Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis

Abstract

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes^1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.

Extended Data Figure 1.

Glucagon acutely stimulates hepatic gluconeogenesis by increasing hepatic acetyl-CoA content and pyruvate carboxylase flux. (a) Body weight (n=11). (b) Liver InsP₃R-I protein expression (n=5). Blots in Figures 1f and 2a, and Extended Data Figures 1b, 1d, 1f, 3f, 3g, and 4a were stripped and re-probed for all proteins of interest. *P<0.05 vs. InsP₃R-I KO 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. On the right, Liver InsP₃R-I phosphorylation in mice infused with glucagon (n=5). The blot for total InsP₃R-I is duplicated from Extended Data Figure 1b. (d) Liver CRTC2 phosphorylation (n=5). The CRTC2 phosphorylation gel was stripped and re-probed to assess HSP90 (loading control). (e) Liver CAMKIV phosphorylation ± a 2 hr acute infusion of glucagon (n=5). (f) Liver glycogen content (n=5 WT–glucagon, otherwise n=6). No differences were observed using one-way ANOVA with Bonferroni’s multiple comparisons test. (g)-(i) Plasma [m+1], [m+2], and [m+7] plasma glucose enrichment during a 120 min infusion of [3-¹³C] lactate and [²H₇] glucose (n=5 WT and 6 KO, with the exception of panel (i), in which n=4 WT+glucagon at 100 and 110 min). (j)-(k) Plasma total amino acid and alanine concentrations (n=5 WT and 6 KO). In panels j-k, groups were compared before and after glucagon by the 2-tailed paired Student’s t-test, and genotypes were compared by the 2-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 flux (n=4) in isolated hepatocytes. (p)-(q) In vitro glucose production (n=9) and V_PC flux (n=4) in isolated hepatocytes with and without 150 pM insulin. Basal data (no insulin) are duplicated from panels n and o. (r)-(s) In vitro glucose production (n=8) and V_PC flux (n=3) in isolated hepatocytes with and without a malic enzyme inhibitor. **P<0.01, ****P<0.0001 vs. WT-glucagon-ME inhibitor. (t)-(v) Plasma c-peptide, glucagon, and glucose concentrations in mice (n=6 WT and 7 KO) treated with somatostatin, basal insulin, and glucagon. Comparisons before and after glucagon used the 2-tailed paired Student’s t-test. (w)-(x) Endogenous glucose production and V_PC (n=6 WT and 7 KO). In all panels, unless otherwise stated, comparisons with and without glucagon, insulin, or malic enzyme inhibitor, and WT vs. KO were performed using the 2-tailed unpaired Student’s t-test. In all panels where comparisons were performed (i.e. all panels with the exception of g-i), if no p-value is shown, groups were not significantly different. In all panels, the mean±S.E.M. are shown.

Extended Data Figure 2.

Glucagon-stimulated glucose production requires activation of the PLC and PKA pathways, converging to activate InsP₃ signaling. In vitro glucose production and V_PC flux in isolated hepatocytes with and without ET-18-OCH₃ (n=3), U-73122 (n=3, with the exception of KO-glucagon-U-73122, in which n=2), H-89 (n=6), vasopressin (n=3), 2-APB (n=3), caffeine (n=3), KN-93 (n=6), and thapsigargin (n=3). In all panels, *P<0.05, **P<0.01, ***P<0.001 vs. WT-glucagon-drug; §P<0.05, §§P<0.01, §§§P<0.001 vs. WT +glucagon -drug by the 2-tailed unpaired Student’s t-test. If no statistical comparison is denoted, the groups were not significantly different. The mean±S.E.M. is shown.

Extended Data Figure 3.

Glucagon stimulates hepatic glucose production independently of transcriptional regulation and plays a key role in maintenance of blood glucose during a prolonged fast. (a)-(c) Liver PC, PEPCK, and G6Pase mRNA expression (n=5). (d)-(e) Liver PC and PEPCK protein (n=5 with the exception of KO+glucagon, where n=6). (f)-(g) Liver pACC/ACC and pAMPK/AMPK (n=5). Blots in Figures 1f and 2a, and Extended Data Figures 1b, 1d, 1f, 3f, 3g, and 4a were stripped and re-probed for all proteins of interest. (h) Plasma NEFA (n=5, with the exception of KO+glucagon, where n=6). (i) Liver malonyl-CoA (n=6). (j)-(l) Plasma glucose, insulin, and glucagon concentrations in 48 hr fasted mice (in all panels j-o, n=5). (m)-(o) Liver long-chain-, acetyl-, and malonyl-CoA content. In all panels, genotypes and groups +/− glucagon were compared using the 2-tailed unpaired Student’s t-test. If no statistical comparison is denoted, the groups were not significantly different. In all panels, the mean±S.E.M.

Extended Data Figure 4.

Glucagon acutely stimulates gluconeogenesis by activating intrahepatic, but not white adipose tissue, lipolysis. (a) Liver HSL phosphorylation (n=5). Blots in Figures 1f and 2a, and Extended Data Figures 1b, 1d, 1f, 3f, 3g, and 4a were stripped and re-probed for all proteins of interest. In panels a-c, groups were compared using the 2-tailed unpaired Student’s t-test. (b)-(c) In vitro NEFA and glycerol production from isolated hepatocytes (n=14 WT and 15 KO). (d) Plasma NEFA concentrations in mice treated with somatostatin, replacement basal insulin, and glucagon (n=5 WT and 6 KO). No significant differences were observed between genotypes using the 2-tailed unpaired Student t-test, or before vs. after glucagon using the 2-tailed paired Student’s t-test. (e)-(f) NEFA production and V_PC in isolated hepatocytes incubated in the ATGL inhibitor atglistatin (n=6). (g)-(n) NEFA production from isolated hepatocytes treated with ET-18-OCH₃ (n=3), U-73122 (n=3), H-89 (n=6), vasopressin (n=3), KN-93 (n=6), 2-APB (n=3), caffeine (n=3), and thapsigargin (n=3). In all panels, *P<0.05, **P<0.01, ***P<0.001 versus the same genotype -glucagon -drug; §P<0.05, §§ P<0.01, §§§ P<0.001 versus the same genotype +glucagon -drug by the 2-tailed unpaired Student’s t-test. If no statistical comparison is denoted, the groups were not significantly different. Error bars represent the S.E.M.

Extended Data Figure 5.

Glucagon requires InsP₃-mediated intrahepatic lipolysis to promote V_PC and hepatic gluconeogenesis. (a) Body weight in mice treated with an adeno-associated virus to knock down liver ATGL (n=6). (b) Representative western blots. Blots from the same tissue (liver or WAT) were stripped and reprobed for all proteins shown. (c) WAT and liver ATGL protein expression (n=4, with the exception of KO+ATGL knockdown, in which n=6). (d)-(e) Hepatic PC and PEPCK protein expression (n=6 WT and KO+ATGL knockdown, in other groups, n=4). (f)-(h) Plasma glucagon, NEFA and glycerol concentrations in mice treated with an adeno-associated virus to knock down ATGL in a liver-specific manner (n=6, with the exception of WT+ATGL knockdown and KO, in which n=5). Groups were compared before and after glucagon by the 2-tailed unpaired Student’s t-test. (i)-(j) Liver glycogen and malonyl-CoA content (n=6 other than WT+ATGL knockdown, in which n=5). All comparisons were performed using the 2-tailed unpaired Student’s t-test, unless otherwise stated. If no statistical comparison is denoted, the groups were not significantly different. Error bars represent the S.E.M.

Extended Data Figure 6.

Glucagon stimulates mitochondrial oxidation through hepatocellular calcium signaling. (a) Representative mitochondrial response to glucagon, which was added where denoted by the “glucagon” bar. (b) Maximum mitochondrial response to glucagon (n=324 WT and 167 KO). Groups in panels b, d, f, and h were compared by the 2-tailed unpaired Student’s t-test. (c) Representative cytosolic response to glucagon. (d) Maximum cytosolic response to glucagon (n=146 WT and 175 KO). (e) Representative mitochondrial response to the InsP₃R agonist vasopressin. (f) Amplitude of the cytosolic response to vasopressin (n=73 WT and 42 KO). (g) Representative cytosolic response to vasopressin. (h) Amplitude of the cytosolic response to vasopressin (n=119 WT and 53 KO). (i) Representative mitochondrial responses to glucagon (added where denoted by the bar) in WT hepatocytes incubated in the PKA inhibitor H-89 or the PLC inhibitor U-73122. (j) Amplitude of the mitochondrial response (n=79 control, 39 H-89, and 127 U-73122). Groups were compared to the control in panels j and l, and m using the 2-tailed unpaired Student’s t-test. (k) Representative cytosolic responses to glucagon±H-89 or U-73122. (l) Amplitude of the cytosolic response (n=182 control, 27 H-89, and 187 U-73122). (m) Percentage of cells with a cytosolic response (>110% baseline) to glucagon±H-89 or U-73122 (n=11 control, 8 H-89, and 3 U-73122). (n)-(o) Hepatic PDH and PK flux in vivo (n=5 WT-glucagon, 6 WT+glucagon, 6 KO-glucagon, 5 KO+glucagon). In panels n-o, groups were compared (with vs. without glucagon, and WT vs. KO) using the 2-tailed unpaired Student’s t-test. (p) Liver V_CS in mice infused with somatostatin, basal insulin, and glucagon (n=5). (q) In vitro oxygen consumption in isolated hepatocytes incubated±100 nM glucagon (n=113 WT-glucagon, 144 KO-glucagon, 149 WT+glucagon, 210 KO+glucagon). (r) Liver triglyceride content (without glucagon infusion) (n=11). In all panels, if no statistical comparison is denoted, the groups were not significantly different. Error bars represent the S.E.M.

Extended Data Figure 7.

Chronic increases in mitochondrial oxidation with a 10-day glucagon infusion lead to reversal of NAFLD and improvements in glucose tolerance. (a) Plasma glucagon concentrations on the last day of infusion. In all panels in this figure, n=6. (b) Hepatic V_CS flux. In panels (b)-(g), measurements were performed while the glucagon infusion continued. (c)-(d) V_PC V_EGP⁻¹ and V_PC V_CS⁻¹ ratios. (e)-(g) Hepatic V_PDH, V_FAO, and V_PK rates. (h) Food intake during the glucagon infusion, determined twice during the 10 day infusion (days 4 and 9) by weighing the food in the cage; the data points are the averages of the two food intake measurements for each animal. (i) Body weight after 10 days of glucagon or saline infusion. (j)-(k) 6 hr fasted plasma glucose and insulin concentrations measured two hours after cessation of the glucagon infusion. (l)-(n) Liver TAG (n=6), DAG (n=8), and ceramide (n=8) concentrations. (o) Hepatic PKCε translocation (n=6 control and 7 glucagon). (p)-(r) Liver glycogen (n=6), acetyl-CoA (n=8), and malonyl-CoA content (n=8). (s)-(t) Plasma glucose concentrations and area under the curve during an intraperitoneal glucose tolerance test which began 2 hrs after completing a 10 day continuous infusion of glucagon or saline (n=6). In panels s and u, *P<0.05, **P<0.01, ***P<0.001. Data are the mean±S.E.M. (u)-(v) Plasma insulin and insulin area under the curve during the GTT. In all panels, error bars represent the S.E.M, and groups were compared using the 2-tailed unpaired Student’s t-test. If no statistical comparison is denoted, the groups are not significantly different.

Extended Data Figure 8.

Chronic glucagon treatment reverses NAFLD and glucose intolerance in WT but not InsP₃R-I KO mice. (a) Body weight (n=10 WT-glucagon, 11 WT+glucagon, 8 KO-glucagon, and 8 KO+glucagon). (b)-(c) Food and water intake. In panels (b)-(g), n=8 WT-glucagon, 9 WT+glucagon, 7 KO-glucagon, 8 KO+glucagon. (d) Energy expenditure. (e)-(f) Oxygen consumption and carbon dioxide production. (g) Activity. (h) Plasma NEFA. In panels (h)-(j), n=10 WT-glucagon, 11 WT+glucagon, 8 KO-glucagon, 8 KO+glucagon. (i)-(j) Glucose and insulin area under the curve during an intraperitoneal glucose tolerance test. In all panels, the mean±S.E.M. is shown. Statistical comparisons were performed using the 2-tailed unpaired Student’s t-test. If no statistical comparison is denoted, the groups were not significantly different.

Figure 1.

Glucagon acutely stimulates hepatic gluconeogenesis by increasing hepatic acetyl-CoA content and V_PC. (a)-(c) Plasma glucose, insulin, and glucagon concentrations before and at the end of a 2 hr intravenous infusion of glucagon (n=7). (d)-(f) Hepatic cAMP concentrations, protein kinase A activity, and CAMKII phosphorylation (n=5, with the exception of WT –glucagon in panel (e), in which n=4). Blots in Figures 1f and 2a, and Extended Data Figures 1b, 1d, 1f, 3f, 3g, and 4a were stripped and re-probed for all proteins of interest. (g)-(h) HGP (n=6 WT and 5 KO) and V_PC (n=5 WT and 6 KO). (i)-(j) Hepatic long-chain- (n=5 WT-glucagon, 6 WT+glucagon, and 6 KO) and acetyl-CoA content (n=6). In all panels, the mean±S.E.M. is shown. Groups were compared before and after glucagon (panels a-c, g, and h) by the 2-tailed paired Student’s t-test, and separate animals (+/− glucagon in panels d-f, i, and j, and WT vs. KO animals in all panels) were compared by the 2-tailed unpaired Student’s t-test.

Figure 2.

Glucagon requires InsP₃-mediated intrahepatic lipolysis to promote V_PC and HGP. (a) ATGL S406 phosphorylation (n=5). Blots in Figures 1f and 2a, and Extended Data Figures 1b, 1d, 1f, 5f, 5g, and 7a were stripped and re-probed for all proteins of interest. (b) Glucose production in hepatocytes (n=6 mice per group) incubated in the ATGL inhibitor atglistatin and/or glucagon. ****P<0.0001 vs. WT-glucagon-atglistatin, §§§§ vs. WT+gluagon-atglistatin. (c)-(d) Plasma glucose and insulin concentrations in mice treated with an adeno-associated virus to knock down ATGL in a liver-specific manner. In panels (c)-(h), n=6 WT, 5 WT+ATGL knockdown, 6 KO±ATGL knockdown). (e)-(f) Liver long-chain- and acetyl-CoA concentrations following a 2 hr glucagon infusion. (g)-(h) Hepatic glucose production and hepatic V_PC. In all panels, the mean±S.E.M. is shown. Groups were compared before and after glucagon (panels c and d) by the 2-tailed paired Student’s t-test, and groups in all other panels (as well as the four separate groups in panels c and d) were compared by the 2-tailed unpaired Student’s t-test.

Figure 3.

Chronic increases in mitochondrial oxidation with a continuous 3.5 week glucagon infusion reverse of hepatic steatosis and improve glucose tolerance in an InsP₃R-I-dependent manner. (a)-(b) Liver V_CS and V_FAO with acute glucagon infusion (n=5 WT-glucagon, 6 KO-glucagon, 6 WT+glucagon, 5 KO+glucagon). (c) Liver TAG concentrations in HFD mice chronically infused with glucagon (n=9 WT-glucagon, 8 WT+glucagon, 8 KO-glucagon, 7 KO+glucagon). (d) Liver ceramide (n=6). (e) Liver DAG (n=9 -glucagon, 7 +glucagon). (f) PKCε translocation (n=6). (g)-(h) Plasma glucose and insulin during a glucose tolerance test (n=10 WT-glucagon, 11 WT+glucagon, 8 KO-glucagon, 8 KO+glucagon). In all panels, the mean±S.E.M. is shown. Groups were compared using the 2-tailed unpaired Student’s t-test.