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J. Biol. Chem., Vol. 277, Issue 36, 32899-32904, September 6, 2002
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From the
Received for publication, March 12, 2002, and in revised form, July 1, 2002
The activities of either the mitochondrial or
cytosolic glycerol phosphate dehydrogenase (mGPD, cGPD) plus that of
glycerol kinase are required for the use of glycerol in aerobic
metabolism and gluconeogenesis. A knockout mouse lacking mGPD has
reduced body weight and fertility but shows remarkably normal liver and muscle metabolite levels. The BALB/cHeA mouse strain, which lacks cGPD,
breeds well and is phenotypically normal, although it demonstrates metabolite abnormalities in certain tissues. Crosses were made between
these two strains, and mice were generated that lacked both
dehydrogenases. These mice, although active and nursing well for
several days, failed to grow, and usually died within the first week.
Liver glycerol phosphate levels were elevated 30-fold, whereas liver
ATP, ADP, and AMP levels were reduced by 30-40%. Plasma glycerol was
elevated 30- to 50-fold to 30-50 mM, and urine glycerol exceeded 0.45 M (4% w/v). GPD-deficient mice were
hypoglycemic, had a 50% increase in plasma free fatty acids, and
developed ketonuria within the first day of life. Uncoupling protein-1
mRNA in brown adipose tissue was reduced 60%. These mice share
some features of both glycerol kinase deficiency and hereditary
fructose intolerance, suggesting the phenotype may be due to the
combined effects of the loss of a gluconeogenic substrate, the osmotic
effects of glycerol, and the metabolic effects of the accumulation of a
phosphorylated metabolite.
Mammalian cells contain two glycerol phosphate dehydrogenase
enzymes. The NADH-dependent cytosolic enzyme (EC 1.1.1.8)
catalyzes the conversion of dihydroxyacetone phosphate
(DHAP)1 to glycerol
phosphate. This reaction is reversible, with a strong preference under
physiologic conditions for the production of glycerol phosphate. In the
mouse, this enzyme is encoded by Gdc1. An embryonic form of
the enzyme is encoded by Gdc2 but has not been found in
liver or kidney during gestation, although it persists in brain of the
neonate for several weeks (1) and in epididymal white fat for at least
5 days after birth (2). The FAD-dependent mitochondrial GPD
(EC 1.1.99.5) is encoded by a single gene, Gdm1, on
chromosome 2 (3) and is located on the outer surface of the
mitochondrial inner membrane. mGPD catalyzes the irreversible conversion of glycerol phosphate to DHAP, with transfer of electrons from bound FAD through ubiquinone to complex III of the electron transport chain. These two enzymes form the glycerol phosphate shuttle,
which cycles glycerol phosphate and DHAP to oxidize NADH formed in the
cytosol. A spontaneous mutation in Gdc1 in the inbred strain
BALB/cHeA results in an altered mRNA size and the loss of cGPD
enzyme activity (4). cGPD-deficient BALB/cHeA animals are viable and
fertile, although they demonstrate some evidence of an inhibition of
glycolysis at glyceraldehyde phosphate dehydrogenase in skeletal muscle
(5). We (40) and others (6) have produced knockout mice-deficient in
mGPD. The mGPD knockout mice have decreased adiposity and reduced
fertility and viability (40). Alterations are seen in liver metabolites
(decreased ATP and increased glycerol phosphate) only in nursing pups.
The glycerol phosphate dehydrogenases also form an important link
between carbohydrate and lipid metabolism. DHAP derived from
carbohydrates is converted by cGPD to glycerol phosphate, which can be
acylated to form phospholipids or triglycerides. An alternate pathway,
acylation of DHAP directly by dihydroxyacetone phosphate
acyltransferase (7), apparently compensates for the loss of cGPD in
mice of the BALB/cHeA strain, which are able to grow and produce normal
fat depots, even when placed on a fat free (triglyceride/glycerol free)
diet at weaning (5). In tissues that contain glycerol kinase,
metabolism of glycerol phosphate by mGPD should allow the conversion of
glycerol to DHAP without perturbation of the cytosolic NAD/NADH ratio.
DHAP is then available for either the oxidative pathway or
gluconeogenesis. Under conditions of glycerol loading, mGPD appears to
play a major role in glycerol oxidation (8-10). In the absence of
mGPD, glycerol phosphate metabolism is handled solely by cGPD, with
remarkably few metabolic abnormalities seen primarily in nursing pups
(40). In the absence of both the cytosolic and mitochondrial enzymes,
the link between carbohydrate and fat metabolism is extremely limited.
Triglyceride glycerol can be produced from carbohydrate indirectly
through the DHAP-acyltransferase pathway, however, glycerol and
glycerol phosphate derived from fat are not able to enter the oxidative
or gluconeogenic pathways.
We report here that mice deficient in both GPDs are unable to use
glycerol, developing hypoglycemia, ketonuria, glyceroluria, elevated
liver glycerol phosphate, profound growth failure, and death within a
week of birth.
Animals--
BALB/cHeA (cGPD-null) and C57BL/6J mice were
obtained from The Jackson Laboratory, Bar Harbor, ME. The mGPD
knockouts were constructed as previously described (40). Briefly, exons
5 and 6 were replaced by a Neomycin marker gene. Targeted RW4 cells (129X1/SvJ origin) were injected into blastocysts derived from the
C57BL/6J strain, and resulting chimeras were bred to C57BL/6J animals
to produce heterozygous animals. Homozygous F2 or F3 mGPD knockout
animals were bred with the BALB/cHeA strain to produce animals
heterozygous for both the cytosolic and mitochondrial GPD deficiencies.
These animals were intercrossed and typed. To simplify further
breeding, animals homozygous for the cGPD deficiency and heterozygous
for the mGPD knockout (co/omo/+) were
intercrossed to produce animals deficient in both enzymes (co/omo/o). Additional crosses were performed
to produce a c+/+m+/+ stock.
Genotyping--
Tail DNA was prepared (11), and mGPD was typed
using a three primer PCR (40). Typing for cGPD status was performed
initially by screening for the BALB/c allele of the linked marker
D15Mit242, which gives a 104-bp product from both the 129X1/SvJ and
C57BL/6J alleles of the mGPD knockout mouse line, and a 90-bp product
from the BALB/cHeA allele. Mouse MapPair primers were purchased from Research Genetics, Inc. (Huntsville, AL) and used according to methods
provided. PCR products were separated on 3.5% Metaphor-agarose (FMC
BioProducts, Rockland, ME) according to the manufacturer's instructions. cGPD status was confirmed in selected cases by enzyme assay on liver homogenates using standard spectrophotometric methods (12).
Diet--
Mice were routinely fed a standard chow diet (Teklad
8604, 4% fat, Harlan Teklad, Madison, WI). Mice on the C57BL/6J
background were maintained on breeder chow (Teklad 8626, 10% fat).
Expression Analysis--
Total RNA was prepared from the
interscapular brown adipose tissue using TRI-Reagent (Molecular
Research Center, Inc., Cincinnati, OH) according to directions
provided. Real-time quantitative reverse transcription polymerase chain
reaction was performed using the ABI Prism 7700 sequence detection
system and UCP1 primers as described previously (13).
Biochemical Assays--
Enzymes and metabolites were measured as
previously described (5), except that, due to the small size of pups
and organs, mouse pups were killed by beheading, and tissues for
metabolites (50-100 mg) were removed as rapidly as possible and
directly homogenized in 6% perchloric acid at 4 °C. Free fatty
acids were measured using the Free fatty acids, Half-micro test kit
(Roche Molecular Biochemicals, Indianapolis, IN), according to
directions. Glucose was measured using a One-Touch Profile instrument
(Lifescan, Milpitas, CA). Urine was collected by postmortem aspiration
from the bladder using an insulin syringe. Acetoacetic acid was
assessed semi-quantitatively on 6-10 µl of urine using Ketostix
(Bayer Corp.) according to directions. Pooled urine was used for assay
of electrolyte content by the University of Wisconsin Hospitals
Clinical Laboratory, and individual urine samples were assayed for
organic acids by gas chromatography/mass spectroscopy (GC/MS) at the
University of Wisconsin Biochemical Genetics Laboratory (Madison, WI).
Protein assays were performed according to the Lowry method (14) on the
sonicated pellets derived from perchloric acid precipitation. Separate
determinations were made of the protein content of whole liver tissue
for calculations of molarity.
Mice Lacking Both GPDs Exhibit Growth Failure and Die Prior to
Weaning--
In a cross between the mGPD knockout strain
(c+/+mo/o) and the BALB/cHeA strain
(co/om+/+), 0 of 56 F2 animals were deficient
in both the cytosolic and mitochondrial GPDs
(co/omo/o) and GPD mo/o mice were
underrepresented (6/56, expected 14/56, p < 0.05). In
subsequent crosses between animals lacking cGPD and heterozygous for
the mGPD knockout (co/omo/+ × co/omo/+), 0 of 61 weaned animals were mGPD
knockouts (expected = 15, p < 0.001). Examination
of young litters revealed discrepancies in size within a few days of
birth (Fig. 1A). Runted
animals, usually weighing less than 2 g at 5 days of age
(versus a normal weight of 3.5 g), were confirmed to be
homozygous for the mGPD deletion and lacking cGPD activity. These
animals were noted to have little subcutaneous fat, although most were
active and well fed for several days. By 5-7 days of age most of these
GPD-deficient animals stopped feeding and were lethargic. Fig.
1A may be misleading with regard to weight gain, because
most of the GPD-deficient animals died within 2-7 days of birth, and
those surviving longer tended to be larger than average. Similar
results were found in co/omo/o pups derived
from co/+mo/o intercrosses (data not shown).
Analysis of pups within the first 4 days showed that mice deficient in
both GPDs were born in expected numbers (23/86) and were of normal size
at birth. Weights on day 1 did not differ significantly: the mean
weight ± S.D. of co/omo/o pups was
1.39 ± 0.11 g (n = 9) and that of
co/omo/+ was 1.43 ± 0.18 g
(n = 16).
Histology--
Consistent with the gross appearance of the
GPD-deficient pups, histology showed little, if any, subcutaneous fat
in 5- to 7-day-old co/omo/o animals. Brown
adipose tissue appeared normal. Other tissues showed no difference from
healthy siblings, with the exception of decreased extramedullary
hematopoesis in the livers of the co/omo/o animals.
GPD-deficient Animals Are Hypoglycemic--
Blood glucose values
rose with age and weight in all pups tested, except those lacking both
GPDs (Fig. 1, B and C). Data from c+/+m+/+ pups did not differ from
co/omo/+ and co/om+/+
animals and are not shown. There were several sporadic high blood glucose values in the co/omo/o pups, but most
had low glucose values that remained near neonatal levels. Some
variation in glucose values may be expected, because we did not attempt
to control for feeding.
GPD-deficient Animals Are Ketotic--
Urine acetoacetate was
positive in all co/omo/o pups tested
(n = 19) and ranged from trace-small (5-15 mg/dl) to
moderate-large (40-80 mg/dl). Ketones were negative in all other pups
tested, including 29 cGPD-null, 5 mGPD knockout, 4 wild type (BALB × C57BL/6J) controls, and 3 C57BL/6J controls. Both acetoacetate and
3-hydroxybutyrate were identified by GC/MS in urine samples from two
co/omo/o animals but not in urine from two
control animals (co/omo/+ and
co/om+/+).
GPD-deficient Animals Have Low Liver Adenylates and Elevated
Glycerol Phosphate and Free Fatty Acids--
ATP values were reduced
40% in the co/omo/o pups versus
mGPD-positive littermates, and ADP and AMP values were also
proportionately reduced, with total adenylates reduced by 31-39%
(Table I). ATP values were increased in
cGPD-null pups (co/om+/+) versus
wild controls (c+/+m+/+). Liver glycerol
phosphate values were doubled in co/omo/+
versus co/om+/+ and were elevated
16- to 32-fold in co/omo/o littermates. Despite
the elevated liver glycerol phosphate, plasma free fatty acids were
slightly elevated in the GPD-deficient animals, although the range of
free fatty acid values in the GPD-deficient animals (0.38-1.35
mM) slightly exceeded that of littermate controls (0.4-1.0
mM) in both high and low values.
GPD-deficient Animals Develop an Elevated Plasma Glycerol and Lose
Glycerol in Their Urine--
Plasma and urine glycerol were elevated
in all pups lacking both GPDs, and these values increased with age
(Fig. 2). Values of plasma glycerol in
normal animals averaged 0.8 mM, ranged from 0.2 to 1.3 mM, and did not change appreciably with age, whereas values
in co/omo/o pups ranged from 10.8 to 74 mM (Fig. 2A). These values are similar to those
reported in the glycerol kinase knockout mouse, which also has growth
failure and dies in 3-4 days (15). In urine, no glycerol value in the
co/om(+or o)/+ controls exceeded 0.4 mM, whereas values in the co/omo/o
pups ranged from a low of 57 mM on day 1 to a high of 478 mM (4% glycerol) on day 4 (Fig. 2B). On GC/MS,
glycerol was the most prominent peak in urine from
co/omo/o animals, but a minor peak in controls
(data not shown). Although elevated urine glycolate was reported in
glycerol kinase knockout animals (15), no glycolate was detected in
our samples.
GPD-deficient Animals Have Low UCP1 mRNA Levels--
mGPD has
been proposed to play a role in thermogenesis (16, 17), however, we
were unable to find a significant alteration in thermogenesis in mGPD
knockout animals, which have normal UCP1 mRNA levels
(40). We evaluated UCP1 mRNA in brown adipose tissue of
3- to 5-day-old pups from co/omo/+
intercrosses. Pups lacking both GPDs had UCP1 mRNA
levels that were reduced 60% compared with littermate controls.
UCP1 mRNA levels (arbitrary units ± S.E., number
in parentheses) were as follows: GPD
co/om+/+, 6698 Urinalysis and Hematology--
Table
II shows the results of urine
electrolytes performed on pooled urine from 1- to 3-day-old
co/omo/o pups and two groups of controls.
Results are consistent with sodium loss due to ketoacidosis, an osmotic
diuresis, or proximal tubular dysfunction. Analysis of these data is
limited by the lack of information on milk intake, urine output, and
other electrolyte losses. The electrolyte composition of mouse milk
itself is uncertain, because very different values have been reported
using microelectrode methods (Na+ 76.9, K+
32.5, Cl Gluconeogenesis and the Importance of Glycerol--
Glyceride
glycerol is an especially important gluconeogenic precursor in the
neonatal mouse, because 80% of calories from mouse milk are derived
from fat, 16-17% from protein, and only 2-5% from lactose (20,
22-24). Thus total calories available from dietary glycerol (~4%)
equal calories from lactose. Because fatty acids cannot be converted to
glucose and dietary protein is needed for growth, glyceride glycerol
could provide 15-25% of glucose production in the fed state, and
possibly more when fasting.
In GPD-deficient pups, liver glycerol kinase produces glycerol
phosphate, which cannot be metabolized to dihydroxyacetone phosphate.
Other substrates are therefore required for gluconeogenesis, however,
gluconeogenesis from glycerol is energetically less expensive than that
from amino acids and citric acid cycle intermediates. For example,
production of one glucose molecule from pyruvate consumes 4 ATP, 2 GTP,
and 2 mitochondrial NADH. Oxidation of each NADH could otherwise
produce 3 ATPs, so the net ATP loss is 12 ATP per glucose molecule
created. Synthesis of one glucose molecule from glycerol requires 2 ATP
and produces either 2 FADH2 or 2 cytosolic NADH (equivalent
to 2-3 ATP each), for a net gain of 2-4 ATP. In a rapidly growing
animal, increased gluconeogenesis from amino acids and citric acid
cycle intermediates may therefore deplete both the substrates and the
energy required for growth (Fig. 3).
Mechanism and Consequences of Ketogenesis--
Control of
ketogenesis occurs primarily through the inhibition of carnitine
palmitoyl transferase I by malonyl CoA (25) and the inhibition of
mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase by
succinyl-CoA-dependent autosuccinylation (26, 27). In the
presence of an elevated glucagon/insulin ratio, both inhibitions are
released. The neonatal rodent, with its low sugar and high fat diet,
has elevated glucagon and low levels of insulin to maintain gluconeogenesis and fatty acid oxidation (28). In the combined GPD
deficiency, loss of a major gluconeogenic substrate might be expected
to exaggerate this response (Fig. 3). In addition, low levels of
adenylates and depletion of Pi could limit coupled respiration, slowing fatty acid oxidation in the liver of the GPD-deficient pups, and therefore increasing the proportion of acetyl-CoA used for the production of ketones (Fig. 3). The loss of
ketones in the urine is coupled with an obligatory loss of cations in
the urine, generally Na+, K+, and
NH Effect of Glycerol Phosphate Accumulation--
In the absence of
both GPDs, there is apparently no mechanism to prevent the accumulation
of glycerol phosphate in tissues that contain glycerol kinase. In liver
and kidney, glycerol phosphate is therefore produced, but cannot be
converted into dihydroxyacetone phosphate and cannot be used for
gluconeogenesis or aerobic metabolism. Although this glycerol phosphate
could be used for lipid synthesis, lipid does not accumulate in the
liver of GPD-deficient mice, most likely due to metabolic conditions
favoring fatty acid degradation. It is not clear what limits the
accumulation of glycerol phosphate, however, glycerol phosphate is
known to be a potent product inhibitor of glycerol kinase (29).
Glycerol phosphate levels found in the liver of GPD-deficient animals
(Table I) correspond to concentrations of ~3-5 mM
(calculated from the protein content of mouse pup liver of 160 mg/g of
wet weight). Plasma glycerol increases up to 74 mM (normal
0.2-1.3 mM), whereas urine glycerol eventually exceeds 400 mM (normal
Accumulation of liver glycerol phosphate may at least partially explain
the low ATP and total adenylates seen in the GPD-deficient animals. In
the rat, an intraperitoneal bolus of glycerol (9) or liver perfusion
with ethanol (31) lead to the accumulation of liver glycerol phosphate
to 4-14 mM. This glycerol phosphate increase is
accompanied by a fall in free Pi (9, 31) and a fall in
liver ATP and total adenylates. The loss of adenylates is due to
activation of AMP deaminase by a fall in Pi levels, and it
is accompanied by a rise in the production of breakdown products of
adenylate (31). A chronic accumulation of phosphorylated metabolites
may be more deleterious. In hereditary fructose intolerance, the
accumulation of fructose 1-phosphate also leads to a fall in ATP and
Pi in liver and kidney. Adenylate degradation leads to the
release of Mg2+ into the blood and the elevation of serum
urate. In affected individuals, severe or prolonged exposure to
fructose can lead to renal proximal tubular dysfunction or renal
failure and to liver dysfunction, fibrosis, or liver failure. Even with
minimal fructose exposure, stunted growth is common (32). These
findings are consistent with the results in our GPD-deficient animals, however, low ATP levels are also seen in the livers of mGPD knockout pups (40), without a large increase in glycerol phosphate. Additional factors may be involved, such as an increased ATP consumption in
gluconeogenesis from amino acids.
Elevated glycerol phosphate may also be directly linked to the
hypoglycemia observed in the GPD-deficient pups. Boluses of glycerol or
glycerol phosphate interfere with gluconeogenesis from alanine,
especially in malnourished animals (33). The mechanism is not clear. Of
the gluconeogenic enzymes, fructose-1,6-bisphosphatase is the most
susceptible to inhibition by glycerol phosphate, with an
I50 of 20 mM in the rat (34) and 60 mM in the Chinese hamster (35). At the level of glycerol
phosphate seen in our animals, a maximum inhibition of 10-30% might
be expected for this enzyme. Gluconeogenesis may also be affected by
the low ATP levels in the face of the increased ATP demand for
gluconeogenesis from amino acids.
Comparison of the GPD-deficient Mouse and the Glycerol Kinase-null
Mouse--
Recently a glycerol kinase knockout mouse was reported (15)
that shows a number of similarities to the GPD-deficient mouse. The
glycerol kinase-deficient mouse has a normal birth weight but fails to
gain weight, has plasma glycerol levels of ~40 mM, elevated free fatty acid levels, and dies by 3-4 days of age. Unlike
the GPD-deficient mice, the glycerol kinase knockout mice are not
ketotic and have normal blood glucose. These differences could be
attributed to the inability of the glycerol kinase-null mouse to
accumulate glycerol phosphate, however, hypoglycemia and episodic
ketoacidosis have been reported in patients with isolated glycerol
kinase deficiency, although the human diet, including human milk,
contains a higher percentage of both sugars and protein than does mouse
milk. The lack of hypoglycemia and ketosis in the glycerol kinase
knockout is likely attributable to the autonomous glucocorticoid
secretion seen in this mouse, which should increase protein catabolism
and provide the amino acid substrate needed for adequate
gluconeogenesis (albeit at the expense of muscle and other tissues).
Glucocorticoid treatment has been used to treat hypoglycemic ketosis of
childhood (36) and bovine ketosis (37), two conditions in which a
paucity of gluconeogenic substrates has been postulated as the cause of
symptomatic hypoglycemia and ketoacidosis. Autonomous glucocorticoid
secretion has not been reported in human glycerol kinase deficiency,
and caution should therefore be used in interpretation of the findings in the glycerol kinase-null mouse.
Conclusions--
Mice deficient in both the cytosolic and
mitochondrial GPDs usually die within the first week of life.
Hypoglycemia, ketonuria, and alterations in liver glycerol phosphate
levels and ATP occur during a time period in which the mice are still
active and well fed (as evidenced by stomachs full of milk), and all
likely contribute to the eventual decompensation. Death may result from
a combination of hypothermia, ketoacidosis, and resultant electrolyte
disturbances; protein deficiency due to the obligatory use of amino
acids for gluconeogenesis; glycerol-induced osmotic diuresis and
dehydration; and organ failure secondary to ATP and Pi depletion.
Although glycerol kinase and the GPDs are essential for normal glycerol
metabolism, compensatory genetic factors clearly play a role in the
phenotype resulting from their loss. In the combined GPD-deficient mice
death occurs within 1-2 days on an inbred BALB/cHeA background,2 whereas pups
often survive 5-7 days on the genetically mixed background. In humans,
the phenotype of individuals with isolated glycerol kinase deficiency
ranges from episodic ketoacidosis, hypoglycemia, and seizures to
completely being asymptomatic. This spectrum can be seen within a
single family and is therefore likely affected by other genetic or
environmental factors (38, 39).
Studies of mice lacking both GPDs, on various genetic backgrounds,
could help to define the compensatory mechanisms responsible for the
maintenance of normoglycemia.
We thank Heather Drought for excellent
technical assistance, Paul Lyne and Dr. Jon Wolff of the
University of Wisconsin Biochemical Genetics Laboratory for
the GC/MS analysis, Dr. Avery J. Cooley for histologic analysis, and
Dr. Leonard A. Fahien for critical reading of the manuscript.
*
This work was supported by National Institutes of Health
(NIH) Grant DK 28348 and the Oscar C. Rennebohm Foundation (to
M. J. M. and L. J. B.) and by NIH Grant HD008431 (to L. P. K. and R. A. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Pediatrics,
University of Wisconsin, 3550 MSC, 1300 University Ave., Madison, WI
53706. Tel.: 608-263-7668; Fax: 608-262-9300; E-mail: ljbrown2@facstaff.wisc.edu.
Published, JBC Papers in Press, July 1, 2002, DOI 10.1074/jbc.M202409200
2
R. A. Koza, unpublished data.
The abbreviations used are:
DHAP, dihydroxyacetone phosphate;
FAD, flavine-adenine dinucleotide;
GC/MS, gas chromatography/mass spectroscopy;
(c- or m)GPD, (cytosolic or
mitochondrial) glycerol phosphate dehydrogenase;
Pi, inorganic phosphate;
UCP1, uncoupling protein-1.
Lethal Hypoglycemic Ketosis and Glyceroluria in Mice Lacking
Both the Mitochondrial and the Cytosolic Glycerol Phosphate
Dehydrogenases*
§,,
Department of Pediatrics, University of
Wisconsin, Madison, Wisconsin 53706 and the ¶ Pennington
Biomedical Research Center, Baton Rouge, Louisiana 70808
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Growth failure and hypoglycemia in
GPD-deficient mice. A, weight gain in GPD
co/omo/o pups (black triangles,
n = 4-9 animals per point) is compared with
co/omo/+ and co/om+/+
littermates (open circles, n = 5-23 animals
per point). Means ± S.E. are shown. Comparisons were performed by
the Student's t test. *, p < 0.05; **,
p < 0.001. B, GPD-deficient mice showed
only a limited increase in blood glucose with age. Symbols as in
A. n = 6-18 animals per point, except
n = 3 for the 5-day co/omo/o.
C, weights of individual animals are shown to demonstrate
the correlation between glucose and weight in control littermates
(R2 = 0.77), and the lack of correlation in the
GPD-deficient pups (R2 = 0.02), in which the
glucose generally remained below 60 mg/dl. Symbols are as in
A.
Liver metabolites and plasma free fatty acids
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Fig. 2.
Urine and plasma glycerol. GPD
co/omo/o pups are show by solid
triangles, whereas mGPD-positive littermates
(co/omo/+ and co/om+/+)
are shown by open circles. Urine glycerol in GPD-deficient
pups ranged from 57 to 478 mM, whereas values in all other
mice tested were less than 0.4 mM and did not increase with
age. Plasma glycerol values in the co/omo/o
animals ranged from 10.8 to 74 mM, whereas those of the
co/omo/+ and co/om+/+
littermates were 0.2-1.3 mM.
± 372 (4);
co/omo/+, 6188 ± 433 (13);
co/omo/o, 2483 ± 335 (8) (,
p < 0.01; , p < 0.001 versus co/omo/o). This reduction may
reflect the general poor condition of the GPD-deficient animals or the
hormonal consequences of hypoglycemia. Chronic hypoglycemia would be
expected to elevate corticosterone, which has been shown to inhibit
UCP1 transcription (18, 19). Low UCP1 levels could impair
the ability of the GPD-deficient pups to maintain body temperature.
41.6) (20) and atomic absorption methods
(Na+ 23.5, K+ 50.4) (21). The GPD-deficient
animals were also found to have elevated plasma proteins (114 ± 3% of littermate controls, n = 11 controls, 7 GPD-deficient, p < 0.02) and a tendency toward elevated hematocrit levels (109%, n = 3 each),
consistent with the presence of severe dehydration in these
animals.
Urine electrolytes
20 °C. Samples were pooled after
animals were genotyped. Number of samples pooled (age): 10 co/omo/o (1-3 days), 8 co/om(o or +)/+
(1-3 days), 2 co/om+/+ (6 days). Na+,
K+, Cl are in mM, osmolarity is in
mosM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 3.
Proposed metabolic alterations in combined
GPD deficiency. 1, glycerol phosphate levels rise due
to the loss of the GPDs. Phosphate derived from ATP is trapped as
glycerol phosphate (G3P), and glucose production is
decreased. Glycerol levels rise along with G3P levels, and glycerol is
excreted in the urine. 2, Pi is depleted,
resulting in degradation of adenylates. 3, low blood glucose
levels result in increased consumption of amino acids for
gluconeogenesis. 4, oxaloacetate (OAA) and malate
are diverted from the citric acid cycle toward gluconeogenesis,
depleting cycle intermediates while increasing ATP demands.
5, reductions in ADP and Pi slow oxidative
phosphorylation, resulting in elevated mitochondrial NADH.
6, low insulin and high glucocorticoid and epinephrine
levels result in hydrolysis and decreased synthesis of triglycerides
(TG), increasing free fatty acids (FFA).
7, low insulin and high glucagon levels lead to inhibition
of acetyl CoA-carboxylase, releasing the malonyl-CoA inhibition of
carnitine palmitoyl transferase I, and accelerating fatty acid
degradation to acetyl CoA. 8, elevated acetyl-CoA, combined
with decreased flux through the citric acid cycle, increases production
of acetoacetyl-CoA. Lowering of the succinyl-CoA concentration relieves
inhibition of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA
synthase, increasing the production of acetoacetate and
3-hydroxybutyrate (3-OHB). Loss of these compounds in the
urine increases loss of NH 
0.4 mM). Urine glycerol is
almost completely reabsorbed by the kidney up to plasma levels of 1 mM (30). Above this level the urine glycerol concentration
depends on the plasma glycerol concentration and the degree of water
reabsorption from the urine. High urine glycerol levels could lead to
an osmotic diuresis, salt loss, and dehydration, consistent with our findings.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Kozak, L. P.,
and Jensen, J. T.
(1974)
J. Biol. Chem.
249,
7775-7781 2.
Cook, J. R.,
and Kozak, L. P.
(1982)
Dev. Biol.
92,
440-448[CrossRef][Medline]
[Order article via Infotrieve]
3.
Koza, R. A.,
Kozak, U. C.,
Brown, L. J.,
Leiter, E. H.,
MacDonald, M. J.,
and Kozak, L. P.
(1996)
Arch. Biochem. Biophys.
336,
97-104[CrossRef][Medline]
[Order article via Infotrieve]
4.
Prochazka, M.,
Kozak, U. C.,
and Kozak, L. P.
(1989)
J. Biol. Chem.
264,
4679-4683 5.
MacDonald, M. J.,
and Marshall, L. K.
(2000)
Arch. Biochem. Biophys.
384,
143-153[CrossRef][Medline]
[Order article via Infotrieve]
6.
Eto, K.,
Tsubamoto, Y.,
Terauchi, Y.,
Sugiyama, T.,
Kishimoto, T.,
Takahashi, N.,
Yamauchi, N.,
Kubota, N.,
Murayama, S.,
Aizawa, T.,
Akanuma, Y.,
Aizawa, S.,
Kasai, H.,
Yazaki, Y.,
and Kadowaki, T.
(1999)
Science
283,
981-985 7.
Hajra, A. K.
(1968)
J. Biol. Chem.
243,
3458-3465 8.
Berry, M. N.,
Kun, E.,
and Werner, H. V.
(1973)
Eur. J. Biochem.
33,
407-417[Medline]
[Order article via Infotrieve]
9.
Burch, H. B.,
Lowry, O. H.,
Meinhardt, L.,
Max, P., Jr.,
and Chyu, K.-J.
(1970)
J. Biol. Chem.
245,
2092-2102 10.
Williamson, D. H.,
Veloso, D.,
Ellington, E. V.,
and Krebs, H. A.
(1969)
Biochem. J.
114,
575-584[Medline]
[Order article via Infotrieve]
11.
Laird, P. W.,
Zijderveld, A.,
Linders, K.,
Rudnicki, M. A.,
Jaenisch, R.,
and Berns, A.
(1991)
Nucleic Acids Res.
19,
4293 12.
Passonneau, J. V.,
and Lowry, O. H.
(1993)
Enzymatic Analysis: A Practical Guide
, p. 262, Humana Press, Totowa, NJ
13.
Koza, R. A.,
Hohmann, S. M.,
Guerra, C.,
Rossmeisl, M.,
and Kozak, L. P.
(2000)
J. Biol. Chem.
275,
34486-34492 14.
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 15.
Huq, A. H. M. M.,
Lovell, R. S., Ou, C.-N.,
Beaudet, A. L.,
and Craigen, W. J.
(1997)
Human Mol. Genet.
6,
1803-1809 16.
Lee, Y.-P.,
and Lardy, H. A.
(1965)
J. Biol. Chem.
240,
1427-1436 17.
Lardy, H., Su, C.-Y.,
Kneer, N.,
and Wielgus, S.
(1989)
in
Hormones, Thermogenesis, and Obesity
(Lardy, H.
, and Stratman, F., eds)
, pp. 415-426, Elsevier, New York
18.
Moriscot, A.,
Rabelo, R.,
and Bianco, A. C.
(1993)
Am. J. Physiol.
265,
E81-E87 19.
Soumano, K.,
Desbiens, S.,
Rabelo, R.,
Bakopanos, E.,
Camirand, A.,
and Silva, J. E.
(2000)
Mol. Cell. Endocrinol.
165,
7-15[CrossRef][Medline]
[Order article via Infotrieve]
20.
Jost, B.,
Vilotte, J.-L.,
Duluc, I.,
Rodeau, J.-L.,
and Freund, J.-N.
(1999)
Nature Biotech.
17,
160-164[CrossRef][Medline]
[Order article via Infotrieve]
21.
Delzer, P. R.,
and Meyer, R. A.
(1983)
Calcif. Tissue Int.
35,
750-754[CrossRef][Medline]
[Order article via Infotrieve]
22.
Knight, C. H.,
Maltz, E.,
and Docherty, A. H.
(1986)
Comp. Biochem. Physiol.
84A,
127-133[CrossRef]
23.
Teter, B. B.,
Sampugna, J.,
and Keeney, M.
(1992)
Lipids
27,
912-916[CrossRef][Medline]
[Order article via Infotrieve]
24.
Nagasawa, H.,
Naito, T.,
and Kataoka, K.
(1989)
Proc. Soc. Exp. Biol. Med.
191,
78-81[Abstract]
25.
McGarry, J. D.,
and Foster, D. W.
(1980)
Ann. Rev. Biochem.
49,
395-420[CrossRef][Medline]
[Order article via Infotrieve]
26.
Lowe, D. M.,
and Tubbs, P. K.
(1985)
Biochem. J.
232,
37-42[Medline]
[Order article via Infotrieve]
27.
Hegardt, F. G.
(1999)
Biochem. J.
338,
569-582
28.
Girard, J.,
Ferré, P.,
Pégorier, J. P.,
and Duée, P.-H.
(1992)
Physiol. Rev.
72,
507-562 29.
Robinson, J.,
and Newsholme, E. A.
(1969)
Biochem. J.
112,
455-464[Medline]
[Order article via Infotrieve]
30.
Lin, E. C. C.
(1977)
Ann. Rev. Biochem.
46,
765-795[CrossRef][Medline]
[Order article via Infotrieve]
31.
Masson, S.,
Desmoulin, F.,
Sciaky, M.,
and Cozzone, P.
(1993)
Biochemistry
32,
1025-1031[CrossRef][Medline]
[Order article via Infotrieve]
32.
Gitzelmann, R.,
Steinmann, B.,
and Van den Berghe, G.
(1995)
in
The Metabolic and Molecular Bases of Inherited Disease
(Scriver, C. R.
, Beaudet, A. L.
, Sly, W. S.
, and Valle, D., eds)
, pp. 905-934, McGraw Hill, Inc., New York
33.
Wapnir, R. A.,
and Stiel, L.
(1985)
Biochem. Med.
33,
141-148[CrossRef][Medline]
[Order article via Infotrieve]
34.
Wapnir, R. A.,
Lifshitz, F.,
Sekaran, C.,
Teichberg, S.,
and Moak, S. A.
(1982)
Metabolism
31,
1057-1064[CrossRef][Medline]
[Order article via Infotrieve]
35.
Wapnir, R. A.,
and Stiel, L.
(1987)
Biochem. Med. Metab. Biol.
37,
228-234[CrossRef][Medline]
[Order article via Infotrieve]
36.
Haymond, M. W.,
and Pagliara, A. S.
(1983)
Clin. Endocrinol. Metab.
12,
447-462[CrossRef][Medline]
[Order article via Infotrieve]
37.
Ballard, F. J.,
Hanson, R. W.,
and Kronfeld, D. S.
(1968)
Biochem. Biophys. Res. Commun.
30,
100-104[CrossRef][Medline]
[Order article via Infotrieve]
38.
Sjarif, D. R.,
Sinke, R. J.,
Duran, M.,
Beemer, F. A.,
Kleijer, W. J.,
Ploos van Amstel, J. K.,
and Poll-The, B. T.
(1998)
J. Med. Genet.
35,
650-656[Abstract]
39.
Walker, A. P.,
Muscatelli, F.,
Stafford, A. N.,
Chelly, J.,
Dahl, N.,
Blomquist, H. K.,
Delanghe, J.,
Willems, P. J.,
Steinmann, B.,
and Monaco, A. P.
(1996)
Am. J. Hum. Genet.
58,
1205-1211[Medline]
[Order article via Infotrieve]
40.
Brown, L. J.,
Koza, R. A.,
Everett, C.,
Reitman, M. L.,
Marshall, L.,
Fahien, L. A.,
Kozak, L. P.,
and MacDonald, M. J.
(2002)
J. Biol. Chem.
277,
32892-32898
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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