Relationship Between Gluconeogenesis and Glutathione Redox State in Rabbit
Kidney-Cortex Tubules
Katarzyna Winiarska, Jakub Droz ˙ak, Michal We ˛ grzynowicz, Adam K. Jagielski, and Jadwiga Bryla
The intracellular glutathione redox state and the rate of glucose formation were studied in rabbit kidney-cortex tubules. In the
presence of substrates effectively utilized for glucose formation, ie, aspartate glycerol octanoate, alanine glycerol
octanoate, malate, or pyruvate, the intracellular reduced glutathione/oxidized glutathione (GSH/GSSG) ratios were signifi-
cantly higher than those under conditions of negligible glucose production. Changes in the intracellular GSH/GSSG ratio
corresponded to those in glucose-6-phosphate content and reduced nicotinamide adenine dinucleotide phosphate/oxidized
nicotinamide adenine dinucleotide phosphate (NADPH/NADP
) ratio obtained from malate/pyruvate measurements. Glu-
coneogenesis stimulation by extracellular adenosine triphosphate (ATP) or inosine caused an elevation of the intracellular
GSH/GSSG and NADPH/NADP
ratios, as well as glucose-6-phosphate level. Surprisingly, in the presence of 5 mmol/L
glucose, both the intracellular GSH/GSSG and NADPH/NADP
ratios and glucose-6-phosphate content were almost as low
as under conditions of negligible glucose synthesis. L-buthionine sulfoximine (BSO)-induced decline in both the intracellular
glutathione level and redox state resulted in inhibition of gluconeogenesis accompanied by accumulation of phosphotrioses
and a decrease in fructose-1,6-bisphosphate content, while cysteine precursors altered neither GSH redox state nor the rate
of glucose formation. In view of the data, it seems likely that: (1) intensive gluconeogenesis rather than extracellular glucose
is responsible for maintaining a high intracellular GSH/GSSG ratio due to effective glucose-6-phosphate delivery for NADPH
generation via the pentose phosphate pathway; (2) a decline in the intracellular glutathione level and/or redox state causes
a decrease in glucose synthesis resulting from a diminished flux through aldolase; (3) induced by cysteine precursors,
elevation of the intracellular GSH level does not affect the rate of glucose formation, probably due to no changes in the
intracellular GSH/GSSG ratio.
© 2003 Elsevier Inc. All rights reserved.
G
LUTATHIONE (L--glutamyl-L-cysteinylglycine) is the
predominant nonprotein thiol in mammalian cells. Its
intracellular concentrations are in the range of 0.5 to 10
mmol/L (see Hammond et al
1
for review). Under physiologic
conditions, more than 98% of intracellular glutathione exists as
the reduced thiol form (GSH), while the rest is present mainly
as the oxidized disulfide form (GSSG) or mixed disulfides.
Reduction of GSSG to GSH is catalyzed by GSH reductase
utilizing nicotinamide adenine dinucleotide phosphate
(NADPH) as reducing equivalent. Glutathione plays several
vital roles: it scavenges free radicals, regulates gene expression
and enzyme activities, controls the process of cell death, de-
toxifies xenobiotics, and maintains other antioxidants in their
reduced forms. Disturbed glutathione status has been reported
to accompany many diseases, eg, diabetes,
2,3
neurodegenera-
tive diseases,
4
viral infections,
5
cirrhosis, and alcoholic dis-
ease.
6
It is commonly accepted that exposition to high glucose
concentrations can induce oxidative stress, which is considered
to be the main cause of diabetic complications.
7,8
Moreover,
following hyperglycemia, the susceptibility of healthy humans
to oxidative stress is increased.
9
To elucidate the mechanisms
of glucose-induced oxidative stress, several hypotheses have
been proposed, including glucose auto-oxidation, glycation of
proteins, and formation of advanced glycation end products
(AGEs) (see Bonnefont-Rousselot et al
7
for review). High
glucose has been reported to diminish GSH levels as a result of
decreased expression of -glutamylcysteine synthetase, the key
enzyme of glutathione synthesis,
10,11
and inhibition of glucose-
6-phosphate dehydrogenase, an enzyme responsible for
NADPH delivery for GSSG reduction.
12
On the other hand, it
is necessary to point out that NADPH generation via the
oxidative phase of pentose phosphate pathway is a glucose-
consuming process.
13
In view of these observations, the aim of this study was to
investigate the intracellular glutathione status with respect to
glucose formation. As the kidney, in addition to liver, makes a
significant contribution to glucose whole body metabolism
14
and the intracellular localization of gluconeogenic enzymes in
rabbit kidney is similar to that in humans,
15
rabbit kidney-
cortex tubules were chosen to be the subject of this investiga-
tion.
MATERIALS AND METHODS
Isolation and Incubation of Kidney-Cortex Tubules
Male California strain rabbits (2 to 3 kg body weight) were used
throughout the experiments. All animal use procedures were approved
by the First Warsaw Local Commission for the Ethics of Experimen-
tation on Animals. Animals were fed ad libitum with standard rabbit
chow and had free access to water. Rabbits were anesthetized with
pentobarbital (30 mg/kg body weight). Kidney-cortex tubules were
isolated according to the method described by Guder at al
16
and
modified by Zablocki et al.
17
Freshly isolated renal tubules (about 10
mg dry weight) were incubated for 1 hour at 37°C, under the atmo-
sphere of 95% O
2
+ 5% CO
2
, in 2 mL Krebs-Ringer bicarbonate buffer
in 25-mL plastic Erlenmeyer flasks (Nalgene, Rochester, NY) sealed
with rubber stoppers. Amino acids and glycerol were added to the
incubation medium at 2 mmol/L concentrations, malate and pyruvate at
From the Institute of Biochemistry, Warsaw University, Warsaw,
Poland.
Submitted August 12, 2002; accepted December 17, 2002.
Supported by Grants No. 4P05A 12017 and BW 1455/17/99 from the
State Committee for Scientific Research.
Address reprint requests to Jadwiga Bryla, PhD, Ul. I. Miecznikowa
1, 02-089 Warsaw, Poland.
© 2003 Elsevier Inc. All rights reserved.
0026-0495/03/5206-0050$30.00/0
doi:10.1016/S0026-0495(03)00035-0
739 Metabolism, Vol 52, No 6 (June), 2003: pp 739-746