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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:1394-1398 (2005)
© 2005 The Gerontological Society of America

Effects of Caloric Restriction and Growth Hormone Resistance on the Expression Level of Peroxisome Proliferator-Activated Receptors Superfamily in Liver of Normal and Long-Lived Growth Hormone Receptor/Binding Protein Knockout Mice

Michal M. Masternak1,, Khalid A. Al-Regaiey1,4, Marc Michael Del Rosario Lim1, Vanesa Jimenez-Ortega1,2, Jacob A. Panici1, Michael S. Bonkowski1, John J. Kopchick3 and Andrzej Bartke1

1 Departments of Internal Medicine and Physiology, Geriatrics Research, Southern Illinois University School of Medicine, Springfield.
2 Complutense University School of Medicine, Department of Biochemistry & Molecular Biology II, Madrid, Spain.
3 Edison Biotechnology Institute, Department of Biomedical Sciences, Ohio University, Athens.
4 Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia.

Address correspondence to Michal M. Masternak, PhD, Southern Illinois University, School of Medicine, Geriatrics Research, Department of Internal Medicine, 801 N. Rutledge St., Room 4389, P.O. Box 19628, Springfield, IL 62794-9628. E-mail: mmasternak{at}siumed.edu


   Abstract
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Growth hormone receptor/binding protein knockout (GHR-KO) mice live approximately 40% longer than their normal siblings do. These mice have dramatically reduced plasma levels of insulin-like growth factor 1 (IGF1) and enhanced insulin sensitivity. We examined the expression level of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors family genes in the livers of normal and GHR-KO mice fed ad libitum or subjected to long-term 30% caloric restriction (CR). The levels of PPAR{gamma} and PPAR{alpha} messenger RNA and proteins and the levels of retinoid X receptors messenger RNA were elevated in long-lived GHR-KO mice as compared to normal mice with no major effect of CR in either genotype. These findings suggest that enhanced insulin sensitivity of GHR-KO mice may be related to the altered actions of PPARs family members in the liver. The results also indicate that CR may increase insulin sensitivity through a different mechanism.

GROWTH hormone receptor/binding protein knockout (GHR-KO) mice are characterized by resistance or insensitivity to GH action and decreased insulin-like growth factor 1 (IGF1) levels (1). The deficiency of GH and IGF1, as well as GH resistance is known to extend longevity in mice (2). For example, Ames dwarf mice (Prop1df) and Snell dwarf mice (Pit1dw) that lack GH, prolactin, and thyroid-stimulating hormone (TSH) live more that than 40% longer than their normal siblings do (2–4). The GHR-KO (KO in text and figures) mice are also characterized by markedly extended longevity compared to normal siblings (5). In our colony, the average life span of GHR-KO mice is approximately 36 months as compared to 26 months in normal animals. The maximal life span is approximately 50 months in GHR-KO mice compared to 40 months in normal mice. GH-resistant KO mice have dramatically reduced plasma IGF1 and insulin levels in addition to low levels of blood glucose. The IGF1 receptor is the mediator of IGF1 action. Studies of IGF1 receptor–heterozygous KO mice have shown that this partial disruption extends female longevity by 33% (6).

The most effective intervention that extends longevity and delays aging is caloric restriction (CR). CR reduces body weight and plasma IGF1, insulin, glucose, and thyroid hormone levels. We have shown that, in long-lived Ames dwarf mice, CR leads to a further extension of life span, similar to the effects of CR in their normal siblings (7). Studies in mutant mice and in animals subjected to CR add to the evidence that GH as well as insulin/IGF1 signaling are important in the control of aging in different species (8).

In this study we analyzed expression of peroxisome proliferator-activated receptors (PPARs) family and the retinoid X receptors (RXRs) genes in the livers of GHR-KO mice and their normal siblings subjected to long term CR. PPARs are members of the nuclear receptor superfamily. These ligand-dependent transcription factors regulate expression of target genes by binding to specific peroxisome proliferator response elements (PPREs) in cis-acting enhancer sites of respective genes. PPARs form heterodimers with RXR and bind to their specific PPRE (9). There are three known isoforms in the PPARs' family, and all are encoded by separate genes: PPAR{gamma}, PPAR{alpha}, and PPARß/{delta} (also known as PPARß or PPAR{delta}). The most studied gene of this superfamily, PPAR{gamma}, is a target receptor for thiazolidinediones. They also are insulin sensitizers, and are used as a treatment of type 2 diabetic patients (10–13). Thiazolidinediones improve glucose homeostasis, primarily by enhancing the action of insulin in skeletal muscle (14).

PPAR{alpha} is highly expressed in the liver. Its main function is to regulate fatty acid (FA) oxidation by regulating the expression of genes encoding enzymes involved in the FA metabolic pathway, including ß and {omega} oxidation. PPAR{alpha} interacts with PPREs present in the promoter regions of genes encoding enzymes involved in this metabolic pathway (15). PPAR{alpha} is also known to control the levels of high density lipoprotein cholesterol through regulating apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) gene expression. PPAR{alpha}-deficient mice have increased level of total and high density lipoprotein cholesterol (16). It is also known that PPAR{alpha} downregulates triglyceride levels in animals and men.

The third gene in the PPARs family, PPARß/{delta}, is still not well explored. However, it is known that it functions in epidermal maturation and skin-wound healing (17,18). Treatment of obese mice with a PPARß/{delta} agonist results in dramatic lipid depletion in tissues (19), indicating that PPARß/{delta} regulates lipid metabolism. Moreover, PPARß/{delta}-deficient mice subjected to a high fat diet had reduced energy uncoupling, which predisposes individuals to obesity. These findings suggest that PPARß/{delta} regulates FA oxidation and energy production, which can make it an important factor as a fat burner (19).

RXRs are characterized by a high potential to heterodimerize with many nuclear receptors. PPARs are among those nuclear receptors which need to form heterodimers with RXRs to be active and bind their PPREs. RXR isoforms are encoded by three separate genes: RXR{alpha}, RXRß, and RXR{gamma} (20). In this study, we analyzed the expression of PPARs and RXR family members in livers collected from normal and long-lived GHR-KO mice fed either ad libitum or subjected to long-term CR. We hypothesize that these genes function in insulin action and fat metabolism and contribute to the long-lived phenotype of mice.


   MATERIALS AND METHODS
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 Materials and Methods
Results and Discussion
 References
 
Animals
Normal and GHR-KO mice used in the present study were produced in our breeding colony derived from animals provided by J. J. K. (Ohio University, Athens). All animal protocols for this study were approved by the Southern Illinois University Laboratory Animal Care and Use Committee. The animals were maintained under temperature- and light-controlled conditions (20–23°C, 12-h light/dark cycle). KO (–/–) males and heterozygous (+/–) females were mated to produce GHR-KO mice. Animals were group housed according to sex and phenotype. At 8 weeks of age, animals were matched for average body weight (BW) within the phenotype and then were either ad libitum fed (AL) or placed on CR. AL animals were allowed unlimited access to food. CR animals were subjected to 30% CR as described previously (21). Thus, there were four experimental groups: normal-AL (N-AL), N-CR, KO-AL, and KO-CR. Each group contained eight males. At the age of 21 months, the animals were anesthetized using isoflurane, blood was taken by cardiac puncture, and mice were killed by decapitation. Livers were collected and rapidly frozen on dry ice.

Extraction of Messenger RNA and Complementary DNA Synthesis
After homogenizing the liver in phosphate-buffered saline, total RNA was extracted using the phenol–chloroform procedure of Chomczynski and Sacchi (22). Total RNA quantity and quality were analyzed on agarose gel using electrophoresis. The synthesis of complementary DNA was performed from 2 µg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol.

Real-Time Polymerase Chain Reaction
The messenger RNA (mRNA) expression was analyzed by real-time polymerase chain reation using the Smart Cycler instrument (Cepheid, Sunnyvale, CA) with iQ SYBR Green Supermix (Bio-Rad). Details of the procedure and the list of primers were reported previously (23).

Western Blots
After homogenizing the livers in phosphate-buffered saline, 250 µl was taken for RNA extraction and the remaining homogenate was used for protein extraction. Western blot reaction was performed using PPAR{gamma}, PPAR{alpha}, and PPAR{delta} antibodies (Sigma, St. Louis, MO) according to a protocol described earlier (24). To control for equal lane loading, monoclonal anti-ß-actin antibody (Sigma) was used.

Statistical Analysis
Data are expressed as mean ± standard error of the mean. To evaluate the effects of the phenotype and diet, two-way analysis of variance was used with Fisher's protected least significant difference (PLSD) as a post hoc test. A t test was used to evaluate the effects of diet within the phenotypes. A value of p <.05 was considered significant.


   RESULTS AND DISCUSSION
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KO mice showed a statistically significant increase in the expression of PPAR{gamma}, at both mRNA and protein levels, in comparison to normal siblings (p <.004 and p <.0001, respectively) (Figure 1). Increased expression of PPAR{gamma}, presumably acting as a contributory factor, is consistent with high insulin sensitivity of these hyper insulin-sensitive KO mutants. PPAR{gamma} action in liver is probably a factor responsible for the coexistence of low glucose and dramatically reduced insulin levels in these animals. Our findings were similar in liver of other long-lived, highly insulin-sensitive animals [Ames dwarf (df/df) mutant mice (25)], and we proposed that enhanced sensitivity to insulin represents one of the mechanisms responsible for extended longevity of KO mice. CR is known to increase insulin sensitivity. We recently reported that CR increased insulin sensitivity in both normal and KO mice (23,24). However, in the present study, CR did not affect PPAR{gamma} levels in the liver of normal or KO mice, again resembling the findings in df/df mutants (25). These data indicate that improvement of the insulin sensitivity in KO and normal animals by CR is not mediated by PPAR{gamma} activation in the liver.



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  		Figure 1. Expression of peroxisome proliferator-activated receptors (PPARs) superfamily genes in liver tissue of normal (N) and growth hormone receptor/binding protein knockout (KO) mice fed ad libitum (AL) or subjected to 30% caloric restriction (CR). The data from real-time polymerase chain reaction were normalized by the housekeeping gene ß-2-microglobulin. Equal loading of protein for western blots was verified using ß-actin. Means ± standard error of the mean. a,b,c: Values that do not share the same letter in the superscript are significantly different (p <.05)

 
Both phenotype and diet affected the level of both mRNA and protein for PPAR{alpha} expression (p <.002, p <.018 and p <.0003, p <.006, respectively) (Figure 1). Increased expression of PPAR{alpha} in KO mice compared to that in normal siblings suggests changes of FA oxidation. FA metabolism plays an important role in insulin action. Increased PPAR{alpha} level could increase usage of FA which, in turn, could prevent obesity and insulin resistance. Increased expression of PPAR{alpha} could also be responsible for the reduced total cholesterol level in plasma of KO mice (23). CR increased the levels of PPAR{alpha} mRNA and proteins in normal animals comparable to levels measured in the long-lived mutants. Moreover, CR led to a further increase in the level of PPAR{alpha} in KO mice. We believe that these changes in the hepatic expression of PPAR{alpha} correlate with, and presumably contribute to, the increased sensitivity of CR animals to insulin, reported in our previous studies (23, 24). Higher fat utilization could also help to reduce the cholesterol level in N-CR mice to the level present in KO-AL and KO-CR mice (23).

Analysis of PPARß/{delta} expression data using two-way analysis of variance revealed significant effects of CR at the mRNA level (p <.03) in contrast to no changes in protein levels. However, t tests indicate that CR decreased both the mRNA and protein level of PPARß/{delta} in normal animals (p <.016 and p <.023, respectively). In contrast, there was no CR effect on PPARß/{delta} expression in KO animals. It is interesting that the levels of PPARß/{delta} protein were decreased in KO mice in comparison to those in the normal controls (p <.022), with no effects of the phenotype on the corresponding mRNA expression (Figure 1).

The lower level of PPARß/{delta} in KO mice compared to that in normal siblings is very surprising. Previous findings in the PPARß/{delta}-deficient mice that showed reduced energy uncoupling suggest that this condition could be expected to be harmful rather than beneficial (19). We suspect that it may be balanced by PPAR{alpha} action on lipid metabolism. Moreover, reduced PPARß/{delta} in N-CR mice in comparison to N-AL mice can indicate that reduced caloric intake can exert negative feedback on PPARß/{delta} level.

The results of real-time polymerase chain reaction analysis of RXR{gamma}, RXR{alpha}, and RXRß/{delta} revealed a consistent and significant phenotype effect. KO animals expressed more mRNA for these proteins than did their normal siblings (p <.014, p <.015, and p <.002, respectively). CR had no effect on the expression of the RXRs genes (Figure 2). Increased levels of RXRs in KO animals imply a greater likelihood of forming heterodimers with PPARs genes which would enhance activation of PPARs in long-lived KO mice.



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  		Figure 2. Expression of retinoid X receptors (RXRs) genes in liver tissue of normal (N) and growth hormone receptor/binding protein knockout (KO) mice fed ad libitum (AL) or subjected to 30% caloric restriction (CR). The data from real-time polymerase chain reaction were normalized by housekeeping gene, ß-2-microglobulin. Means ± standard error of the mean. a,b: Values that do not share the same letter in the superscript are significantly different (p <.05)

 
Summary
The present data indicate that the altered expression of the PPARs family members in the liver could contribute to the effects of GH resistance and CR on the molecular pathways of FA metabolism and insulin action. It is interesting that the results of this study, in comparison with the results of our previous study of the effects of CR on PPARs genes in skeletal muscle of GHR-KO and normal mice, reveal major differences between these two insulin target tissues (23). A better understanding of the regulation of lipid metabolism and the interrelationship to insulin action in different tissues by the PPARs family should help in elucidating the actions of longevity genes and CR on aging.


   Acknowledgments
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 Results and Discussion
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This project was supported by National Institute on Aging (NIA) grants AG 19899 and U19 AG023122, by the Ellison Medical Foundation, and by the Southern Illinois University Geriatrics Medicine and Research Initiative. J. J. K. is supported, in part, by the State of Ohio's Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by DiAthegen, LLC, and by NIA grant AG19889.

We thank Marty Wilson for laboratory assistance. We also thank Anna Masternak and Dr. Robert G. Struble for helping with the editing of the manuscript.


   Footnotes
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Decision Editor: James R. Smith, PhD

Received April 4, 2005

Accepted May 11, 2005


   References
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 Abstract
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 References
 

  1. Zhou Y, Xu BC, Maheshwari HG, et al. A mammalian model of Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci U S A. 1997;94:13215-13220.[Abstract/Free Full Text]
  2. Bartke A, Heiman M, Turyn D, Dominici F, Kopchick JJ. The role of growth hormone signaling as the control of ageing. In: Rainer HS, Mocchegiani E, eds. The Neuroendocrine Immune Network in Ageing: NeuroImmune Biology, 4. Elsevier; 2004:123–137.
  3. Brown-Borg HM, Borg KE, Meliska CJ, Bartke A. Dwarf mice and the ageing process. Nature. 1996;384:33.[Medline]
  4. Flurkey K, Papaconstantinou J, Miller RA, Harrison DA. Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production. Proc Natl Acad Sci U S A. 2001;98:6736-6741.[Abstract/Free Full Text]
  5. Coschigano KT, Clemmons D, Bellush LL, Kopchick JJ. Assessment of growth parameters and life span of GHR/BP gene disrupted mice. Endocrinology. 2000;141:2608-2613.[Abstract/Free Full Text]
  6. Holzenberger M, Dupont J, Ducos B, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2003;421:182-187.[Medline]
  7. Bartke A, Wright JC, Mattison JA, Ingram DK, Miller RA, Roth GS. Extending the lifespan of long-lived mice. Nature. 2001;412:414.[Medline]
  8. Tatar M, Bartke A, Antebi A. The endocrine regulation of aging by insulin-like signals. Science. 2003;299:1346-1351.[Abstract/Free Full Text]
  9. Moller DE, Berger JP. Role of PPARs in the regulation of obesity-related insulin sensitivity and inflammation. Int J Obesity. 2003;27:S17-S21.
  10. Knouff C, Auwerx J. PPAR{gamma} calls for activation in moderation: lessons from genetics and pharmacology. Endocr Rev. 2004;25:899-918.[Abstract/Free Full Text]
  11. Petersen KF, Krssak M, Inzucchi S, Cline GW, Dufour S, Shulman GI. Mechanism of troglitazone action in type 2 diabetes. Diabetes. 2000;49:827-831.[Abstract]
  12. Kraegen EW, James DE, Jenkins AB, Chisholm DJ, Storlien LH. A potent in vivo effect of ciglitazone on muscle insulin resistance induced by high fat feeding of rats. Metabolism. 1989;38:1089-1093.[Medline]
  13. Zierath JR, Ryder JW, Doebber T, et al. Role of skeletal muscle in thiazolidinedione insulin sensitizer (PPARgamma agonist) action. Endocrinology. 1998;139:5034-5041.[Abstract/Free Full Text]
  14. Norris AW, Chen L, Fisher SJ, et al. Muscle-specific PPAR{gamma}-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones. J Clin Invest. 2003;112:608-618.[Medline]
  15. Schoonjans K, Martin G, Staels B, Auwerex J. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol. 1997;8:159-166.[Medline]
  16. Peters JM, Hennuyer N, Staels B, et al. Alterations in lipoprotein metabolism in peroxisome proliferators-activated receptor {alpha}-deficient mice. J Biol Chem. 1997;272:27307-27312.[Abstract/Free Full Text]
  17. Michalik L, Desvergene B, Tan NS, et al. Impaired skin wound healing in peroxisome proliferators-activated receptor (PPAR){alpha} and PPARß mutant mice. J Cell Biol. 2001;154:799-814.[Abstract/Free Full Text]
  18. Tan NS, Michalik L, Noy N, et al. Critical roles of PPARß/{delta} in keratinocyte response to inflammation. Genes Dev. 2001;15:3263-3277.[Abstract/Free Full Text]
  19. Wang YX, Lee C, Tiep S, et al. Peroxisome-proliferator-activated receptor {delta} activates fat metabolism to prevent obesity. Cell. 2003;113:159-170.[Medline]
  20. IJpenberg A, Tan NS, Gelman L, et al. In vivo activation of PPAR target genes by RXR homodimers. EMBO J. 2004;23:2083-2091.[Medline]
  21. Mattison JA, Wright C, Bronson RT, Roth GS, Ingram DK, Bartke A. Studies of aging in Ames dwarf mice: effects of caloric restriction. J Am Aging Assoc. 2000;23:9.
  22. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline]
  23. Masternak MM, Al-Regaiey KA, Del Rosario Lim MM, et al. Caloric restriction results in decreased expression of peroxisome proliferator-activated receptors (PPARs) superfamily in muscle of normal and long-lived GHR-KO mice. J Gerontol A Biol Sci Med Sci. 2005;60A:1238-1245.[Abstract/Free Full Text]
  24. Al-Regaiey K, Masternak MM, Bonkowski M, Sun L, Bartke A. Long-lived growth hormone receptor knockout mice: interaction of reduced IGF-1/insulin signaling and caloric restriction. Endocrinology. 2005;146:851-860.[Abstract/Free Full Text]
  25. Masternak MM, Al-Regaiey K, Bonkowski MS, et al. Divergent effects of caloric restriction on gene expression in normal and long-lived mice. J Gerontol A Biol Sci Med Sci. 2004;59:784-788.



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