Extended Data Fig. 10: Role of the ISR in biguanide-induced Gdf15 expression.
From: GDF15 mediates the effects of metformin on body weight and energy balance
a, b, mRNA levels in kidney (a) and colon (b) isolated from obese mice 24 h after a single oral dose of metformin (600 mg kg−1). Data are mean ± s.e.m. (n = 5 per group, except forn = 4 for colon metformin Slc22a1). P values (95% CI) by two-tailed t-test. Gdf15 mRNA fold induction 24 h after metformin (600 mg kg−1) is positively correlated with Chop mRNA induction in both kidney (a, right) and colon (b, right). Black line shows linear regression analysis. c–g, Immunoblot analysis of ISR components (c) and Gdf15 mRNA expression (d) in wild-type mouse embryonic fibroblasts (MEF) treated with vehicle control (Con), metformin (Met, 2 mM) or phenformin (Phen, 5 mM) or tunicamycin (Tn, 5 μg ml−1, used as a positive control) for 6 h. e–g, Gdf15 mRNA expression in ATF4 knockout (KO) MEFs (e), in control siRNA and CHOP siRNA transfected wild-type MEFs treated with Tn or Phen for 6 h (f), or in wild-type MEFs pre-treated for 1 h with either the PERK inhibitor GSK2606414 (GSK, 200 nM) or eIF2α inhibitor ISRIB (ISR, 100 nM) then co-treated with phenformin for a further 6 h (g). mRNA expression is presented as fold expression relative to its respective control treatment (set at 1) or phenformin-treated samples (set as 100) with normalization to Hprt gene expression. Data are mean ± s.e.m. from two (c, d) or at least three (e–g) independent experiments. P values (95% CI) by two tailed t-test relative to phenformin-treated control wild-type and control siRNA-treated samples. h, GDF15 protein in supernatant of mouse derived 2D duodenal organoids treated with metformin in the absence or presence of ISRIB (1 μM). Data are expressed as mean ± s.e.m. from two independent experiments. At least duplicate protein measurements for each sample. P values by two-way ANOVA with Sidak’s correction for multiple comparisons. i, GDF15 protein in supernatants of mouse-derived 2D duodenal organoids from wild-type and Chop-null mice treated with metformin from two independent experiments. At least duplicate protein measurements for each sample. Data are mean ± s.e.m.; P values (95% CI) by two-tailed t-test.
