Nature | Vol 579 | 12 March 2020 | 279Article
Glucagon stimulates gluconeogenesis by
INSP3R1-mediated hepatic lipolysis
Rachel J. Perry1,2, Dongyan Zhang^1 , Mateus T. Guerra^1 , Allison L. Brill^2 , Leigh Goedeke^1 ,
Ali R. Nasiri^1 , Aviva Rabin-Court^1 , Yongliang Wang^1 , Liang Peng^1 , Sylvie Dufour^1 , Ye Zhang^1 ,
Xian-Man Zhang^1 , Gina M. Butrico^1 , Keshia Toussaint^1 , Yuichi Nozaki^1 , Gary W. Cline^1 ,
Kitt Falk Petersen^1 , Michael H. Nathanson^1 , Barbara E. Ehrlich2,3 & Gerald I. Shulman1,2 ✉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.On the basis of the clear role for glucagon in the pathogenesis of
diabetes, glucagon-blocking therapies have been pursued as adjuncts
to therapies for this condition. Glucagon antagonism^4 –^9 and knock-
down of the glucagon receptor^10 ,^11 have shown glucose-lowering effects;
however, these agents increase liver enzymes^7 ,^8 by an unknown mecha-
nism. Conversely, a dual agonist for glucagon-like peptide-1 and gluca-
gon receptor increases energy expenditure and promotes weight loss,
an effect associated with reductions in blood glucose^12. Together, these
data suggest a role for glucagon in promoting hepatic mitochondrial
fat oxidation, in addition to its known effect of stimulating hepatic
gluconeogenesis.
Glucagon stimulates hepatic glycogenolysis and gluconeogenesis,
the latter of which is believed to occur largely through transcriptional
regulation. Hepatic calcium signalling is integral to the transcriptional
regulation of gluconeogenesis: inhibition or deletion of liver Ca2+/
calmodulin-dependent protein kinase II (CAMKII) results in reduced
expression of proteins in the gluconeogenic pathway in mouse livers
and associated reductions in plasma glucose and insulin concentra-
tions^13 –^15. Type-1 INSP3R is the isoform that is primarily responsible for
mitochondrial calcium signalling in hepatocytes^16. It has previously
been shown^14 that knocking down Insp3r1 reduces glucose production
in isolated hepatocytes; however, no studies have fully characterized
the role and mechanism of INSP3R signalling in glucagon-stimulated
hepatic gluconeogenesis in vivo.
To examine the potential calcium dependence of the acute response
to glucagon, we studied mice with Insp3r1 knocked out specifically
in the liver, along with their weight-matched wild-type littermates
(Extended Data Fig. 1a). The Insp3r1-knockout mice exhibited a marked
reduction in hepatic INSP3R1 protein expression (Extended Data
Fig. 1b; full gels for all blots are shown in Supplementary Fig. 1). We
examined subcellular fractions in primary hepatocytes and found that
INSP3R1 was associated with mitochondria, whereas the type-2 INSP3R
isoform (INSP3R2) was not; the type-3 INSP3R isoform (INSP3R3) was
not detected at all (Extended Data Fig. 1c), consistent with previous
data that show that INSP3R1 is primarily responsible for mitochondrial
calcium signals^16. We then treated mice—which were fasted overnight,
and therefore depleted of glycogen—with an acute infusion of glucagon,
which modestly increased plasma glucose and insulin concentrations in
wild-type—but not Insp3r1-knockout—mice and increased phosphoryla-
tion of INSP3R1 (Fig. 1a–c, Extended Data Fig. 1c). Phosphorylation of
CREB-regulated transcriptional coactivator 2 (CRTC2) was decreased
with glucagon infusion in both wild-type and Insp3r1-knockout mice
(Extended Data Fig. 1d), consistent with previous literature^14. Glucagon
increased cAMP concentrations and the activity of protein kinase A
(PKA) in both wild-type and Insp3r1-knockout mice, dissociating these
mediators from glucagon-induced changes in plasma glucose con-
centrations (Fig. 1d, e). Consistent with a previous report^13 , glucagon
stimulated the phosphorylation of CAMKII but not of CAMKIV in an
INSP3R1-dependent manner (Fig. 1f, Extended Data Fig. 1e). These data
imply that INSP3R1 activation and CAMKII activity are required for the
acute effect of glucagon in stimulating hepatic glucose production
(HGP), which can be attributed almost entirely to gluconeogenesis in
this hepatic glycogen-depleted state^17. Indeed, we measured increases
of approximately 25% in in vivo HGP, which could be attributed to anhttps://doi.org/10.1038/s41586-020-2074-6
Received: 24 February 2018
Accepted: 15 January 2020
Published online: 4 March 2020
Check for updates(^1) Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA. (^2) Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA. (^3) Department
of Pharmacology, Yale School of Medicine, New Haven, CT, USA. ✉e-mail: [email protected]