282 | Nature | Vol 579 | 12 March 2020
Article
stimulation in isolated hepatocytes from wild-type, but not Insp3r1-
knockout, mice (Extended Data Fig. 6q). These data demonstrate that
glucagon mediates these effects in a cell-autonomous manner.
Liver triglyceride concentrations did not differ between chow-fed
wild-type and Insp3r1-knockout mice (Extended Data Fig. 6r), which
suggests that basal physiological levels of glucagon may not increase
mitochondrial fat oxidation in ad libitum fed mice to a sufficient
extent to alter hepatic triglyceride content. However, we hypothesized
that the chronic increases in hepatic mitochondrial oxidation induced
by chronic glucagon treatment would reverse nonalcoholic fatty liver
disease and improve whole-body insulin sensitivity. To investigate
this, we performed chronic glucagon infusions in awake rats with diet-
induced obesity, and found that after ten days of treatment chronic
glucagon infusion doubled the rate of hepatic mitochondrial oxidation
(which could be attributed to increased rates of hepatic fat oxidation)
(Extended Data Fig. 7a–g). Two hours after the withdrawal of glucagon,
rats exhibited lower fasting plasma glucose and insulin concentrations
that were associated with 50–90% reductions in hepatic triglyceride and
diacylglycerol concentrations as well as marked reductions in protein
kinase C-ε (PKCε) translocation—despite similar food intake, body
weight and hepatic ceramide content (Extended Data Fig. 7h–o, Sup-
plementary Tables 3, 4). Consistent with chronic increases in hepatic
glycogenolysis, chronic glucagon infusion also resulted in reductions in
liver glycogen content. In contrast to acute glucagon infusion, chronic
glucagon infusion with withdrawal of glucagon treatment four hours
before the tissues were isolated lowered both the hepatic acetyl- and
malonyl-CoA content (Extended Data Fig. 7p–r); the reduction in mal-
onyl-CoA content potentially contributes to the observed reductions
in hepatic lipid concentrations due to suppression of hepatic lipogen-
esis^25. Consistent with their lower hepatic lipid and acetyl-CoA content,
rats that were chronically infused with glucagon manifested improved
glucose tolerance and insulin sensitivity (Extended Data Fig. 7s–v).
To determine whether INSP3R1-dependent calcium signalling medi-
ates the ability of chronic hyperglucagonaemia to reverse nonalcoholic
fatty liver disease, we performed a four-week continuous infusion of
glucagon in wild-type and Insp3r1-knockout mice fed a high-fat diet.
Despite unchanged body weight, food and water intake, and energetics,
wild-type mice treated with glucagon exhibited 50–80% reductions
in hepatic triglyceride and diacylglycerol (but not ceramide) content
and in PKCε translocation that resulted in a marked improvement in
glucose tolerance, whereas each of these parameters was unchanged
in Insp3r1-knockout mice (Fig. 3c–h, Extended Data Fig. 8a–j, Supple-
mentary Tables 5, 6).
Collectively, our studies reveal that glucagon stimulates intrahepatic
lipolysis through an INSP3R1- and CAMKII-dependent process that
increases hepatic acetyl-CoA content (which allosterically activates
VPC^18 ), and that this phenomenon explains the acute transcription-
independent ability of glucagon to acutely stimulate gluconeogenesis
in vivo. In addition, glucagon stimulates hepatic mitochondrial oxida-
tion through INSP3R1-mediated calcium signalling, and this process
can be exploited to reverse nonalcoholic fatty liver disease and hepatic
insulin resistance with short-term continuous glucagon treatment.ace0100200300******* * *03060
Time (min)90 120 03060
Time (min)(^590120)
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Insp3r1 KOWT Liver-speci c
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Insp3r1 KO- Glucagon + Glucagon
- Glucagon + Glucagon
P = 0.004 P = 0.002 P = 0.006 P = 0.005VFAO
(μmol kg–1 min–1)VCS
(μmol kg–1 min–1)Liver TAG(mg g–1)Liver DAG(nmol g–1)Liver PKCε(membrane/cytosol)Plasma glucose(mM)
Plasma insulin(pM)Liver ceramide(nmol g–1)P < 0.0001P = 0.02
P = 0.002 P < 0.05
P = 0.04CytosolCytosolMembraneMembraneFig. 3 | Chronic increases in mitochondrial oxidation with a continuous
3.5-week glucagon infusion reverse hepatic steatosis and improve glucose
tolerance in an INSP3R1-dependent manner. a, b, Liver VCS and the rate of fatty
acid oxidation (VFAO) with acute glucagon infusion. n = 5 wild t y pe − glucagon,
6 knockout − glucagon, 6 wild type + glucagon and 5 knockout + glucagon. c,
Liver triacylglycerol (TAG) concentrations in high-fat-diet-fed mice chronically
infused with glucagon. n = 9 wild type − glucagon, 8 wild type + glucagon,
8 knockout − glucagon and 7 knockout + glucagon. d, Liver ceramide. n = 9 wild
type − glucagon, 7 wild type + glucagon, 9 knockout − glucagon and 8 knockout
+ glucagon. e, Liver diacylglycerol (DAG). n = 9 without glucagon, 7 with
glucagon. f, PKCε translocation (n = 6). g, h, Plasma glucose and insulin during a
glucose tolerance test. n = 10 wild type without glucagon (WT vehicle),
11 wild type with glucagon (WT glucagon), 8 knockout (KO) vehicle,
8 knockout glucagon). In all panels, mean ± s.e.m. is shown. *P < 0.05, **P < 0.01,
***P < 0.001. Groups were compared using two-tailed unpaired Student’s t-test.
All n values refer to numbers of mice.