Extended Data Fig. 4: Effects of defective hepatic glutaminolysis on whole-body and hepatic metabolism.

(a) Association of Gls2 variants with several Metabolic Syndrome traits from publicly available GWAS HuGEAMP datasets. (b) H&E staining of paraffin-embedded serial sections (left panel, scale bar, 200 mm) and triglyceride content (right panel) of the liver of Apoe^−/−Gls2^shRNA mice compared to controls (n = 9-10 mice per group; P values displayed; two-tailed t test) or Ldlr^−/−Gls2^−/− mice compared to respective controls (n = 8-13 mice per group; P values displayed; two-tailed t test). (c) Plasma ALT, (d) plasma urea and (e) plasma triglyceride levels in Apoe^−/−Gls2^shRNA mice and controls (n = 9-10 mice per group, except ALT levels, for which some measurement failed; P values displayed; two-tailed t test) and Ldlr^−/−Gls2^−/− mice compared to controls (n = 8-13 mice per group; P values displayed; two-tailed t test). (f) Glucose tolerance test was performed on atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice. Blood glucose concentrations were measured at the indicate time points. The glucose tolerance index was calculated as the product of areas under glucose curves (n = 9 mice per group; P = 0.86; two-tailed t test). (g) Plasma insulin levels were measured in atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice (left panel) (n = 9 mice per group; P values displayed; two-tailed t test). Paraffin-embedded serial sections obtained from the adipose tissue of these mice (right panel). Representative H&E staining revealed similar cellularity. (h) Respiratory quotient (RQ) measured by indirect calorimetry in atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice (n = 4 mice per group; no statistical difference was regardless of the light-on or light-off phase; two-tailed t test). (i) Plasma triglyceride levels were determined at 0, 1, 2 and 4 hours after the olive oil gavage in these mice (right panel represents area under the curve) (n = 10 mice per group; P = 0.79; two-tailed t test). All values are mean ± s.e.m. and are representative of at least one independent experiment. P values were determined by two-tailed Student’s t-test. (j) Schematic representation of glycolysis and neoglucogenesis pathways (upper left panel), triglyceride synthesis pathway (upper right panel) and VLDL production (lower panel). (k) RNAseq analysis with focus on enzymatic regulators expressed in the liver of Ldlr^−/−Gls2^−/− and Apoe^−/−Gls2^−/− mice and their respective controls. (l) RNAseq analysis with focus on of genes involved in VLDL production and hydrolysis depicted in the upper schematic representation.