Estimates of Krebs cycle activity and contributions of gluconeogenesis to hepatic glucose production in fasting healthy subjects and IDDM patients
Diabetologia
Estimates of Krebs cycle activity and contributions of gluconeogenesis to hepatic glucose production in fasting healthy subjects and IDDM patients
B.R. Landau 0
V. Chandramouli 0
W.C. Schumann 0
K. Ekberg 0
K. Kumaran 0
S.C. Kalhan 0
J. Wahren 0
0 1Department of Medicine,Case Western Reserve University , Cleveland,Ohio , USA 2Department of ClinicalPhysiology,KarolinskaHospital, Stockholm,Sweden 3Department of Pediatrics,Case Western Reserve University , Cleveland,Ohio , USA
Summary Normal subjects, fasted 60 h, and patients with insulin-dependent diabetes mellitus (IDDM), withdrawn from insulin and fasted overnight, were given phenylacetate orally and intravenously infused with [3-14C]lactate and 13C-bicarbonate. Rates of hepatic gluconeogenesis relative to Krebs cycle rates were estimated from the 14C distribution in glutamate from urinary phenylacetylglutamine. Assuming the 13C enrichment of breath C O 2 was that of the CO 2 fixed by pyruvate, the enrichment to be expected in blood glucose, if all hepatic glucose production had been by gluconeogenesis, was then estimated. That estimate was compared with the actual enrichment in blood glucose, yielding the fraction of glucose production due to gluconeogenesis. Relative rates were similar in the 60-h fasted healthy subjects and the diabetic patients. Conversion of oxaloacetate to phosphoenolpyruvate was two to eight times Krebs cycle flux and decarboxylation of pyruvate to acetyl-CoA, oxidized in the cycle, was less than one30th the fixation by pyruvate of CO2. Thus, in estimating the contribution of a gluconeogenic substrate to glucose production by measuring the incorporation of label from the labelled substrate into glucose, dilution of label at the level of oxaloacetate is relatively small. Pyruvate cycling was as much as onehalf the rate of conversion of pyruvate to oxaloacetate. Glucose and glutamate carbons were derived from oxaloacetate formed by similar pathways if not from a common pool. In the 60-h fasted subjects, over 80 % of glucose production was via gluconeogenesis. In the diabetic subjects the percentages averaged about 45 %. [Diabetologia (1995) 38: 831-838]
Gluconeogenesis; Krebs cycle; fasting; insulin-dependent diabetes mellitus; liver
9 Springer-Verlag1995
It is a continuing challenge to develop a method for
reliably estimating the contribution of
gluconeogenesis to glucose production in humans, so that it can
be applied under both physiological and
pathological conditions [
1, 2
]. Measurements of incorporation
of label into glucose on administration of a labelled
gluconeogenic substrate are limited by the need to
know the amount of label in the substrate entering
the gluconeogenic process and the extent of dilution
of label during that process, notably at the level of
oxaloacetate, an intermediate common to the Krebs
cycle and gluconeogenesis.
Phosphoenolpyruvate (PEP) is the necessary
intermediate formed from oxaloacetate via pyruvate
in the production of glucose (Fig. 1). We therefore
gave l~C-labelled bicarbonate to fasted normal
subjects, assuming the specific activity of expired 14CO2
to be the specific activity of 14C fixed in the
conversion of oxaloacetate to pyruvate [
3
]. We did that
because of evidence that expired 14CO2 is a good
measure of mitochondrial t4CO2. We estimated the
extent of dilution at the level of oxaloacetate from the
distribution of 14C from [3-14C]lactate in hepatic
etketoglutarate, estimated from the distribution of 14C
Fatty
V=~ acids
CO,/
A~tyF-COA
, ~[ ,
=Fum.ar~ate
c,,,=~
V ~V~
CO
n-Katoglutarnte
COs
in glutamine conjugated to phenylacetate. Our
method, while time consuming, provides details on the
metabolic rates occurring in liver while offering a direct
measure of the contribution of gluconeogenesis. We
have now used that approach to examine the rate of
gluconeogenesis relative to Krebs cycle flux and the
contribution of gluconeogenesis to glucose
production in insulin-dependent diabetic (IDDM) patients,
withdrawn from insulin so as to develop mild ketosis.
These findings have been compared with those
observed in fasted normal subjects also in mild ketosis.
Subjects, materials and methods
Subjects. A healthy man and woman, ages 35 and 28 years, with
body mass indices 26.0 and 22.0 kg/m2, respectively, and fasted
for 60 h, were studied. Five males with IDDM, ages 19 to
23 years, with a mean body mass index of 22.9 kg/m2 (range
18.0 to 26.8) were also studied. The haemoglobin Ale
concentration in four of the diabetic subjects ranged from 6.9 to
8.0 % and in one subject, MP, it was 13.9 %. Insulin was
withdrawn 24 h before the study and food was withheld for 12 h.
The diabetic subjects were encouraged to drink water
throughout the period of insulin withdrawal. Approval for the study
was obtained from the Human Investigation Committees at
the Karolinska Hospital, Stockholm and University Hospitals
of Cleveland. Informed consent was obtained from each
subject.
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