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Article

Methods

Sample size was determined based on a power calculation to detect
moderate to large differences between groups^26. The experiments
were not formally randomized, but littermates (wild-type and knock-
out) were used in parallel for each experiment. Investigators were not
blinded to allocation during experiments but were blinded during
outcome assessment.

Animals
All protocols were approved by the Yale University Animal Care and Use
Committee. Liver-specific Insp3r1-knockout mice were generated as
previously described^16 , and in all experiments littermates were studied
at 10–12 weeks of age. Mice were fed standard chow (Teklad no. 2018)
and housed on a 12-h light/dark cycle in the Yale Animal Resources
Center. To knock down ATGL in a liver-specific manner, an adeno-
associated virus targeting ATGL (Vector BioLabs, 10^12 genome copies
per mouse) was administered by retro-orbital injection four weeks
before studies. Male mice were used for in vivo studies and both sexes
were used for in vitro studies. One week before in vivo studies, mice
underwent surgery under isoflurane anaesthesia to place a catheter
in the jugular vein. Postsurgical recovery was demonstrated by con-
firming that mice had regained their presurgical body weight before
any in vivo studies. Mice were fasted overnight before in vivo stud-
ies, unless otherwise stated. In the acute glucagon-infusion studies,
mice were given an intravenous infusion of glucagon (5 ng per kg body
weight per minute (kg−1 min−1)) for two hours, with tissue and plasma
samples obtained after two hours of infusion. Mice were killed using
intravenous pentobarbital at the conclusion of the terminal study.
In the somatostatin infusion study, a 90-min jugular venous infusion of
somatostatin (4 μg kg−1 min−1, 1:1 mix of somatostatin 14 and somatosta-
tin 28) concurrently with [3-^13 C]lactate and [^2 H 7 ]glucose, as described
in ‘In vivo studies’, was administered. During the somatostatin infusion,
mice were also infused with insulin (2 mU kg−1 min−1) and glucagon
(6 ng kg−1 min−1). In the acute high-dose glucagon experiment, mice
were injected with 600 μg kg−1 glucagon and euthanized 20 min later,
after isoflurane anaesthesia. Blood was collected by cardiac puncture,
and the liver was excised and freeze-clamped.
Mice infused chronically with glucagon were fed a high-fat diet
(Research Diets D12492) for 4 weeks, after which they were implanted
with an Alzet pump providing glucagon continuously (0.15 ng min−1) for
another 3.5 weeks, during which time they were continued on a high-fat
diet. Mice underwent CLAMS metabolic cage analysis to assess energet-
ics, as well as food and water intake, during the second week of glucagon
infusion. After an overnight fast, three weeks after implantation of the
glucagon pumps, mice underwent an intraperitoneal glucose tolerance
test and were subsequently re-fed. Forty-eight hours later, after a 6-h
fast, mice were euthanized under isoflurane anaesthesia for measure-
ment of hepatic lipid content as described in ‘Biochemical analysis’.
For the chronic glucagon-infusion studies in rats, 300 g male
Sprague–Dawley rats were obtained from Charles River Laboratories
and fed a safflower-oil-based high-fat diet (60% calories from fat) (Dyets
no. 112245) for 4 weeks. During week 3 of the diet, rats underwent sur-
gery under isoflurane anaesthesia to place catheters in the jugular
vein and carotid artery, and recovery was confirmed by regaining the
presurgical body weight before in vivo studies. After four weeks on a
high-fat diet, rats were placed in a soft plastic harness to protect their
catheters and infused continuously for 10 days with glucagon (5 ng kg−1
min−1, to a total volume 5 ml kg−1 day−1). The glucagon infusion was either
continued throughout the terminal study (PINTA) or discontinued two
hours before the start of the terminal study (glucose tolerance test
with hepatic lipid, acetyl-CoA or glycogen measurements), in separate
groups of rats, as specified in the figure legends. Rats were fasted for 8 h
before being euthanized with an intravenous injection of pentobarbital.

In vivo studies

In all in vivo mouse studies, blood was collected from the tail vein,

with the exception of portal-vein glucagon measurements, for which

a needle was inserted into the portal vein of anaesthetized mice to

collect blood. In the rat studies, blood was collected from the jugular

venous catheter. In both species, samples were immediately centri-

fuged (12,000 rpm) to separate plasma from red blood cells. We used

PINTA to measure hepatic mitochondrial fluxes in both rats and mice^27.

In brief, rodents were infused with a 2-h primed (5 min, 3×)-continuous

infusion of [3-^13 C]lactate (40 μmol kg−1 min−1) and either [1,2,3,4,5,6,

6-^2 H 7 ]glucose (0.1 mg kg−1 min−1) or [3-^3 H]glucose (0.1 μCi kg−1 min−1).

At the conclusion of the study, animals were euthanized with an intra-

venous injection of pentobarbital.

In mice, hepatic glycogenolysis was assumed to be negligible owing

to their prolonged (16 h) fasted state and their low hepatic glycogen

content (Extended Data Figs. 1f, 5i, 7p). HGP was measured by determin-

ing the specific activity of [^3 H]glucose in plasma using a scintillation

counter, and the VPC/VHGP ratio was calculated using the equation

V

V

G

=

2

XFE

PC

HGP^2

in which G2 represents the [m + 2]glucose enrichment corrected for any

[m + 2]glucose synthesized from^13 C 2 -labelled trioses: corrected [m + 2]

glucose = G2 = measured [m + 2]glucose − 2 × C 4 C 5 C6[m + 2 ]glucose,

and XFE represents the fractional triose enrichment:

XFE=

1

1+2×GG^12

in which G1 represents the measured [m + 1]glucose, and G2 is as

described above. To calculate absolute VPC, we multiplied the measured

HGP by the ratio VPC/VHGP. The ratio of hepatic VPC/VCS was calculated as

V

V =

[5-C]glucose

2×[4-C]glucose

PC −1

CS

13

13

and the absolute VCS was calculated by dividing VPC by VPC/VCS. The deri-

vations of each equation are described in detail in a previous study^27.

We corrected for the possible contribution of [^13 C]bicarbonate to label

the TCA cycle, as previously described^28.

We measured the ratio

V

V

=[4-C]glutamate

[3-C]alanine

PDH

CS

13

13

and calculated the absolute VPDH by multiplying this ratio by the meas-

ured VCS. Finally, the ratio of VPK—assuming minimal malic enzyme flux—

to (VPC + VPDH) was calculated as (ref.^29 )

V

VV+ =

[2-C]alanine

[5-C]glucose

PK

PC PDH

13

13

Absolute VPK rates were then determined by multiplying VPK/(VPC + VPDH)

by the sum of VPC and VPDH. As has previously been described^27 , VPC/VCS

can be expanded to account for pyruvate recycling(ref.^29 ):

VV

V

+

= [5-C]glucose

2×[4-C]glucose

PC −1

1

2 PK

CS

13

13

In this study, we measured a maximum VPK/(VPC + VPDH) of 0.4, indicating

that the maximal VPK/VPC is 0.4. A VPK/VPC at this maximal value would

generate a 17% underestimation of VPC/VCS. Ex vivo NMR analysis was