4; 1:1,000; cat. no. ab97971; Abcam), rabbit anti-CYP27A1 (1:2,000; cat. no. ab126785; Abcam), rabbit anti-CYP39A1 (1:1,000; cat. no. ab129334; Abcam), rabbit anti-isopentenyl-diphosphate δ isomerase 1 (IDI1; 1:3,000; cat. no. ab97448; Abcam), rabbit anti-lanosterol synthase [anti-oxidosqualene-lanosterol cyclase (OSC); 1:1,000; cat. no. ab80364; Abcam], rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1; 1:1,000; cat. no. ab195046; Abcam), rabbit anti-farnesyl diphosphate synthase (FDPS; 1:2,000; cat. no. ab153805; Abcam), rabbit anti-mevalonate diphosphate decarboxylase (MVD; 1:1,000; cat. no. ab96226; Abcam), rabbit anti-mevalonate kinase (MVK; 1:1,000; cat. no. ab154515; Abcam), rabbit anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR; 1:1,000; cat. no. ab174830; Abcam), rabbit anti-low density lipoprotein receptor (LDLR; 1:1,000; cat. no. ab52818; Abcam), rabbit anti-c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252s; CST Biological Reagents Co., Ltd.), mouse anti-phosphorylated (p)-JNK (1:2,000; cat. no. 9255s; CST Biological Reagents Co., Ltd.), rabbit anti-p-insulin receptor substrate 1 (p-IRS1; Tyr608) mouse (1:1,000; cat. no. 09-432; MilliporeSigma), rabbit anti-IRS-1 (1:1,000; cat. no. 2382S; CST Biological Reagents Co., Ltd.), rabbit anti-p-AKT (Ser473) (1:1,000; cat. no. 4060S; CST Biological Reagents Co., Ltd.), rabbit anti-AKT (1:1,000; cat. no. 4685S; CST Biological Reagents Co., Ltd.), mouse anti-tribbles pseudokinase 3 (TRB3; 1:100; cat. no. sc-390242; Santa Cruz Biotechnology, Inc.), rabbit anti-c-Jun (1:1,000; cat. no. ab32137; Abcam), mouse anti-p-c-Jun (1:200; cat. no. sc-822; Santa Cruz Biotechnology, Inc.) and goat anti-KLB (0.5 µg/ml; cat. no. AF5889; R&D Systems China Co., Ltd.).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Sodium sulphate ameliorates hypercholesterolemia via the upregulation of Cyp7a1 in hepatocytes and alleviates hepatic insulin resistance via the downregulation of Trib3 in mice with high cholesterol diets

doi: 10.3892/etm.2024.12650

Figure Lengend Snippet: Sodium sulphate ameliorates hypercholesterolemia and hepatic insulin resistance in mice fed an HCD. Sodium sulphate inhibits the highly expressed TRB3 in the hepatocytes of mice fed an HCD, to attenuate the hepatic insulin resistance of these mice. In the enterocytes of the ileum, FGF15 expression is downregulated following the administration of sodium sulphate to mice fed an HCD. In addition, the hepatic expression of KLB, which is the co-receptor of FGFR4 for FGF15 binding, is significantly downregulated by sodium sulphate, and reduces the activation of JNK, which is downstream of the FGF15/FGFR4-KLB signaling pathway. The activation of c-Jun, which is the major target of p-JNK, is notably reduced, and the expression of Cyp7a1 , which is inhibited by p-c-Jun, is significantly increased, which enhances the conversion of cholesterols to bile acids in these mice. HCD, high cholesterol diet; TRB3, tribbles homolog 3; FGF, fibroblast growth factor; FGFR, FGF receptor; KLB, Klotho β; JNK, c-Jun N-terminal kinase; p-, phosphorylated; IRS1, insulin receptor substrate 1; PDK1, 3-phosphoinositide-dependent kinase 1; FXR, farnesoid X receptor.

Article Snippet: The primary antibodies comprised: Rabbit anti-GAPDH (1:1,000; cat. no. 2118S; CST Biological Reagents Co., Ltd.), mouse anti-CYP7A1 (1:1,000; cat. no. 2683295; MilliporeSigma), rabbit anti-hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 7 (HSD3B7; 1:1,000; cat. no. ab190223; Abcam), rabbit anti-aldo-keto reductase family 1 member D1 (AKR1D1; 1:1,000; cat. no. ab101393; Abcam), rabbit anti-acyl-CoA oxidase 2 (1:1,000; cat. no. ab197808; Abcam), rabbit anti-hydroxysteroid 17-β dehydrogenase 4 (HSD17B4; 1:1,000; cat. no. ab97971; Abcam), rabbit anti-CYP27A1 (1:2,000; cat. no. ab126785; Abcam), rabbit anti-CYP39A1 (1:1,000; cat. no. ab129334; Abcam), rabbit anti-isopentenyl-diphosphate δ isomerase 1 (IDI1; 1:3,000; cat. no. ab97448; Abcam), rabbit anti-lanosterol synthase [anti-oxidosqualene-lanosterol cyclase (OSC); 1:1,000; cat. no. ab80364; Abcam], rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1; 1:1,000; cat. no. ab195046; Abcam), rabbit anti-farnesyl diphosphate synthase (FDPS; 1:2,000; cat. no. ab153805; Abcam), rabbit anti-mevalonate diphosphate decarboxylase (MVD; 1:1,000; cat. no. ab96226; Abcam), rabbit anti-mevalonate kinase (MVK; 1:1,000; cat. no. ab154515; Abcam), rabbit anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR; 1:1,000; cat. no. ab174830; Abcam), rabbit anti-low density lipoprotein receptor (LDLR; 1:1,000; cat. no. ab52818; Abcam), rabbit anti-c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252s; CST Biological Reagents Co., Ltd.), mouse anti-phosphorylated (p)-JNK (1:2,000; cat. no. 9255s; CST Biological Reagents Co., Ltd.), rabbit anti-p-insulin receptor substrate 1 (p-IRS1; Tyr608) mouse (1:1,000; cat. no. 09-432; MilliporeSigma), rabbit anti-IRS-1 (1:1,000; cat. no. 2382S; CST Biological Reagents Co., Ltd.), rabbit anti-p-AKT (Ser473) (1:1,000; cat. no. 4060S; CST Biological Reagents Co., Ltd.), rabbit anti-AKT (1:1,000; cat. no. 4685S; CST Biological Reagents Co., Ltd.), mouse anti-tribbles pseudokinase 3 (TRB3; 1:100; cat. no. sc-390242; Santa Cruz Biotechnology, Inc.), rabbit anti-c-Jun (1:1,000; cat. no. ab32137; Abcam), mouse anti-p-c-Jun (1:200; cat. no. sc-822; Santa Cruz Biotechnology, Inc.) and goat anti-KLB (0.5 µg/ml; cat. no. AF5889; R&D Systems China Co., Ltd.).

Techniques: Expressing, Binding Assay, Activation Assay

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: HFD‐induced insulin resistance and altered lipid metabolism‐related genes are improved in Cyr61 knockdown (AAV‐shCyr61) mice. Blood glucose levels in AAV‐shGFP or AAV‐shCyr61 during (A) GTT and (B) ITT assays after 6 h of fasting. Relative mRNA expression levels of key genes involved in (C) lipogenesis and (D) fatty acid oxidation in the liver of AAV‐shGFP or AAV‐shCyr61 mice. (E) Representative hepatic expression of Sirt6, p‐AMPK, and T‐AMPK were determined using western blotting. (F) Mice were intraperitoneally injected with insulin after 6 h of fasting. Representative hepatic expression of IRS1 and AKT was determined using western blotting. Statistical significance was determined using the two‐way ANOVA. * p < .05, ** p < .01, *** p < .001 vs CD/AAV‐GFP. # p < .05, ## p < .01, ### p < .001 vs HFD/AAV‐GFP. AAV, adeno‐associated virus; AKT, protein kinase B; AUC, area under the curve; Cyr61, cysteine‐rich 61; GTT, glucose tolerance test; IRS1, insulin receptor substrate 1; ITT, insulin tolerance test; p‐AMPK, phosphorylated AMP‐activated protein kinase; Sirt6, sirtuin 6.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Expressing, Western Blot, Injection, Virus