X-linked glycerol kinase deficiency in the mouse leads to growth retardation, altered fat metabolism, autonomous glucocorticoid secretion and neonatal death
X-linked glycerol kinase deficiency in the mouse leads to growth retardation, altered fat metabolism, autonomous glucocorticoid secretion and neonatal deathA. H. M. Mahbubul Huq1, Rhonda S. Lovell1, Ching-Nan Ou2, Arthur L. Beaudet1,2 and William J. Craigen1,2,*
Departments of 1Molecular and Human Genetics and 2Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Received May 8, 1997;Revised and Accepted July 15, 1997
Glycerol kinase is an X chromosome-encoded enzyme involved in the metabolism of endogenous and dietary glycerolipids. The physiological significance of its activity in mammals is not well understood. Glycerol kinase deficiency in humans occurs as an isolated enzyme deficiency or as part of a contiguous gene deletion syndrome in variable association with Duchenne muscular dystrophy and adrenal hypoplasia congenita. Isolated glycerol kinase deficiency has an inconstant phenotype, ranging from asymptomatic hyperglycerolemia to a severe metabolic disorder with growth and psychomotor retardation. Although intragenic mutations were reported recently, the pathophysiological basis for the phenotypic variability remains unknown. To understand better the physiological significance of glycerol kinase and the pathophysiology of its deficiency, we generated glycerol kinase-deficient mice by gene targeting. Mutant male mice appear normal at birth, but exhibit postnatal growth retardation, altered fat metabolism with profound hyperglycerolemia and elevated free fatty acids, autonomous glucocorticoid synthesis and death by 3-4 days of age. Heterozygous females are healthy and biochemically normal. The biochemical features observed in glycerol kinase-deficient mice provide the basis for further investigations into the pathogenesis of the human disorder.
Glycerol kinase (ATP:glycerol-3-phosphotransferase, EC 2.7.1.30; Gyk in the mouse, GK in humans) is an X chromosome-encoded enzyme involved in the metabolism of endogenous and dietary glycerolipids (1 ). GK deficiency in humans occurs as an isolated enzyme deficiency or as part of a contiguous gene deletion syndrome in variable association with Duchenne muscular dystrophy and/or adrenal hypoplasia congenita. Isolated GK deficiency has an inconstant phenotype, ranging from asymptomatic hyperglycerolemia to a severe metabolic disorder with growth and psychomotor retardation (2 ). Glycerol kinase is one of a group of structurally related kinases of intermediary metabolism that have been termed `ambiquitous', based on the ability to bind the mitochondrial outer membrane reversibly (3 ). Mitochondrial binding is mediated by a voltage-dependent anion channel (also known as mitochondrial porin) that provides the kinase with direct access to ATP derived from oxidative phosphorylation (4 ). Although enzymatic differences in the bound kinases are recognized (5 -7 ), the physiological significance of mitochondrial binding remains unknown.
The isolation of cDNAs encoding glycerol kinase recently has been reported from human and mouse sources (8 -11 ). The amino acid sequence is highly conserved in evolution, with ~50% sequence identity between the proteins of bacteria and mammals (8 ). Glycerol kinase transcripts undergo alternative splicing in the brain (8 ,12 ), although the functional significance of this is unknown. In addition, there are autosomal glycerol kinase-like sequences that potentially encode proteins and whose expression is restricted to testes (12 ; A.H.M.M.Huq and W.J.Craigen, unpublished results). Since these sequences lack introns, they probably arose by retrotransposition, similarly to the X-linked genes phosphoglycerate kinase (Pgk) and the E1[alpha] subunit of pyruvate dehydrogenase, presumably to provide enzyme function during spermatogenesis, when many X-linked genes undergo X-inactivation (13 ).
Although three intragenic mutations at the GK locus were reported recently in humans with isolated glycerol kinase deficiency (14 ), the physiological basis for the phenotypic variability remains unknown. To begin to understand the physiological role of glycerol kinase and the pathophysiology of GK deficiency, we have generated Gyk-deficient mice by gene targeting. Here we report the initial characterization of these animals. In contrast to GK-deficient humans, the affected male mice have a uniformly lethal outcome and demonstrate perturbations in glucose and lipid metabolism, while heterozygous female mice are phenotypically normal. We suggest that abnormal energy homeostasis secondary to perturbed fat metabolism is the cause of death in affected males.
A mouse Gyk cDNA (8 ) was used to isolate a region of the mouse Gyk genomic locus corresponding to the human GK exons 13-17 (12 ) (Fig. 1 A). Exons corresponding to those previously characterized in the human gene were identified by DNA sequencing. DNA fragments from the region were used to construct a replacement-type targeting vector for homologous recombination. Successful targeting was predicted to delete exons 13, 14 and a portion of 15, potentially leading to an in-frame deletion of 101 amino acids via aberrant splicing from exon 12 to exon 16, with a corresponding reduction in the size of the mRNA. Eight correctly targeted embryonic stem (ES) cell clones were identified by Southern blotting, and one clone was used for microinjection into blastocysts. Heterozygous female offspring from founder chimeras were identified (Fig. 1 B) and bred to C57BL/6J males, and the offspring examined for an abnormal phenotype. Southern blot analysis of mutant males demonstrated that only a single integration at the Gyk locus was present (data not shown). Assay of the liver, kidney and brown fat of mutant males demonstrated <5% Gyk activity (Fig. 1 C). Likewise, RNA blot analysis of liver tissue showed that the mutant male mice had no detectable Gyk mRNA (Fig. 2 ), confirming that a null mutation was generated.
Heterozygous female mice are fertile and have no obvious phenotype, including normal blood glycerol and glucose concentrations (data not shown). Hemizygous mutant male mice are born in expected numbers and are indistinguishable from wild-type littermates in terms of weight and appearance at birth. However, the mutant mice are growth retarded by day 2 and die at day 3 or 4 (Fig. 3 A), despite apparently normal suckling (milk is invariably present in the stomachs of affected mice). No long-term survivors were identified in litters allowed to go to weaning age, reflecting the uniform, X-linked lethality of the mutation. By detailed necropsy, the mice have no obvious structural abnormalities, although histologically the liver shows mild fatty infiltration (not shown). The adrenal glands appear histologically normal. Hypoglycemia is an inconsistent feature observed in GK deficiency in humans (2 ), whereas, to date, hyperglycerolemia is a constant feature in both symptomatic and asymptomatic humans. All mutant male mice have profound hyperglycerolemia, but no hypoglycemia (Fig. 3 B and C). The lack of hypoglycemia in mutant mice may be due to enhanced activation of gluconeogenic enzymes (see below).
The degree of hyperglycerolemia (>80-fold elevation) reflects the large flux of glycerol through triglyceride and glycerolipid metabolism by 2-3 days of age. Although the glycerol 3-phosphate backbone of glycerolipids is believed to derive primarily from glycolysis (15 ), free fatty acid (FFA) levels were measured to determine whether the availability of glycerol 3-phosphate may be a limiting factor for the reesterification of fatty acids. Plasma FFA levels are increased ~3-fold in affected males (1.5 +- 0.16 mmol/l versus 0.5 +- 0.06 mmol/l, P = 0.01; Fig. 3 D). Organic acid analysis of affected male urine by gas chromatography/mass spectroscopy (GC/MS) demonstrated, in addition to glyceroluria, abnormally elevated long chain fatty acids (e.g. palmitic and stearic acid; data not shown), consistent with the observations made in plasma. These results suggest an important role for Gyk in FFA re-esterification, although enhanced FFA synthesis cannot be excluded. In addition, high levels of glycolic acid were detected by GC/MS, suggesting that glycerol is diverted into glycolate/oxalate production.
A consequence of FFA elevation is, in part, activation of gluconeogenesis and ketogenesis (16 ,17 ). In addition, increased concentrations of FFA are known to stimulate insulin release from pancreatic beta cells (18 ,19 ). By GC/MS, no urine ketone bodies were detected, and we were unable to measure insulin levels accurately due to the small plasma volumes available.
The Gyk cDNA encodes a protein nearly identical (99% amino acid identity) to a rat liver protein termed ATP-stimulated glucocorticoid receptor translocation promoter (ASTP) that is reported to promote the translocation of activated glucocorticoid receptors to the cell nucleus (8 ,20 ), suggesting that Gyk may have two distinct functions. If this were the case, both hyperglycerolemia and an end-organ resistance to the action of glucocorticoids would be anticipated. After birth, the expression of various genes necessary for gluconeogenesis is induced by a complex interaction of a few physiologic regulators, including glucocorticoids, insulin, glucagon and cAMP (21 ). To test for the concomitant loss of ASTP activity, we examined the RNA expression of several of these genes. Surprisingly, the prototypic glucocorticoid-inducible enzyme tyrosine aminotransferase (22 ) is induced >8-fold in affected males above the postnatal induction seen in control mice (Fig. 2 ). The gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) is also induced ~2.7-fold above control animals, indicating that glucocorticoid induction is intact and inconsistent with Gyk functioning as an activator of glucocorticoid receptor nuclear translocation. Because of this excessive expression of glucocorticoid-inducible enzymes, corticosterone levels were then determined in mutant males and wild-type male littermates. The plasma corticosterone level is significantly increased to ~3-fold that seen in control animals (191.1 +- 44.7 ng/ml versus 63.3 +- 5.9 ng/ml; P = 0.007, Fig. 3 E).
A rise in glucocorticoids may be expected in response to the stress associated with illness, or with end-organ resistance to hormone action. For example, end-organ resistance to glucocorticoid action due to disruption of the glucocorticoid receptor gene is associated with an ~2.5-fold elevation in corticosterone and a 15-fold elevation in adrenocorticotrophic hormone (ACTH) (23 ). Levels of ACTH were determined in affected males and unaffected male littermates. Gyk-deficient mice exhibit low-normal ACTH levels (172 +- 72 pg/ml versus 229 +- 74 pg/ml, P = 0.059; Fig. 3 F), not consistent with end-organ resistance but rather indicative of autonomous glucocorticoid secretion. It has been shown both in vitro and in vivo that FFA elevations lead to increased corticosterone secretion, while glycerol infusion has no similar in vivo effect (24 ). In cultured rat adrenocortical cells, FFAs directly stimulate corticosterone release by a cAMP-independent mechanism (ACTH-directed secretion is a cAMP-dependent activity), and stimulation requires oxidation of fatty acids (25 ). In contrast, in vivo studies show stimulation of both ACTH and corticosterone by FFAs (24 ). Our results suggest that a similar ACTH-independent mechanism can be seen in vivo.
Here we report the generation and characterization of a Gyk-deficient mouse strain. The data presented provide evidence for a disruption in normal glucose and lipid homeostasis in Gyk-deficient mice. Mutant mice fail to grow, exhibit hyperglycerolemia and glyceroluria, and have increased levels of circulating FFA and glucocorticoids without a concomitant increase in ACTH. Since a single line of mice was studied, it is formally possible that the observed severe phenotype reflects a co-existing mutation in the ES cell clone used to generate the line, but given that the biochemical phenotype is concordant with Gyk deficiency, the phenotype is uniform on a mixed strain background through five generations and inheritance of the phenotype follows an X-linked pattern, this possibility would appear unlikely.
The observation that the blood levels of both glycerol and FFA are increased suggests there is incomplete fatty acid re-esterification due to a lack of glycerol 3-phosphate. Since glycerolipid synthesis can occur either from glycerol via glycerol kinase, from dihydroxyacetone phosphate (DHAP) via glycerol 3-phosphate dehydrogenase or, to a lesser extent, directly from acyl dihydroxyacetone phosphate (26 ), these observations suggest that glycerol phosphorylation plays a significant role in FFA esterification.
The induction of gluconeogenic gene expression probably leads to enhanced hepatic glucose production in response to increased circulating FFA and glucocorticoids [and perhaps low levels of glycerol 3-phosphate (27 )] (Fig. 4 ). The observed induction is well in excess of that seen after birth in control animals, and indicates that there is no clear defect in glucocorticoid inducibility, a finding not readily consistent with a deficiency of ASTP activity if both activities are encoded by the same gene. We have demonstrated previously that the mouse Gyk cDNA encodes glycerol kinase activity (8 ), providing further evidence that Gyk is not simply the ASTP activity without glycerol kinase activity.
A phage genomic library from the 129/SvJ mouse strain (Stratagene) was screened using a Gyk cDNA probe (8 ). A single phage clone containing a 9 kb XbaI fragment was characterized by restriction digestion and DNA sequencing. This established the intron-exon boundaries for the mouse sequences corresponding to the human exons 13-17 (12 ). A 0.9 kb XbaI-EcoRI genomic fragment containing a portion of exon 15 and all of exon 16 was subcloned into the XbaI-EcoRI sites of pBluescript KS(-) (Stratagene). A 5.1 kb XbaI-HindIII fragment was blunt-ended with the Klenow fragment and subcloned into the HincII site of pBluescript KS(-). A 1.4 kb Pol2shortneobpa (36 ) HindIII cassette was introduced into the HindIIIsite of the plasmid polylinker between the two genomic fragments, and the HSV-tk gene inserted into an adjoining NotI site.
The targeting plasmid was linearized at the XhoI site in the plasmid polylinker between the 5.1 kb genomic fragment and the vector backbone, and electroporated into ES cells (AB2.1) under standard conditions (37 ). After 24 h, G418 (0.2 mg/ml) and FIAU (0.2 [mu]M) were added and selection carried out for 6-10 days. Following digestion with HindIII,the colonies were screened by the `mini-Southern' procedure (38 ) using as a probe a 0.6 kb genomic XbaIfragment containing exon 17. A total of 300 colonies were screened and eight homologous recombination events detected (not shown).
Chimeric mice were generated by injection of the Gyk-disrupted ES cells into C57BL/6J blastocysts and implantation into pseudopregnant C57BL/6J foster mothers (37 ). Chimeras identified by the presence of an agouti coat color were test-mated with C57BL/6J males and females. Agouti offspring were tested for the mutant Gyk gene by Southern blotting. The heterozygous female mice were bred to C57BL/6J males to generate hemizygous male mice lacking the functional Gyk gene.
Total RNA was extracted using guanidinium isothiocyanate from mouse liver as described (39 ). RNA was fractionated on a 1% agarose-formaldehyde gel, and transferred to Hybond N+ membranes (Amersham). Hybridization was carried out at 65oC in 5* SSPE, 0.5% SDS, 5* Denhardt's solution, and the membrane washed twice at 37oC in 0.1* SSC, 0.1% SDS for 15 min and exposed for 6-12 h at -70oC. RNA loading and integrity were assessed by ethidium staining of 18S and 28S RNA and by hybridization with the human [beta]-actin cDNA. Signals were quantified using a Betagen Betascope 603 (Intelligenetics), and the values expressed as a ratio with the [beta]-actin signal. Unaffected male littermates served as controls, and the experiments were carried out on two litters to confirm the observations. Statistical analysis was performed with StatView software using the unpaired t-test for determination of significance.
Mouse liver, kidney and brown fat tissues obtained from 3-day-old wild-type and mutant mice were homogenized and the total cellular extract used for measuring Gyk activity. Gyk activity was determined by measuring the conversion of [3H]glycerol to [3H]glycerol 3-phosphate using a filter binding assay (40 ). The protein content was measured by BCA protein assay reagent (Pierce, Rockford, IL). Statistical analysis was performed using the unpaired t-test for determination of significance.
Mothering mice were maintained on a normal postpartum diet. Weight gain in pups was assessed daily. Plasma samples were collected using heparinized collection tubes (Chase Scientific Glass, Norcross, GA). Because of the small sample sizes available from the pups, all biochemical determinations had to be scaled appropriately. Glucose and triglyceride levels were analyzed using an EKTACHEM 950 automatic chemistry analyzer (Johnson and Johnson Clinical Diagnostics, Rochester, NY). Glycerol was measured using a modified Sigma semi-automatic triglyceride reagent kit (Sigma procedure No. 320-UV) by eliminating the saponification step and adapting it to a COBAS-BIO centrifugal analyzer (Roche Diagnostic System, Montclair, NJ). FFAs were measured using a diagnostic kit (Free fatty acids, Half-micro test, Boehringer Mannheim Biochemicals, Indianapolis, IN). Corticosterone and ACTH levels were determined by a modified radioimmunoassay method using commercially available diagnostic kits (ICN Biochemicals, Costa Mesa, CA).
The authors thank Isabel Lorenzo for technical assistance, Dr Gretchen Darlington for cDNA probes, Dr M. Finegold for expert opinion in reviewing the liver histology, the Baylor Biochemical Genetics lab for organic acid analysis, and Drs D. Nelson, J. Belmont and E. R. B. McCabe for reviewing the manuscript. This project was supported by a Basil O'Connor award from the March of Dimes (W.J.C.) and the Mental retardation Research Center at Baylor College of Medicine (NICHD 2PO30-HD24064) (W.J.C.).
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*To whom correspondence should be addressed. Tel: +1 713 798 8305; Fax: +1 713 7985386; Email: wcraigen@bcm.tmc.edu
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