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Department of Medicine, University of Rochester, Rochester, NY 14642 and * Department of Medicine, University of Tubingen, 72076 Tubingen, Germany
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • glutamine • gluconeogenesis • kidney • liver • glucose metabolism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It not only participates as an
important intermediate in numerous metabolic pathways, but also acts as
a regulator of certain key physiologic processes (e.g., glycogen
synthesis, gluconeogenesis and lipolysis) (Stumvoll et al. 1999
). Preeminent among these functions is its role as an
interorgan carrier of nitrogen and carbon (Nurjhan et al. 1995
, Welbourne 1987
).
Until quite recently, studies in humans have used
15N-labeled glutamine to assess its fluxes;
consequently, virtually nothing was known about the fate of its carbon.
During the past few years, we have used a combination of isotopic
(14C-labeled glutamine) and net-balance
techniques to assess various aspects of glutamine carbon metabolism in
humans (Nurjhan et al. 1995
, Perriello et al. 1995
and 1997
, Stumvoll et al. 1996
). These
studies indicate the following: 1) in postabsorptive, normal
volunteers, plasma glutamine turnover (rates of appearance and
disappearance) averages 5–6 µmol/(kg·min), slightly
greater than that calculated using an 15N-labeled
glutamine tracer (Kreider et al. 1997
); and
2) 40–60% of plasma glutamine disappearance is due to
oxidation (Nurjhan et al. 1995
, Perriello et al. 1997
, Stumvoll et al. 1996
), 10–20% to
gluconeogenesis (Nurjhan et al. 1995
, Perriello et al. 1997
, Stumvoll et al. 1996
) and most of
the remainder (~15%) to protein synthesis (Perriello et al. 1997
) and incorporation into other macromolecules. Skeletal
muscle (~50%) (Nurjhan et al. 1995
, Stumvoll et al. 1996
), kidney (~15%) (Stumvoll et al. 1998b
) and liver (5–10%) (Stumvoll et al. 1998b
) account for nearly 75% of glutamine uptake from plasma.
The bulk of glutamine entering plasma (~70%) is due to de novo
synthesis (Nurjhan et al. 1995
, Stumvoll et al. 1996
), some of which is the consequence of the conversion of
glucose to glutamine (Perriello et al. 1995
). Skeletal
muscle is the predominant source of plasma glutamine, accounting for
65–75% of its rate of appearance in plasma (Nurjhan et al. 1995
, Stumvoll et al. 1996
).
In this paper, we review our recent work (Meyer et al. 1998
, Stumvoll et al. 1998a
and 1998b
)
concerning the effects of insulin, epinephrine and glucagon on systemic
glutamine kinetics, the incorporation of glutamine into glucose by
liver and kidney and the metabolism of glutamine by the kidney.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data presented here were published previously (Meyer et al. 1998
, Stumvoll et al. 1998a
and 1998b
); the
reader is referred to these papers for a detailed description of the
study designs and analytical procedures. In brief, all subjects were
normal volunteers of both genders who had fasted overnight. At ~0530
h, primed continuous infusions of [6-3H] glucose and
[U-14C] glutamine were begun; at least 4 h were
allowed for achievement of isotopic steady state. At ~0730 h, a renal
vein catheter was inserted under fluoroscopy; shortly thereafter, a
primed infusion of p-aminohippuric acid was started for
calculation of renal blood flow. After the collection of at least three
baseline blood samples at 30-min intervals for relevant substrate and
hormone concentrations and for [3H] glucose,
[14C] glucose and [14C] glutamine specific
activities from an arterialized dorsal hand vein and the renal vein,
subjects were, depending on the protocol, infused with insulin [0.6
mU/(kg · min) for 4 h] along with sufficient glucose
to maintain euglycemia using the glucose clamp technique (Meyer et al. 1998
), or glucagon [5 ng/(kg · min) for
3 h] (Stumvoll et al. 1998a
), or epinephrine [270
pmol/(kg · min) for 3 h] (Stumvoll et al. 1998b
).
Calculations.
Renal plasma flow
(RPF)4
was determined by the p-aminohippuric acid clearance
technique (Brun 1951
) and renal blood flow (RBF) was
calculated as RPF/(1 - hematocrit). Fractional extraction (FX) of
glucose across the kidney was calculated as ([6-3H]
glucose specific activity (SA)art x glucoseart
- [6-3H] glucoseSArenal vein x glucoserenal vein)/([6-3H] glucose
SAart x glucoseart). Renal
glucose uptake (RGU) was calculated as RBF x glucoseart x FX, and renal glucose net balance (NB)
as RBF x (glucoseart - glucoserenal
vein). Renal glucose release (RGR) was calculated as RGU - NB. Analogous equations were used for glutamine, except that renal
plasma flow was used.
Systemic appearance (Ra) and removal (Rd) of glucose from the
circulation was determined with steady-state equations under basal
conditions and subsequently during infusion of epinephrine with
non-steady–state equations. Hepatic glucose release (HGR) was
calculated as the difference between the overall plasma appearance of
glucose and renal glucose release. Systemic glutamine rates of
appearance and disappearance were calculated using steady-state
equations under basal conditions and a modification of DeBodo’s
equation (DeBodo et al. 1963
) during the
non-steady–state using a pool fraction of 0.75 and a volume of
distribution of 430 mL/kg body weight (Kreider et al. 1997
).
The proportion of systemic glucose appearance in the steady state due
to whole-body glutamine gluconeogenesis was calculated as
([14C]glucose
SAart/[14C]glutamine SAart)
x 100/1.2 using the standard precursor/product calculation
(Kreisberg et al. 1970
). The division by 1.2 corrects
for differences in carbons (i.e., glutamine has 5, glucose has 6
carbons). Total glutamine gluconeogenesis was calculated as the
proportion of glucose Ra due to glutamine multiplied by glucose Ra.
During the non-steady state, whole-body glutamine
gluconeogenesis was calculated using the equation of Chiasson et al. (1977)
. These calculations assume that all glutamine
carbons are incorporated into glucose, that there is no fixation of
14CO2 derived from oxidation of glutamine and
that there is no dilution of 14C derived from glutamine by
unlabeled glucose in the Krebs cycle. The first and last assumptions
are not valid and will result in underestimations that could be
considerable (~40%) (Consoli et al. 1987
, Katz 1985
, Krebs et al. 1966
). On the other hand,
Hankard et al. (1997)
showed recently that only 4% of
[14C] glutamine incorporation into plasma glucose
involves fixation of 14CO2 derived from
glutamine.
Renal gluconeogenesis from glutamine was calculated as RBF x ([14C]glucose SArenal vein x glucoserenal vein - (1 - FX) x ([14C]glucose SAart x glucoseart)/{1.2 x ([14C]glutamine
SArenal vein)} (BenGalim et al. 1980
).
Hepatic gluconeogenesis was calculated as the difference between
systemic and renal gluconeogenesis for glutamine.
Statistical analysis.
Data are expressed as means ± SEM Unless stated otherwise, paired two-tailed Student’s t tests were used to compare data obtained before and after hormone infusions using the mean of baseline determinations and generally the mean of determinations during the last hour of the hormone infusions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During infusion of insulin, plasma insulin concentrations averaged 219
± 13 pmol/L (P < 0.001 vs. basal 36 ± 4
pmol/L) and plasma glucose concentrations were maintained at 4.97
± 0.03 mmol/L. Systemic glucose release decreased ~50%
(P < 0.001) (Fig. 1
). Although the absolute decrement in HGR was greater than that for RGR,
in terms of the percentage of suppression, HGR decreased to a lesser
extent than did RGR (47.3 ± 6.0 vs. 60.7 ± 4.3%,
P = 0.027).
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) decreased (P = 0.001) because systemic glutamine
disposal increased to a greater extent than systemic glutamine release.
Systemic glutamine clearance increased ~25% from 10.0 ± 0.8 to
12.8 ± 0.6 mL/(kg · min) (P < 0.001). Insulin
had no effect on renal glutamine net balance, fractional extraction,
uptake and release of glutamine (Table 1
).
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|
). Infusion of insulin decreased systemic glutamine gluconeogenesis
~50%. As was the case for glucose release, insulin suppressed renal
glutamine gluconeogenesis to a greater extent than hepatic glutamine
gluconeogenesis (71.6 ± 5.2 vs. 24.7 ± 4.9%, P
< 0.001).
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During infusion of epinephrine, arterial epinephrine concentrations
averaged 3550 ± 387 pmol/L; arterial glucose and insulin
increased significantly (P < 0.001), whereas arterial
glucagon remained unchanged (Fig. 4
). Systemic glucose release, RGR and HGR all increased (Fig. 5
). Arterial glutamine (Fig. 6
) decreased slightly (P < 0.01) because glutamine
removal from plasma increased to a greater extent than did its release
into plasma.
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) increased nearly twofold (P < 0.001) and accounted
for 90.3 ± 4.0% of systemic glutamine gluconeogenesis and 67.3
± 7.9% of renal glutamine uptake during this interval. The
contribution of renal glutamine gluconeogenesis to overall renal
glucose release remained unchanged (12.7 ± 1.7%, P
= 0.13), suggesting that gluconeogenesis from other precursors was
increased. Hepatic glutamine gluconeogenesis decreased 50% during
infusion of epinephrine (P = 0.01) and accounted for
<10% of overall glutamine gluconeogenesis and <1% of overall
hepatic glucose release during this period.
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. Effects of glucagon.
Infusion of glucagon increased plasma glucagon to ~270 ng/L
(Fig. 8
). Plasma glucose increased transiently to a peak at 60 min, and then
decreased to ~4.8 mmol/L at the end of the glucagon infusion
(P = 0.16). Plasma insulin also increased transiently.
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) increased transiently to a peak at 30 min and subsequently decreased
to values not different from baseline (P = 0.48). HGR
increased to a peak at 30 min and subsequently decreased to rates that
were not different from baseline. RGR did not change during infusion of
glucagon.
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) despite no detectable change in plasma glutamine rates of appearance
and disappearance.
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) accounted for 3.7 ± 0.3% of systemic glucose release. Renal
glutamine gluconeogenesis accounted for 74.8 ± 6.2% of systemic
glutamine gluconeogenesis and 12.3 ± 1.4% of RGR. Hepatic
glutamine gluconeogenesis accounted for 25.2 ± 6.2% of systemic
glutamine gluconeogenesis and 1.2 ± 0.3% of HGR.
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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) could readily account for this. The increase in
systemic glutamine disposal could reflect stimulation of protein
synthesis or the substitution of glutamine for free fatty acids as an
oxidative fuel in certain tissues.
Although hepatic glutamine uptake was not measured, renal glutamine
uptake was not affected by insulin. This indicates that the increased
systemic glutamine disposal was due to extrarenal tissues and,
furthermore, that the decrease in renal glutamine gluconeogenesis was
not due to decreased availability of glutamine. Regarding the latter,
inhibition of gluconeogenic enzymes by insulin and suppression of
plasma free fatty acids (FFA) (known stimulators of renal
gluconeogenesis) (Krebs et al. 1965
) leading to
diversion of glutamine carbon into the oxidative pathway could be
possible explanations. Clearly it would have been of interest to
measure glutamine oxidation in those experiments.
Epinephrine had effects similar to those of insulin on arterial
glutamine and its plasma kinetics but increased glutamine conversion to
glucose selectively by the kidney. The increase in release of glutamine
into plasma, which was also found by Matthew et al. (1990)
using N15-labeled glutamine, could
reflect increased proteolysis as well as increased conversion of
glucose to glutamine. However, Miles et al. (1984)
reported that proteolysis, as reflected by leucine fluxes, decreases
during infusion of epinephrine and that the increase in plasma alanine
appearance during infusion of epinephrine is due to de novo synthesis.
The increase in systemic glutamine disposal was due to some extent to
an increase in renal glutamine uptake, but this could explain only a
small proportion leaving plasma. The other tissue sites remain to be
determined.
The stimulation of glutamine gluconeogenesis was wholly accounted for by the increase in renal gluconeogenesis, which in turn was almost nearly equal to the increase in renal glutamine uptake. The latter was explained largely by an increase in renal glutamine fractional extraction. Whether this completely explains the stimulatory effects of epinephrine remains to be determined because other mechanisms are possible (e.g., increased FFA or activation of gluconeogenic enzymes).
The lack of effects of epinephrine on hepatic glutamine gluconeogenesis
was unexpected because epinephrine stimulates hepatic alanine
gluconeogenesis (Stumvoll et al. 1998b
). These different
responses of liver and kidney could involve differences in hepatic and
renal amino acid transport systems (Shotwell et al. 1983
) and glutaminase activities (Joseph and McGivan, 1978
) as well as the relative sensitivity of liver and kidney
to epinephrine (e.g., receptor density).
Glucagon infusion decreased arterial glutamine concentration without a
detectable change in either the rate of plasma glutamine appearance or
disappearance, and increased hepatic, but not renal glutamine
conversion to glucose. We believe that lack of statistical power
probably explains the failure to detect changes in systemic glutamine
kinetics. Presumably, glutamine removal from plasma during the glucagon
infusion must have exceeded its release into plasma. Simulation of
glutamine conversion to glucose in liver could reflect the effects of
glucagon on hepatic amino acid transport and/or its stimulation of
hepatic glutaminase (Ochwadt et al. 1965
).
In summary, previous studies have shown that cortisol affects glutamine
metabolism in humans (Darmaun et al. 1988
); the present
studies demonstrate that insulin, glucagon and epinephrine also affect
glutamine metabolism in humans. Furthermore, glucagon and epinephrine
show selectivity in their stimulation of glutamine conversion to
glucose by liver and kidney. The mechanism for these differences and
their physiologic significance remain to be determined. The studies
reported here provide evidence that the human kidney makes more than a
trivial contribution to overall gluconeogenesis under postabsorptive
conditions, e.g., 40–50%, assuming that gluconeogenesis represents
~50% of overall glucose production (Landau et al. 1995
). A similar conclusion can be drawn from the recent work
by Cersosimo et al. (1999a
, 1999b
and 1998)
.
Nevertheless, Ekberg et al. (1999)
recently reported a
contribution of only ~10%. The latter findings should be interpreted
with caution, however, because negative values for renal glucose uptake
and renal glucose fractional extraction were found using
[63H] glucose, but not using
[U-13C] glucose. Indeed, their 95% confidence
limits would be consistent with a renal contribution to overall
gluconeogenesis as great as 20–30%.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by National Institutes of Health/DRR/GCRC grants 5M01-RR00044 and NIDDK-20411.
4 Abbreviations used: FFA, free fatty acids; FX, fractional extraction; HGR, hepatic glucose release; NB, net balance; Ra, rate of glucose appearance; RBF, renal blood flow; Rd, rate of glucose removal; RGR, renal glucose release; RGU, renal glucose uptake; RPF, renal plasma flow; SA, specific activity.
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