Nature | Vol 579 | 12 March 2020 | 281stimulate glucose production and VPC only in wild-type hepatocytes,
which demonstrates that both the PLC and PKA pathways are required
to activate gluconeogenesis in response to glucagon via INSP3R1 sig-
nalling (Extended Data Fig. 2e, f ). To specifically confirm the role of
INSP3R1 in promoting HGP, we treated hepatocytes with vasopressin
(an activator of the INSP3 receptor) and found that this agent reca-
pitulated the effect of glucagon to increase glucose production and
VPC (Extended Data Fig. 2g, h). However, small-molecule inhibitors of
INSP3 (2-aminoethoxydiphenyl borate (2-APB) and caffeine), CAMKII
and CAMKIV (KN-93) and the SERCA pump (thapsigargin) inhibited the
ability of glucagon to stimulate glucose production and VPC (Extended
Data Fig. 2i–p), which suggests that maintaining a normal balance of
calcium throughout the cell is required for normal INSP3R1 signalling.
None of these agents had any effect in Insp3r1-knockout mice, which
indicates that INSP3R2 and INSP3R3 do not restore glucose production
in the absence of INSP3R1 in the liver.
The observed increases in the rate of HGP occurred in the absence
of any effect of glucagon to acutely increase the expression of mRNAs
or proteins associated with hepatic gluconeogenesis in vivo. However,
glucagon infusion increased hepatic long-chain acyl-CoA and acetyl-
CoA content by 35–60% (Fig. 1i, j, Extended Data Fig. 3a–e). These
increases in hepatic long-chain acyl- and acetyl-CoA concentrations
were dissociated from the phosphorylation of both acetyl-CoA car-
boxylase (ACC) and 5′ AMP-activated protein kinase (AMPK), both of
which were increased by glucagon in Insp3r1-knockout, but not in wild-
type, mice (Extended Data Fig. 3f, g). The differences in ACC and AMPK
phosphorylation between genotypes probably reflect the interplay
between the ability of glucagon to promote the phosphorylation of
both of these enzymes and the ability of hyperinsulinaemia (which was
observed only in wild-type mice infused with glucagon) to suppress this
phosphorylation. Hepatic long-chain acyl-CoA and acetyl-CoA content
increased in wild-type mice infused with glucagon despite reduced
plasma non-esterified fatty acids (NEFA) concentrations (Extended
Data Fig. 3h), consistent with glucagon stimulation of intrahepatic
lipolysis but not white adipose tissue lipolysis. As acetyl-CoA is an allos-
teric activator of pyruvate carboxylase (PC)^18 , the increase in hepatic
acetyl-CoA content observed with glucagon infusion could explain
the increases in VPC and HGP that occurred after glucagon treatment in
wild-type mice. These changes occurred independently of any changes
in hepatic malonyl-CoA content (Extended Data Fig. 3i), which indicates
that malonyl-CoA suppression of carnitine palmitoyl transferase I was
not responsible for glucagon stimulation of hepatic mitochondrial
β-oxidation under these conditions.
To further understand the physiological role of glucagon-induced
HGP, we fasted wild-type and Insp3r1-knockout mice for 48 h. In the
starved state and despite 70–90% increases in plasma glucagon con-
centrations in the tail vein and portal vein, Insp3r1-knockout mice
manifested lower plasma glucose and insulin concentrations that
were associated with reductions in hepatic long-chain acyl-CoA and
acetyl-CoA content and no change in hepatic malonyl-CoA content
as compared to wild-type mice (Extended Data Fig. 3j–o). These data
demonstrate a critical role for INSP3R1 in the maintenance of eugly-
caemia during starvation.
We next hypothesized that INSP3R1 stimulation of intrahepatic
lipolysis may explain the observed increases in hepatic acetyl-CoA
content and VPC after glucagon treatment. Consistent with this hypoth-
esis, phosphorylation of hepatic adipose triglyceride lipase (ATGL)
at Ser406—which has previously been shown to regulate the activity
of ATGL^19 —was increased threefold by glucagon in wild-type, but not
Insp3r1-knockout, mouse livers (Fig. 2a). By contrast, phosphorylation
of hormone-sensitive lipase (HSL) was increased by glucagon in both
genotypes, which dissociates HSL activity from glucagon activation of
HGP and VPC (Extended Data Fig. 4a). In vitro studies revealed 60–100%
increases in NEFA and glycerol production with glucagon treatment
in wild-type, but not INSP3R1-deficient, hepatocytes (Extended
Data Fig. 4b, c). Plasma NEFA concentrations were unchanged by gluca-
gon infusion in mice treated with somatostatin (Extended Data Fig. 4d).
These data suggest that although glucagon may promote lipolysis
when infused at markedly supraphysiological doses^20 , in the setting of
physiological concentrations of glucagon and intact β-cell function,
glucagon does not directly affect white adipose tissue lipolysis^21 –^23.
Confirming the requirement for glucagon-stimulated intrahepatic
lipolysis to promote gluconeogenesis, glucagon had no effect in hepato-
cytes treated with a small molecule inhibitor of ATGL (atglistatin).
Similarly, INSP3 agonism recapitulated the effect of glucagon in terms
of stimulating intrahepatic lipolysis, and inhibitors of INSP3, CAMKII
and CAMKIV, PLC, PKA and the SERCA pump inhibited the ability of
glucagon to stimulate intrahepatic lipolysis, glucose production and
VPC (Fig. 2b, Extended Data Fig. 4e–n). To examine the effect of glucagon
stimulation of intrahepatic lipolysis in vivo, we knocked down Atgl
(also known as Pnpla2) in a liver-specific manner in wild-type mice
and their Insp3r1-knockout littermates (Extended Data Fig. 5a–c). Atgl
knockdown abrogated the ability of glucagon to stimulate HGP, VPC and
to increase long-chain acyl- and acetyl-CoA content in wild-type mice
(Fig. 2c–h), which demonstrates a critical role for the stimulation of
intrahepatic lipolysis in mediating the increases in each parameter that
result from glucagon infusion. These alterations in hepatic gluconeo-
genesis were again dissociated from changes in hepatic glycogen con-
tent or the expression of gluconeogenic proteins in the liver. In addition,
the alterations in the rate of hepatic gluconeogenesis were dissociated
from white adipose tissue lipolysis and from hepatic malonyl-CoA con-
tent (Extended Data Fig. 5d–j). Taken together, these data demonstrate
that glucagon acutely stimulates hepatic gluconeogenesis via INSP3R1
by promoting intrahepatic lipolysis through the stimulation of ATGL,
and thereby increases hepatic acetyl-CoA and gluconeogenesis via
allosteric activation of PC.
We hypothesized that glucagon may also stimulate hepatic mito-
chondrial oxidation via INSP3R1. Glucagon increased both mitochon-
drial and cytosolic calcium signalling in hepatocytes from wild-type
mice, whereas Insp3r1-knockout mice manifested a reduced response
to glucagon, with some residual calcium responsiveness that was prob-
ably attributable to the activity of INSP3R2 and INSP3R3. Vasopressin
caused a similar increase in cytosolic and mitochondrial calcium signal-
ling in wild-type, but not Insp3r1-knockout, mice. However, incubation
of wild-type hepatocytes with a PLC inhibitor completely abrogated
the mitochondrial and cytosolic calcium responses to glucagon. PKA
inhibition reduced the amplitude of the mitochondrial—but not the
cytosolic—calcium response, and lowered the percentage of responding
cells by 75% (Extended Data Fig. 6a–m). Ex vivo positional isotopomer
nuclear magnetic resonance (NMR) tracer analysis (PINTA) revealed
that glucagon stimulated hepatic mitochondrial oxidation (measured
as the rate of citrate synthase flux (VCS)) in vivo, increasing VCS five-
fold in wild-type mice; this could mostly be attributed to increased
mitochondrial fat oxidation (Fig. 3a, b). However, these increases in
hepatic mitochondrial oxidation in response to glucagon did not occur
in Insp3r1-knockout mice. Taken together, these data suggest that gluca-
gon stimulates hepatic mitochondrial oxidation through the activation
of INSP3R1, which results in increased levels of intramitochondrial
calcium and—in turn—stimulation of mitochondrial dehydrogenases^24.
Consistent with this hypothesis, we observed a sevenfold increase in
the rate of hepatic pyruvate dehydrogenase flux (VPDH) in wild-type, but
not Insp3r1-knockout, mice infused with glucagon, without any differ-
ence in the rate of pyruvate kinase flux (VPK) (Extended Data Fig. 6n, o).
Glucagon stimulation of mitochondrial oxidation was not dependent
on alterations in insulin: wild-type mice infused with somatostatin,
basal insulin and approximately 100-pM glucagon exhibited increases
in VCS similar to those of wild-type mice not treated with somatosta-
tin—however, again, no glucagon stimulation of VCS was observed in
their Insp3r1- knockout littermates (Extended Data Fig. 6p). In vitro
studies confirmed an increase in oxygen consumption with glucagon