nt12dreuar3esd

280 | Nature | Vol 579 | 12 March 2020

Article

increased rate of hepatic pyruvate carboxylase flux (VPC) that was
associated with amino acid depletion in the liver without alterations
in hepatic glycogen content (Fig. 1g, h, Extended Data Fig. 1f–m, Sup-
plementary Tables 1, 2). The in vivo HGP data were mirrored by twofold
increases in both HGP and VPC in isolated hepatocytes from wild-type

but not Insp3r1-knockout mice incubated with glucagon (Extended Data Fig. 1n, o). The ability of insulin to suppress HGP and VPC was unaltered in Insp3r1-knockout hepatocytes, and malic enzyme activity was dis- sociated from glucose production by treatment with a malic enzyme inhibitor (Extended Data Fig. 1p–s). Finally, in vivo studies with soma- tostatin and replacement insulin infusion to mimic basal insulin concentrations in the portal vein (85–90 pM in both genotypes) revealed that changes in plasma insulin could not explain the observation that glucagon stimulated HGP and VPC only in wild-type mice (Extended Data Fig. 1t–x). To further explore the calcium-dependent mechanism by which glucagon stimulates HGP and VPC, we treated hepatocytes with two phospholipase C (PLC) antagonists (ET-18-OCH3 or U-73122) and found that both agents inhibited glucose production and VPC in wild-type— but not Insp3r1-knockout—hepatocytes (Extended Data Fig. 2a–d). Similarly, a PKA inhibitor (H-89) abrogated the ability of glucagon to

(^0) Before glucagon After glucagon Before glucagon After glucagon
25
50
75
100
125
HGP
(μmol kg
–1 min
–1)
Liver long-chain acyl-CoA
(nmol g
–1)
Liver acetyl-CoA
(nmol g
–1)
VPC
(μmol kg
–1 min
–1)
P = 0.02 P = 0.02
0
100
200
300
Plasma insuli
n
(pM)
P = 0.03 P = 0.0007
P = 0.03
Before glucagon After glucagon Before glucagon After glucagon
Before glucagon After glucagon – Glucagon + Glucagon
5
6
7
8
9
Plasma glucos
e
(mM)
a P = 0.003 P = 2 × 10 –5
c
e
0
25
50
75
100
125 P = 0.008 P = 0.01
40
50
60
70
80
90 P = 0.008 P = 0.02
0
50
100
150
200
250 P = 0.001 P = 0.0006
g

Glucagon + Glucagon

Glucagon + Glucagon – Glucagon + Glucagon

0

1

2

3

4

5 P = 0.0008 P = 0.001

Glucagon
WT KO

+ Glucagon WT KO

pCAMKII CAMKII

0

1,000

2,000

3,000

Liver cAMP(pmol g

–1)

P = 0.006

P = 0.00 4

i

b

d

f

h

j

0

50

100

150

200

Plasma glucagon

(pM)

Liver PKA activity

(U ml

–1)

Liver pCAMKII/

CAMKII

P = 0.02 P = 0.01

P = 0.03

0

2

4

6

P = 0.004

P = 0.003

WT Liver-specic Insp3r1 KO

Fig. 1 | Glucagon acutely stimulates hepatic gluconeogenesis by increasing
hepatic acetyl-CoA content and VPC. a–c, Plasma glucose, insulin and glucagon
concentrations before and at the end of a 2-h intravenous infusion of glucagon
(n = 7). d–f, Hepatic cAMP concentrations, PK A activity and CAMKII
phosphorylation (pCAMKII) (n = 5, with the exception of wild type without (–)
glucagon in e (n = 4)). Blots in f, and in Fig. 2a, Extended Data Figs. 1b, c, e, 3f, g, 4a
were stripped and reprobed for all proteins of interest. g, h, HGP (n = 6 wild t y pe
and 5 knockout) and VPC (n = 5 wild type and 6 knockout). i, j, Hepatic long-chain
acyl- (n = 5 − glucagon, 6 with (+) glucagon) and acetyl-CoA content (n = 6). In all
panels, mean ± s.e.m. is shown. Groups were compared before and after
glucagon treatment (a–c, g and h) by two-tailed paired Student’s t-test, and
separate mice (±glucagon in d–f, i, j, and wild-type versus knockout mice in all
panels) were compared by two-tailed unpaired Student’s t-test. All n values refer
to numbers of mice.

0

1

2

3

4

pATGL ATGL

(^0) Before glucagon After glucagon
50
100
150
200
250
P < 0.0001
P < 0.0001
P < 0.0001
P = 0.007
P < 0.05
P < 0.05
P < 0.05
0
1
2
3
Glucagon
Atglistatin

– –+ +– ++

****

§§§§

P < 0.0001

40

60

80

100

120 P = 0.002 P < 0.0001

P < 0.0001

0

25

50

75

100

125

P < 0.0001 P < 0.0001 P < 0.0001

60

80

100

120 P < 0.0001 P < 0.0001

P < 0.0001

0

50

100

150

200

250 P < 0.01

P < 0.001

P < 0.01

a

c

e

g

b

d

f

h

Before glucagon After glucagon

Before glucagon After glucagon Before glucagon After glucagon

5

6

7

8

9

10

Liver pATGL

/ATGL

Plasma glucose

(mM)

Liver long-chain acyl-CoA

(nmol g

–1)

HGP (μmol kg

–1 min

–1)

Liver acetyl-CoA

(nmol g

–1)

Plasma insulin

(pM)

Glucose production

(fold basal)

Glucagon + Glucagon

WT KO WT KO

P = 0.0003

P < 0.0001 P < 0.0001 P = 0.0003 P < 0.0001

P = 0.01

P = 0.0002 WT Liver-specic Insp3r1 KO

VPC (μmol kg

–1 min

–1)

WT Liver-specic Insp3r1 KO

WT WT + liver-specic Atgl knockdown Liver-specic Insp3r1 KO Liver-specic liver-specic Atgl Insp3r1knockdown KO +

Fig. 2 | Glucagon requires INSP3-mediated intrahepatic lipolysis to promote VPC and HGP. a, Phosphorylation of ATGL at Ser406 (n = 5). The blots here, as well as those in Fig. 1f, Extended Data Figs. 1b, c, e, 5f, g, 7a, were stripped and reprobed for all proteins of interest. b, Glucose production in hepatocytes (n = 6 mice per group) incubated with the ATGL inhibitor atglistatin and/or glucagon. ****P < 0.0001 versus wild type − glucagon − atglistatin, §§§§P < 0.0001 versus wild type + glucagon − 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 c–h, n = 6 wild type, 5 wild type + Atgl knockdown and 6 knockout ± Atgl knockdown. e, f, Liver long-chain acyl- and acetyl-CoA concentrations following a 2-h glucagon infusion. g, h, HGP and VPC. In all panels, mean ± s.e.m. is shown. Groups were compared before and after glucagon treatment (c, d) by two-tailed paired Student’s t-test, and groups in all other panels (as well as the four separate groups in c, d) were compared by two-tailed unpaired Student’s t-test. All n values refer to numbers of mice.

nt12dreuar3esd

Get our desktop app

Company

Features

Documentation

Resources