Hypertension and abnormal fat distribution but not insulin resistance in mice with P465L PPARγ (original) (raw)
Mice with the PPARγ P465L mutation. We mutated codon 465 of the mouse Pparg gene (equivalent to codon 467 in human PPARG) from CCC (proline) to CTG (leucine) via gene targeting in mouse ES cells, and generated mice carrying the P465L substitution (Figure 1A). Southern blots (Figure 1B) and nucleotide sequence analyses (data not shown) confirmed the mutation in the Pparg gene. Wild-type and heterozygous PpargP465L/+ littermates were born at the expected mendelian ratio (34:61) from mating of heterozygous pairs, but no homozygous PpargP465L/P465L mice were born. Thus, homozygosity for the P465L mutation is lethal in utero, establishing that the mutant protein is effectively nonfunctional. For the following studies, we used F1 offspring derived from crosses between 129/SvEv heterozygous PpargP465L/+ mice and C57BL/6 wild-type mice.
Generation of mice with the PPARγ P465L mutation. (A) Crossovers (indicated by the large X marks) between the wild-type mouse Pparg locus with Pro465 in exon 6 (6P) (top diagram) and targeting construct with Leu465 (6L) (second diagram) resulted in the targeted allele in ES cells (third diagram). The ACN (Cre-Neo) cassette, flanked by loxP sequences, was excised out of the mutant allele upon germline transmission (bottom diagram). B, X, Xb, and St indicate the _Bam_HI, _Xho_I, _Xba_I, and _Stu_I restriction enzyme sites, respectively. tAce, testis-specific Ace promoter; TK, thymidine kinase. (B) Southern blot analysis of genomic DNA. The targeted allele was identified by a 5′ probe that hybridizes to an 11-kb fragment in wild-type (+/+) DNA and to a 7.7-kb fragment in heterozygous DNA that includes the P465L mutation (L/+). (C) PPARγ mRNA of the wild-type (white bars) and mutant allele (black bar) in gonadal adipose tissue from wild-type and PpargP465L/+ mice (n = 8 each). The PPARγ mRNA amount is expressed relative to that of wild-type allele in wild-type mice. (D) Rosiglitazone-induced PEPCK expression in gonadal adipose tissue explants. Tissues isolated from four wild-type (open squares) and four PpargP465L/+ (filled squares) mice were incubated in cultured media containing various concentrations of PPARγ agonist rosiglitazone as indicated. The levels of PEPCK mRNA are relative to the wild-type basal level. *P < 0.01 and **P < 0.005 compared with the respective basal levels.
Precision quantitative RT-PCR revealed that expression of the wild-type allele in adipose tissues of PpargP465L/+ mice was, as expected, about 50% of that in wild-type littermates (Figure 1C). The mutant P465L transcripts in PpargP465L/+ mice were present at the same level as the wild-type transcripts, showing that the mutant allele is transcribed normally. Total amounts of PPARγ transcripts were not different between PpargP465L/+ and wild-type mice. Similarly, there was no compensatory alteration in the gene expression of RXRα, PPARα, or PPARΔ (data not shown).
Cultured gonadal adipose tissue isolated from wild-type mice responded to rosiglitazone by increasing expression of the PPARγ target gene encoding phosphoenolpyruvate carboxykinase (PEPCK) in a dose-dependent manner (Figure 1D). In contrast, activation of PEPCK expression was markedly impaired in cultured adipose tissue from PpargP465L/+ mice (Figure 1D), indicating that rosiglitazone-dependent activation of wild-type PPARγ is dominantly suppressed by the P465L protein in vivo.
Abnormal body fat distribution in PpargP465L/+ mice. PpargP465L/+ mice developed normally and appeared healthy. Their food and water intake, urine output, rectal temperature, and hematological parameters, and the macroscopic and microscopic features of their major organs were normal (data not shown). Fasting plasma lipids, including total cholesterol, triglyceride, and FFA, were not altered in PpargP465L/+ mice (Table 1). The growth curves of both male and female PpargP465L/+ mice were the same as those of wild-type mice fed regular chow or a high-fat diet (Figure 2A). Organ and total weights of abdominal fat pads (gonadal, mesenteric, retroperitoneal, and inguinal, normalized by body weight) were not significantly different between mice of the two genotypes.
Altered adipose tissue distribution in PpargP465L/+ mice. (A) Body weights of male mice (open squares, wild-type, n = 9–10; filled squares, PpargP465L/+, n = 9–10) and female mice (open circles, wild-type, n = 7–9; filled circles, PpargP465L/+, n = 8) fed regular chow (left panel) or a high-fat diet (right panel). (B) Adipose tissue mass in 10-week-old female wild-type (white bars) and PpargP465L/+ (black bars) mice fed regular chow. Data are expressed as percent body weights. Gon, Mes, Ret, Ing, and Pec represent gonadal, mesenteric, retroperitoneal, inguinal, and pectoral white adipose tissue, respectively; BAT indicates interscapular BAT. *P < 0.05 and **P < 0.01, compared with wild-type littermates. (C) Morphology of gonadal and inguinal white adipose and brown adipose tissues from 10-week-old female mice fed regular chow. +/+, wild-type; L/+, PpargP465L/+. (D) Distribution of cell size in gonadal (left panel), inguinal (middle panel), and retroperitoneal (right panel) adipose tissues. Open circles, wild-type; filled circles, PpargP465L/+. (E) UCP1 mRNA relative to the level of gonadal adipose tissue from wild-type mice. *P < 0.05 between PpargP465L/+ and wild-type littermates (n = 16 each). The y axis is in a log scale. (F) Relative ratio of mRNA for PPARg target genes in gonadal and inguinal adipose tissues of PpargP465L/+ mice to those of wild-type mice (n = 16 each). *P < 0.05 and **P < 0.005 between PpargP465L/+ and wild-type littermates.
Metabolic parameters in PpargP465L/+ (L/+) and WT male mice
Despite the similar total body fat in PpargP465L/+ and wild-type mice, we found significant differences in their fat distribution (Figure 2B). The interscapular brown adipose tissue (BAT) weight relative to body weight of PpargP465L/+ mice was about 80% that of wild-type mice (P < 0.02), and the gonadal fat mass in PpargP465L/+ mice was about 70% (P < 0.004). In contrast, the weight of the inguinal fat pad in PpargP465L/+ mice was increased to about 140% that of wild-type mice (P < 0.01). The pectoral subcutaneous fat pad in PpargP465L/+ mice was similarly increased (P < 0.02). Consequently, the ratio of intra-abdominal fat weight to extra-abdominal fat weight (the sum of mesenteric, gonadal, and retroperitoneal fat depots versus inguinal fat) in PpargP465L/+ mice was substantially reduced to 63% that of wild-type mice (P < 0.0001). A high-fat diet led to fat deposition in both mutant and wild-type mice and increased this difference in fat distribution (47% of wild-type; P < 0.0001).
Microscopically, the cellularity of adipose tissues in wild-type mice was heterogeneous. In contrast, the adipocytes in PpargP465L/+ mice were relatively uniform and the number of small adipocytes was reduced (Figure 2C). The size distributions of adipocytes in the gonadal and inguinal fat from PpargP465L/+ mice showed significant shifts toward larger cells compared with the distributions from wild-type mice (Figure 2D). Retroperitoneal, mesenteric, and pectoral fat showed a similar but less marked trend. These results suggest that the increase in inguinal fat mass of PpargP465L/+ mice is mainly due to an increase in triacylglycerol storage, while the decrease in gonadal fat mass is due to a decrease in cell number. Both the gonadal and inguinal fat pads of PpargP465L/+ mice showed a decrease in the number of multilocular adipocytes (Figure 2C). Consistent with this, we found significant reductions in mRNA for the brown fat–specific protein uncoupling protein 1 (UCP1) in gonadal and inguinal adipose tissues to 37% (P < 0.02) and 29% (P < 0.03) that of wild-type mice, respectively (Figure 2E). The amount of UCP1 mRNA in interscapular BAT did not differ between PpargP465L/+ and wild-type mice (Figure 2E).
To examine the in vivo effects of the P465L mutation on the expression of PPARγ target genes, we measured their steady-state mRNA levels in adipose tissues. Despite the impaired PEPCK activation in culture described above, expression of the gene encoding PEPCK in both gonadal and inguinal adipose tissues of PpargP465L/+ mice was indistinguishable from that of wild-type mice (Figure 2F). In contrast, mRNA levels for adipocyte fatty acid–binding protein (aP2; reduced to 0.58× wild-type levels, P < 0.002) and glucose transporter 4 (Glut4; reduced to 0.64× wild-type levels, P < 0.05) were significantly decreased in gonadal but not inguinal adipose tissue in PpargP465L/+ mice. Conversely, expression of the gene encoding lipoprotein lipase (LPL) was not affected in gonadal adipose tissue but was significantly increased in inguinal adipose tissue of PpargP465L/+ mice (increased to 3.48× wild-type levels, P < 0.05). Thus, the presence of the P465L mutation does not lead to a uniform change, but exerts adipose depot–specific effects on the basal expression of PPARγ target genes in vivo.
These results demonstrate that the P465L mutation in PPARγ causes abnormal fat distribution, leading to a preferential deposition of fat in subcutaneous fat pads rather than in intra-abdominal fat pads, and reduces the number of small adipocytes, including mutilocular adipocytes, in these white fat depots.
Normal insulin sensitivity in PpargP465L/+ mice. The human P467L mutation is associated with severe insulin resistance, hyperinsulinemia, and diabetes. However, the plasma glucose concentrations in PpargP465L/+ mice after fasting were normal compared with those of wild-type mice (Table 1). Their plasma insulin levels were slightly but not significantly higher than those of wild-type mice. Feeding the mice a diet high in fat for 4 weeks substantially increased plasma glucose and insulin levels in both PpargP465L/+ and wild-type mice compared with feeding the mice regular chow. The PpargP465L/+ mice fed the high-fat diet had slightly lower glucose and higher insulin levels than did their wild-type littermates fed a similar diet (Table 1). These differences were not statistically significant, however.
To assess the dynamic response of PpargP465L/+ mice to increased glucose concentrations, we performed the intraperitoneal glucose tolerance test (IPGTT). PpargP465L/+ mice cleared glucose faster than their wild-type littermates did, regardless of diet, indicating improved glucose tolerance in the mutant mice (Figure 3A). This improved glucose tolerance was accompanied by significantly elevated plasma insulin levels (P < 0.05 at 15 minutes) during the IPGTTs of PpargP465L/+ mice fed a high-fat diet but not of mice fed regular chow. The insulin resistance index of PpargP465L/+ mice calculated from the IPGTT was slightly lower in the group fed regular chow but higher in the group fed a high-fat diet than that of wild-type mice (Table 1). However, these differences were not significant. Consistent with these findings, the intraperitoneal insulin tolerance test (IPITT) revealed that the hypoglycemic response to an acute administration of insulin (0.5 U/kg) was normal in PpargP465L/+ mice fed regular chow or a high-fat diet (Figure 3B).
Increased glucose tolerance but normal insulin sensitivity in PpargP465L/+ mice. (A) Plasma glucose and insulin levels during the IPGTT in 14- to 16-week-old male mice fed regular chow (left panels; n = 15–16) or a high-fat diet (right panels; n = 7–11). Open squares, wild-type; filled squares, PpargP465L/+. *P < 0.05 versus wild-type. (B) IPITTs of 14- to 16-week-old female mice fed regular chow (left panel; n = 7–8) or a high-fat diet (right panel; n = 6–7). Open squares, wild-type; filled squares, PpargP465L/+. Data are expressed as the percentage of the plasma glucose before insulin injection. (C) Whole-body metabolic parameters during the hyperinsulinemic-euglycemic clamp experiment. Steady-state glucose infusion rates (top left) and insulin-stimulated whole-body glucose turnover rates (top right) were obtained for 10- to 12-week-old male mice fed regular chow (RC; n = 5) or a high-fat diet (HF; n = 5–8). White bars, wild-type; black bars, Pparg465L/+. *P < 0.05 for diet effect. Basal (bottom left) and clamped (bottom right) rates of HGP in wild-type (white bars) and Pparg465L/+ (black bars) mice fed regular chow (RC; n = 5) or a high-fat diet (HF; n = 5–8). (D) Morphometric analysis of pancreatic islets. Mean islet area (left), pancreatic endocrine mass (middle), and islet number (right) were measured in sections from 14- to 16-week-old male mice fed regular chow (RC; n = 4) or a high-fat diet (HF; n = 4). White bars, wild-type; black bars, Pparg465L/+. *P < 0.05 and **P < 0.001 compared with wild-type littermates.
To further examine the insulin sensitivity of PpargP465L/+ mice in vivo, we established a 2-hour hyperinsulinemic-euglycemic clamp in conscious mice fed regular chow or a high-fat diet for 3 weeks. The rates of glucose infusion required to maintain the euglycemic clamp were not different for PpargP465L/+ mice fed regular chow compared with their wild-type littermates (Figure 3C). Feeding mice a high-fat diet blunted the insulin response during the clamps to the same extent in both groups, as reflected by their significantly lower but equal steady-state glucose infusion rates. Insulin-stimulated whole-body glucose turnover rates were not different in mice of the two genotypes fed regular chow and were similarly reduced after mice were fed a high-fat diet. No genotype effect was observed in insulin-stimulated whole-body glycolysis, glycogen/lipid synthesis (data not shown), and hepatic glucose production (HGP) in basal and clamped states (Figure 3C). These results indicate that the PpargP465L/+ mutation does not alter peripheral or hepatic insulin sensitivity and suggest that the increased glucose tolerance of PpargP465L/+ mice is due mainly to their elevated plasma insulin rather than to an altered insulin sensitivity.
We next examined pancreatic islet morphology. While there was no change in the weight of pancreas (data not shown), the mean islet area and endocrine mass in 4-month-old PpargP465L/+ mice were both slightly increased in the group fed regular chow and were significantly increased in the group fed a high-fat diet (Figure 3D). The number of islets was similar in PpargP465L/+ and wild-type mice. Thus, increased endocrine mass and islet size may be responsible for the observed elevation of plasma insulin levels during glucose overload in PpargP465L/+ mice.
Elevated BP in PpargP465L/+ mice. The P465L mutation in PPARγ caused a significant increase of about 8 mmHg in the BP of both male and female mice, as measured by a tail cuff method (P < 0.01 for genotype effect but not significant for gender effect by two-way ANOVA; Figure 4A). The pulse rates of PpargP465L/+ mice were not different from those of their wild-type littermates (Table 1). Consistent with the tail-cuff measurements, telemetric BP monitoring showed higher than average systolic/diastolic BP during the light cycle in the PpargP465L/+ mice (132/94 mmHg versus wild-type 110/82 mmHg) (Figure 4B). BP during the dark cycle was similarly elevated in PpargP465L/+ mice (144/103 mmHg versus wild-type 124/95 mmHg) (Figure 4B). Thus, the diurnal rhythm of BP is maintained in PpargP465L/+ mice and the elevation of their BP in the active phase is not disproportionate.
Elevated BP in PpargP465L/+ mice. (A) BP of 14- to 16-week-old wild-type (white bars) and PpargP465L/+ (black bars) mice by tail-cuff measurement. Numbers of mice are inside bars. P < 0.01 for genotype effect by ANOVA. (B) Four-day telemetric recordings of systolic and diastolic BP in 24-week-old female mice. Results are expressed as mean of four wild-type (dashed lines) and four PpargP465L/+ (solid lines) mice averaged with 12 values each hour. Bolded bars on the x axis represent the dark cycles. (C) Responses to changes in dietary salt intake. BP of 14- to 16-week-old male wild-type (white bar) and PpargP465L/+ (black bar) mice fed a high-salt diet for 4 weeks (left panel). Numbers inside bars indicate sample size. *P < 0.05. Changes in daily food consumption (middle panel) and urinary sodium excretion (UNaV; right panel) after mice were switched to a high-salt diet at day 0 for 10- to 12-week-old male wild-type (open squares, n = 7) and PpargP465L/+ (filled squares, n = 6) mice. (D) Relative ratio of the expression of RAS genes in PpargP465L/+ mice to those in wild-type mice (n = 16 each). Left panel, organs of major RAS expression: K, kidney; L, liver; AG, adrenal gland. Middle panel, inguinal adipose tissue. Right panel, gonadal adipose tissue. *P < 0.05 and **P < 0.005 between PpargP465L/+ and wild-type mice.
Because PPARγ is expressed in the renal medulla (23), we next examined the possibility that PpargP465L/+ mice may have abnormal salt and water reabsorption in the kidney and may display salt-sensitive hypertension. Feeding mice a high-salt diet containing 8% NaCl for 4 weeks did not change the BP of either PpargP465L/+ or wild-type mice, and the genotype effect on BP was maintained (Figure 4C). We further tested the salt sensitivity of PpargP465L/+ mice during the immediate adaptation to an increased dietary salt load before homeostatic adjustments take place. Changes in daily food consumption were not different, indicating mice had the same salt intake. Urinary sodium excretion was increased considerably on initiation of high-salt feeding. However, the daily sodium excretion as well as 4-day cumulative sodium excretion after initiation of the high-salt diet was not different between mice of the two genotypes. Thus PpargP465L/+ mice have normal salt and water handling in the kidney and the elevation of BP in PpargP465L/+ mice is not salt sensitive.
Because the RAS plays a crucial role in the maintenance of BP, we measured the steady-state mRNA levels of genes of the RAS. The mRNA levels for AGT, renin, and aldosterone synthase (AS) in the liver, kidney, and adrenal gland, the major sites of their expression, respectively, were not affected by the presence of the P465L mutation (Figure 4D). PPARγ activators have been shown to transcriptionally suppress the angiotensin II type I receptor (AT1R) (24), but we found that the AT1R mRNA levels in kidney were not different in PpargP465L/+ versus wild-type mice. Consistent with the mRNA levels, plasma renin concentration (42 ± 13 ng of angiotensin I/ml/h in PpargP465L/+ versus 44 ± 14 in wild-type; P = 0.89), plasma AGT levels (equivalent to 622 ± 7 ng of angiotensin I/ml in PpargP465L/+ versus 608 ± 20 in wild-type; P = 0.50), and plasma aldosterone (426 ± 73 pg/ml in PpargP465L/+ versus 408 ± 79 in wild-type; P = 0.87) concentrations were not different between mice of the two genotypes. Thus, the systemic RAS does not appear to be altered in P465L PPARγ–mediated hypertension.
Adipose tissue also produces and secretes vasoactive precursor substances including AGT, which may contribute to the development of hypertension (17). Expression of the gene encoding for renin in both gonadal and inguinal adipose tissues was low and was not significantly different in mice of the two genotypes (Figure 4D). The mRNA levels for angiotensin II type II receptor (AT2R), which is reported to antagonize AT1R by inducing vasodilation and inhibiting cell growth and proliferation (25), were not different in mice of the two genotypes. In contrast, the level of AGT mRNA in the inguinal fat of PpargP465L/+ mice was 2.5 times that of wild-type mice (P < 0.05). The level of AGT mRNA in the gonadal fat of PpargP465L/+ mice was not different from that of wild-type mice. Conversely, a significant increase in the AT1R gene expression was observed in the gonadal fat (2.1× wild type, P < 0.005) but not in the inguinal fat of PpargP465L/+ mice. Thus the genes of the RAS are altered in adipose tissues of PpargP465L/+ mice in a depot-specific fashion. These data suggest that the elevated BP of PpargP465L/+ mice could be affected by changes in the local RAS of adipose tissues.
Effects of age, diet, and gender. Diets containing high fat as well as aging are known to affect weight gain, fat deposition, and insulin sensitivity, which could ultimately affect BP. We therefore examined the phenotypes of mice at 9 months of age that were either maintained on regular chow or fed a high-fat diet for 22 weeks (Table 1). We found significant age and diet effects on body weight, fat mass, plasma glucose, and insulin, and insulin resistance index, but the changes induced by diet and age were similar in both PpargP465L/+ and wild-type mice. Neither age nor diet influenced BP. Except for the hyperinsulinemic clamp experiments, which were done only in male mice, we studied both male and female mice. The P465L effects on BP, fat distribution, and insulin sensitivity were the same in both male and female mice without any interaction between gender and genotype by ANOVA.