Sulfonylureas exert antidiabetic action on adipocytes by inhibition of PPARγ serine 273 phosphorylation

Abstract

Objective: Sulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic β-cells. In addition, effects of SUs on adipocytes by activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) have been described, which might explain their insulin-sensitizing potential observed in patients. However, there is a discrepancy between the impact of SUs on antidiabetic action and their rather moderate in vitro effect on PPARγ transcriptional activity. Recent studies have shown that some PPARγ ligands can improve insulin sensitivity by blocking PPARγ Ser-273 phosphorylation without having full agonist activity. It is unknown if SUs elicit their antidiabetic effects on adipocytes by inhibition of PPARγ phosphorylation. Here, we investigated if binding of SUs to PPARγ can interfere with PPARγ Ser-273 phosphorylation and determined their antidiabetic actions in vitro in primary human white adipocytes and in vivo in high-fat diet (HFD) obese mice.

Methods: Primary human white preadipocytes were differentiated in the presence of glibenclamide, glimepiride and PPARγ ligands rosiglitazone and SR1664 to compare PPARγ Ser-273 phosphorylation, glucose uptake and adipokine expression. Transcriptional activity at PPARγ was determined by luciferase assays, quantification of PPARγ Ser-273 phosphorylation was determined by Western blotting and CDK5 kinase assays. In silico modelling was performed to gain insight into the binding characteristics of SUs to PPARγ. HFD mice were administered SUs and rosiglitazone for 6 days. PPARγ Ser-273 phosphorylation in white adipose tissue (WAT), body composition, glucose tolerance, adipocyte morphology and expression levels of genes involved in PPARγ activity in WAT and brown adipose tissue (BAT) were evaluated.

Results: SUs inhibit phosphorylation of PPARγ at Ser-273 in primary human white adipocytes and exhibit a positive antidiabetic expression profile, which is characterized by up regulation of insulin-sensitizing and down regulation of insulin resistance-inducing adipokines. We demonstrate that SUs directly bind to PPARγ by in silico modelling and inhibit phosphorylation in kinase assays to a similar extend as rosiglitazone and SR1664. In HFD mice SUs reduce PPARγ phosphorylation in WAT and have comparable effects on gene expression to rosiglitazone. In BAT SUs increase UCP1 expression and reduce lipid droplets sizes.

Conclusions: Our findings indicate that a part of SUs extra-pancreatic effects on adipocytes in vitro and in vivo is probably mediated via their interference with PPARγ phosphorylation rather than via classical agonistic activity at clinical concentrations.

Keywords: Brown adipose tissue; PPARγ; Sulfonylureas; White adipose tissue.

Figure 1

Transcriptional activity of sulfonylureas, rosiglitazone and SR1664, and effects on primary human white preadipocytes during differentiation. (A) Transcriptional activity (luciferase assays) of a PPAR response element (PPRE) in HEK293T cells following treatment with increasing concentrations (0.01–10 μM) of glimepiride (Glim), glibenclamide (Glib), rosiglitazone (Rosi) and SR1664 (n = 4). Primary human white preadipocytes were differentiated in the presence of 2.5 μM Glim, Glib, Rosi and SR1664 until Day 21. (B) Red O staining of differentiated adipocytes (upper panel). Triglyceride (TG) content was quantified using an enzymatic assay and normalized to the protein content of the sample (lower graph, n = 4). (C) mRNA expression of adipogenic markers aP2, adiponectin, CD36 and leptin was determined by qRT-PCR (n = 5). (D) aP2 protein expression was assessed by Western blotting. β-Actin Western blot was performed to control for loading (upper panels). Quantification of relative aP2 expression vs. β-Actin (n = 3, lower graph) (E) mRNA expression of pro-inflammatory cytokines CXCL1, CXCL2, CXCL5, IL6 and MCP1 was determined by qRT-PCR (n = 5). Data are represented as means +/− SEM. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001 vs NT or as indicated, One-way ANOVA with Dunnett's or Tukey's post-hoc test. NT, not treated.

Figure 2

Sulfonylureas, rosiglitazone and SR1664 have antidiabetic effects and block PPARγ Ser-273 phosphorylation in vitro. (A) Total glucose uptake (n = 6) and (B) GLUT4 mRNA expression (n = 3) of differentiated primary human white adipocytes pre-incubated with 2.5 μM glimepiride (Glim), glibenclamide (Glib), rosiglitazone (Rosi) and SR1664 for 45 min prior to treatment with 5 ng/mL human TNFα for 48 h. (C) Western blot against phosphorylated PPARγ at Ser-273 (pPPARγ) of differentiated primary human white adipocytes pre-treated with 2.5 μM Glim, Glib, Rosi and SR1664 for 45 min prior to treatment with 5 ng/mL human TNFα for 60 min. PPARγ and β-Actin Western blots were performed to control for loading (left panels). Quantification of relative PPARγ phosphorylation vs. PPARγ expression is shown in the right graph (n = 4). (D) In vitro CDK5 kinase assay on PPARγ LBD incubated with 2.5 μM Glim, Glib, Rosi and SR1664. Kinase assays were subjected to Western blotting with a CDK substrate specific antibody, PPARγ or CDK5. (E) In vitro CDK5 kinase assay on Histone H1 incubated with 2.5 μM Glim, Glib, Rosi and SR1664. Kinase assays were subjected to Western blotting with a CDK substrate specific antibody, Histone H1 or CDK5. Data are represented as means +/− SEM. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001 vs TNFα, One-way ANOVA with Dunnett's post-hoc test. IB, immunoblot; NT, not treated.

Figure 3

Sulfonylureas inhibit PPARγ Ser-273 phosphorylation in a concentration-dependent manner in vitro. In vitro CDK5 kinase assays on PPARγ LBD incubated with increasing concentrations (0.01–10 μM) of (A) rosiglitazone, (B) SR1664, (C) glibenclamide and (D) glimepiride. Kinase reactions were subjected to Western blotting against phosphorylated PPARγ at Ser-273, PPARγ or CDK5 (A-D, upper panels). ADP content of the same kinase reactions was determined by a chemoluminescent assay (A-D, lower graphs). Data are represented as means +/− SEM (n = 4). IC₅₀ values were determined from the fitted concentration-response curves. NT, not treated.

Figure 4

White adipose tissue (WAT) analysis of high-fat diet (HFD) mice after 6 days treatment with glimepiride (Glim), glibenclamide (Glib) and rosiglitazone (Rosi). (A) Western blot against phosphorylated PPARγ at Ser-273 (pPPARγ). PPARγ and β-Actin Western blots were performed to control for loading (left), quantification of relative PPARγ phosphorylation vs. PPARγ expression (right). (B) Immunohistochemistry of GLUT4 and F4/80 expression in WAT, crown-like structures are marked with an arrow. Control slides (Ctr) were stained with hematoxylin and secondary antibodies; scale bar 200 μm. (C) Relative F4/80, aP2, adiponectin and Glut4 mRNA expression in WAT was determined by qRT-PCR. (D) Adiponectin serum levels and (E) body composition of HFD mice. Data are represented as means +/− SEM (n = 5). ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001 vs Vehicle, One-way ANOVA with Dunnett's post-hoc test.

Figure 5

Brown adipose tissue (BAT) analysis of high-fat diet (HFD) mice after 6 days treatment with glimepiride (Glim), glibenclamide (Glib) and rosiglitazone (Rosi). (A) Hematoxylin/eosin (H&E) staining (right panels) and immunohistochemistry of UCP1 expression in BAT (left panels); scale bar 100 μm. (B) Quantification of lipid droplet sizes per 20x magnification frame/mouse. Large lipid droplets were defined as surface >300 μm². (C) BAT weight per g body weight (bw). (D) Relative Ucp1, Prdm16 and Pgc1a mRNA expression was determined by qRT-PCR in BAT. Data are represented as means +/− SEM (n = 5). ∗, p ≤ 0.05 vs Vehicle, One-way ANOVA with Dunnett's post-hoc test.

Figure 6

Docking of ligands onto PPARγ structure and structural alignment of bound (2hfp) and unbound (model) ligand binding regions. Top 5 docking poses for (A) rosiglitazone, (B) SR1664, (C) glibenclamide and (D) glimepiride. In each panel, the protein backbone is depicted in grey colored ribbon format with the ligand shown as stick models colored atom wise. The Ser-273 residue is depicted in each panel as a red colored stick model depiction. In case of docking poses belonging to different regions/ensembles they have been numbered. (E) Structural alignment bound (2hfp; green ribbon depiction) and unbound (model; red ribbon depiction) ligand binding regions. The inset image shows a closer view of the loop bearing the Ser-273 residue. The Ser-273 residue is depicted in stick model format.