Effect of fat on insulin-stimulated carbohydrate metabolism in normal man

Abstract

We have examined the onset and duration of the inhibitory effect of an intravenous infusion of lipid/heparin on total body carbohydrate and fat oxidation (by indirect calorimetry) and on glucose disappearance (with 6,6 D2-glucose and gas chromatography-mass spectrometry) in healthy men during euglycemic hyperinsulinemia. Glycogen synthase activity and concentrations of acetyl-CoA, free CoA-SH, citrate, and glucose-6-phosphate were measured in muscle biopsies obtained before and after insulin/lipid and insulin/saline infusions. Lipid increased insulin-inhibited fat oxidation (+40%) and decreased insulin-stimulated carbohydrate oxidation (-63%) within 1 h. These changes were associated with an increase (+489%) in the muscle acetyl-CoA/free CoA-SH ratio. Glucose disappearance did not decrease until 2-4 h later (-55%). This decrease was associated with a decrease in muscle glycogen synthase fractional velocity (-82%). The muscle content of citrate and glucose-6-phosphate did not change. We concluded that, during hyperinsulinemia, lipid promptly replaced carbohydrate as fuel for oxidation in muscle and hours later inhibited glucose uptake, presumably by interfering with muscle glycogen formation.

Full text

Effects
of
Fat
on
Insulin-stimulated
Carbohydrate
Metabolism
in
Normal
Men
G.
Boden,
F.
Jadali,
J.
White,*
Y.
Liang,*
M.
Mozzoll,
X.
Chen,
E.
Coleman,
and
C.
Smith'
Division
of
Endocrinology/Metabolism
and
General
Clinical
Research
Center,
Departments
of
Surgery*
and
Biochemistry,
Temple
University
School
of
Medicine,
and
Diabetes
Research
Center,
University
of
Pennsylvania,$
Philadelphia,
Pennsylvania
19140
Abstract
We
have
examined
the
onset
and
duration
of
the
inhibitory
effect
of
an
intravenous
infusion
of
lipid/heparin
on
total
body
carbohydrate
and
fat
oxidation
(by
indirect
calorimetry)
and
on
glucose
disappearance
(with
6,6
D2-glucose
and
gas
chromatog-
raphy-mass
spectrometry)
in
healthy
men
during
euglycemic
hyperinsulinemia.
Glycogen
synthase
activity
and
concentra-
tions
of
acetyl-CoA,
free
CoA-SH,
citrate,
and
glucose-6-phos-
phate
were
measured
in
muscle
biopsies
obtained
before
and
after
insulin/lipid
and
insulin/saline
infusions.
Lipid
increased
insulin-inhibited
fat
oxidation
(+40%)
and
decreased
insulin-
stimulated
carbohydrate
oxidation
(-63%)
within
1
h.
These
changes
were
associated
with
an
increase
(+489%)
in
the
mus-
cle
acetyl-CoA/free
CoA-SH
ratio.
Glucose
disappearance
did
not
decrease
until
24
h
later
(-55%).
This
decrease
was
asso-
ciated
with
a
decrease
in
muscle
glycogen synthase
fractional
velocity
(-82%).
The
muscle
content
of
citrate
and
glucose-6-
phosphate
did
not
change.
We
concluded
that,
during
hyperin-
sulinemia,
lipid
promptly
replaced
carbohydrate
as
fuel
for
oxi-
dation
in
muscle
and
hours
later
inhibited
glucose
uptake,
pre-
sumably
by
interfering
with
muscle
glycogen
formation.
(J.
Clin.
Invest.
1991.
88:960-966.)
Key
words:
acetyl-coenzymee
carbohydrate
oxidation
*
carbohydrate
storage
*
citrate
*
muscle
glycogen
synthase
Introduction
More
than
25
years
ago
Randle
et
al.
(1)
demonstrated
that
increased
availability
of
fatty
acids
enhanced
fat
oxidation
(FAT
OX)'
and
decreased
carbohydrate
oxidation
(CHO
OX)
and
glucose
uptake
in
perfused
rat
heart
and
to
a
lesser
extent
in
rat
diaphragm.
Based
on
these
findings,
they
proposed
a
glucose-fatty
acid
cycle
presumed
to
be
of
fundamental
impor-
tance
for
the
control
of
blood
glucose
and
free
fatty
acid
(FFA)
concentrations
and
insulin
sensitivity
(1).
Inasmuch
as
FFA
Address
reprint
requests
to
Dr.
Boden,
Temple
University
Hospital,
3401
North
Broad
Street,
Philadelphia,
PA
19140.
Receivedfor
publication
10
December
1990
and
in
revisedform
29
May
1991.
1.
Abbreviations
used
in
this
paper:
CHO
OX
and
CHO
STOR,
carbo-
hydrate
oxidation
and
storage,
respectively;
FAT
OX,
fat
oxidation;
GR,
and
GRd,
rate
of
glucose
appearance
and
disappearance,
respec-
tively;
GIR,
glucose
infusion
rates;
GS,
glucogen
synthase;
G-6-P,
glu-
cose-6-phosphate;
NIDDM,
non-insulin-dependent
diabetes
mellitus;
npRQ,
non-protein
respiratory
quotient.
were
known
to
be
frequently
elevated
in
obesity
and
non-insu-
lin-dependent
diabetes
mellitus
(NIDDM),
they
also
postu-
lated
that
increased
FAT
OX
contributed
to
the
impaired
glu-
cose
tolerance
commonly
associated
with
these
conditions
(2,
3).
Subsequently,
many
(4-7),
but
not
all
(8),
investigators
were
unable
to
reproduce
the
fatty
acid-mediated
inhibition
of
glu-
cose
uptake
in
rat
diaphragm
and
striated
muscle
that
Randle
et
al.
had
observed
in
perfused
rat hearts.
(Effects
of
fatty
acids
on
CHO
OX
were
not
examined
in
these
studies.)
More
re-
cently,
several
groups
(9-17)
have
examined
glucose-fatty
acid
interactions
in
vivo
during
hyperinsulinemia,
when
most
of
the
glucose
uptake
occurs
in
muscle
(18).
Practically
all
found
that
raising
FFA
increased
FAT
OX
and
decreased
CHO
OX.
Some
also
found
inhibitory
effects
of
lipid
on
glucose
uptake
(1
1,
16)
but
most
did
not
(12,
14,
15,
17).
Thus,
although
there
seems
to
be
growing
acceptance
of
the
idea
that
increasing
FAT
OX
will
reduce
CHO
OX,
at
least
in
vivo
and
under
hyperinsulinemic
conditions,
there
remains
much
controversy
as
to
whether
fatty
acids
also
inhibit
glucose uptake.
Contributing
to
this
uncer-
tainty
is
the
fact
that
none
of
the
critical
enzyme
and
substrate
changes
predicted
by
the
glucose-fatty
acid
cycle
to
occur
in
response
to
increased
FAT
OX
have
been
demonstrated
in
hu-
man
muscle.
Furthermore,
it
is
not
known
whether
the
time
of
onset
and
the duration
of
the
fatty
acid-induced
alterations
are
the
same
or
are
different
for
CHO
OX
and
glucose uptake.
Noteworthy
in
this
respect
are
recent
findings
by
Bonnadonna
et
al.
(15),
who
suggested
that
the
fatty
acid
effects
depended
on
the
time
of
the
fat
infusion.
In
the
present
study
we
have
therefore
investigated
the
on-
set
and
duration
of
the
inhibitory
effect
of
intravascular
lipoly-
sis,
produced
by
intravenous
infusion
of
triglycerides
and
hepa-
rin,
on
total
body
CHO
OX
and
glucose
disappearance
rates
(GRd)
in
normal
men
during
euglycemic
hyperinsulinemia.
In
addition,
we
have
obtained
muscle
biopsies
from
these
individ-
uals
before
and
after
lipid
and
saline
infusions
for
measure-
ment
of
glycogen
synthase
(GS)
activity
and
of
several
impor-
tant
intermediates
of
fat
and
CHO
metabolism.
Methods
Subjects
18
healthy,
normal
weight
men
were
studied.
Their
ages,
weights,
and
heights
are
shown
in
Table
I.
None
of
the
subjects
had
a
family
history
of
diabetes
or
other
endocrine
disorders
and
none
were
taking
any
medications.
Their
weights
were
stable for
at
least
2
mo
and
their
diets
contained
a
minimum
of
250
g/day
of
carbohydrates
for
at
least
2
d
before
the
men
were
studied.
Informed
written
consent
was
obtained
from
all
after
explanation
of
the
nature,
purpose,
and
potential
risks
of
each
study.
The
study
protocol
was
approved
by
the
Institutional
Re-
view
Board
for
Human
Research
of
Temple
University.
Studies
were
performed
in
the
General
Clinical
Research
Center
of
Temple
Univer-
sity
Hospital
and
began
between
8
and
9
a.m.
after
an-overnight
fast.
The
subjects
were
studied
reclining
in
bed.
A
short
polyethylene
cath-
eter
was
inserted
into
an
antecubital
vein
for
infusion
of
all
test
sub-
960
Boden
et
al.
J.
Clin.
Invest.
©
The
American
Society
for
Clinical
Investigation,
Inc.
0021-9738/91/09/0960/07
$2.00
Volume
88,
September
1991,
960-966

Table
L
Study
Subjects
Study
Age
Weight
Height
yr
kg
cm
1
(n
=
6)
26.0±1.2
79.4±4.4
180.3±3.1
2
(n
=
6)
25.2±1.5
82.6±5.0
181.8±2.7
3
(n
=
6)
26.2±1.8
82.5±3.0
181.5±3.1
stances.
Another
catheter
was
placed
into
a
forearm
vein
of
the
same
arm
to
supply
the
Biostator
(Ames
Lifescience
Instruments,
Elkhart,
IN)
with
a
continuous
flow
(2
ml/h)
of
blood.
A
third
catheter
was
inserted
into
a
contralateral
forearm
vein
for
blood
sampling.
This
arm
was
kept
at
70'C
with
a
heating
blanket
to
arterialize
venous
blood.
Experimental
design
Study
1.
Six
subjects
were
studied.
6,6
D2-glucose
(Tracer
Technolo-
gies,
Somerville,
MA)
was
infused
i.v.
for
7
h
(-60
to
360
min)
starting
with
a
bolus
of
30
Amol/kg
followed
by
a
continuous
infusion
of
0.3
umol/kg-
min.
At
0
min,
LIPOSYN
II
(Abbott
Laboratories,
North
Chicago,
IL),
a
20%
triglyceride
emulsion
(10%
safflower,
10%
soy
bean
oil,
and
2.14
g
of
glycerol
per
100
ml)
plus
heparin
(0.4
U/kg
min)
were
infused
at
a
rate
of
1.5
ml/min
for
6.5
h.
Regular
human
insulin
(Hu-
mulin
R,
Eli
Lilly
&
Co.,
Indianapolis,
IN)
was
infused
i.v.
at
a
rate
of
1
mU/kg
min
for
6.5
h
starting
at
0
min.
Glucose
concentrations
were
clamped
at
-
85
mg/dl
by
a
feedback-controlled
glucose
infusion
(Biostator).
The
first
muscle
biopsy
was
performed
before
the
start
of
the
infusions
(between
-90
and
-60
min);
a
second
biopsy
was
ob-
tained
at
the
end
of
the
studies
(between
360
and
390
min).
Study
2.
Six
subjects
were
studied.
The
protocol
for
study
2
was
identical
to
that
of
study
1
except
that
saline
(1.5
ml/min
for
6.5
h)
was
infused
instead
of
lipid/heparin.
Study
3.
Six
subjects
were
studied.
Infusions
of
6,6
D2-glucose
and
insulin
were
as
in
study
1.
Lipid/heparin
was
infused
for
2
h
starting
at
0
min
at
a
rate
of
1.5
ml/min.
At
120
min
the
lipid/heparnn
infusion
was
discontinued
and
saline
was
infused
for
another
4
h.
Glucose
turnover
Glucose
turnover
was
determined
with
6,6
D2-glucose.
Plasma
glucose
was
isolated
from
blood
drawn
at
30-min
intervals
for
determination
of
isotope
enrichment
as
described
(19)
with
a
gas
chromatograph-mass
spectrometer
(model
4610-B,
Finnigan-MAT,
San
Jose,
CA).
The
penta-acetyl
derivative
of
glucose
was
measured
by
the
electron
impact
mode
at
70
eV.
Ions
were
measured
at
mfe
242
and
244,
respectively.
Rates
of
glucose
appearance
(GR.)
and
disappearance
(GRd)
were
calcu-
lated
from
the
isotope
enrichment
for
30-min
intervals
using
Steele's
equation
for
non-steady-state
conditions
(20).
In
all
three
studies,
GR.
during
hyperinsulinemia
were
frequently
lower
than
glucose
infusion
rates
(GIR)
resulting
in
negative
values
for
hepatic
glucose
production.
This
problem
has
recently
been
attributed
to
errors
in
Steele's
non-
steady-state
equation
(21).
We
have
assumed
that
endogenous
glucose
production
was
completely
suppressed
whenever
the
isotopically
deter-
mined
GR.
was
equal
or
smaller
than
GIR.
Rates
of
CHO
storage
(CHO
STOR)
were
obtained
by
subtracting
rates
of
CHO
OX
(see
below)
from
GRd.
Indirect
calorimetry
Respiratory
gas
exchange
rates
were
determined
as
previously
de-
scribed
before
and
at
30-min
intervals
during
lipid/heparin
or
saline
infusions
with
a
metabolic
measurement
cart
(Beckman
Instruments,
Inc.,
Palo
Alto,
CA)
(22).
Rates
of
protein
oxidation
were
estimated
from
urinary
nitrogen
excretion
after
correction
for
changes
in
urea
nitrogen
pool
size
(23).
Rates
of
protein
oxidation
were
used
to
deter-
mine
the
non-protein
respiratory
quotient
(npRQ).
It
was
assumed
that
for
each
gram
of
N
excreted
in
the
urine,
6.02
liters
of
02
were
consumed
and
4.75
liters
of
CO2
were
produced
(RQ
=
0.79).
Rates
of
CHO
OX
and
FAT
OX
were
determined
with
the
npRQ
tables
of
Lusk,
which
are
based
on
an
npRQ
of
0.707
for
100%
FAT
OX
and
1.00
for
100%
CHO
OX.
Muscle
biopsies
and
extractions
Biopsies
were
obtained
from
the
lateral
aspect
of
the
vastus
lateralis
muscle
-
15
cm
above
the
patella
from
six
of
six
subjects
in
study
1
and
from
four
of
six
subjects
in
study
2.
The
skin
was
cleaned
with
betadine
and
anesthetized
with
1%
lidocaine
without
epinephrine
in
a
field
block
pattern
(at
2
X
3
in.).
(We
have
found
that
injection
of
lidocaine
too
close
to
the
biopsy
site
interfered
with
the
measurement
of
acetyl-CoA.)
After
an
incision
(-
1
in.)
was
made
through
the
skin,
subcutaneous
tissue,
and
fascia,
-
150
mg
of
muscle
was
mobilized
and
excised.
The
muscle
was
dropped
immediately
into
isopentane,
kept
at
its
freezing
point
(-
160'C)
by
liquid
nitrogen.
The
frozen
mus-
cle
was
stored
at
-70°
until
it
was
aliquoted
into
three
separate
por-
tions.
One
portion
was
extracted
with
fluoride
buffer
according
to
Hagg
et
al.
(24)
for
measurement
of
glycogen
synthase.
A
second
portion
was
extracted
with
perchloric
acid
according
to
Allred
and
Guy
(25)
for
measurement
of
glucose-6
phosphate
(G-6-P)
and
citrate.
The
third
portion
was
extracted
with
methanol-TCA
for
measurement
of
acetyl-
CoA
and
free
CoA-SH.
GS
assay
GS
was
assayed
by
a
modification
of
the
method
of
Thomas
et
al.
(26).
Reactions
were
started
by
addition
of
30-jAI
aliquots
of
the
muscle
ex-
tract
to
60
1d
of
a
reaction
mixture
containing
20
mM
EDTA,
25
mM
sodium
fluoride,
50
mM
Tris-HCl,
1%
glycogen,
0.7
gCi
[U-"C]UDP
glucose,
0.3
mM
UDP
glucose,
and
0-10
mM
G-6-P.
The
reaction
was
terminated
after
15
min
by
precipitating
75-Ml
aliquots
of
the
reaction
mixture
on
2
x
2-cm
squares
of
filter
paper
which
were
dropped
into
cold
66%
ethanol,
washed,
dried,
and
counted.
GS
activity
was
calcu-
lated
as
micromoles
of
UDP
glucose
incorporated
into
glycogen
per
minute
per
milligram
of
protein.
Results
are
expressed
as
the
fractional
velocity
of
GS
activity,
i.e.,
the
activity
of
GS
at
0.
1
mM
G-6-P
divided
by
the
activity
at
10
mM
G-6-P.
This
is
an
indicator
of
the
active
form
of
GS
and
believed
to
be
a
sensitive
parameter
of
in
vivo
GS
activity
(27).
Metabolite
assays
HPLC
analysis
of
Acetyl-CoA
and
free
CoA-SH.
Frozen
muscle
sam-
ples
were
minced
in
200-400
ul
of
methanol-TCA
(10%)
at
-20°C.
After
5
min
at
-20°C,
10
volumes
of
ice
cold
10%
TCA
in
H20
was
added
to
the
sample,
followed
by
sonication
in
ice
for
1
min.
The
supernatant
of
the
TCA
extract
was
washed
five
times
with
ether,
which
raised
the
pH
to
-
5.0
(28).
The
acid-free
extract
was
then
Iyophilized
and
stored
at
-20°C
until
analysis.
HPLC
analysis
was
performed
as
described
(29)
using
a
Waters
Associates
(Milford,
MA)
Nova-Pak
C18
5-Mm
reverse-phase
column.
The
two
mobile-phase
solvents
were
0.1
M
KH2PO4
(A)
and
0.1
M
KH2PO4
containing
40%
acetonitrile
(B),
both
at
pH
5.0.
Acetyl-CoA
and
free
CoA-SH
were
determined
at
260
nm.
Citrate
was
assayed
in
a
coupled
end-point
assay
with
citrate
lyase
and
malate
dehydrogenase
(30).
G-6-P
was
assayed
in
a
coupled
end-
point
assay
with
hexokinase
and
G-6-P
dehydrogenase
(31).
Analytical
procedures
Plasma
glucose
was
measured
with
a
glucose
analyzer
(Beckman
In-
struments,
Inc.,
Palo
Alto,
CA).
Serum
insulin
(32)
was
determined
by
radioimmunoassay.
Blood
urea
nitrogen
(33),
was
measured
colori-
metrically.
Urinary
nitrogen
was
measured
by
the
method
of
Kjeldahl
(34).
Lactate
(35)
and
pyruvate
(36)
were
measured
enzymatically.
Statistical
analysis
and
calculations
All
data
were
expressed
as
the
mean±SEM.
Statistical
significance
was
assessed
using
MANOVA
and
Student's
two-tailed
paired
or
unpaired
t
test.
Effect
of
Fat
on
Insulin-stimulated
Carbohydrate
Metabolism
961

Results
Insulin,
glucose,
and
GIR
(Fig.
1)
In
all
three
studies,
serum
insulin
was
raised
about
eightfold
from
7.5±1.0
to
a
mean
of
58±5
,gU/ml
by
infusion
of
insulin
(1
mU/kg
.
min).
Plasma
glucose
was
clamped
at
88±2
mg/dl
(CV
11.2%).
For
the
first
3
h
of
insulin
infusion,
GIR
were
comparable
during
insulin/saline
or
insulin/lipid
infusions.
The
inhibitory
effect
of
lipid
became
statistically
significant
at
210
min
when
GIR
had
risen
to
6.3±0.4
mg/kg.
min
with
sa-
line,
while
remaining
at
3.7±0.5
mg/kg-
min
with
lipid
(P
<
0.05).
When
lipid
was
replaced
by
saline
at
120
min
in
study
3,
3
h
elapsed
before
the
inhibitory
effect
of
lipid
disappeared
and
GIR
increased
significantly.
100T
75
±
50
+
LUJ
F-
I-
-I
25t
150
100
LUI
F-
=c
LUJ
I~-
F-
LUJ
40
I.-
LUI
I-
=
0o
1
oooT
0.800
0.600
0.400
0.200
u-.-.
0*
O.OO.
1.000
0.800
0.600
0.400
_
0.000
6-
3
2
0.2
0.1
50-
U
I
~I
90
*
8
T7//
1'_
_;
_
6
T
I
~~~~~~~T
A~~~~
T
0~~I
0-
2
a/
1
0O
l
em
.
Go
.
ox
0
60
120
180
MINUTES
Figure
1.
Peripheral
venous
insulin
and
glucose
co
(GIR)
in
healthy
men
during
study
1
(lipid
infusion
mic
hyperinsulinemia,
*,
n
=
6),
study
2
(saline
int
cemic
hyperinsulinemia,
a,
n
=
6),
and
study
3
(euj
sulinemia
plus
lipid
from
0
to
2
h
and
saline
from
*P
<
0.05,
**P
<
0.01
comparing
studies
2
with
I
is--,
I
L
,~
m
0-0~
6-1
AI\
0--
I
I
I
\iA-
Am;w*
A
0
60
120 180
240
300
360
MINUTES
Figure
2.
Acetoacetate,
fl-OH-butyrate,
lactate,
and
pyruvate
concen-
trations
in
studies
1-3.
In
study
1,
lipid/heparin
was
infused
from
0
to
360
min.
In
study
2,
saline
was
infused
from
0
to
360
min.
In
study
3,
lipid/heparin
was
infused
from
0
to
120
min
and
saline
from
120
to
360
min.
Symbols
and
numbers
of
experiments
as
in
Fig.
1.
All
studies
were
performed
under
euglycemic
hyperinsulinemic
condi-
tions.
Mean±SE.
*P
<
0.05,
**P
<
0.05,
***P
<
0.005
comparing
studies
1
with
2
and
3
with
2.
Ketone
bodies,
lactate,
and
pyruvate
(Fig.
2)
Insulin/lipid
infusion
in
study
1
resulted
in
small
increases
in
240
300
360
plasma
concentrations
of
acetoacetate
(from
0.02
to
0.12
mM,
P
<
0.001)
and
fl-hydroxybutyrate
(from
0.04
to
0.34
mM,
P
oncentrations
and
<
0.001).
The
accumulation
of
ketone
bodies
observed
during
i
during
euglyce-
studies
1
and
3
were,
however,
too
small
to
affect
the
calculated
fusion
during
eugly-
rates
of
CHO
OX
and
FAT
OX.
After
lipid
was
replaced
by
Olycemic
hyperin-
saline
at
120
min
in
study
3,
both
ketone
bodies
returned
to
2
to
4
h,
o,
n
=
6).
basal
levels
within
1
h.
Insulin/saline
or
insulin/lipid
infusions
and
3
with
1.
had
no
effect
on
plasma
lactate
and
lactate/pyruvate
ratios
(not
962
Boden
et
al.
-I
Z
=
z
-I
.j
=
a
0
z
LUJ
VI
z
z
0
%w
0.3
-
-60

shown).
Pyruvate
concentrations
were
slightly
higher
during
insulin/saline
as
compared
to
insulin/lipid
infusions
between
30
and
90
min
(P
<
0.05).
Onset
and
duration
of
effect
of
lipid
on
FA
T
OX,
and
CHO
OX
(Fig.
3)
Panel
1.
During
insulin/lipid
infusion,
FAT
OX
rose
from
0.63±0.13
mg/kg
.
min
at
0
min
to
0.91
±0.1
mg/kg
.
min
at
60
min
(P
<
0.05)
and
continued
to
rise
to
1.36±0.12
mg/kg
.
min
at
360
min.
During
insulin/saline
infusion,
FAT
OX
decreased
from
0.70±0.28
mg/kg.
min
at
0
min
to
0.40±0.23
mg/
kg
min
at
360
min.
After
lipid
was
replaced
by
saline
in
study
3,
FAT
OX
fell
significantly
within
30
min
from
1.3±0.18
(at
120
min)
to
0.93±0.08
mg/kg
.
min
(at
150
min)
(P
<
0.05)
and
then
continued
to
fall
to
reach
0.39±0.14
mg/kg
min
at
360
min.
Panel
2.
During
insulin/saline
CHO
OX
increased
from
0.85±0.5
(O
min)
to
1.93±0.14
mg/kg
-
min
30
min
later
and
then
continued
to
rise
to
reach
3.15±0.33
mg/kg
.
min
at
360
min.
During
insulin/lipid
infusion
CHO
OX
decreased
slowly
lWC
z
m
z
to
X
a
K
C
C
I-
Cu
%w
2.000-
1.500
-
1.000
.
0.500
+
0
To
\
I
I
0/
o'S
I
-0
U.UU
in
i
4
lo
~
~ ~ ~ ~
*
I
I
jl
IQf
2
T°
T-A^-.0e-
ii
.
onI
,
0
400
*
-
300
200-A
100-
-60
from
1.6±0.33
(O
min)
to
0.71±0.06
mg/kg
min
(360
min).
After
lipid
was
replaced
by
saline
in
study
3,
CHO
OX
in-
creased
within
60
min
to
values
indistinguishable
from
those
seen
during
insulin/saline
infusions.
Panel
3.
To
better
illustrate
these
directional
changes,
CHO
OX
was
normalized
with
basal
values
(O
min)
being
set
at
100%.
As
can
be
seen,
the
difference
between
insulin/lipid
and
insulin/saline
infusions
became
statistically
significant
at
30
min.
Onset
and
duration
of
lipid
on
glucose
uptake
(GRd)
(Fig.
4)
In
contrast
to
its
early
effect
on
CHO
OX,
insulin/lipid
infu-
sion
had
no
effect
on
GRd
or
GR.
for
at
least
3
h.
After
3
h,
GRd
and
GRd
plateaued
at
4
mg/kg-
min
during
insulin/lipid,
whereas
it
continued
to
rise
to
between
7
and
8
mg/kg.
min
during
insulin/saline
infusion.
The
difference
between
insulin/
lipid
and
insulin/saline
infusions,
however,
did
not
become
statistically
significant
until
330
min.
Similarly,
after
lipid
was
replaced
by
saline
in
study
3,
GRd
and
GRd
remained
inhibited
for
3
h
before
rising.
Correlations
between
GRd,
FAT
OX,
and
CHO
OX
GRd
did
not
correlate
with
FAT
OX
(r
=
-0.19,
P
<
0.04)
and
correlated
only
weakly
with
CHO
OX
(r
=
41,
P
<
0.0001).
GS,
acetyl-CoA,
free
CoA-SH,
G-6-P,
and
citrate
in
muscle
biopsies
(Fig.
5)
GS
activity,
expressed
as
fractional
velocity,
i.e.,
GS
activity
at
a
subsaturating
(0.1
mM)
G-6-P
concentration
divided
by
GS
8
6
4-
2
z
N
I
10
8
6
J
4-
2'-
060
120
180
240
300
360
MINUTES
Figure
3.
Rates
of
FAT
OX
and
CHO
OX
in
healthy
men
during
lipid
or
saline
infusions.
Symbols
and
number
of
experiments
were
as
in
Fig.
1.
In
the
bottom
panel,
CHO
OX
was
normalized
by
setting
0
min
values
as
100%.
Shown
are
mean±SE.
*P
<
0.05,
**P
<
0.01,
***P
<
0.005
comparing
studies
1
with
2
and
3
with
1.
v-6
-60
T
,I
r
/'
X;
9~
I/-~
a
v-a
=8e
i
00-
0
60
120
180
240
300
360
MINUTES
Figure
4.
GR.
and
GRd
in
healthy
men
during
lipid
or
saline
infusions.
Symbols
and
number
of
experiments
were
as
in
Fig.
1.
Shown
are
mean±SE.
*P
<
0.05,
**P
<
0.01
comparing
studies
2
with
1
and
3
with
1.
Effect
of
Fat
on
Insulin-stimulated
Carbohydrate
Metabolism
963
10T
*
_
-4

0.7-
0.6-
0.51
0.4
0.3
0.2
0.1
GLYCOGEN
SYNTHASE
c
E
0)
Acetyl-CoA
CoA
0
k
)
0
IN
0
180
-
160-
140
-
120-
100
80
60
40
20
,\i
G-6-P
CITRATE
Figure
5.
Upper
panel:
glycogen
synthase
activity
in
muscle
biopsies
expressed
as
fractional
velocity
(activity
at
0.1
mM
G-6-P
divided
by
activity
at
10
mM
G-6-P)
before
infusions
(open
bars,
n
=
10),
after
6
h
of
euglycemic
hyperinsulinemia
(solid
bars,
n
=
4)
and
after
6
h
of
lipid
plus
euglycemic
hyperinsulinemia
(cross-hatched
bars,
n
=
6).
Middle
panel:
acetyl-CoA
and
free
CoA-SH
and
the
acetyl-CoA/free
CoA-SH
ratio
in
muscle
biopsies.
Symbols
and
number
of
experi-
ments
as
in
the
upper
panel.
Lower
panel:
G-6-P
and
citrate
concen-
trations
in
muscle
biopsies.
Mean±SE.
*P
<
0.05,
**P
<
0.005
com-
parison
with
the
preceding
column.
activity
at
a
saturating
(10
mM)
concentration
of
G-6-P,
in-
creased
from
0.14±0.05
before
to
0.44±0.12
after
insulin/sa-
line
infusion
(study
2)
(P
<
0.02).
After
insulin/lipid
infusion
(study
1)
GS
fractional
velocity
was
0.08±0.02;
i.e.,
it
failed
to
rise
in
response
to
insulin.
GS
fractional
velocity
correlated
positively
with
GRd
(r
=
0.61)
(Fig.
6).
Acetyl-CoA
concentration
was
1.65±0.6
pmol/,gg
DNA
after
insulin/saline
and
7.12±0.90
pmol/,tg
DNA
after
insulin/
lipid
infusion
(+432%,
P
<
0.03)
whereas
the
acetyl-CoA/free
CoA-SH
ratio
was
0.28±0.04
during
insulin/saline
and
1.37±0.28
during
insulin/lipid
(+489%,
P
<
0.03).
Insulin/saline
or
insulin/lipid
infusions
had
no
significant
effects
on
G-6-P
or
on
citrate
concentrations.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
GS
ACTIVITY
(Fractional
Velocity)
Figure
6.
Correlation
between
GS
activity
(fractional
velocity)
and
GRd.
Data
from
studies
1
and
2.
Discussion
Effect
of
lipid
on
FAT
OX
and
CHO
OX.
Lipid
infusion
was
followed
within
60
min
by
an
increase
in
FAT
OX
and
by
a
decrease
in
insulin-stimulated
total
body
CHO
OX
(study
1).
This
effect
was
reversed,
also
within
1
h,
after
FAT
OX
had
declined
after
replacement
of
insulin/lipid
infusion
by
insulin/
saline
(study
3).
These
observations
confirm
and
expand
reports
by
others
which,
while
not
specifically
investigating
time/effect
relation-
ships,
did
show
that
fat
infusion
increased
total
body
FAT
OX
and
decreased
insulin-stimulated
CHO
OX
within
the
usual
2-h
studies
(10-17).
Thus,
there
is
convincing
evidence
demon-
strating
that
elevation
FAT
OX
leads
to
a
prompt
decrease
in
insulin-stimulated
CHO
OX
in
vivo.
These
findings
are
com-
patible
with
that
part
of
Randle's
glucose-fatty
acid
cycle
hy-
pothesis
which
postulated
that
rising
plasma
FFA
concentra-
tions
increased
FAT
OX
and
inhibited
CHO
OX
in
striated
muscle
via
a
rise
in
the
mitochondrial
acetyl-CoA/CoA
ratio
and
inhibition
of
pyruvate
dehydrogenase.
In
further
support
of
this
hypothesis,
we
have
demonstrated
in
this
study
that
lipid
infusions
produced
large
increments
in
acetyl-CoA
(+432%)
and
in
the
acetyl-CoA/free
CoA-SH
ratio
(+489%)
in
human
skeletal
muscle.
Effect
of
lipid
on
GRd.
Randle's
hypothesis,
however,
also
postulated
that
increased
FAT
OX
caused
muscle
citrate
and
G-6-P
concentrations
to
rise
(the
latter
by
inhibition
of
phos-
phofructokinase
1)
and
glucose
uptake
to
fall
(by
inhibition
of
hexokinase).
In
this
study,
we
found
that
lipid
induced
incre-
ments
in
FAT
OX
did
neither
increase
citrate
nor
G-6-P
con-
centrations
in
muscle
and
did
not
reduce
GRd
(GRd
is
equiva-
lent
with
glucose
uptake)
for
at
least
3
h.
On
the
other
hand,
once
established,
the
inhibition
of
GRd
lasted
for
several
hours
even
after
FAT
OX
had
fallen
(in
study
3).
It
must
be
pointed
out,
however,
that
the
validity
of
our
GRd
values
depended
on
the
accuracy
of
the
glucose
turnover
measurements.
As
men-
tioned
(see
Methods)
the
stable
isotope
method
used
underesti-
mated
Gpa
(and
thus
GRd)
during
hyperinsulinemia.
The
error,
however,
appeared
to
be
relatively
minor
(negative
values
for
hepatic
glucose
output
ranged
from
0.5
to
1.0
mg/kg
min).
Moreover,
the
error
was
similar
in
all
three
studies
and
thus
did
not
invalidate
our
observation
that
lipid
inhibited
CHO
OX
964
Boden
et
al.
0
0,--
ZeN
0-
-Jo
l-,
z
0-
~-0
E
0z
0.
'-a
76
E
c
I

first
and
GRd
hours
later.
The
long
delay
between
the
rise
in
FAT
OX
and
the
inhibition
of
GRd,
the
lack
of
correlation
between
FAT
OX
and
GRd
and
the
lack
of
increases
in
muscle
citrate
and
G-6-P
concentrations
cannot
easily
be
explained
by
the
Randle
hypothesis
and
suggest
that
the
effects
of
fat
on
rat
heart
muscle
are
different
from
those
on
human
striated
mus-
cle.
Others
have
arrived
at
similar
conclusions.
Lillioja
et
al.
(37),
studying
lipid
turnover
in
nondiabetic
Pima
Indian
women
during
hyperinsulinemic
clamps,
concluded
that
the
Randle
cycle
was
probably
operative
for
the
regulation
of
CHO
OX
but
not
for
the
regulation
of
the
nonoxidative
component
of
GRd.
Yki-Yarvinnen
et
al.
(38)
found
that
the
rate-limiting
step
for
GRd
during
hyperinsulinemia
appeared
to
be
beyond
the
glucose
transport
step.
What
caused
the
late
decrease
in
GRd?
We
found
that
GRd
correlated
poorly
with
FAT
OX
and
CHO
OX
but
closely
with
GS
activity.
GS
activity
is
generally
considered
to
be
the
rate-
limiting
step
in
glycogen
synthesis
(39),
which
has
recently
been
demonstrated
to
account
for
nearly
all
of
CHO
storage
during
euglycemic
hyperinsulinemia
(40).
It
appeared,
there-
fore,
that
GRd
declined
eventually
because
of
a
problem
with
glycogen
synthesis,
which
developed
after
several
hours
of
insu-
lin/lipid
infusion.
The
mechanism
by
which
lipid
inhibited
GS
activity
was
not
explored.
It
is,
however,
known
that
as
muscle
glycogen
stores
fill
up,
GS
activity
decreases,
at
least
partially
owing
to
a
decrease
in
the
active
form
of
GS
(41-43).
This
may
have
oc-
curred
in
our
study
where
a
marked
fall
in
the
active
form
of
GS
was
seen
after
several
hours
of
lipid
infusion.
Bjorntorp
et
al.
(44)
have
estimated
on
the
basis
of
forearm
glucose
uptake
studies
that
at
rest,
when no
utilization
of
glycogen
has
oc-
curred,
the
uptake
of
glucose
into
human
muscle
was
limited
to
-
20
g/d.
We
have
calculated
that
after
51/2
h
of
insulin/lipid
infusion,
i.e.,
at
the
time
when
GRd
started
to
decrease
sigifi-
cantly,
-
23.4
g
of
glucose
had
been
prevented
from
being
oxidized
and
presumably
had
been
shunted
for
the
most
part
into
muscle
glycogen
formation.
We
would,
therefore,
like
to
propose
the
following
hypothesis:
during
the
initial
3-4
h
of
insulin/lipid
infusion,
when
GRd
was
normal
but
CHO
OX
was
diminished,
the
glucose
which
entered
the
muscle,
but
could
not
be
oxidized,
was
stored
as
glycogen.
At
least,
we
found
no
evidence
that
glucose
was
shunted
in
any
substantial
amount
into
other
forms
of
nonoxidative
glucose
disposal
such
as
lac-
tate
production
or
lipid
synthesis
(plasma
lactate
levels,
pre-
sumably
reflecting
lactate
production,
did
not
change
and
npRQ
values
never
exceeded
1.0).
Blood
lactate
levels,
how-
ever,
may
not
accurately
reflect
lactate
turnover
and
the
possi-
bility
cannot
be
ruled
out
that
there
was
a
small
accumulation
of
lactate
in
muscle
or
that
some
lactate
was
used
for
gluconeo-
genesis.
After
4-5
h,
muscle
storage
capacity
was
saturated
and
GRd
had
to
decrease
since
all
major
pathways
of
glucose
utiliza-
tion
were
now
blocked.
There
are,
however,
alternative
path-
ways
by
which
lipid
could
have
affected
GRd.
For
instance,
long
chain
acyl-CoA
was
likely
to
increase
in
muscle
and
may
have
inhibited
GS
activity.
Palmityl-CoA
has
been
shown
to
dissoci-
ate
active
tetrameric
liver
glycogen
synthase
into
monomers
which
were
unable
to
bind
to
the
primer
glycogen
(45).
Further-
more,
we
cannot
rule
out
the
possibility
that
lipids
may
have
inhibited
glucose
transport
directly.
Hissin
et
al.
have
shown
that
feeding
of
high
fat/low
CHO
diets
for
21
days
to
rats
re-
duced
the
number
of
glucose
transporters
in
adipocytes
plasma
membranes
and
decreased
glucose
transport
(46).
Clearly,
fur-
ther
studies
are
needed
to
determine
which
of
these
mecha-
nisms
was
operative.
The
delayed
inhibitory
effect
of
lipid
infusion
on
GRd
may
explain
why
the
in
vitro
studies
(4-7),
which
were
short,
lasting
only
from
45
to
150
min,
failed
to
show
lipid
effects
on
glucose
uptake.
In
addition,
it
may
help
to
reconcile
several
contradic-
tory
in
vivo
reports.
For
instance,
several
groups
have
failed
to
detect
effects
of
lipid
infusions
on
glucose
uptake
during
hyper-
insulinemic
clamps
(12,
14,
15,
17),
while
others
have
found
only
marginal
(-
15%)
inhibition
(10).
All
these
studies
lasted
for
only
2
h.
According
to
our
data,
2
h
is
not
sufficient
for
the
inhibitory
effect
of
FAT
OX
on
GRd
to
develop.
By
compari-
son,
groups
who
infused
lipid
for
3-4
h
did
observe
inhibition
of
glucose
uptake
(1
1,
16).
Bonnadonna
et
al.
(15),
in
the
only
other
study
on
the
time
dependency
of
lipid
and
glucose
inter-
action,
reported
that
lipid
infusion,
when
started
simulta-
neously
with
the
insulin
clamp,
resulted
in
inhibition
of
insu-
lin-mediated
glucose
disposal
after
3-4
h.
In
contrast,
when
the
lipid
infusion
was
started
2
h
after
commencement
of
the
insu-
lin
clamp
and
continued
for
another
2
h,
there
was
no
inhibi-
tion
of
glucose
disposal.
They
interpreted
their
findings
as
indi-
cating
that
2
h
of
hyperinsulinemia
had
rendered
the
body
relatively
refractory
to
the
action
of
lipid
on
glucose
disposal.
Our
findings
suggested
that
lipids
infused
for
4
h
allowed
suffi-
cient
time
for
the
inhibition
of
glucose
disposal
to
develop
whereas
lipid
infused
for
only
2
h
did
not.
Clinical
relevance.
Patients
with
NIDDM
commonly
have
long
histories
of
excessive
caloric
intake
(frequently
in
the
form
of
fat),
lack
of
physical
exercise,
increased
plasma
FFA
concen-
trations,
and
increased
FAT
OX.
Impaired
CHO
STOR
has
been
recognized
as
one
of
the
earliest
problems
(47-49).
As
shown
in
this
and
other
studies,
many
of
these
abnormalities
including
impaired glycogen
synthesis
can
be
produced
by
fat
infusion
in
healthy
individuals.
It
is,
therefore,
likely
that
an
excess
of
FFA
may
contribute
to
the
insulin
resistance
asso-
ciated
with
NIDDM
in
susceptible
individuals.
Of
particular
interest,
in
this
respect,
is
a
recent
report
by
Pascoe
and
Storlien
(50),
describing
development
of
fasting
hyperglycemia
in
rats
with
mildly
compromised
,-cell
function
after
being
fed
a
high
fat
diet
for
only
1
wk.
In
summary,
we
have
shown
in
normal
men
that
insulin/
lipid
infusion
caused
prompt
inhibition
of
CHO
OX
which
was
followed
after
several
hours
by
a
fall
in
GRd.
This
fall
in
GRd
was
associated
with
a
drastic
decrease
in
muscle
GS
activity.
On
the
basis
of
these
observations
we
concluded
that
lipid
reduced
GRd
by
interfering
with
glycogen
synthesis.
Putative
mechanism
in-
cluded
inhibition
of
GS
by
saturation
of
glycogen
storage
or
by
long
chain
acyl-CoA.
Acknowledgments
We
thank
the
nurses
of
the
General
Clinical
Research
Center
for
help
with
the
studies,
Brenda
Blyler
for
technical
help,
and
Constance
Harris
for
typing
the
manuscript.
This
study
was
supported
by
National
Institutes
of
Health
grants
AG-07988
(G.
Boden),
DK-22
122
(Y.
Liang),
RR-349
(General
Clini-
cal
Research
Center),
and
a
grant-in-aid
from
the
American
Diabetes
Association,
Philadelphia
affiliate
(F.
Jadali).
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