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1.  The effect of calorie restriction on insulin signaling in skeletal muscle and adipose tissue of Ames dwarf mice 
Aging (Albany NY)  2014;6(10):900-912.
Long-living Ames dwarf (df/df) mice are homozygous for a mutation of the Prop1df gene. As a result, mice are deficient in growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). In spite of the hormonal deficiencies, df/df mice live significantly longer and healthier lives compared to their wild type siblings. We studied the effects of calorie restriction (CR) on the expression of insulin signaling genes in skeletal muscle and adipose tissue of normal and df/df mice. The analysis of genes expression showed that CR differentially affects the insulin signaling pathway in these insulin target organs. Moreover, results obtained in both normal and Ames dwarf mice indicate more direct effects of CR on insulin signaling genes in adipose tissue than in skeletal muscle. Interestingly, CR reduced the protein levels of adiponectin in the epididymal adipose tissue of normal and Ames dwarf mice, while elevating adiponectin levels in skeletal muscle and plasma of normal mice only.
In conclusion, our findings suggest that both skeletal muscle and adipose tissue are important mediators of insulin effects on longevity. Additionally, the results revealed divergent effects of CR on expression of genes in the insulin signaling pathway of normal and Ames dwarf mice.
PMCID: PMC4247389  PMID: 25411241
Ames dwarf; insulin; adipose tissue; skeletal muscle; adiponectin; obesity
2.  Growth hormone, inflammation and aging 
Pathobiology of Aging & Age Related Diseases  2012;2:10.3402/pba.v2i0.17293.
Mutant animals characterized by extended longevity provide valuable tools to study the mechanisms of aging. Growth hormone and insulin-like growth factor-1 (IGF-1) constitute one of the well-established pathways involved in the regulation of aging and lifespan. Ames and Snell dwarf mice characterized by GH deficiency as well as growth hormone receptor/growth hormone binding protein knockout (GHRKO) mice characterized by GH resistance live significantly longer than genetically normal animals. During normal aging of rodents and humans there is increased insulin resistance, disruption of metabolic activities and decline of the function of the immune system. All of these age related processes promote inflammatory activity, causing long term tissue damage and systemic chronic inflammation. However, studies of long living mutants and calorie restricted animals show decreased pro-inflammatory activity with increased levels of anti-inflammatory adipokines such as adiponectin. At the same time, these animals have improved insulin signaling and carbohydrate homeostasis that relate to alterations in the secretory profile of adipose tissue including increased production and release of anti-inflammatory adipokines. This suggests that reduced inflammation promoting healthy metabolism may represent one of the major mechanisms of extended longevity in long-lived mutant mice and likely also in the human.
doi:10.3402/pba.v2i0.17293
PMCID: PMC3417471  PMID: 22953033
Ghowth hormone; obesity; inflammation; calorie restriction; aging
3.  The contribution of visceral fat to improved insulin signaling in Ames dwarf mice 
Aging Cell  2014;13(3):497-506.
Ames dwarf (Prop1df, df/df) mice are characterized by growth hormone (GH), prolactin, and thyrotropin deficiency, remarkable extension of longevity and increased insulin sensitivity with low levels of fasting insulin and glucose. Plasma levels of anti-inflammatory adiponectin are increased in df/df mice, while pro-inflammatory IL-6 is decreased in plasma and epididymal fat. This represents an important shift in the balance between pro- and anti-inflammatory adipokines in adipose tissue, which was not exposed to GH signals during development or adult life. To determine the role of adipose tissue in the control of insulin signaling in these long-living mutants, we examined the effects of surgical removal of visceral (epididymal and perinephric) adipose tissue. Comparison of the results obtained in df/df mice and their normal (N) siblings indicated different effects of visceral fat removal (VFR) on insulin sensitivity and glucose tolerance. The analysis of the expression of genes related to insulin signaling indicated that VFR improved insulin action in skeletal muscle in N mice. Interestingly, this surgical intervention did not improve insulin signaling in df/df mice skeletal muscle but caused suppression of the signal in subcutaneous fat. We conclude that altered profile of adipokines secreted by visceral fat of Ames dwarf mice may act as a key contributor to increased insulin sensitivity and extended longevity of these animals.
doi:10.1111/acel.12201
PMCID: PMC4032618  PMID: 24690289
adiponectin; adipose tissue; Ames dwarf; insulin; obesity
4.  Metabolic effects of intra-abdominal fat in GHRKO mice 
Aging Cell  2011;11(1):73-81.
SUMMARY
Mice with targeted deletion of the growth hormone receptor (GHRKO mice) are GH resistant, small, obese, hypoinsulinemic, highly insulin sensitive and remarkably long-lived. To elucidate the unexpected coexistence of adiposity with improved insulin sensitivity and extended longevity, we examined effects of surgical removal of visceral (epididymal and perinephric) fat on metabolic traits related to insulin signaling and longevity. Comparison of results obtained in GHRKO mice and in normal animals from the same strain revealed disparate effects of visceral fat removal (VFR) on insulin and glucose tolerance, adiponectin levels, accumulation of ectopic fat, phosphorylation of insulin signaling intermediates, body temperature and respiratory quotient (RQ). Overall, VFR produced the expected improvements in insulin sensitivity and reduced body temperature and RQ in normal mice and had opposite effects in GHRKO mice. Some of the examined parameters were altered by VFR in opposite directions in GHRKO and normal mice, others were affected in only one genotype or exhibited significant genotype × treatment interactions. Functional differences between visceral fat of GHRKO and normal mice were confirmed by measurements of adipokine secretion, lipolysis and expression of genes related to fat metabolism. We conclude that in the absence of GH signaling the secretory activity of visceral fat is profoundly altered and unexpectedly promotes enhanced insulin sensitivity. The apparent beneficial effects of visceral fat in GHRKO mice may also explain why reducing adiposity by calorie restriction fails to improve insulin signaling or further extend longevity in these animals.
doi:10.1111/j.1474-9726.2011.00763.x
PMCID: PMC3257405  PMID: 22040032
GHRKO; insulin; adipose tissue
5.  Caloric Restriction and Growth Hormone Receptor Knockout: Effects on Expression of Genes Involved in Insulin Action in the Heart 
Experimental gerontology  2006;41(4):417-429.
Blockade of growth hormone (GH), decreased insulin-like growth factor-1 (IGF1) action and increased insulin sensitivity are associated with life extension and an apparent slowing of the aging process. We examined expression of genes involved in insulin action, IR, IRS1, IRS2, IGF1, IGF1R, GLUT4, PPARs and RXRs in the hearts of normal and GHR−/− (KO) mice fed ad libitum or subjected to 30% caloric restriction (CR). CR increased the cardiac expression of IR, IRS1, IGF1, IGF1R and GLUT4 in normal mice and IRS1, GLUT4, PPARα and PPARβ/δ in GHR-KO animals. Expression of IR, IRS1, IRS2, IGF1, GLUT4, PPARγ and PPARα did not differ between GHR-KO and normal mice. These unexpected results suggest that CR may lead to major modifications of insulin action in the heart, but high insulin sensitivity of GHR-KO mice is not associated with alterations in the levels of most of the examined molecules related to intracellular insulin signaling.
doi:10.1016/j.exger.2006.01.009
PMCID: PMC3082456  PMID: 16524678
Caloric restriction; aging; GHR-KO; insulin; fatty acid
6.  Effects of Growth Hormone and Thyroxine Replacement Therapy on Insulin Signaling in Ames Dwarf Mice 
Ames dwarf (Prop1df, df/df) mice lack growth hormone (GH), prolactin, and thyrotropin and live remarkably longer than their normal siblings. Significance of reduced activity of the somatotropic and thyroid axes during development and adulthood on longevity are unknown. Because enhanced insulin sensitivity and reduced insulin levels are among likely mechanisms responsible for increased longevity in these mutants, we compared the effects of GH and thyroxine (T4) replacement on various parameters related to insulin signaling in young and old male df/df mice. The results suggest that altered plasma adiponectin and insulin-like growth factor-1 (IGF-1) and hepatic IGF-1, insulin receptor (IR), IR substrate-1, peroxisome proliferator–activated receptor (PPAR) γ, and PPARγ coactivator-1 α may contribute to increased insulin sensitivity in Ames dwarfs. The stimulatory effect of GH and T4 treatment on plasma insulin and inhibitory effect on expression of hepatic glucose transporter-2 were greater in old than in young dwarfs. These results indicate that GH and T4 treatment has differential impact on insulin signaling during development and adulthood.
doi:10.1093/gerona/glq018
PMCID: PMC2844062  PMID: 20200088
Ames dwarf; Aging; Insulin; Growth hormone
7.  The Effects of Growth Hormone (GH) Treatment on GH and Insulin/IGF-1 Signaling in Long-Lived Ames Dwarf Mice 
The disruption of the growth hormone (GH) axis in mice promotes insulin sensitivity and is strongly correlated with extended longevity. Ames dwarf (Prop1df, df/df) mice are GH, prolactin (PRL), and thyrotropin (TSH) deficient and live approximately 50% longer than their normal siblings. To investigate the effects of GH on insulin and GH signaling pathways, we subjected these dwarf mice to twice-daily GH injections (6 μg/g/d) starting at the age of 2 weeks and continuing for 6 weeks. This produced the expected activation of the GH signaling pathway and stimulated somatic growth of the Ames dwarf mice. However, concomitantly with increased growth and increased production of insulinlike growth factor-1, the GH treatment strongly inhibited the insulin signaling pathway by decreasing insulin sensitivity of the dwarf mice. This suggests that improving growth of these animals may negatively affect both their healthspan and longevity by causing insulin resistance.
doi:10.1093/gerona/glp172
PMCID: PMC2796883  PMID: 19906822
Ames dwarf; Aging; Insulin; Growth hormone
8.  Is Altered Expression of Hepatic Insulin-Related Genes in Growth Hormone Receptor Knockout Mice Due to GH Resistance or a Difference in Biological Life Spans? 
Growth hormone receptor knockout (GHRKO) mice live about 40%–55% longer than their normal (N) littermates. Previous studies of 21-month-old GHRKO and N mice showed major alterations of the hepatic expression of genes involved in insulin signaling. Differences detected at this age may have been caused by the knockout of the growth hormone receptor (GHR) or by differences in biological age between GHRKO and N mice. To address this question, we compared GHRKO and N mice at ages corresponding to the same percentage of median life span to see if the differences of gene expression persisted. Comparison of GHRKO and N mice at ∼50% of biological life span showed significant differences in hepatic expression of all 14 analyzed genes. We conclude that these changes are due to disruption of GHR gene and the consequent suppression of growth hormone signaling rather than to differences in “biological age” between mutant and normal animals sampled at the same chronological age.
doi:10.1093/gerona/glp111
PMCID: PMC2759569  PMID: 19706698
Aging; GHRKO; Insulin
9.  Insulin Sensitivity as a Key Mediator of Growth Hormone Actions on Longevity 
Reduced insulin sensitivity and glucose intolerance have been long suspected of having important involvement in aging. Here we report that in studies of calorie restriction (CR) effects in mutant (Prop1df and growth hormone receptor knockout [GHRKO]) and normal mice, insulin sensitivity was strongly associated with longevity. Of particular interest was enhancement of the already increased insulin sensitivity in CR df/df mice in which longevity was also further extended and the lack of changes in insulin sensitivity in calorically restricted GHRKO mice in which there was no further increase in average life span. We suggest that enhanced insulin sensitivity, in conjunction with reduced insulin levels, may represent an important (although almost certainly not exclusive) mechanism of increased longevity in hypopituitary, growth hormone (GH)-resistant, and calorie-restricted animals. We also report that the effects of GH treatment on insulin sensitivity may be limited to the period of GH administration.
doi:10.1093/gerona/glp024
PMCID: PMC2667133  PMID: 19304940
Insulin; Longevity; GHRKO; Ames dwarf
10.  PIOGLITAZONE DOES NOT IMPROVE INSULIN SIGNALING IN MICE WITH GROWTH HORMONE OVER-EXPRESSION 
The Journal of endocrinology  2013;219(2):109-117.
Type 2 diabetes and obesity are very serious health problems in both developed and developing countries. Increased level of growth hormone (GH) is known to promote insulin resistance. Transgenic (Tg) mice over-expressing bovine GH are short-living and characterized, among others, by hyperinsulinemia and increased insulin resistance in comparison to normal (N) mice. Pioglitazone (PIO) is a member of the thiazolidinediones – group of insulin-sensitizing drugs which are selective agonists of peroxisome proliferator-activated receptor gamma (PPARγ). The aim of the study was to analyze the effects of PIO on the insulin signaling pathway in Tg and N mice. Plasma levels of insulin and glucose as well as hepatic levels of proteins involved in insulin signaling were analyzed by ELISA or western blot methods. Treatment with PIO decreased plasma level of glucose in N mice only. Similarly, PIO increased insulin sensitivity (expressed as the Relative Insulin Sensitivity Index; RISI) only in N mice. In the liver, PIO decreased the phosphorylation of IRS1 at a serine residue (Ser307-pS-IRS1), that inhibits insulin action, and had a tendency to increase tyrosine phosphorylation of IRS2 (Tyr-pY-IRS2) only in N mice but did not affect either of these parameters in Tg mice. Levels of total and phosphorylated mTOR were increased in Tg mice. Moreover, the AKT2 level was decreased by PIO in N mice only. In conclusion, the lack of improvement of insulin sensitivity in insulin-resistant Tg mice during PIO treatment suggests that chronically elevated GH level can inhibit the beneficial effects of PIO on insulin signaling.
doi:10.1530/JOE-13-0124
PMCID: PMC3811004  PMID: 23946430
pioglitazone; insulin signaling; growth hormone; transgenic mice
11.  Circulating blood leukocyte gene expression profiles: Effects of the Ames dwarf mutation on pathways related to immunity and inflammation 
Experimental gerontology  2007;42(8):772-788.
Aging is associated with a decline of immune competence and an increase in markers of inflammation. There is considerable evidence that inflammatory processes play a role in aging and the determination of lifespan. Hypopituitary Ames dwarf mice have extended longevity and exhibit many symptoms of delayed aging, although various aspects of immune function are suppressed in the mutants. In the present study, the expression of genes related to immunity and inflammation was compared in peripheral blood leukocytes (PBL) from Ames dwarf and normal mice using Affymetrix Gene Chip arrays. Among the more than 3000 probe sets that were differentially expressed, 273 were identified as being associated with immunity and/or inflammation. Pathway analysis revealed interactions among 91 of these probe sets, centered on casp3, bcl2, il4, prkca, mapk14, and TGFβ1. Ames dwarf mice had reduced leukocyte expression of casp3 and TGFβ and increased expression of Bcl2. Alterations in the expression of these genes suggest likely functional changes in apoptosis, B and T cell homeostasis, prostaglandin synthesis, humoral immunity, chemokine activity, complement activation, hemostasis and wound healing pathways. Collectively, these results suggest that activation of both anti-inflammatory pathways and an anti-clotting mechanism combined with reduced turnover of leukocytes may contribute to delayed aging and extended longevity of Ames dwarf mice. We are also aware that alterations in gene expression in PBLs can be due to different composition of PBL populations when comparing Ames dwarf to WT animals, and it will be interesting to investigate these genes in particular PBL populations in the future. However, whole leucocytes population represents the function of immune system in these organisms.
doi:10.1016/j.exger.2007.04.004
PMCID: PMC2045642  PMID: 17611063
Ames dwarf; aging; PBL; inflammation
12.  PPARs in Calorie Restricted and Genetically Long-Lived Mice 
PPAR Research  2006;2007:28436.
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptors superfamily. The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. These transcription factors regulate different physiological functions such as energy metabolism (including lipid and carbohydrate metabolism), insulin action, and immunity and inflammation, and apparently also act as important mediators of longevity and aging. Calorie restriction (CR) is the most effective intervention known to delay aging and increase lifespan. Calorie restriction affects the same physiological functions as PPARs. This review summarizes recent findings on the effects of CR and aging on the expression of PPARγ, α, and β/δ in mice and discusses possible involvement of PPARs in mediating the effects of murine longevity genes. The levels of PPARs change with age and CR appears to prevent these alterations which make “PPARs-CR-AGING” dependence of considerable interest.
doi:10.1155/2007/28436
PMCID: PMC1779582  PMID: 17389764
13.  Growth hormone action predicts age-related white adipose tissue dysfunction and senescent cell burden in mice 
Aging (Albany NY)  2014;6(7):575-586.
The aging process is associated with the development of several chronic diseases. White adipose tissue (WAT) may play a central role in age-related disease onset and progression due to declines in adipogenesis with advancing age. Recent reports indicate that the accumulation of senescent progenitor cells may be involved in age-related WAT dysfunction. Growth hormone (GH) action has profound effects on adiposity and metabolism and is known to influence lifespan. In the present study we tested the hypothesis that GH activity would predict age-related WAT dysfunction and accumulation of senescent cells. We found that long-lived GH-deficient and -resistant mice have reduced age-related lipid redistribution. Primary preadipocytes from GH-resistant mice also were found to have greater differentiation capacity at 20 months of age when compared to controls. GH activity was also found to be positively associated with senescent cell accumulation in WAT. Our results demonstrate an association between GH activity, age-related WAT dysfunction, and WAT senescent cell accumulation in mice. Further studies are needed to determine if GH is directly inducing cellular senescence in WAT or if GH actions on other target organs or alternative downstream alterations in insulin-like growth factor-1, insulin or glucose levels are responsible.
PMCID: PMC4153624  PMID: 25063774
adipose tissue; aging; cellular senescence; growth hormone
14.  Decreased Levels of Proapoptotic Factors and Increased Key Regulators of Mitochondrial Biogenesis Constitute New Potential Beneficial Features of Long-lived Growth Hormone Receptor Gene–Disrupted Mice 
Decreased somatotrophic signaling is among the most important mechanisms associated with extended longevity. Mice homozygous for the targeted disruption of the growth hormone (GH) receptor gene (GH receptor knockout; GHRKO) are obese and dwarf, are characterized by a reduced weight and body size, undetectable levels of GH receptor, high concentration of serum GH, and greatly reduced plasma levels of insulin and insulin-like growth factor-I, and are remarkably long lived. Recent results suggest new features of GHRKO mice that may positively affect longevity—decreased levels of proapoptotic factors and increased levels of key regulators of mitochondrial biogenesis. The alterations in levels of the proapoptotic factors and key regulators of mitochondrial biogenesis were not further improved by two other potential life-extending interventions—calorie restriction and visceral fat removal. This may attribute the primary role to GH resistance in the regulation of apoptosis and mitochondrial biogenesis in GHRKO mice in terms of increased life span.
doi:10.1093/gerona/gls231
PMCID: PMC3708518  PMID: 23197187
Aging; Longevity; Dwarf mice; Apoptosis; Mitochondrial biogenesis; GHRKO mice; Calorie restriction; Visceral fat removal.
15.  Upregulation of the Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Receptor Axis in the Heart and the Kidney of Growth Hormone Receptor knock-out Mice 
Objective
Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR−/−) mouse.
Design
RAS components were evaluated in the heart and the kidney of GHR−/− and control mice by immunohistochemistry and western blotting (n=12 for both groups).
Results
The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR−/− mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR−/− mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western Blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues.
Conclusions
The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR−/− mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.
doi:10.1016/j.ghir.2012.08.003
PMCID: PMC3698955  PMID: 22947377
Angiotensin-(1-7); AT1 receptor; Mas receptor; Growth hormone; Renin-angiotensin system
18.  RENAL PRO-APOPTOTIC PROTEINS ARE REDUCED BY GROWTH HORMONE RESISTANCE BUT NOT BY VISCERAL FAT REMOVAL 
Biological chemistry  2011;392(5):475-481.
The growth hormone (GH) receptor knockout (GHRKO) mice are highly insulin sensitive and long-lived. Surgical visceral fat removal (VFR) improves insulin signaling in normal mice and rats and extends longevity in rats. We have previously demonstrated decreased expression of certain pro-apoptotic genes in kidneys of GHRKO mice, and suggested that this may contribute to the increased longevity of these animals. The aim of the present study was to examine the level of the following proteins: caspase-3, caspase-9, caspase-8, bax, bad, phospho-bad (p-bad), bcl-2, Smac/DIABLO, Apaf-1, phospho-p53 (pp53) and cytochrome c (cyc) in male GHRKO and normal (N) mice subjected to VFR or sham surgery, at approximately 6 months of age. The kidneys were collected 2 months after VFR. Results: Caspase-3, caspase-8, bax, bad, Smac/DIABLO, Apaf-1 and pp53 levels were decreased in GHRKO mice as compared to N animals. VFR did not change the level of any of the examined proteins. Conclusion: Decreased renal levels of pro-apoptotic proteins may contribute to extended lifespan due to targeted disruption of GH receptor (Ghr) gene but are apparently not involved in mediating the effects of VFR.
doi:10.1515/BC.2011.051
PMCID: PMC3703833  PMID: 21391871
apoptosis; caspases; GHRKO mice; kidney; proteins; visceral fat removal
19.  DECREASED EXPRESSION LEVEL OF APOPTOSIS-RELATED GENES AND/OR PROTEINS IN SKELETAL MUSCLES, BUT NOT IN HEARTS, OF GROWTH HORMONE RECEPTOR KNOCKOUT MICE 
The long-lived growth hormone (GH) receptor knockout (GHRKO; KO) mice are GH resistant due to targeted disruption of the GH receptor (Ghr) gene. Apoptosis is a physiological process in which cells play an active role in their own death and is a normal component of the development and health of multicellular organisms. Aging is associated with the progressive loss of strength of skeletal and heart muscles. Calorie restriction (CR) is a well known experimental model to delay aging and increase lifespan. The aim of the study was to examine the expression of the following apoptosis-related genes: caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 (cyc1) in the skeletal muscles and hearts of female normal and GHRKO mice, fed ad libitum or subjected to 40% CR for 6 months, starting at 2 months of age. Moreover, skeletal muscle caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 (pp53) and cytochrome c (cyc) protein expression levels were assessed.
Results
Expression of caspase-3, caspase-9, bax and Smac/DIABLO genes and proteins was decreased in GHRKO’s skeletal muscles. The Apaf-1 protein expression also was diminished in this tissue. In contrast, bcl-2 and pbad protein levels were increased in skeletal muscles in knockouts. No changes were demonstrated for the examined genes expression in GHRKO’s hearts except for the increased level of cyc1 mRNA. CR did not alter the expression of the examined genes and proteins in skeletal muscles of knockouts vs. normal (N) mice. In heart homogenates, CR increased caspase-3 mRNA level as compared to ad libitum (AL) mice.
Conclusion
decreased expression of certain pro-apoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by calorie restriction.
doi:10.1258/ebm.2010.010202
PMCID: PMC3703836  PMID: 21321312
20.  Key Regulators of Mitochondrial Biogenesis are Increased in Kidneys of Growth Hormone Receptor Knockout (GHRKO) Mice 
Cell biochemistry and function  2011;29(6):459-467.
The growth hormone (GH) receptor knockout mice (GHRKO) are remarkably long-lived and highly insulin sensitive. Alterations in mitochondrial biogenesis are associated with aging and various metabolic derangements. We have previously demonstrated increased gene expression of key regulators of mitochondriogenesis in kidneys, hearts and skeletal muscles of GHRKO mice. The aim of the present study was to quantify the protein levels of the following regulators of mitochondriogenesis: peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), AMP-activated protein kinase α (AMPKα), phospho-AMPKα (p-AMPKα), sirtuin-3 (SIRT-3), endothelial nitric oxide synthase (eNOS), phospho-eNOS (p-eNOS), nuclear respiratory factor-1 (NRF-1) and mitofusin-2 (MFN-2) in skeletal muscles and kidneys of GHRKOs in comparison to normal mice. We were also interested in the effects of calorie restriction (CR) and visceral fat removal (VFR) on these parameters. Both CR and VFR improve insulin sensitivity and can extend lifespan. Results: The renal levels of PGC-1α, AMPKα, p-AMPKα, SIRT-3, eNOS, p-eNOS and MFN-2 were increased in GHRKOs. In the GHRKO skeletal muscles, only MFN-2 was increased. Levels of the examined proteins were not affected by CR (except for PGC-1α and p-eNOS in skeletal muscles) or VFR. Conclusion: GHRKO mice have increased renal protein levels of key regulators of mitochondriogenesis and this may contribute to increased longevity of these knockouts.
doi:10.1002/cbf.1773
PMCID: PMC3682479  PMID: 21755522
mitochondrial biogenesis; GHRKO mice; skeletal muscles; kidneys; proteins; calorie restriction; visceral fat
21.  Post-transcriptional regulation of IGF1R by key microRNAs in long-lived mutant mice 
Aging cell  2011;10(6):1080-1088.
Summary
Long-lived mutant mice, both Ames dwarf and growth hormone receptor gene disrupted or knockout (GHRKO) strains, exhibit heightened cognitive robustness and altered IGF1 signaling in the brain. Here we report, in both these long-lived mice, that three up-regulated lead microRNAs, miR-470, −669b, and −681, are involved in post-transcriptional regulation of genes pertinent to growth hormone (GH)/IGF1 signaling. All three are most prominently localized in the hippocampus, and correspond to reduced expression of key IGF1 signaling genes: IGF1, IGF1R, and PI3 kinase. The decline in these genes’ expression translates into decreased phosphorylation of downstream molecules AKT and FoxO3a. Cultures transfected with either miR-470, −669b, or −681 show repressed endogenous expression of all three genes of the IGF1 signaling axis, most significantly IGF1R, while other similarly up-regulated microRNAs, including let-7g and miR-509, do not induce the same levels of repression. Transduction study in IGF1-responsive cell cultures shows significantly reduced IGF1R expression, and AKT to some extent, most notably by miR-681. This is accompanied by decreased levels of downstream phosphorylated forms of AKT and FoxO3a upon IGF1 stimulation. Suppression of IGF1R by the three microRNAs is further validated by IGF1R 3′UTR reporter assays. Taken together, our results suggest that miR-470, miR-669b, and miR-681 are all functionally able to suppress IGF1R and AKT, two upstream genes controlling FoxO3a phosphorylation status. Their up-regulation in GH signaling-deficient mutant mouse brain suggests reduced IGF1 signaling at the post-transcriptional level, for numerous gains of neuronal function in these long-lived mice.
doi:10.1111/j.1474-9726.2011.00751.x
PMCID: PMC3587961  PMID: 21967153
microRNA; aging; IGF1; IGF1R; growth hormone; Ames dwarf mice and GHRKO mice; miR-470; miR-669b; miR-681; cognitive robustness and longevity
22.  Expression of Key Regulators of Mitochondrial Biogenesis in Growth Hormone Receptor Knockout (GHRKO) Mice is Enhanced but is Not Further Improved by Other Potential Life-Extending Interventions 
Mitochondrial biogenesis is essential for cell viability. Growth hormone receptor knockout (GHRKO), calorie restriction, and surgical visceral fat removal constitute experimental interventions to delay aging and increase life span. We examined the expression of known regulators of mitochondriogenesis: peroxisome proliferator–activated receptor γ co-activator 1α (PGC-1α), adenosine monophosphate (AMP)–activated protein kinase (AMPK), sirtuin-1 (SIRT-1) and sirtuin-3 (SIRT-3), endothelial nitric oxide synthase (eNOS), nuclear respiratory factor-1, mitochondrial transcription factor A (TFAM), and mitofusin-2 (MFN-2) in the skeletal muscles and hearts of control and calorie-restricted female GHRKO mice and in the kidneys of male GHRKOs after visceral fat removal or sham surgery. Expression of PGC-1α in skeletal muscles, AMPK, SIRT-1, SIRT-3, eNOS, and MFN-2 in the heart and PGC-1α, AMPK, SIRT-3, eNOS, and MFN-2 in kidneys was increased in GHRKO mice but was not affected by calorie restriction or visceral fat removal. GHRKO mice have increased expression of key regulators of mitochondriogenesis, which is not improved further by calorie restriction or visceral fat removal.
doi:10.1093/gerona/glr080
PMCID: PMC3172561  PMID: 21788651
GHRKO mice; Mitochondrial biogenesis; Gene expression; Calorie restriction; Visceral fat removal
23.  Connecting serum IGF-1, body size, and age in the domestic dog 
Age  2010;33(3):475-483.
Many investigations in recent years have targeted understanding the genetic and biochemical basis of aging. Collectively, genetic factors and biological mechanisms appear to influence longevity in general and specifically; reduction of the insulin/IGF-1 signaling cascade has extended life span in diverse species. Genetic alteration of mammals for life extension indicates correlation to serum IGF-1 levels in mice, and IGF-1 levels have been demonstrated as a physiological predictor of frailty with aging in man. Longevity and aging data in the dog offer a close measure of the natural multifactorial longevity interactions of genetic influence, IGF-1 signaling, and environmental factors such as exposure, exercise, and lifestyle. The absence of genetic alteration more closely represents the human longevity status, and the unique species structure of the canine facilitates analyses not possible in other species. These investigations aimed to measure serum IGF-1 in numerous purebred and mixed-breed dogs of variable size and longevity in comparison to age, gender, and spay/neuter differences. The primary objective of this investigation was to determine plasma IGF-1 levels in the adult dog, including a wide range of breeds and adult body weight. The sample set includes animals ranging from just a few months of age through 204 months and ranging in size from 5 to 160 lb. Four groups were evaluated for serum IGF-1 levels, including intact and neutered males, and intact and spayed females. IGF-1 loss over time, as a function of age, decreases in all groups with significant differences between males and females. The relationship between IGF-1 and weight differs depending upon spay/neuter status, but there is an overall increase in IGF-1 levels with increasing weight. The data, currently being interrogated further for delineation of IGF-1 receptor variants and sex differences, are being collected longitudinally and explored for longevity associations previously unavailable in non-genetically modified mammals.
doi:10.1007/s11357-010-9182-4
PMCID: PMC3168604  PMID: 20865338
Longevity; IGF-1; Canine; Aging; Insulin signaling
24.  Decreased thyroid follicle size in dwarf mice may suggest the role of growth hormone signaling in thyroid growth regulation 
Thyroid Research  2012;5:7.
Background
Altered somatotrophic signaling is among the most important potential mechanisms of extended longevity. Ames dwarf (df/df) mice are homozygous for mutation at the Prop-1 gene, leading to a lack of growth hormone (GH), prolactin and thyroid stimulating hormone (TSH). Mice homozygous for targeted disruption of the growth hormone receptor/growth hormone binding protein gene are known as GH receptor knockout (GHRKO) mice or “Laron dwarf”. Both, df/df and GHRKO mice, are characterized by reduced body size, low plasma insulin and insulin-like growth factor-I (IGF-I), remarkably extended longevity, and severe (in df/df mice) or mild (in GHRKO mice) thyroid hypofunction. Recently, by crossing df/df and GHRKO mice, double-mutant Ames dwarf/GHRKO (df/KO) mice were created. Interestingly, these mice are smaller than Ames dwarfs or GHRKOs, and also have reduced insulin and IGF-I levels. The aim of the study was to investigate if and to what extent certain thyroid morphological parameters, such as inner follicular surface area, inner follicular perimeter, as well as the follicular epithelium thickness are changed in the examined dwarf mice.
Methods
This quantification was performed in thyroids collected from df/df, GHRKO and df/KO female mice, at approximately 5–6 months of age. We used a computerized plotting programme that combines a live microscopic image of the slide with an operator-generated overlay.
Results
Inner follicular surface area and inner follicular perimeter were decreased in all examined kinds of dwarf mice as compared to normal animals. Furthermore, decreases in these two parameters were more pronounced in df/df and df/KO than in GHRKO mice. Concerning the follicular epithelium thickness, only a tendency towards decrease of this parameter was found in all three kinds of dwarf mice.
Conclusions
Parameters characterizing thyroid follicle size are decreased in all three examined models of dwarf mice, which may explain decreased thyroid hormone levels in both basal mutants (Ames dwarfs and GHRKOs). df/df mutation seems to predominate over GHRKO genetic intervention concerning their effects on thyroid growth. Beside TSH, also GH signaling seems to constitute a crucial element in the regulation of thyroid growth and, possibly, function.
doi:10.1186/1756-6614-5-7
PMCID: PMC3464137  PMID: 22897932
Ames dwarf mice; GHRKO mice; Thyroid follicle; Inner follicular surface area; Inner follicular perimeter; Follicular epithelium thickness
25.  RasGrf1: genomic imprinting, VSELs, and aging 
Aging (Albany NY)  2011;3(7):692-697.
Increase in life span in RasGrf1-deficient mice revealed that RasGrf1 deficiency promotes longevity. Interestingly, RasGrf1 is one of parentally imprinted genes transcribed from paternally-derived chromosome. Erasure of its imprinting results in RasGrf1 downregulation and has been demonstrated in a population of pluripotent adult tissues-derived very small embryonic like stem cells (VSELs), stem cells involved in tissue organ rejuvenation. Furthermore, based on recent observation that RasGrf1 signaling molecule is located downstream from insulin (Ins) and insulin like growth factor-1 (Igf-1) receptors, the extended life-span of RasGrf1−/− mice may support beneficial effect of reduced Ins/Igf-1 signaling on longevity. Similarly, downregulation of RasGrf1 in VSELs renders them resistant to chronic Ins/Igf-1 signaling and protects from premature depletion from adult tissues. Thus, the studies in RasGrf1−/− mice indicate that some of the imprinted genes may play a role in ontogenetic longevity and suggest that there are sex differences in life span that originate at the genome level. All this in toto supports a concept that the sperm genome may have a detrimental effect on longevity in mammals. We will discuss a role of RasGrf1 on life span in context of genomic imprinting and VSELs.
PMCID: PMC3181169  PMID: 21765200
Aging; longevity; IGF-1; RasGrf1; VSEL

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