1 The Jackson Laboratory, Bar Harbor, Maine
2 Group on Molecular and Cell Biology of Lipids, Canadian Institutes of Health Research, Edmonton, Alberta, Canada
3 Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
4 Linco Research, St. Charles, Missouri
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The most common forms of human type 2 diabetes are polygenic in origin with contributions from both sides of the pedigree. In mice, obesity-associated diabetes (diabesity) also has a complex polygenic basis. Admixing genomes of unrelated strains of inbred mice provides insight as to how a complex disease such as type 2 diabetes can become more common as genetic heterogeneity increases in an outbreeding human population. As strains of laboratory mice become increasingly more inbred, genomes are selected that are capable of sustaining reproductive fitness in the face of increased mutational load. As mutations occur that affect the expression or function of multiple quantitative trait loci (QTLs), continued reproductive fitness requires co-adaptation within the overall genetic architecture for sets of QTLs that must interact to maintain metabolic homeostasis. Variation among inbred strains in physiologic parameters associated with glucose homeostasis (e.g., nonfasting plasma glucose or insulin concentrations) reflects strain-dependent systemic adaptations to strain-unique genetic polymorphisms at multiple QTLs. To model how a genetic outcross can destabilize such metabolic adaptations, we previously crossed two unrelated inbred strains, nonobese nondiabetic (NON) and New Zealand obese (NZO). The NON/Lt strain carries QTLs conferring latent type 2 diabetes susceptibility, whereas NZO/HlLt mice carry numerous QTLs contributing to male diabesity in a threshold fashion (1). Whereas diabesity spontaneously developed in 0% of NON/Lt males and in 50% of NZO/HlLt males, the two sets of parental QTLs synergized in a way that 90100% of F1 males exhibited the diabesity trait (1). Genetic analysis subsequently identified seven QTLs contributed by the NZO parent and two QTLs contributed by the NON parent (1,2).
The hyperglycemia, hyperinsulinemia, and hyperlipidemia that developed in diabetic F1 males were effectively suppressed by chronic treatment with the thiazolidinedione (TZD) compound, rosiglitazone, incorporated into the diet. However, these antidiabetic effects were accompanied by marked exacerbation of an underlying hepatosteatosis (3). Because choline deficiency is known to produce hepatosteatosis in mice (4,5), we tested the lipid composition of milk from untreated F1 lactating dams; this analysis showed a marked deficiency in all classes of phosphatidylcholine (S. Watkins, Lipomics, Sacramento, CA, personal communication). This result suggested to us that the genetic milieu of the F1 mice combined QTLs that adversely affect the two phosphatidylcholine biosynthetic pathways in liver and that this effect is influenced by treatment with TZDs.
In mouse liver, the phosphatidylethanolamine methyltransferase (PEMT) pathway contributes 30% and the CDP-choline pathway, including choline kinase and CTP-cholinephosphate cytidylyltransferase, contributes
70% of the total hepatic phosphatidylcholine. Male mice with a targeted mutation in the gene encoding PEMT are sensitized to high-fat/high-cholesterolinduced hepatosteatosis, with hepatocytes showing a marked defect in export of VLDLs containing triglycerides and apolipoprotein B100 (68). Similarly, genetic disruption of the gene encoding cholinephosphate cytidylyltransferase-
decreased secretion of both HDLs and VLDLs (9). Hence, polygene combinations producing impaired hepatic phosphatidylcholine biosynthesis would be predicted to predispose to hepatosteatosis risk in response to pharmacologic agents that can increase triglyceride transport into the liver. The activity of the PEMT reaction seems to be governed largely by the supply of the substrates phosphatidylethanolamine and S-adenosylmethionine (10). Because the phosphatidylethanolamine is derived from diacylglycerol via the CDP-ethanolamine pathway, the amount of diacylglycerol could influence the activity of PEMT. The regulation of phosphatidylcholine biosynthesis via the CDP-choline pathway in liver and other tissues and cells focuses on the cholinephosphate cytidylyltransferase reaction, which is considered to be the rate-limiting step (10). The active form of cholinephosphate cytidylyltransferase is found associated with membranes, and the inactive form is in a soluble form largely located in the nucleus. There is a rapid movement of the enzyme between these two locations that is regulated by the level of phosphatidylcholine and diacylglycerol in the membranes. If the level of phosphatidylcholine is high, there is release of active cholinephosphate cytidylyltransferase from the membrane into the inactive reservoir. If phosphatidylcholine levels are low and/or diacylglycerol levels are high in membranes, cholinephosphate cytidylyltransferase is translocated to membranes where it is activated. The activity of cholinephosphate cytidylyltransferase is also regulated at the level of transcription best documented in studies on the cell cycle (11). The activity of choline kinase is generally not considered to regulate the biosynthesis of phosphatidylcholine (10).
Our objective is to use mouse models of diabesity to understand how natural allelic variation at multiple genetic loci can affect responsiveness to drug-mediated therapy. The polygenic obesity/diabesity syndromes in mice are particularly relevant because the "metabolic syndrome" associated with most human diabetogenic obesities also has a complex genetic basis. To elucidate the pharmacogenetic basis for the adverse responses of the F1 liver to TZD exposure, we compared the hepatic activities of the phosphatidylcholine biosynthetic enzymes in both parental strains with the F1 and in a panel of genetically characterized recombinant congenic strains (RCSs). The RCSs were produced by backcrossing the F1 for two cycles onto the parental NON/Lt background, with selection for different subsets of diabesity QTLs sorting into each RCS (12). For example, RCS10 contains the greatest number of diabesity contributions from both parental backgrounds (on chromosomes 1, 4, 5, 11, 12, and 18) and develops the highest (F1-like) diabetes frequency (90100%) of any of the RCSs. In contrast, RCS1, selected to contain the diabetogenic QTLs mainly on chromosomes 1 and 15, showed only 50% diabetes frequency. RCS2, a strain lacking most of the NZO-derived diabesity contribution on chromosome 15 while otherwise carrying the same genetic composition as RCS1, showed an even lower (25%) diabesity frequency (12). In the present study, we show that hybridizing the two parental genomes produced an unusual lowering of hepatic phosphatidylcholine biosynthetic enzyme activities in control F1 males and that rosiglitazone treatment further reduced these enzymatic activities. Screening a selected panel of RCSs showed that only one, NONcNZO8, developing a diabesity frequency of 75%, exhibited the extreme hepatosteatotic response to rosiglitazone when compared with F1 males. The results demonstrate how susceptibility genes from both parental genomes contribute additively or codominantly to a complex disease and further demonstrate the utility of the RCS panel for identifying genetic architectures that might predict positive versus adverse responses to drug therapy.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinical markers.
Nonfasting plasma glucose sampled at 8 A.M. was measured by a Glucose 2 analyzer (Beckman Instruments, Fullerton, CA). Plasma insulin in the same samples was measured by Luminex bead assay (Linco).
Histology studies.
Mice were necropsied at 22 weeks of age (14 weeks on the diet). Livers were fixed in Bouins solution, and sections were stained by a standard protocol with periodic acid-Schiff reagent. Percentage of fatty area in liver was determined morphometrically using the MetaMorph Offline program (Universal Imaging, Downingtown, PA).
Cell fractionation and enzyme assays.
Livers (1 g each) from nonfasted males at 22 weeks necropsy were cut into small pieces, then homogenized in a glass-Teflon homogenizer in 5 vol of 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, 1.0 mmol/l phenylmethylsulfonylfluoride, 1.0 mmol/l EDTA, 2.0 mmol/l dithiothreitol, and 0.25 mol/l sucrose. Homogenates were centrifuged at 600g for 5 min to remove unbroken cells. This supernatant ("total homogenate") was further centrifuged at 12,000g for 10 min to obtain a crude mitochondria pellet. The mitochondrial-free supernatant fraction was transferred to a new tube and centrifuged at 100,000g for 1 h to obtain the postmicrosomal fraction (cytosol) from the supernatant and microsomes from the pellet. Protein concentration was determined (Total Protein; Sigma, St. Louis, MO) and normalized before performing enzyme assays and Western blot analyses. All assays were performed at saturating concentrations of substrate. PEMT activity in 100-µg protein aliquots of total homogenate was assayed as described previously (15) at pH 9.2 with 2 mmol/l phosphatidyldimethylethanolamine (Avanti Polar Lipids, Alabaster, AL) as substrate and 200 µmol/l S-adenosyl-L-methionine (Sigma) and S-adenosyl-L-[Me-3H]methionine (2 µCi/reaction) (code TRK865; Amersham Pharmacia, Piscataway, NJ) as cofactor. The specific activity was estimated by the nanomoles of [3H]phosphatidylcholine formed per minute per milligram of protein. Choline kinase activity was determined in 400-µg protein aliquots of 100,000g supernatant as described previously (16) at pH 8.7, with 10 mmol/l MgCl2, 10 mmol/l ATP, 0.25 mmol/l choline chloride (Sigma), and [Me-3H]choline chloride (2 µCi/reaction) (code TRK 593; Amersham Pharmacia). Specific activity was estimated by the nanomoles of [3H]phosphocholine formed per minute per milligram of protein. Cholinephosphate cytidylyltransferasespecific activity was measured in total homogenates with [3H]phosphocholine (15 µCi/reaction, 78 µCi/µmol) as substrate as described previously (7). Cholinephosphate cytidylyltransferasespecific activity was estimated by nanomoles of [3H]CDP-choline formed per minute per milligram of protein.
Western blot analysis.
An aliquot (100 µg protein) of the total homogenate was resolved by SDS-PAGE (10% gel), then transferred on to a polyvinylidine fluoride membrane and incubated with rabbit antibody against PEMT2 (1:1,000) (15). For choline kinase analysis, a 100-µg protein aliquot of the 100,000g high-speed supernatant (postmicrosomal fraction) of liver homogenates was incubated with affinity-purified subunit-specific rabbit antisera (100x dilution for anticholine kinase- and 200x dilution for anticholine kinase-ß) (17), a gift of Dr. K. Ishidate (Tokyo Medical University, Tokyo). Horseradish peroxidaseconjugated anti-rabbit IgG and enhanced chemiluminescence were used for subsequent signal detection procedures (Roche, Indianapolis, IN).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
After 14 weeks of rosiglitazone treatment, NON males on control diet maintained normoglycemia. Rosiglitazone treatment did not produce an increase in body weight, although plasma insulin was significantly reduced (Table 1). However, with the exception of RCS1, most RCS strains, similar to the F1 males, responded to rosiglitazone-stimulated body weight gain (Table 1). As previously reported, RCS2 showed resistance to diabesity development, whereas RCS1, RCS8, and RCS10 all exhibited diabesity syndromes of differential severity as manifested by increases in both plasma glucose and insulin concentrations (Table 1). Also, in all three, the insulin-sensitizing action of rosiglitazone was reflected by significant decreases in plasma insulin. Nevertheless, differences in antihyperglycemic effects were noted. Drug-mediated plasma glucose decreases were significant in both RCS1 and RCS10 mice, but the decline in treated RCS8 mice did not achieve significance (Table 1). Moreover, plasma insulin was much more variable in control RCS8 males. Although rosiglitazone treatment reduced mean plasma insulin concentrations and reduced variability in the measurement, the difference again failed to achieve statistical significance. This suggested that the diabesity syndrome developing in RCS8 was different from that in RCS1 and RCS10.
|
Only RCS8 responds to rosiglitazone-mediated inhibition of phosphatidylcholine biosynthetic enzymes.
Data in Fig. 4 show that, among the panel of RCSs tested, only RCS8 responded to rosiglitazone with the same marked inhibitory effect on hepatic phosphatidylcholine biosynthetic enzymes shown for the rosiglitazone-treated F1 males described in Fig. 2A. In comparison with NON, all RCSs tested showed lower hepatic PEMT and choline kinase activities. After rosiglitazone treatment, a significant increase in choline kinase activity was observed in rosiglitazone-treated NON males. All of the control dietfed RCSs, regardless of their constitutive sensitivity to diabesity development on this diet, exhibited the reduced PEMT and choline kinase activities, but not cholinephosphate cytidylyltransferase activity characteristic of the diabesity-developing F1. Yet only RCS8 showed the further depression in activity levels of all three enzymes in response to chronic rosiglitazone treatment (Fig. 4). This close correlation between the histologic documentation of heightened sensitivity of RCS8 to drug-exacerbated lipidosis and the RCS8-specific loss of activity for enzymes in both pathways of phosphatidylcholine biosynthesis clearly provide a mechanism for this RCS-specific drug sensitivity. It should be noted that a considerably higher concentration of troglitazone in the diet (2 g/kg diet) produced the same degree of hepatosteatosis in RCS8 males as did the rosiglitazone feeding at 50 mg/kg diet reported here. The same troglitazone diet had no effect on parental NON/Lt males (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Troglitazone, the first TZD marketed for treatment of type 2 diabetes, was withdrawn because of extreme hepatotoxicity in a very small percentage of patients taking the compound (19). Drugs of the later generation TZDs, of which rosiglitazone is an example, have been shown to reduce hepatic triglycerides in most patients rather than promote triglyceride accumulation and steatosis (20). Thus, availability of the panel of genetically characterized RCS models of diabesity with differential TZD sensitivities is useful for understanding the genetic basis for susceptibility to adverse drug responses. Such pharmacogenetic knowledge might distinguish that small percentage of patients who should not take this class of compounds. RCS8 and RCS10 exhibit comparable levels of obesity and basal hepatic lipidosis with a control diet, but only RCS8 experiences a drug-exacerbated steatosis as extreme as originally described in the F1. Interestingly, RCS10 rather than RCS8 is more comparable with the F1 in terms of the numbers of shared diabesity QTLs and, consequently, diabetes frequency (90100%) and disease severity defined by nonfasting plasma glucose levels. RCS8 develops a lower frequency of diabesity with hyperglycemia not establishing consistently before 20 weeks of age (12). Thus, it was surprising that the moderate shifts in the glycemic status of RCS8 males before 20 weeks were not as responsive to rosiglitazone treatment as were the more hyperglycemic RCS10 males. Because the NON/Lt strain is completely resistant to rosiglitazone-exacerbated steatosis, the steatosis-promoting gene or genes must be NZO in origin. We surmise that this NZO-derived locus or loci promoting the phenotype of drug-exacerbated steatosis may be distinct from the set of NZO-contributed diabesity QTLs that synergize with certain NON-derived QTLs to increase frequency of diabetic hyperglycemia development in the F1 male. This inference is reinforced by the fact that the three steatosis-resistant RCSs collectively carry the spectrum of known diabesity QTLs. RCS8 has been fixed for known diabesity QTLs on chromosomes 1 and 11 (from NZO) and chromosomes 4 and 18 (from NON). The most notable genetic difference distinguishing RCS8 from the others is its NZO origin of chromosome 16 in its entirety. The cholinephosphate cytidylyltransferase- subunit is encoded on this chromosome. The active form of cholinephosphate cytidylyltransferase activity resides on cellular membranes. Subcellular fractionation showed that rosiglitazone-mediated reduction in cholinephosphate cytidylyltransferase activity in total homogenate was primarily in a 100,000g cytosol and to a lesser extent, in the crude mitochondrial fraction (data not shown). We have sequenced cholinephosphate cytidylyltransferase-
cDNA from NON and NZO and have not found coding polymorphisms. This is consistent with our finding of no differences in cholinephosphate cytidylyltransferase enzymatic activities in NON, NZO, and F1 liver fractions from males fed a control diet. However, cholinephosphate cytidylyltransferase-
differs from the cholinephosphate cytidylyltransferase-ß subunit (X chromosome linked and NON derived in all of the RCSs) in having a nuclear localization domain. Possibly, the rosiglitazone-specific suppression of cholinephosphate cytidylyltransferase total activity in all subcellular fractions in F1 liver may entail a drug-specific interaction with cholinephosphate cytidylyltransferase-
. Genetic disruption of cholinephosphate cytidylyltransferase-
produces a hepatic phenotype similar to that observed in rosiglitazone-treated F1; e.g., 85% reduction of total cholinephosphate cytidylyltransferase activity, reduced phosphatidylcholine levels, and triglyceride accumulation (9). In RCS8, not only is cholinephosphate cytidylyltransferase activity reduced, but also this partial loss of cholinephosphate cytidylyltransferase activity is accompanied by significant losses in both choline kinase and PEMT biosynthetic functions. In the case of the reductions in choline kinase enzymatic activity, our unpublished data suggest that, in the specific post-translational environment in the F1, coexpression of polymorphic alleles from NON and NZO reduce overall choline kinase catalysis by impairing
/ß heterodimer formation or, alternatively, that the NZO/NON heterodimeric combinations are less thermodynamically favored compared with within-strain subunit dimeric combinations. At least four other loci on chromosome 16 associated with hepatic triglyceride metabolism and steatosis represent potential candidates. Among these are lipid defect 1 (lpd, 45 cM) and lipase, member H (Liph, 14.8 cM) (21,22). A QTL contributing to plasma glucose independent of body weight and mapping to chromosome 16 has also been reported in TallyHo mice, another diabesity model (23). A triglyceride hydrolase-encoding gene also maps to chromosome 16 (24). Finally, high expression of SOCS-1 (suppressor of cytokine signaling), another chromosome 16-encoded gene product has recently been shown in mice to contribute to heightened sensitivity to hepatic steatosis (25).
Although the PEMT pathway only accounts for 30% of phosphatidylcholine biosynthesis in liver, it is required for secretion of lipoproteins (8), so that PEMT inhibition significantly impairs the bulk incorporation of triglycerides into lipoprotein particles for export (26). In livers of mice with a genetically disrupted Pemt allele fed a standard diet, a diet deficient in choline, or a choline-enriched diet, there was hepatic steatosis and significantly higher frequency of apoptotic cells compared with wild-type controls (5). Similarly, reductions in multiple classes of plasma lipoproteins were also observed in mice with a disrupted cholinephosphate cytidylyltransferase- gene (9). Thus, the remarkable finding that rosiglitazone treatment inhibited all three phosphatidylcholine biosynthetic enzymes assayed easily explains our previous analysis of the lipid metabolome, showing that rosiglitazone treatment removed plasma triglyceride into the liver where it accumulated rather than being metabolized in a normal manner (3). This rosiglitazone-mediated inhibition of enzymes in both arms of the phosphatidylcholine biosynthetic pathway would very likely impair lipoprotein assembly and export and hence promote the observed hepatic triglyceride accumulation. Moreover, regardless of the mechanism for the decrease in cholinephosphate cytidylyltransferase and PEMT activity as a result of rosiglitazone treatment, less diacylglycerol would be used in the biosynthesis of phosphatidylcholine. Under such conditions, the diacylglycerol would be acylated to triacylglycerol and this could lead to the observed hepatic steatosis.
It remains to be established whether this unusual pharmacogenetic effect is mediated via direct effects of peroxisome proliferatoractivated receptor- (PPAR
) at the hepatocyte or indirectly by primary drug effects on another tissue such as white fat. We previously were unable to detect upregulation in PPAR
1 and PPAR
2 gene transcription in livers of rosiglitazone-treated F1 males undergoing severe steatosis (3). We used computational means (TRANSFAC database for transcription factor binding sites [http://www.biobase.de/pages/products/customer.html]) to search for upstream consensus PPAR
binding sites in the gene sequences for PEMT, choline kinase, and cholinephosphate cytidylyltransferase in ENSEMBL. Although none were found, we cannot exclude the possibility of nonconventional PPAR
binding sites capable of negative regulation (27,28). That reduced hepatic phosphatidylcholine production is a major component of rosiglitazone-exacerbated steatosis in F1 and RCS8 mice is inferred from results with a ß3-adrenergic receptor agonist, CL316,243 (Fig. 2). We previously demonstrated that this compound completely suppressed hyperglycemia and hyperlipidemia in NZO/HlLt males and eliminated the mild hepatic lipidosis (13). When PEMT and choline kinase activities were measured in F1 males whose diabesity syndrome was also effectively suppressed by CL316,243, the absence of steatosis correlated with a drug-mediated increase in mean activity levels rather than a decrease (Fig. 2). The increased activities were reflected by increased choline kinase and PEMT protein expression (Fig. 2). In contrast, rosiglitazone treatment failed to increase either activity and in fact suppressed choline kinase protein in Western blots (Fig. 2).
The insulin-resistant NZO parental strain donating obesity/diabesity QTLs to the RCS panel shows a dissociation of insulins effects on genes involved in glucose and lipid metabolism. Insulin-suppressible genes associated with hepatic gluconeogenesis and glycogenolysis are not suppressed, but insulin-inducible genes associated with lipogenesis are markedly upregulated (29). A combined gene transcription and lipid metabolome analysis of rosiglitazone-treated F1 livers indicated that lipid uptake and rosiglitazone-stimulated de novo lipid biosynthesis were not adequately compensated by lipid export (3). In the case of the RCSs tested in this report, the physiologic profiling of plasma glucose and insulin responses to rosiglitazone in Table 1 suggest different degrees of insulin resistance. Unlike RCS10, whose insulin resistance was clearly diminished by rosiglitazone treatment as reflected by a restoration of normoglycemia, RCS8 appeared less responsive despite a considerably milder mean hyperglycemia over the time period studied.
In summary, the RCS analysis reported herein has allowed biochemical dissection of the marked sensitivity of the F1 diabesity model to TZD-exacerbated steatosis. Our results indicate a constitutive impairment of hepatic phosphatidylcholine biosynthetic enzyme functions that is further exacerbated by TZD treatment. The fact that, unlike RCS8, the NON/Lt parental background strain, RCS1, RCS2, and RCS10 do not exhibit these extreme responses to TZD establishes this RCS panel as potentially useful pharmacogenetic screening tools. Clearly, there are well-known differences between humans and mice in terms of rosiglitazone effects on liver fat accumulation (20). Rosiglitazone treatment of humans is associated with decreases, not increases, in liver fat (20). In mice, inability to express PPAR in liver protects from rosiglitazone-mediated steatosis (30,31). Despite these differences distinguishing mice from humans, understanding the genetic basis for these differential drug responses in mice may provide both physiologic and genetic insights to guide selection of patients capable of tolerating long-term drug treatments without adverse side effects.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Pam Stanley and Sandra Ungarian for technical assistance. We thank Drs. Rick Woychick and Jürgen Naggert at The Jackson Laboratory for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
Address correspondence and reprint requests to Edward H. Leiter, PhD, The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. E-mail: ehl{at}jax.org
Received for publication December 17, 2004 and accepted in revised form February 18, 2005
PEMT, phosphatidylethanolamine methyltransferase; PPAR, peroxisome proliferatoractivated receptor-
; QTL, quantitative trait locus; RCS, recombinant congenic strain; TZD, thiazolidinedione
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|