Leptin Deficiency Unmasks the Deleterious Effects of Impaired Peroxiso
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《糖尿病学杂志》
1 Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Cambridge, U.K
2 Ingenium Pharmaceuticals, Martinsried, Germany
3 Department of Normal Human Morphology, Faculty of Medicine, Ancona University, Ancona, Italy
4 GSF-National Research Center for Environment and Health, Institute of Inhalation Biology, Muenchen-Neuherberg, Germany
5 Genetics Unit, Department of Biochemistry, University of Oxford, Oxford, U.K
6 Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, U.K
7 TNO-Prevention and Health, Division VBO, Leiden, the Netherlands
gWAT, gonadal white adipose tissue; PPAR, peroxisome proliferator–activated receptor; scWAT, subcutaneous white adipose tissue; WAT, white adipose tissue
ABSTRACT
Peroxisome proliferator–activated receptor (PPAR) is a key transcription factor facilitating fat deposition in adipose tissue through its proadipogenic and lipogenic actions. Human patients with dominant-negative mutations in PPAR display lipodystrophy and extreme insulin resistance. For this reason it was completely unexpected that mice harboring an equivalent mutation (P465L) in PPAR developed normal amounts of adipose tissue and were insulin sensitive. This finding raised important doubts about the interspecies translatability of PPAR-related findings, bringing into question the relevance of other PPAR murine models. Here, we demonstrate that when expressed on a hyperphagic ob/ob background, the P465L PPAR mutant grossly exacerbates the insulin resistance and metabolic disturbances associated with leptin deficiency, yet reduces whole-body adiposity and adipocyte size. In mouse, coexistence of the P465L PPAR mutation and the leptin-deficient state creates a mismatch between insufficient adipose tissue expandability and excessive energy availability, unmasking the deleterious effects of PPAR mutations on carbohydrate metabolism and replicating the characteristic clinical symptoms observed in human patients with dominant-negative PPAR mutations. Thus, adipose tissue expandability is identified as an important factor for the development of insulin resistance in the context of positive energy balance.
a thermodynamic point of view, the main factors involved in the development of obesity are food intake and energy expenditure, assuming that accumulation of fat is a passive event that follows a situation of positive energy balance. However, accumulation of fat in adipose tissue is an active process that requires the coordinated regulation of mechanisms involved in adipocyte differentiation and growth.
An important molecular regulator of adipose tissue development and expansion is peroxisome proliferator–activated receptor (PPAR) (1,2). PPAR is essential for adipogenesis, as shown by the inability of PPAR-null embryonic stem cells to undergo adipogenesis (3). PPAR is a modulator of insulin sensitivity. The link between PPAR-induced adipocyte differentiation and insulin sensitivity in humans is strengthened by the metabolic characterization of a small number of individuals with naturally occurring, dominant-negative mutations in PPAR. One such mutation, P467L, has been identified in the heterozygous state, and patients with this mutation suffer from severe insulin resistance, partial lipodystrophy, impaired postprandial lipid metabolism, fatty liver, and hypertension (4–6). To overcome the limitations imposed by the rarity of the human mutations, the function of PPAR has been studied extensively using mouse models with tissue/isoform-specific ablation of PPAR or knockin of PPAR mutations (7–17). These models have confirmed an important role for PPAR in controlling adipose tissue differentiation and function, as inducible knockout of PPAR in developed adipose tissue results in progressive reduction in fat depots (8,12) and heterozygosity for a PPAR-null mutation (18) reduces whole-body adipose tissue mass.
The first mouse model expressing a human-related mutation in PPAR (P465L), however, produced a surprising phenotype that has brought into question the translatability of results obtained using PPAR mouse models to humans. Particularly puzzling was the observation that the severe insulin resistance and partial lipodystrophy identified in human patients harboring dominant-negative PPAR mutations were not reproduced in a mouse model with the equivalent mutation (P465L) (15), a finding we confirm here using a new independently generated P465L PPAR mouse line. Because substantial evidence suggests that PPAR acts via similar biochemical mechanisms to promote lipogenesis and enhance insulin sensitivity in rodents and humans (19), we wondered why the effects of this mutant on fat deposition and insulin sensitivity were so different in human and mouse. In our opinion, this is a fundamental question that may be relevant to the interpretation of other discrepant metabolic phenotypes between humans and rodents (20–22).
Here, we show that when the P465L PPAR mutation is expressed in a lean background with limited requirements for adipose tissue expandability, the animals have normal adipose tissue mass and insulin sensitivity, even when challenged with high-fat feeding, a finding reported previously (15). Under these conditions the P465L PPAR mouse shows impaired postprandial lipid clearance and altered adipose tissue distribution, despite having normal adipose mass and insulin sensitivity. We hypothesized that the discrepancy in fat deposition and insulin sensitivity between human patients and mice expressing the PPAR mutations may be due to species-related differences in the balance between adipose tissue storage demands and adipose tissue expandability. Forcing a mismatch between adipose tissue storage capacity and energy availability may be key for the development of insulin resistance mediated by defective PPAR function; thus, we expressed the P465L PPAR mutation in a hyperphagic mouse background (ob/ob). In this context of positive energy balance, activity of the P465L PPAR mutant did not allow adequate adipose tissue expansion to fulfill storage requirements and severe insulin resistance developed, replicating the characteristic clinical symptoms observed in human patients with dominant-negative PPAR mutations.
RESEARCH DESIGN AND METHODS
Generation of P465L/wt and P465L/ob mice.
A targeting construct with a C-to-T point mutation in exon 6 of the PPAR gene was generated by site-directed mutagenesis. A neomycin selection cassette flanked by two FRT sites was incorporated into intron 6 with two thymidine kinase genes positioned at the 5' and 3' ends of the flanking sequence (Fig. 1A). The targeting construct was transfected into a TBV2 (129/SvP) ES cell line and homologously recombined clones were selected for using G418/ganciclovir and identified by Southern blot analysis (Fig. 1B). Chimeras with successful germ-line transmission were obtained and bred to C57/BL6 mice to produce mice heterozygous for the point mutation. Removal of the neomycin selection cassette in intron 6 was performed by breeding mice heterozygous for the P465L PPAR mutation to FLP transgenic mice.
Animals were genotyped using a three-primer PCR strategy that identified the wild-type allele (498 bp) and the recombined allele containing the P465L mutation (641 bp) and, if present, detected the P465L allele with the neomycin cassette (391 bp) (Fig. 1C). Homozygosity for P465L PPAR resulted in embryonic lethality. Expression levels of the wild-type and mutant alleles were determined by a real-time PCR strategy using a common primer set with unique probes that differed by the single base pair in exon 6.
To generate mice with the P465L PPAR mutation on an ob/ob background, mice heterozygous for the P465L PPAR mutation were crossed to mice heterozygous for a point mutation in the leptin gene (ob) (23). Mice heterozygous for both P465L PPAR and the leptin mutation were further crossed to generate four experimental genotypes: wild type for PPAR and leptin (wt/wt), heterozygous for P465L PPAR and wild type for leptin (P465L/wt), wild type for PPAR and homozygous for the leptin mutation (ob/ob), and heterozygous for P465L PPAR and homozygous for the leptin mutation (P465L/ob). All animals used were of mixed C57/BL6/129SvJ background and unless otherwise stated were fed a standard mouse chow ad libitum, with 12-h light/dark cycles. All comparisons were made using littermate controls. Experimental protocols requiring the use of animals were approved by the U.K. home office and the University of Cambridge.
Body composition and food consumption.
Body mass was recorded for 12 weeks, and food consumption of individually housed animals was monitored from weaning for 10 days. Body composition was determined in dead animals using the Lunar PIXImus two-bone densitometer (dual-energy X-ray absorptiometry; GE Medical Systems, Madison, WI). Individual fat-pad mass was measured at dissection.
Histology.
Tissues were dissected and fixed in 10% formalin, or animals were perfused with 4% paraformaldehyde. Tissue was either cryoprotected (20% sucrose) and frozen in chilled isopentane or embedded in paraffin and sectioned using a cryostat (Leica CM1900; Leica Microsystems, Milton Keynes, U.K.) or standard microtome (Leica RM2125RT; Leica Microsystems), respectively. Cryosectioned tissue was stained for lipid with oil red O.
Morphometric analysis of adipocytes included calculation of adipocyte area (square micrometers) from gonadal and inguinal subcutaneous adipose tissue depots. Image analysis software (analySIS) was used to analyze the images and calculate adipocyte area. Two images per section (10x magnification), two sections per mouse, and four mice per genotype were used to calculate the average adipocyte area for the two adipose tissue depots.
Biochemistry.
Biochemical analysis of serum or tissue extracts included measurements of glucose, total cholesterol, HDL, triglycerides, insulin (DRG Diagnostics International), free fatty acids (Roche, Welwyn Garden City, U.K.), adiponectin (B-Bridge International), and leptin (R&D Systems, Abingdon, U.K.). Triglyceride content of liver was determined using a previously described extraction method (24).
Gene expression profiling.
Individual tissues (gonadal white adipose tissue [gWAT], skeletal muscle, and liver) were dissected and frozen immediately in liquid nitrogen. Total RNA was extracted using RNA STAT-60 (Tel-Test). cDNA was reverse transcribed from 500 ng of total RNA (Promega, Southampton, U.K.). Gene expression levels were determined using real-time PCR (TaqMan; Applied Biosystems, Warrington, U.K.) with primers and probes designed to specific mRNAs (Sigma, Haverhill, U.K.). PCR conditions were as follows: 1 cycle of 50°C for 2 min and 95°C for 10 min and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Values were normalized against 18 S RNA and expressed as relative arbitrary units.
Evaluation of carbohydrate metabolism.
Blood glucose was measured (OneTouch Ultra; LifeScan Johnson & Johnson, High Wycombe, U.K.) from the tail vein of 3-week-old mice before weaning (preweaning) and in fed and fasted 4-week-old mice. Glucose tolerance tests were performed in nondiabetic mice after an overnight (16 h) fast. Blood glucose levels were measured (OneTouch Ultra) before and after (10, 20, 30, 60, 120, and 180 min) an intraperitoneal injection of D-glucose (1 g/kg). Insulin tolerance tests were performed after a 6-h fast. Blood glucose was measured before and after (10, 20, 30, and 60 min) an intraperitoneal injection of insulin (0.75 units/kg). Values are expressed relative to basal (6-h fast) blood glucose. Hyperinsulinemic-euglycemic clamps were performed on adult wild-type (n = 10) and P465L PPAR mice (n = 10) only. Clamps, subsequent tissue preparation, and calculations were performed as described previously (25,26).
Energy expenditure.
Oxygen consumption rates were determined in 6-week-old mice using closed calorimetry. The metabolic rate was determined over a 5-h period and expressed as VO2 (milliliters per minute per kilogram0.75).
Primary culture of preadipocytes.
Preadipocytes were isolated from gWAT and differentiated as described previously (27) with the inclusion of 10% fetal bovine serum in the differentiation media. Adipose tissue pads from three mice were pooled for wt/wt and P465L/wt mice in three separate experiments (Fig. 3E). In ob/ob and P465L/ob mice, fat pads from individual mice were isolated at 5 weeks of age and differentiated as above. Differentiation success was determined after 8 days of differentiation by oil red O staining and/or measurement of aP2 mRNA expression in adipocytes (Fig. 3E and online appendix Fig. 1 [available at http://diabetes.diabetesjournals.org]).
Lipid clearance.
To assess postprandial lipid clearance, animals were fasted overnight and dosed orally with 200 μl of olive oil. Serum triglycerides and free fatty acids were determined before the lipid dose and at 1, 2, 4, and 8 h after the lipid load.
High-fat feeding.
A high-fat diet (45% kcal as fat; Research Diets, New Brunswick, NJ) was fed to wild-type and P465L PPAR animals for 16 weeks after weaning.
Statistical analysis.
Data are presented as means ± SE. Statistical significance between genotypes was determined using a one-way ANOVA and between relevant pairs of genotypes using unpaired Student’s t tests. Significance was declared for P values <0.05 (GraphPad Instat 3, Microsoft Excel).
RESULTS
Characterization of the P465L PPAR allele.
Southern and PCR analysis of genomic DNA identified the recombined P465L PPAR allele (Fig. 1A–C). Subsequent sequencing of genomic DNA confirmed the presence of the C-to-T point mutation in exon 6 of the PPAR gene.
Homozygous transmission of P465L PPAR results in embryonic lethality.
Heterozygous P465L PPAR matings produced an altered Mendelian ratio (wt/wt 28%, P465L/wt 72%, and P465L/P465L 0%), demonstrating that homozygous transmission of the P465L PPAR allele was embryonic lethal. Previous in vitro studies have demonstrated that the dominant-negative activity of the ligand-binding domain PPAR mutants was associated with impaired coactivator recruitment and transactivation (6,28,29). Quantification of allelic expression in heterozygous animals revealed that transcription of the mutant allele was not impaired (Fig. 1D). Generation of mice with P465L PPAR on an ob/ob background produced the four experimental genotypes at the expected (2 = 0.2517) Mendelian ratio (wt/wt 6.4%, P465L/wt 13.5%, ob/ob 13.5%, and P465L/ob 15.1%).
P465L PPAR promotes abnormal adipose tissue distribution and prevents adipose tissue expansion when expressed on an obese background.
P465L PPAR expressed on a lean background does not affect overall body mass (Fig. 2A and Table 1) or total body fat content (Fig. 2B), although it does cause a redistribution of white adipose tissue (WAT), with a significantly decreased ratio of gWAT to inguinal subcutaneous WAT (scWAT) mass when compared with wild-type controls (Fig. 3C). Additionally, preadipocytes isolated from WAT of mice with the P465L PPAR mutation (P465L/wt and P465L/ob) had impaired differentiation capability in vitro compared with animals wild type for PPAR (wt/wt and ob/ob) (Fig. 3E and online appendix Fig. 1).
When P465L PPAR was expressed on an ob/ob background, there was a significant decrease in body mass that was evident after 5 weeks compared with ob/ob controls (Fig. 2A). By 12 weeks, body mass of P465L/ob mice was 14% (females, Fig. 2A) and 12% (males, data not shown) lower than that of ob/ob controls. This change in body weight was due to reduced fat mass (Fig. 2B). P465L/ob mice consumed the same amount of oxygen and were as hyperphagic as ob/ob controls (Fig. 2C and D). By 5 weeks, P465L/ob mice had reduced scWAT and gWAT (Fig. 3A), and white adipoctyes from these depots were smaller compared with those from ob/ob mice (Fig. 3B), suggesting that adipocytes were unable to expand appropriately in response to the increased energy availability.
P465L PPAR prevents appropriate expression of lipogenic genes.
Gene expression profiling in WAT revealed that the fatty acid binding protein aP2 and the fatty acid carrier CD36 were significantly reduced in gWAT of mice carrying the P465L PPAR mutation on both wild-type and ob/ob backgrounds (Fig. 3D) compared with controls.
P465L PPAR only confers insulin resistance in the context of severe positive energy balance.
Unlike human patients with dominant-negative PPAR mutations, P465L PPAR mice on a lean background displayed normal insulin sensitivity as demonstrated by glucose tolerance tests (Fig. 4A) and hyperinsulinemic clamps (Fig. 4B and C). Similarly, the P465L mutation did not exacerbate high-fat feeding–induced insulin resistance as shown by glucose and insulin tolerance tests (Fig. 4D and E). In contrast, when the P465L mutation is expressed on an ob/ob background, mice develop severe insulin resistance. As early as 5 weeks of age, the P465L/ob mice are more insulin resistant than the ob/ob mice, demonstrated by extremely high blood glucose and serum insulin levels in the fed state (Table 2) and a severely blunted response to an insulin tolerance test (Fig. 4F). Of interest, adiponectin was decreased to a similar extent in mice carrying the P465L PPAR mutation on either a wild-type or ob/ob background, suggesting that the failure in insulin sensitivity was not related to quantitative differences in adiponectin plasma levels (Table 1).
Impaired postprandial lipid clearance despite normal insulin sensitivity in P465L PPAR mice.
Despite showing normal body mass and insulin sensitivity on a lean wild-type background, the P465L PPAR mice had a significant increase in serum triglyceride levels in the fed state with a high-fat diet compared with wild-type controls (Table 1). An oral lipid load test showed that P465L/wt mice were unable to clear triglycerides and fatty acids as efficiently as wild-type controls (Fig. 5A and B). This defective clearance of lipids was magnified in the P465L/ob mice as indicated by significantly increased serum triglyceride levels compared with ob/ob controls in 5-week-old fed mice (Table 1).
Lipid accumulation in nonadipose tissues.
P465L/wt mice fed a chow diet did not reveal morphological signs suggestive of fatty liver. However, when fed a high-fat diet for 16 weeks, the P465L/wt mice developed increased liver weight (Table 1) and increased liver triglyceride content compared with wild-type controls (Fig. 5C). In addition, the P465L/ob mice had increased liver mass compared with ob/ob control mice by 12 weeks of age (Table 1). Gene expression profiling of P465L/ob liver revealed increased expression of lipogenic SREBP1c compared with ob/ob controls and, as in ob/ob mice, a sixfold increase in CD36 expression compared with wild-type controls. Also, an increase in glucose 6-phosphatase gene expression suggests an increase in hepatic glucose production in P465L/ob mice compared with ob/ob controls (Fig. 5D).
In contrast with the increased lipid deposition in liver, skeletal muscle of P465L/ob mice were protected against fat deposition. P465L/ob mice had a reduced amount of intramyocellular lipid compared with ob/ob controls, as shown by reduced oil red O staining (Fig. 5E). Furthermore, gene expression profiling of skeletal muscle revealed decreased expression of the lipogenic genes CD36 and SREBP1c, whereas we observed no changes in the expression of several oxidative genes (carnitine palmitol transferase I [CPTI], acyl-CoA synthetase [ACS], long- chain acyl CoA dehydrogenase [LCAD], and uncoupling protein 2 [UCP2]) compared with ob/ob controls (Fig. 5F).
DISCUSSION
Expression of the human dominant-negative PPAR mutation (P465L) in mouse produced an unexpected phenotype of normal adipose tissue mass and maintained insulin sensitivity (15). These observations were in stark contrast to the clinical phenotype described in humans and brought into question the relevance of mouse models to study PPAR and insulin sensitivity in humans. Here, we show that when expressed on a genetic background of severe obesity (ob/ob), dominant-negative P465L PPAR is associated with severe insulin resistance, impaired postprandial lipid metabolism, and reduced body fat and thus replicates the clinical characteristics of patients with equivalent dominant-negative mutations in PPAR (5,6).
PPAR is a potent regulator of lipid and carbohydrate metabolism, and biochemical mechanisms and target genes of PPAR are similar in rodents and humans (19,30). We speculate that the discrepant phenotypes in mice and humans arise from species-related differences in the contribution of specific organs, particularly adipose tissue, to energy homeostasis and the development of insulin resistance.
Unexpectedly, total body-fat content in P465L/wt mice was similar to that in wild-type controls when fed either a chow or a high-fat diet. Despite this observation, the effects of the P465L mutation on adipocyte differentiation were not completely homogeneous across the adipose depots. The gonadal adipose tissue depot was preferentially affected by defective PPAR activity compared with the other depots, a finding consistent with the previous P465L PPAR mouse model (15,31).
Biochemical effects of P465L PPAR were present in the lean phenotype as indicated by impaired transactivation of lipogenic genes, CD36 and aP2, in gonadal adipose tissue of P465L/wt mice compared with wt/wt controls. Despite these changes, the adipose tissue of P465L/wt mice was able to store lipid. In contrast, when challenged with the severe positive energy balance of the ob/ob background, the reduction in lipogenic gene expression (CD36 and aP2) in P465L/ob mice compared with that in ob/ob controls was associated with insufficient expansion of the adipose tissue. This was observed by a reduction in total body-fat mass and adipocyte area in P465L/ob compared with ob/ob mice, suggesting that defective PPAR prevents not only adipocyte recruitment but also adipocyte hypertrophy. Given that the adipogenic potential of preadipocytes isolated from mice with the P465L mutation is markedly impaired in vitro, the paradoxically normal appearance of the adipose tissue in the P465L PPAR mouse suggests that compensatory mechanisms are able to promote differentiation of adipocytes in vivo despite impairment of PPAR function. These mechanisms are not robust enough to compensate when adipose tissue expandability is challenged with excess energy availability (ob/ob background) and thus the defect in PPAR activity becomes functionally relevant.
As previously observed (15) and confirmed here, the P465L mutation expressed on a wild-type background does not confer insulin resistance even when challenged with high-fat feeding. The same phenotype was observed in dominant-negative L466A PPAR mice, which only developed a mild degree of insulin resistance when fed a high-fat diet for as long as 8 months (16). This conserved insulin sensitivity contrasts markedly with the severe insulin resistance observed in young, human patients with dominant-negative mutations in PPAR (5). Here, we show that the severe insulin resistance associated with the dominant-negative PPAR mutations in human patients is replicated in mice when expressed under conditions of extreme positive energy balance. It is well established that ob/ob mice develop insulin resistance and diabetes (23), but here we show that when adipose tissue expandability is limited as in the P465L/ob animals, there is marked insulin resistance, significantly worse than that in the ob/ob mice, a phenomenon that becomes evident as early as 4 weeks of age.
As in the human patients (32), circulating levels of PPAR-regulated adiponectin were significantly lower in mice with the P465L PPAR mutation on both backgrounds. Reduced adiponectin plasma levels do not correlate with impaired insulin sensitivity in the P465L/wt mice, suggesting that reduced circulating total adiponectin is not the main factor conferring insulin resistance.
Postprandial lipid clearance is impaired in P465L/wt mice, demonstrating that lipid handling is fundamentally altered by the PPAR mutation independently of adipose tissue differentiation or insulin sensitivity. This defect in postprandial lipid clearance is accentuated in P465L/ob mice, as indicated by a twofold increase in serum triglycerides in P465L/ob mice compared with ob/ob controls at 5 weeks of age. This phenotype replicates the elevated postprandial hypertriglyceridemia observed in human patients with dominant-negative PPAR mutations, resulting from an inability of adipose tissue to trap and store free fatty acids (5).
The P465L PPAR mice show increased liver triglyceride accumulation compared with controls when challenged with a high-fat diet. Development of fatty liver is also potentiated on the ob/ob background in contrast with previous evidence showing that liver-specific disruption of PPAR prevents hepatic steatosis in ob/ob mice (7). This apparent discrepancy may be reconciled by the fact that in liver-specific PPAR knockout mice the partitioning of fat toward the fully functional adipose tissue is probably facilitated. Impaired lipid clearance in the P465L PPAR mice exposes nonadipose tissues to increased plasma lipid concentrations. The accumulation of triglyceride-rich lipoproteins in the postprandial phase can be considered an independent risk factor for coronary heart disease and atherosclerosis (33), which is particularly relevant to human health as humans are in a postprandial state most of the day. Our results indicate that PPAR-related defects in adipose tissue function should be considered in those patients with marked postprandial hypertriglyceridemia.
We speculated that defects in insulin sensitivity may be linked to an insufficient fatty acid buffering capacity of adipose tissue. Lack of leptin action represents a paradigm of positive energy balance resulting from the combined effect of increased food intake, decreased energy expenditure, and peripheral impairment of catabolic processes such as fatty acid oxidation (34–36). Under these conditions, the adipose tissue is challenged to accommodate the energy surplus, resulting in copious fat and hypertrophic adipocytes. We show that P465L PPAR impairs lipid handling by restricting the adipogenic and lipogenic capacity of adipose tissue. Therefore, the ob/ob adipose tissue is able to expand more efficiently than that of the P465L/ob mouse in response to the increased energy demands. This restriction in adipose tissue expandability triggers the development of severe insulin resistance to levels above those produced by obesity alone.
With energy intake similar to that of ob/ob mice, P465L/ob mice are able to accumulate fat and become obese yet maintain a body mass 14% lower than that of ob/ob controls. Therefore, the P465L/ob mice could be considered inappropriately insulin resistant and dyslipidemic for their degree of obesity. Our results suggest that defective adipose tissue expansion mechanisms may lay behind the phenotype of human patients with an inappropriately high level of insulin resistance and hypertriglyceridemia for their degree of obesity. Although PPAR expressed outside adipose tissue may directly contribute to insulin sensitivity, our data favor the concept that the primary site of PPAR action is the adipose tissue where it acts as a nutrient sensor facilitating the expansion of the adipose tissue to safely store available energy, protecting peripheral tissues from the accumulation of metabolically toxic lipid intermediates (37) and thus improving insulin sensitivity.
A synergic effect between PPAR inactivation and lack of leptin may account for the severe insulin resistance in the P465L/ob mice. Short-term leptin therapy in ob/ob mice is known to improve insulin sensitivity before changes in body weight occur, suggesting that leptin improves insulin resistance either directly by acting on peripheral tissues enhancing oxidation or indirectly by a leptin-induced reduction in nutrient input. Our data suggest that a mismatch between nutrient load and storage capabilities is critical to the metabolic status of the P465L/ob mice. Experiments to tease apart the direct and indirect effects of leptin will be required to conclusively determine the mechanism of severe insulin resistance in the P465L/ob mice.
In summary, our analysis of the P465L PPAR mutation in mice indicates that genetic manipulations of PPAR in mice induce biochemical defects similar to those in humans but that may only become physiologically relevant in mice when challenged with an extreme positive energy balance. Similar considerations related to the role of adipose tissue controlling energy homeostasis in mice and humans may be relevant for the translatability of other metabolic findings between these two species.
ACKNOWLEDGMENTS
This work was supported by funding from Diabetes UK, the NOVO-European Association for the Study of Diabetes (EASD), and Hepatic and Adipose Tissue and Functions in the Metabolic Syndrome (HEPADIP) Contract FP6 (LSHM-CT2005-018734) and salary support from the Natural Sciences and Engineering Research Council of Canada (to S.L.G.) and the O. Arlotti Trust, Ferrara (to E.D.N.).
We acknowledge Sylvia Shelton, Margaret Blount, Martin Dale, Helen Westby, and Janice Carter for their exceptional technical assistance.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 Ingenium Pharmaceuticals, Martinsried, Germany
3 Department of Normal Human Morphology, Faculty of Medicine, Ancona University, Ancona, Italy
4 GSF-National Research Center for Environment and Health, Institute of Inhalation Biology, Muenchen-Neuherberg, Germany
5 Genetics Unit, Department of Biochemistry, University of Oxford, Oxford, U.K
6 Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, U.K
7 TNO-Prevention and Health, Division VBO, Leiden, the Netherlands
gWAT, gonadal white adipose tissue; PPAR, peroxisome proliferator–activated receptor; scWAT, subcutaneous white adipose tissue; WAT, white adipose tissue
ABSTRACT
Peroxisome proliferator–activated receptor (PPAR) is a key transcription factor facilitating fat deposition in adipose tissue through its proadipogenic and lipogenic actions. Human patients with dominant-negative mutations in PPAR display lipodystrophy and extreme insulin resistance. For this reason it was completely unexpected that mice harboring an equivalent mutation (P465L) in PPAR developed normal amounts of adipose tissue and were insulin sensitive. This finding raised important doubts about the interspecies translatability of PPAR-related findings, bringing into question the relevance of other PPAR murine models. Here, we demonstrate that when expressed on a hyperphagic ob/ob background, the P465L PPAR mutant grossly exacerbates the insulin resistance and metabolic disturbances associated with leptin deficiency, yet reduces whole-body adiposity and adipocyte size. In mouse, coexistence of the P465L PPAR mutation and the leptin-deficient state creates a mismatch between insufficient adipose tissue expandability and excessive energy availability, unmasking the deleterious effects of PPAR mutations on carbohydrate metabolism and replicating the characteristic clinical symptoms observed in human patients with dominant-negative PPAR mutations. Thus, adipose tissue expandability is identified as an important factor for the development of insulin resistance in the context of positive energy balance.
a thermodynamic point of view, the main factors involved in the development of obesity are food intake and energy expenditure, assuming that accumulation of fat is a passive event that follows a situation of positive energy balance. However, accumulation of fat in adipose tissue is an active process that requires the coordinated regulation of mechanisms involved in adipocyte differentiation and growth.
An important molecular regulator of adipose tissue development and expansion is peroxisome proliferator–activated receptor (PPAR) (1,2). PPAR is essential for adipogenesis, as shown by the inability of PPAR-null embryonic stem cells to undergo adipogenesis (3). PPAR is a modulator of insulin sensitivity. The link between PPAR-induced adipocyte differentiation and insulin sensitivity in humans is strengthened by the metabolic characterization of a small number of individuals with naturally occurring, dominant-negative mutations in PPAR. One such mutation, P467L, has been identified in the heterozygous state, and patients with this mutation suffer from severe insulin resistance, partial lipodystrophy, impaired postprandial lipid metabolism, fatty liver, and hypertension (4–6). To overcome the limitations imposed by the rarity of the human mutations, the function of PPAR has been studied extensively using mouse models with tissue/isoform-specific ablation of PPAR or knockin of PPAR mutations (7–17). These models have confirmed an important role for PPAR in controlling adipose tissue differentiation and function, as inducible knockout of PPAR in developed adipose tissue results in progressive reduction in fat depots (8,12) and heterozygosity for a PPAR-null mutation (18) reduces whole-body adipose tissue mass.
The first mouse model expressing a human-related mutation in PPAR (P465L), however, produced a surprising phenotype that has brought into question the translatability of results obtained using PPAR mouse models to humans. Particularly puzzling was the observation that the severe insulin resistance and partial lipodystrophy identified in human patients harboring dominant-negative PPAR mutations were not reproduced in a mouse model with the equivalent mutation (P465L) (15), a finding we confirm here using a new independently generated P465L PPAR mouse line. Because substantial evidence suggests that PPAR acts via similar biochemical mechanisms to promote lipogenesis and enhance insulin sensitivity in rodents and humans (19), we wondered why the effects of this mutant on fat deposition and insulin sensitivity were so different in human and mouse. In our opinion, this is a fundamental question that may be relevant to the interpretation of other discrepant metabolic phenotypes between humans and rodents (20–22).
Here, we show that when the P465L PPAR mutation is expressed in a lean background with limited requirements for adipose tissue expandability, the animals have normal adipose tissue mass and insulin sensitivity, even when challenged with high-fat feeding, a finding reported previously (15). Under these conditions the P465L PPAR mouse shows impaired postprandial lipid clearance and altered adipose tissue distribution, despite having normal adipose mass and insulin sensitivity. We hypothesized that the discrepancy in fat deposition and insulin sensitivity between human patients and mice expressing the PPAR mutations may be due to species-related differences in the balance between adipose tissue storage demands and adipose tissue expandability. Forcing a mismatch between adipose tissue storage capacity and energy availability may be key for the development of insulin resistance mediated by defective PPAR function; thus, we expressed the P465L PPAR mutation in a hyperphagic mouse background (ob/ob). In this context of positive energy balance, activity of the P465L PPAR mutant did not allow adequate adipose tissue expansion to fulfill storage requirements and severe insulin resistance developed, replicating the characteristic clinical symptoms observed in human patients with dominant-negative PPAR mutations.
RESEARCH DESIGN AND METHODS
Generation of P465L/wt and P465L/ob mice.
A targeting construct with a C-to-T point mutation in exon 6 of the PPAR gene was generated by site-directed mutagenesis. A neomycin selection cassette flanked by two FRT sites was incorporated into intron 6 with two thymidine kinase genes positioned at the 5' and 3' ends of the flanking sequence (Fig. 1A). The targeting construct was transfected into a TBV2 (129/SvP) ES cell line and homologously recombined clones were selected for using G418/ganciclovir and identified by Southern blot analysis (Fig. 1B). Chimeras with successful germ-line transmission were obtained and bred to C57/BL6 mice to produce mice heterozygous for the point mutation. Removal of the neomycin selection cassette in intron 6 was performed by breeding mice heterozygous for the P465L PPAR mutation to FLP transgenic mice.
Animals were genotyped using a three-primer PCR strategy that identified the wild-type allele (498 bp) and the recombined allele containing the P465L mutation (641 bp) and, if present, detected the P465L allele with the neomycin cassette (391 bp) (Fig. 1C). Homozygosity for P465L PPAR resulted in embryonic lethality. Expression levels of the wild-type and mutant alleles were determined by a real-time PCR strategy using a common primer set with unique probes that differed by the single base pair in exon 6.
To generate mice with the P465L PPAR mutation on an ob/ob background, mice heterozygous for the P465L PPAR mutation were crossed to mice heterozygous for a point mutation in the leptin gene (ob) (23). Mice heterozygous for both P465L PPAR and the leptin mutation were further crossed to generate four experimental genotypes: wild type for PPAR and leptin (wt/wt), heterozygous for P465L PPAR and wild type for leptin (P465L/wt), wild type for PPAR and homozygous for the leptin mutation (ob/ob), and heterozygous for P465L PPAR and homozygous for the leptin mutation (P465L/ob). All animals used were of mixed C57/BL6/129SvJ background and unless otherwise stated were fed a standard mouse chow ad libitum, with 12-h light/dark cycles. All comparisons were made using littermate controls. Experimental protocols requiring the use of animals were approved by the U.K. home office and the University of Cambridge.
Body composition and food consumption.
Body mass was recorded for 12 weeks, and food consumption of individually housed animals was monitored from weaning for 10 days. Body composition was determined in dead animals using the Lunar PIXImus two-bone densitometer (dual-energy X-ray absorptiometry; GE Medical Systems, Madison, WI). Individual fat-pad mass was measured at dissection.
Histology.
Tissues were dissected and fixed in 10% formalin, or animals were perfused with 4% paraformaldehyde. Tissue was either cryoprotected (20% sucrose) and frozen in chilled isopentane or embedded in paraffin and sectioned using a cryostat (Leica CM1900; Leica Microsystems, Milton Keynes, U.K.) or standard microtome (Leica RM2125RT; Leica Microsystems), respectively. Cryosectioned tissue was stained for lipid with oil red O.
Morphometric analysis of adipocytes included calculation of adipocyte area (square micrometers) from gonadal and inguinal subcutaneous adipose tissue depots. Image analysis software (analySIS) was used to analyze the images and calculate adipocyte area. Two images per section (10x magnification), two sections per mouse, and four mice per genotype were used to calculate the average adipocyte area for the two adipose tissue depots.
Biochemistry.
Biochemical analysis of serum or tissue extracts included measurements of glucose, total cholesterol, HDL, triglycerides, insulin (DRG Diagnostics International), free fatty acids (Roche, Welwyn Garden City, U.K.), adiponectin (B-Bridge International), and leptin (R&D Systems, Abingdon, U.K.). Triglyceride content of liver was determined using a previously described extraction method (24).
Gene expression profiling.
Individual tissues (gonadal white adipose tissue [gWAT], skeletal muscle, and liver) were dissected and frozen immediately in liquid nitrogen. Total RNA was extracted using RNA STAT-60 (Tel-Test). cDNA was reverse transcribed from 500 ng of total RNA (Promega, Southampton, U.K.). Gene expression levels were determined using real-time PCR (TaqMan; Applied Biosystems, Warrington, U.K.) with primers and probes designed to specific mRNAs (Sigma, Haverhill, U.K.). PCR conditions were as follows: 1 cycle of 50°C for 2 min and 95°C for 10 min and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Values were normalized against 18 S RNA and expressed as relative arbitrary units.
Evaluation of carbohydrate metabolism.
Blood glucose was measured (OneTouch Ultra; LifeScan Johnson & Johnson, High Wycombe, U.K.) from the tail vein of 3-week-old mice before weaning (preweaning) and in fed and fasted 4-week-old mice. Glucose tolerance tests were performed in nondiabetic mice after an overnight (16 h) fast. Blood glucose levels were measured (OneTouch Ultra) before and after (10, 20, 30, 60, 120, and 180 min) an intraperitoneal injection of D-glucose (1 g/kg). Insulin tolerance tests were performed after a 6-h fast. Blood glucose was measured before and after (10, 20, 30, and 60 min) an intraperitoneal injection of insulin (0.75 units/kg). Values are expressed relative to basal (6-h fast) blood glucose. Hyperinsulinemic-euglycemic clamps were performed on adult wild-type (n = 10) and P465L PPAR mice (n = 10) only. Clamps, subsequent tissue preparation, and calculations were performed as described previously (25,26).
Energy expenditure.
Oxygen consumption rates were determined in 6-week-old mice using closed calorimetry. The metabolic rate was determined over a 5-h period and expressed as VO2 (milliliters per minute per kilogram0.75).
Primary culture of preadipocytes.
Preadipocytes were isolated from gWAT and differentiated as described previously (27) with the inclusion of 10% fetal bovine serum in the differentiation media. Adipose tissue pads from three mice were pooled for wt/wt and P465L/wt mice in three separate experiments (Fig. 3E). In ob/ob and P465L/ob mice, fat pads from individual mice were isolated at 5 weeks of age and differentiated as above. Differentiation success was determined after 8 days of differentiation by oil red O staining and/or measurement of aP2 mRNA expression in adipocytes (Fig. 3E and online appendix Fig. 1 [available at http://diabetes.diabetesjournals.org]).
Lipid clearance.
To assess postprandial lipid clearance, animals were fasted overnight and dosed orally with 200 μl of olive oil. Serum triglycerides and free fatty acids were determined before the lipid dose and at 1, 2, 4, and 8 h after the lipid load.
High-fat feeding.
A high-fat diet (45% kcal as fat; Research Diets, New Brunswick, NJ) was fed to wild-type and P465L PPAR animals for 16 weeks after weaning.
Statistical analysis.
Data are presented as means ± SE. Statistical significance between genotypes was determined using a one-way ANOVA and between relevant pairs of genotypes using unpaired Student’s t tests. Significance was declared for P values <0.05 (GraphPad Instat 3, Microsoft Excel).
RESULTS
Characterization of the P465L PPAR allele.
Southern and PCR analysis of genomic DNA identified the recombined P465L PPAR allele (Fig. 1A–C). Subsequent sequencing of genomic DNA confirmed the presence of the C-to-T point mutation in exon 6 of the PPAR gene.
Homozygous transmission of P465L PPAR results in embryonic lethality.
Heterozygous P465L PPAR matings produced an altered Mendelian ratio (wt/wt 28%, P465L/wt 72%, and P465L/P465L 0%), demonstrating that homozygous transmission of the P465L PPAR allele was embryonic lethal. Previous in vitro studies have demonstrated that the dominant-negative activity of the ligand-binding domain PPAR mutants was associated with impaired coactivator recruitment and transactivation (6,28,29). Quantification of allelic expression in heterozygous animals revealed that transcription of the mutant allele was not impaired (Fig. 1D). Generation of mice with P465L PPAR on an ob/ob background produced the four experimental genotypes at the expected (2 = 0.2517) Mendelian ratio (wt/wt 6.4%, P465L/wt 13.5%, ob/ob 13.5%, and P465L/ob 15.1%).
P465L PPAR promotes abnormal adipose tissue distribution and prevents adipose tissue expansion when expressed on an obese background.
P465L PPAR expressed on a lean background does not affect overall body mass (Fig. 2A and Table 1) or total body fat content (Fig. 2B), although it does cause a redistribution of white adipose tissue (WAT), with a significantly decreased ratio of gWAT to inguinal subcutaneous WAT (scWAT) mass when compared with wild-type controls (Fig. 3C). Additionally, preadipocytes isolated from WAT of mice with the P465L PPAR mutation (P465L/wt and P465L/ob) had impaired differentiation capability in vitro compared with animals wild type for PPAR (wt/wt and ob/ob) (Fig. 3E and online appendix Fig. 1).
When P465L PPAR was expressed on an ob/ob background, there was a significant decrease in body mass that was evident after 5 weeks compared with ob/ob controls (Fig. 2A). By 12 weeks, body mass of P465L/ob mice was 14% (females, Fig. 2A) and 12% (males, data not shown) lower than that of ob/ob controls. This change in body weight was due to reduced fat mass (Fig. 2B). P465L/ob mice consumed the same amount of oxygen and were as hyperphagic as ob/ob controls (Fig. 2C and D). By 5 weeks, P465L/ob mice had reduced scWAT and gWAT (Fig. 3A), and white adipoctyes from these depots were smaller compared with those from ob/ob mice (Fig. 3B), suggesting that adipocytes were unable to expand appropriately in response to the increased energy availability.
P465L PPAR prevents appropriate expression of lipogenic genes.
Gene expression profiling in WAT revealed that the fatty acid binding protein aP2 and the fatty acid carrier CD36 were significantly reduced in gWAT of mice carrying the P465L PPAR mutation on both wild-type and ob/ob backgrounds (Fig. 3D) compared with controls.
P465L PPAR only confers insulin resistance in the context of severe positive energy balance.
Unlike human patients with dominant-negative PPAR mutations, P465L PPAR mice on a lean background displayed normal insulin sensitivity as demonstrated by glucose tolerance tests (Fig. 4A) and hyperinsulinemic clamps (Fig. 4B and C). Similarly, the P465L mutation did not exacerbate high-fat feeding–induced insulin resistance as shown by glucose and insulin tolerance tests (Fig. 4D and E). In contrast, when the P465L mutation is expressed on an ob/ob background, mice develop severe insulin resistance. As early as 5 weeks of age, the P465L/ob mice are more insulin resistant than the ob/ob mice, demonstrated by extremely high blood glucose and serum insulin levels in the fed state (Table 2) and a severely blunted response to an insulin tolerance test (Fig. 4F). Of interest, adiponectin was decreased to a similar extent in mice carrying the P465L PPAR mutation on either a wild-type or ob/ob background, suggesting that the failure in insulin sensitivity was not related to quantitative differences in adiponectin plasma levels (Table 1).
Impaired postprandial lipid clearance despite normal insulin sensitivity in P465L PPAR mice.
Despite showing normal body mass and insulin sensitivity on a lean wild-type background, the P465L PPAR mice had a significant increase in serum triglyceride levels in the fed state with a high-fat diet compared with wild-type controls (Table 1). An oral lipid load test showed that P465L/wt mice were unable to clear triglycerides and fatty acids as efficiently as wild-type controls (Fig. 5A and B). This defective clearance of lipids was magnified in the P465L/ob mice as indicated by significantly increased serum triglyceride levels compared with ob/ob controls in 5-week-old fed mice (Table 1).
Lipid accumulation in nonadipose tissues.
P465L/wt mice fed a chow diet did not reveal morphological signs suggestive of fatty liver. However, when fed a high-fat diet for 16 weeks, the P465L/wt mice developed increased liver weight (Table 1) and increased liver triglyceride content compared with wild-type controls (Fig. 5C). In addition, the P465L/ob mice had increased liver mass compared with ob/ob control mice by 12 weeks of age (Table 1). Gene expression profiling of P465L/ob liver revealed increased expression of lipogenic SREBP1c compared with ob/ob controls and, as in ob/ob mice, a sixfold increase in CD36 expression compared with wild-type controls. Also, an increase in glucose 6-phosphatase gene expression suggests an increase in hepatic glucose production in P465L/ob mice compared with ob/ob controls (Fig. 5D).
In contrast with the increased lipid deposition in liver, skeletal muscle of P465L/ob mice were protected against fat deposition. P465L/ob mice had a reduced amount of intramyocellular lipid compared with ob/ob controls, as shown by reduced oil red O staining (Fig. 5E). Furthermore, gene expression profiling of skeletal muscle revealed decreased expression of the lipogenic genes CD36 and SREBP1c, whereas we observed no changes in the expression of several oxidative genes (carnitine palmitol transferase I [CPTI], acyl-CoA synthetase [ACS], long- chain acyl CoA dehydrogenase [LCAD], and uncoupling protein 2 [UCP2]) compared with ob/ob controls (Fig. 5F).
DISCUSSION
Expression of the human dominant-negative PPAR mutation (P465L) in mouse produced an unexpected phenotype of normal adipose tissue mass and maintained insulin sensitivity (15). These observations were in stark contrast to the clinical phenotype described in humans and brought into question the relevance of mouse models to study PPAR and insulin sensitivity in humans. Here, we show that when expressed on a genetic background of severe obesity (ob/ob), dominant-negative P465L PPAR is associated with severe insulin resistance, impaired postprandial lipid metabolism, and reduced body fat and thus replicates the clinical characteristics of patients with equivalent dominant-negative mutations in PPAR (5,6).
PPAR is a potent regulator of lipid and carbohydrate metabolism, and biochemical mechanisms and target genes of PPAR are similar in rodents and humans (19,30). We speculate that the discrepant phenotypes in mice and humans arise from species-related differences in the contribution of specific organs, particularly adipose tissue, to energy homeostasis and the development of insulin resistance.
Unexpectedly, total body-fat content in P465L/wt mice was similar to that in wild-type controls when fed either a chow or a high-fat diet. Despite this observation, the effects of the P465L mutation on adipocyte differentiation were not completely homogeneous across the adipose depots. The gonadal adipose tissue depot was preferentially affected by defective PPAR activity compared with the other depots, a finding consistent with the previous P465L PPAR mouse model (15,31).
Biochemical effects of P465L PPAR were present in the lean phenotype as indicated by impaired transactivation of lipogenic genes, CD36 and aP2, in gonadal adipose tissue of P465L/wt mice compared with wt/wt controls. Despite these changes, the adipose tissue of P465L/wt mice was able to store lipid. In contrast, when challenged with the severe positive energy balance of the ob/ob background, the reduction in lipogenic gene expression (CD36 and aP2) in P465L/ob mice compared with that in ob/ob controls was associated with insufficient expansion of the adipose tissue. This was observed by a reduction in total body-fat mass and adipocyte area in P465L/ob compared with ob/ob mice, suggesting that defective PPAR prevents not only adipocyte recruitment but also adipocyte hypertrophy. Given that the adipogenic potential of preadipocytes isolated from mice with the P465L mutation is markedly impaired in vitro, the paradoxically normal appearance of the adipose tissue in the P465L PPAR mouse suggests that compensatory mechanisms are able to promote differentiation of adipocytes in vivo despite impairment of PPAR function. These mechanisms are not robust enough to compensate when adipose tissue expandability is challenged with excess energy availability (ob/ob background) and thus the defect in PPAR activity becomes functionally relevant.
As previously observed (15) and confirmed here, the P465L mutation expressed on a wild-type background does not confer insulin resistance even when challenged with high-fat feeding. The same phenotype was observed in dominant-negative L466A PPAR mice, which only developed a mild degree of insulin resistance when fed a high-fat diet for as long as 8 months (16). This conserved insulin sensitivity contrasts markedly with the severe insulin resistance observed in young, human patients with dominant-negative mutations in PPAR (5). Here, we show that the severe insulin resistance associated with the dominant-negative PPAR mutations in human patients is replicated in mice when expressed under conditions of extreme positive energy balance. It is well established that ob/ob mice develop insulin resistance and diabetes (23), but here we show that when adipose tissue expandability is limited as in the P465L/ob animals, there is marked insulin resistance, significantly worse than that in the ob/ob mice, a phenomenon that becomes evident as early as 4 weeks of age.
As in the human patients (32), circulating levels of PPAR-regulated adiponectin were significantly lower in mice with the P465L PPAR mutation on both backgrounds. Reduced adiponectin plasma levels do not correlate with impaired insulin sensitivity in the P465L/wt mice, suggesting that reduced circulating total adiponectin is not the main factor conferring insulin resistance.
Postprandial lipid clearance is impaired in P465L/wt mice, demonstrating that lipid handling is fundamentally altered by the PPAR mutation independently of adipose tissue differentiation or insulin sensitivity. This defect in postprandial lipid clearance is accentuated in P465L/ob mice, as indicated by a twofold increase in serum triglycerides in P465L/ob mice compared with ob/ob controls at 5 weeks of age. This phenotype replicates the elevated postprandial hypertriglyceridemia observed in human patients with dominant-negative PPAR mutations, resulting from an inability of adipose tissue to trap and store free fatty acids (5).
The P465L PPAR mice show increased liver triglyceride accumulation compared with controls when challenged with a high-fat diet. Development of fatty liver is also potentiated on the ob/ob background in contrast with previous evidence showing that liver-specific disruption of PPAR prevents hepatic steatosis in ob/ob mice (7). This apparent discrepancy may be reconciled by the fact that in liver-specific PPAR knockout mice the partitioning of fat toward the fully functional adipose tissue is probably facilitated. Impaired lipid clearance in the P465L PPAR mice exposes nonadipose tissues to increased plasma lipid concentrations. The accumulation of triglyceride-rich lipoproteins in the postprandial phase can be considered an independent risk factor for coronary heart disease and atherosclerosis (33), which is particularly relevant to human health as humans are in a postprandial state most of the day. Our results indicate that PPAR-related defects in adipose tissue function should be considered in those patients with marked postprandial hypertriglyceridemia.
We speculated that defects in insulin sensitivity may be linked to an insufficient fatty acid buffering capacity of adipose tissue. Lack of leptin action represents a paradigm of positive energy balance resulting from the combined effect of increased food intake, decreased energy expenditure, and peripheral impairment of catabolic processes such as fatty acid oxidation (34–36). Under these conditions, the adipose tissue is challenged to accommodate the energy surplus, resulting in copious fat and hypertrophic adipocytes. We show that P465L PPAR impairs lipid handling by restricting the adipogenic and lipogenic capacity of adipose tissue. Therefore, the ob/ob adipose tissue is able to expand more efficiently than that of the P465L/ob mouse in response to the increased energy demands. This restriction in adipose tissue expandability triggers the development of severe insulin resistance to levels above those produced by obesity alone.
With energy intake similar to that of ob/ob mice, P465L/ob mice are able to accumulate fat and become obese yet maintain a body mass 14% lower than that of ob/ob controls. Therefore, the P465L/ob mice could be considered inappropriately insulin resistant and dyslipidemic for their degree of obesity. Our results suggest that defective adipose tissue expansion mechanisms may lay behind the phenotype of human patients with an inappropriately high level of insulin resistance and hypertriglyceridemia for their degree of obesity. Although PPAR expressed outside adipose tissue may directly contribute to insulin sensitivity, our data favor the concept that the primary site of PPAR action is the adipose tissue where it acts as a nutrient sensor facilitating the expansion of the adipose tissue to safely store available energy, protecting peripheral tissues from the accumulation of metabolically toxic lipid intermediates (37) and thus improving insulin sensitivity.
A synergic effect between PPAR inactivation and lack of leptin may account for the severe insulin resistance in the P465L/ob mice. Short-term leptin therapy in ob/ob mice is known to improve insulin sensitivity before changes in body weight occur, suggesting that leptin improves insulin resistance either directly by acting on peripheral tissues enhancing oxidation or indirectly by a leptin-induced reduction in nutrient input. Our data suggest that a mismatch between nutrient load and storage capabilities is critical to the metabolic status of the P465L/ob mice. Experiments to tease apart the direct and indirect effects of leptin will be required to conclusively determine the mechanism of severe insulin resistance in the P465L/ob mice.
In summary, our analysis of the P465L PPAR mutation in mice indicates that genetic manipulations of PPAR in mice induce biochemical defects similar to those in humans but that may only become physiologically relevant in mice when challenged with an extreme positive energy balance. Similar considerations related to the role of adipose tissue controlling energy homeostasis in mice and humans may be relevant for the translatability of other metabolic findings between these two species.
ACKNOWLEDGMENTS
This work was supported by funding from Diabetes UK, the NOVO-European Association for the Study of Diabetes (EASD), and Hepatic and Adipose Tissue and Functions in the Metabolic Syndrome (HEPADIP) Contract FP6 (LSHM-CT2005-018734) and salary support from the Natural Sciences and Engineering Research Council of Canada (to S.L.G.) and the O. Arlotti Trust, Ferrara (to E.D.N.).
We acknowledge Sylvia Shelton, Margaret Blount, Martin Dale, Helen Westby, and Janice Carter for their exceptional technical assistance.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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