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. 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. dihydroxyacetone phosphate flavine-adenine dinucleotide gas chromatography/mass spectroscopy (cytosolic or mitochondrial) glycerol phosphate dehydrogenase inorganic phosphate uncoupling protein-1 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 (1Kozak L.P. Jensen J.T. J. Biol. Chem. 1974; 249: 7775-7781Abstract Full Text PDF PubMed Google Scholar) and in epididymal white fat for at least 5 days after birth (2Cook J.R. Kozak L.P. Dev. Biol. 1982; 92: 440-448Crossref PubMed Scopus (26) Google Scholar). The FAD-dependent mitochondrial GPD (EC 1.1.99.5) is encoded by a single gene, Gdm1, on chromosome 2 (3Koza R.A. Kozak U.C. Brown L.J. Leiter E.H. MacDonald M.J. Kozak L.P. Arch. Biochem. Biophys. 1996; 336: 97-104Crossref PubMed Scopus (58) Google Scholar) 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 (4Prochazka M. Kozak U.C. Kozak L.P. J. Biol. Chem. 1989; 264: 4679-4683Abstract Full Text PDF PubMed Google Scholar). 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 (5MacDonald M.J. Marshall L.K. Arch. Biochem. Biophys. 2000; 384: 143-153Crossref PubMed Scopus (59) Google Scholar). We (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and others (6Eto 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. Kadowaki T. Science. 1999; 283: 981-985Crossref PubMed Scopus (397) Google Scholar) have produced knockout mice-deficient in mGPD. The mGPD knockout mice have decreased adiposity and reduced fertility and viability (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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 (7Hajra A.K. J. Biol. Chem. 1968; 243: 3458-3465Abstract Full Text PDF PubMed Google Scholar), 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 (5MacDonald M.J. Marshall L.K. Arch. Biochem. Biophys. 2000; 384: 143-153Crossref PubMed Scopus (59) Google Scholar). 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 (8Berry M.N. Kun E. Werner H.V. Eur. J. Biochem. 1973; 33: 407-417Crossref PubMed Scopus (81) Google Scholar, 9Burch H.B. Lowry O.H. Meinhardt L. Max P., Jr. Chyu K.-J. J. Biol. Chem. 1970; 245: 2092-2102Abstract Full Text PDF PubMed Google Scholar, 10Williamson D.H. Veloso D. Ellington E.V. Krebs H.A. Biochem. J. 1969; 114: 575-584Crossref PubMed Scopus (65) Google Scholar). In the absence of mGPD, glycerol phosphate metabolism is handled solely by cGPD, with remarkably few metabolic abnormalities seen primarily in nursing pups (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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. 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 (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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. Tail DNA was prepared (11Laird P.W. Zijderveld A. Linders K. Rudnicki M.A. Jaenisch R. Berns A. Nucleic Acids Res. 1991; 19: 4293Crossref PubMed Scopus (1303) Google Scholar), and mGPD was typed using a three primer PCR (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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 (12Passonneau J.V. Lowry O.H. Enzymatic Analysis: A Practical Guide. Humana Press, Totowa, NJ1993: 262Google Scholar). 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). 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 (13Koza R.A. Hohmann S.M. Guerra C. Rossmeisl M. Kozak L.P. J. Biol. Chem. 2000; 275: 34486-34492Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Enzymes and metabolites were measured as previously described (5MacDonald M.J. Marshall L.K. Arch. Biochem. Biophys. 2000; 384: 143-153Crossref PubMed Scopus (59) Google Scholar), 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 (14Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) 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. 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 to be than results were found in pups derived from not of pups within the first 4 days that mice deficient in both GPDs were in expected and were of normal size at birth. on day not the weight of pups was g = and that of was g = with the of the GPD-deficient subcutaneous fat in to animals. Brown adipose tissue tissues from with the of decreased in the of the with age and weight in pups except lacking both GPDs (Fig. and from c+/+m+/+ pups not from and and are not were several in the but most had that levels. in may be because not to for Urine was in pups = and from to were in pups 5 mGPD 4 × and C57BL/6J and were by in urine samples from two animals but not in urine from two animals (co/omo/+ and ATP were reduced in the pups and and AMP were also with reduced by ATP were increased in pups Liver glycerol phosphate were in and were elevated to in the elevated liver glycerol phosphate, plasma free fatty acids were elevated in the GPD-deficient animals, although the of free fatty acid in the GPD-deficient animals exceeded that of in both and metabolites and plasma free fatty metabolites are as of and free fatty acids as are and the of samples is in of are the was performed and a inbred of animals. were 2–7 days and were from of GPD animals, with the of animals of the GPD which were maintained as a derived from the glycerol phosphate. were performed using the are p p p p p in a Liver metabolites are as of and free fatty acids as are and the of samples is in of are the was performed and a inbred of animals. were 2–7 days and were from of GPD animals, with the of animals of the GPD which were maintained as a derived from the glycerol phosphate. were performed using the are Plasma and urine glycerol were elevated in pups lacking both and these increased with age (Fig. of plasma glycerol in normal animals mm, from to mm, and not with whereas in pups from to (Fig. These are to in the glycerol kinase knockout mouse, which also has growth and in days A.L. PubMed Scopus Google Scholar). In glycerol in the exceeded mm, whereas in the from a of on day to a of (4% on day 4 (Fig. glycerol was the most in urine from animals, but a in not elevated urine was in glycerol kinase knockout animals A.L. PubMed Scopus Google Scholar), was in mGPD has been to play a role in H.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, N. S. H. and Scholar), however, were unable to a in in mGPD knockout animals, which have normal UCP1 mRNA levels (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We UCP1 mRNA in brown adipose tissue of to pups from lacking both GPDs had UCP1 mRNA levels that were reduced with mRNA levels in were as GPD (4Prochazka M. Kozak U.C. Kozak L.P. J. Biol. Chem. 1989; 264: 4679-4683Abstract Full Text PDF PubMed Google (13Koza R.A. Hohmann S.M. Guerra C. Rossmeisl M. Kozak L.P. J. Biol. Chem. 2000; 275: 34486-34492Abstract Full Text Full Text PDF PubMed Scopus (64) Google (8Berry M.N. Kun E. Werner H.V. Eur. J. Biochem. 1973; 33: 407-417Crossref PubMed Scopus (81) Google Scholar) < p < This may the of the GPD-deficient animals or the of be expected to which has been to transcription A. R. J. Google Scholar, K. S. R. E. A. 2000; PubMed Scopus Google Scholar). UCP1 levels the of the GPD-deficient pups to body shows the results of urine performed on urine from to pups and two of are with loss due to an osmotic or of these is by the of on urine and electrolyte The electrolyte of mouse is because have been using methods 1999; PubMed Scopus Google Scholar) and methods R.A. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar) and a in liver ATP and The loss of is due to of AMP by a in and it is by a in the production of products of S. M. PubMed Scopus Google Scholar). A accumulation of phosphorylated metabolites may be In hereditary fructose intolerance, the accumulation of fructose also to a in ATP and in liver and to the of into the and the of In or to fructose can to or and to liver or liver with fructose growth is R. A.L. D. The and Molecular of Inc., Scholar). These are with the results in GPD-deficient animals, however, ATP levels are also seen in the of mGPD knockout pups (40Brown L.J. Koza R.A. Everett C. Reitman M.L. Marshall L. Fahien L.A. Kozak L.P. MacDonald M.J. J. Biol. Chem. 2002; 277: 32892-32898Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), without a increase in glycerol phosphate. 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PubMed Scopus Google Scholar) that shows a of to the GPD-deficient The glycerol mouse has a normal birth weight but to has plasma glycerol levels of mm, elevated free fatty acid and by days of the GPD-deficient mice, the glycerol kinase knockout mice are not and have normal These be to the of the glycerol mouse to glycerol phosphate, however, and have been in with glycerol kinase although the a of both and protein than mouse The of and in the glycerol kinase knockout is to the seen in this mouse, which should increase protein and the acid for at the of muscle and has been used to of Full Text PDF PubMed Scopus Google Scholar) and Biochem. Biophys. Res. 1968; PubMed Scopus Google Scholar), two conditions in which a of gluconeogenic has been as the of and has not been in glycerol kinase and should be used in of the in the glycerol Mice deficient in both the cytosolic and mitochondrial GPDs usually within the first week of life. ketonuria, and in liver glycerol phosphate levels and ATP during a in which the mice are active and well fed by of and to the may from a of and electrolyte protein deficiency due to the use of acids for osmotic and and to ATP and
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