Extended Data Fig. 1: Glucagon acutely stimulates hepatic gluconeogenesis by increasing hepatic acetyl-CoA content and PC flux.

a, Body weight (n = 11). b, Expression of INSP3R1 protein in the liver (n = 5). Blots in b, c, e and Figs. 1f, 2a, Extended Data Figs. 3f, g, 4a were stripped and reprobed for all proteins of interest. *P < 0.05 versus Insp3r1-knockout mice not treated with glucagon. c, INSP3R protein expression in cytosolic (c.) and crude mitochondrial (c.m.) fractions from primary hepatocytes, in which VDAC was examined as a marker for mitochondrial protein content, and calreticulin as a marker for non-mitochondria-associated membrane protein. Right, phosphorylation of INSP3R1 in the liver of mice infused with glucagon (n = 5). d, Phosphorylation of CRTC2 in the liver (n = 5). The CRTC2 phosphorylation gel was stripped and reprobed to assess HSP90 (loading control). e, Phosphorylation of CAMKIV in the liver, with or without a 2-h acute infusion of glucagon (n = 5). f, Liver glycogen content (n = 5 wild type – glucagon, otherwise n = 6). No differences were observed using one-way analysis of variance with Bonferroni’s multiple comparisons test. g–i, Plasma [m + 1], [m + 2] and [m + 7]glucose enrichment during a 2-h infusion of [3-¹³C]lactate and [²H₇]glucose (n = 5 wild type and 6 knockout, with the exception of i, for which n = 4 wild type + glucagon at 100 and 110 min). j, k, Plasma total amino acid and alanine concentrations (n = 5 wild type and 6 knockout). In j, k, groups were compared before and after glucagon treatment by two-tailed paired Student’s t-test, and genotypes were compared by two-tailed unpaired Student’s t-test. l, m, Liver total amino acid and alanine concentrations (n = 5). n, o, In vitro glucose production (n = 9) and V_PC (n = 4) in isolated hepatocytes. p, q, In vitro glucose production (n = 9) and V_PC (n = 4) in isolated hepatocytes with and without 150 pM insulin. Basal data (no insulin) are duplicated from n and o. r, s, In vitro glucose production (n = 8) and V_PC (n = 3) in isolated hepatocytes with and without a malic enzyme (ME) inhibitor. **P < 0.01, ****P < 0.0001 versus wild type − glucagon − ME inhibitor. t–v, Plasma connecting peptide, glucagon and glucose concentrations in mice (n = 6 wild type and 7 knockout) treated with somatostatin, basal insulin and glucagon. Comparisons before and after glucagon treatment used a two-tailed paired Student’s t-test. w, x, Endogenous glucose production and V_PC (n = 6 wild type and 7 knockout). In all panels, comparisons with and without glucagon, insulin or malic enzyme inhibitor, and wild type versus knockout comparisons were performed using a two-tailed unpaired Student’s t-test, unless otherwise stated. In all panels in which comparisons were performed (that is, all panels with the exception of g, i), if no P value is shown, groups were not significantly different. In all panels, mean ± s.e.m. is shown. All n values refer to numbers of mice.

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