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1.  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.
2.  The negative effect of prolonged somatotrophic/insulin signaling on an adult bone marrow-residing population of pluripotent very small embryonic-like stem cells (VSELs) 
Age  2012;35(2):315-330.
It is well known that attenuated insulin/insulin-like growth factor signaling (IIS) has a positive effect on longevity in several animal species, including mice. Here, we demonstrate that a population of murine pluripotent very small embryonic-like stem cells (VSELs) that reside in bone marrow (BM) is protected from premature depletion during aging by intrinsic parental gene imprinting mechanisms and the level of circulating insulin-like growth factor-I (IGF-I). Accordingly, an increase in the circulating level of IGF-I, as seen in short-lived bovine growth hormone (bGH)-expressing transgenic mice, which age prematurely, as well as in wild-type animals injected for 2 months with bGH, leads to accelerated depletion of VSELs from bone marrow (BM). In contrast, long-living GHR-null or Ames dwarf mice, which have very low levels of circulating IGF-I, exhibit a significantly higher number of VSELs in BM than their littermates at the same age. However, the number of VSELs in these animals decreases after GH or IGF-I treatment. These changes in the level of plasma-circulating IGF-I corroborate with changes in the genomic imprinting status of crucial genes involved in IIS, such as Igf-2-H19, RasGRF1, and Ig2R. Thus, we propose that a chronic increase in IIS contributes to aging by premature depletion of pluripotent VSELs in adult tissues.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-011-9364-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-011-9364-8
PMCID: PMC3592960  PMID: 22218782
VSELs; IGF-1; GH; Aging
3.  The effects of weight cycling on lifespan in male C57BL/6J mice 
Objective
With the increasing rates of obesity, many people diet in attempts to lose weight. Since weight loss is seldom maintained in a single effort, weight cycling is a common occurrence. Unfortunately, reports from clinical studies that have attempted to determine the effect of weight cycling on mortality are in disagreement, and to date, no controlled animal study has been performed to assess the impact of weight cycling on longevity. Therefore, our objective was to determine whether weight cycling altered lifespan in mice that experienced repeated weight gain and weight loss throughout their lives.
Methods
Male C57BL/6J mice were placed on one of three lifelong diets: a low fat (LF) diet, a high fat (HF) diet, or a cycled diet in which the mice alternated between 4 weeks on the LF diet and 4 weeks on the HF diet. Body weight, body composition, several blood parameters and lifespan were assessed.
Results
Cycling between the HF and LF diet resulted in large fluctuations in body weight and fat mass. These gains and losses corresponded to significant increases and decreases, respectively, in leptin, resistin, GIP, IGF-1, glucose, insulin, and glucose tolerance. Surprisingly, weight cycled mice had no significant difference in lifespan (801±45 days) as compared to LF fed controls (828±74 days), despite being overweight and eating a HF diet for half of their lives. In contrast, the HF fed group experienced a significant decrease in lifespan (544±73days) compared to LF fed controls and cycled mice.
Conclusions
This is the first controlled mouse study to demonstrate the effect of lifelong weight cycling on longevity. The act of repeatedly gaining and losing weight, in itself, did not decrease lifespan and was more beneficial than remaining obese.
doi:10.1038/ijo.2012.203
PMCID: PMC3609933  PMID: 23229739
weight cycling; weight fluctuation; yo-yo dieting; weight loss; mortality; longevity
4.  Mouse models of growth hormone action and aging: A proteomic perspective 
Proteomics  2012;13(0):674-685.
Growth hormone (GH) is a protein secreted by the anterior pituitary and circulates throughout the body to exert important actions on growth and metabolism. GH stimulates the secretion of insulin-like growth factor-I (IGF-I) which mediates some of the growth promoting actions of GH. The GH/IGF-I axis has recently been recognized as important in terms of longevity in organisms ranging from C. elegans to mice. For example, GH transgenic mice possess short lifespans while GH receptor null (GHR−/−) mice have extended longevity. Thus, the actions of GH (or IGF-I) or lack thereof impacts the aging process. In this review, we summarize the proteomic analyses of plasma and white adipose tissue in these two mouse models of GH action, i.e., GH transgenic and GHR−/− mice. At the protein level, we wanted to establish novel plasma biomarkers of GH action as a function of age and to determine differences in adipose tissue depots. We have shown that these proteomic approaches have not only confirmed several known physiological actions of GH, but also resulted in novel protein biomarkers and targets that may be indicative of the aging process and/or new functions of GH. These results may generate new directions for GH and/or aging research.
doi:10.1002/pmic.201200271
PMCID: PMC3756660  PMID: 23019135
5.  Age- and Sex-Associated Plasma Proteomic Changes in Growth Hormone Receptor Gene–Disrupted Mice 
Growth hormone receptor gene–disrupted (GHR−/−) mice are dwarf, insulin sensitive, and long lived despite being obese. In order to identify characteristics associated with their increased longevity, we studied age-related plasma proteomic changes in these mice. Male and female GHR−/− mice and their littermate controls were followed longitudinally at 8, 16, and 24 months of ages for plasma proteomic analysis. Relative to control littermates, GHR−/− mice had increased levels of apolipoprotein A-4 and retinol-binding protein-4 and decreased levels of apolipoprotein E, haptoglobin, and mannose-binding protein-C. Female GHR−/− mice showed decreased inflammatory cytokines including interleukin-1β and monocyte chemotactic protein-1. Additionally, sex differences were found in specific isoforms of apolipoprotein E, RBP-4, haptoglobin, albumin, and hemoglobin subunit beta. In conclusion, we find plasma proteomic changes in GHR−/− mice that favor a longer life span as well as sex differences indicative of an improved health span in female mice.
doi:10.1093/gerona/glr212
PMCID: PMC3403865  PMID: 22156438
Growth hormone receptor; Plasma; Proteomics; Sex; Aging
6.  Decreased insulin sensitivity and increased oxidative damage in wasting adipose tissue depots of wild-type mice 
Age  2011;34(5):1225-1237.
Unintentional weight loss (wasting) in the elderly is a major health concern as it leads to increased mortality. Several studies have focused on muscle loss, but little is known about the mechanisms giving rise to loss of fat mass at old ages. To investigate potential mechanisms, white adipose tissue (WAT) characteristics and proteomic profiles were compared between adult (10–12-month-old) and aged (22–24-month-old) wild-type mice. Four individual WAT depots were analyzed to account for possible depot-specific differences. Proteomic profiles of WAT depots, along with body weights and compositions, plasma levels of insulin, leptin and adiponectin, insulin tolerance, adipocyte sizes, and products of oxidative damage in each WAT depot were determined. We found that lean mass remained constant while fat mass and insulin tolerance were decreased in old age, as were adipocyte sizes in the WAT depots. Proteomic results showed increased levels of enolase, pyruvate dehydrogenase E1β, NAD+−dependent isocitrate dehydrogenase α, and ATP synthase subunit β, and decreased levels of carbonic anhydrase 3 in WAT of aged mice. These data suggest increased aerobic glucose oxidation in wasting WAT, consistent with decreased insulin signaling. Also, Cu/Zn superoxide dismutase and two chaperones were increased in aged WAT depots, indicating higher stress resistance. In agreement, lipid peroxidation (HNE-His adducts) increased in old age, although protein oxidation (carbonyl groups) showed no increase. In conclusion, features of wasting WAT were similar in the four depots, including decreased adipocyte sizes and alterations in protein expression profiles that indicated decreased insulin sensitivity and increased lipid peroxidation.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-011-9304-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-011-9304-7
PMCID: PMC3448990  PMID: 21953241
Wasting; Aging; White adipose tissue depots; Proteomics; Oxidative damage; Stress resistance; Insulin resistance
8.  The effect of calorie restriction on the presence of apoptotic ovarian cells in normal wild type mice and low-plasma-IGF-1 Laron dwarf mice 
Background
It is known that caloric restriction extends lifespan and can minimize age-related dysfunction of the reproductive system. We became interested in how caloric restriction influences apoptosis, which is a crucial process that maintains ovarian cell homeostasis.
Methods
We examined ovarian cells in: 2.5-year-old wild type mice on caloric restriction (CR) or fed ad libitum (AL) and Laron dwarf mice (GHR-KO) at the same ages on CR or fed AL. Apoptosis was assessed by histochemical analysis on paraffin sections of ovarian tissue.
Results
Morphological and histochemical analysis revealed that CR improved reproductive potential in 2.5-year-old WT littermates and GHR-KO female mice, as indicated by the increased number of ovarian follicles. The level of apoptosis in ovarian tissue was higher in WT mice on a CR diet compared with WT mice on the AL diet. In GHR-KO mice, the level of apoptosis in ovaries was similar for mice on CR and on AL diets and bigger than in WT mice on CR.
Conclusions
Morphological and histochemical analysis revealed a younger biological age of the ovaries in 2-year-old WT littermates and GHR-KO female mice on CR compared with animals fed AL.
doi:10.1186/1757-2215-6-67
PMCID: PMC3849152  PMID: 24063422
Ovary; Laron dwarf mice; Caloric restriction; Apoptosis; Aging
9.  Adiponectin in mice with altered growth hormone action: links to insulin sensitivity and longevity? 
The Journal of endocrinology  2013;216(3):363-374.
Adiponectin is positively correlated with longevity and negatively correlated with many obesity-related diseases. While there are several circulating forms of adiponectin, the high molecular weight (HMW) version has been suggested to have the predominant bioactivity. Adiponectin gene expression and cognate serum protein levels are of particular interest in mice with altered growth hormone (GH) signaling as these mice exhibit extremes in obesity that are positively associated with insulin sensitivity and lifespan as opposed to the typical negative association of these factors. While a few studies have reported total adiponectin levels in young adult mice with altered GH signaling, much remains unresolved, including changes in adiponectin levels with advancing age, proportion of total adiponectin in the HMW form, adipose depot of origin, and differential effects of GH versus IGF1. Therefore, the purpose of this study was to address these issues using assorted mouse lines with altered GH signaling. Our results show that adiponectin is generally negatively associated with GH activity, regardless of age. Further, the amount of HMW adiponectin is consistently linked with the level of total adiponectin and not necessarily with previously reported lifespan or insulin sensitivity of these mice. Interestingly, circulating adiponectin levels correlated strongly with inguinal fat mass, implying the effects of GH on adiponectin are depot-specific. Interestingly rbGH, but not IGF1, decreased circulating total and HMW adiponectin levels. Taken together, these results fill important gaps in the literature related to GH and adiponectin and question the frequently reported associations of total and HMW adiponectin with insulin sensitivity and longevity.
doi:10.1530/JOE-12-0505
PMCID: PMC3756886  PMID: 23261955
adiponectin; high molecular weight adiponectin; growth hormone receptor; growth hormone; growth hormone deficiency; growth hormone antagonist
10.  NOVEL SERUM PROTEIN BIOMARKERS INDICATIVE OF GROWTH HORMONE DOPING IN HEALTHY HUMAN SUBJECTS 
Proteomics  2011;11(17):3565-3571.
The detection of recombinant human growth hormone (rhGH) is difficult due to its short half-life; therefore, novel and robust biomarkers of rhGH abuse are needed. In this study, serum samples derived from subjects treated with rhGH in a randomized, double blind, placebo-controlled crossover study were analyzed by 2-DE coupled with MS. Eight healthy male subjects aged 23.2 ± 0.6 yr were injected with rhGH (2 mg/day) or saline for 7 days with serum samples drawn at days 0, 3, and 8. Protein intensities were quantified and analyzed for differences between rhGH versus placebo treatments. Protein that showed significant changes were identified and confirmed by Western blotting. These included specific isoforms of alpha-1 antitrypsin and transthyretin that increased; and inter-alpha-trypsin inhibitor heavy chain H4, apolipoprotein A-1 and hemoglobin beta chain that decreased. These proteins represent novel biomarkers of short-term rhGH exposure and may lead to a new method for detecting rhGH doping.
doi:10.1002/pmic.201100077
PMCID: PMC3517138  PMID: 21751372
2-DE; biomarker; doping; growth hormone
11.  Growth Hormone Research Society Workshop Summary: Consensus Guidelines for Recombinant Human Growth Hormone Therapy in Prader-Willi Syndrome 
Context:
Recombinant human GH (rhGH) therapy in Prader-Willi syndrome (PWS) has been used by the medical community and advocated by parental support groups since its approval in the United States in 2000 and in Europe in 2001. Its use in PWS represents a unique therapeutic challenge that includes treating individuals with cognitive disability, varied therapeutic goals that are not focused exclusively on increased height, and concerns about potential life-threatening adverse events.
Objective:
The aim of the study was to formulate recommendations for the use of rhGH in children and adult patients with PWS.
Evidence:
We performed a systematic review of the clinical evidence in the pediatric population, including randomized controlled trials, comparative observational studies, and long-term studies (>3.5 y). Adult studies included randomized controlled trials of rhGH treatment for ≥ 6 months and uncontrolled trials. Safety data were obtained from case reports, clinical trials, and pharmaceutical registries.
Methodology:
Forty-three international experts and stakeholders followed clinical practice guideline development recommendations outlined by the AGREE Collaboration (www.agreetrust.org). Evidence was synthesized and graded using a comprehensive multicriteria methodology (EVIDEM) (http://bit.ly.PWGHIN).
Conclusions:
Following a multidisciplinary evaluation, preferably by experts, rhGH treatment should be considered for patients with genetically confirmed PWS in conjunction with dietary, environmental, and lifestyle interventions. Cognitive impairment should not be a barrier to treatment, and informed consent/assent should include benefit/risk information. Exclusion criteria should include severe obesity, uncontrolled diabetes mellitus, untreated severe obstructive sleep apnea, active cancer, or psychosis. Clinical outcome priorities should vary depending upon age and the presence of physical, mental, and social disability, and treatment should be continued for as long as demonstrated benefits outweigh the risks.
doi:10.1210/jc.2012-3888
PMCID: PMC3789886  PMID: 23543664
12.  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
13.  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
14.  Proteomic Changes in the Heart of Diet-Induced Pre-Diabetic Mice 
Journal of proteomics  2011;74(5):716-727.
The development of type 2 diabetes (T2D) is strongly associated with obesity. In humans, T2D increases the risk for end organ complications. Among these, heart disease has been ranked as the leading cause of death. We used a proteomic methodology to test the hypothesis that a pre-diabetic state generated by high-fat diet leads to changes in proteins related to heart function and structure. Over 300 proteins spots were resolved by 2-DE. Fifteen protein spots were found to be altered (7 decreased and 8 increased) in pre-diabetic hearts. The protein spots were then identified by mass spectrometry and immunoblots. Among the decreased proteins, 3 are involved in heart structure (one isoform of desmin, troponin T2 and α-cardiac actin), 3 are involved in energy metabolism (mitochondrial ATP synthase β subunit, adenylate kinase and creatine kinase) and one is a component of the citric acid cycle (isocitrate dehydrogenase 3). In contrast, proteins involved in fatty acid oxidation (two isoforms of peroxisomal enoyl-CoA hydratase) and the citric acid cycle (three isoforms of malate dehydrogenase) were increased in pre-diabetic hearts. The results suggest that changes in the levels of several heart proteins may have implications in the development of the cardiac phenotype associated to T2D.
doi:10.1016/j.jprot.2011.02.018
PMCID: PMC3081671  PMID: 21354350
Type 2 diabetes; obesity; murine models of obesity-type 2 diabetes; heart proteome; heart dysfunction
15.  Plasma proteomic profiles of bovine growth hormone transgenic mice as they age 
Transgenic research  2011;20(6):1305-1320.
Attenuation of the growth hormone (GH)/ insulin-like growth factor-1 (IGF-1) axis results in extended lifespan in many organisms including mice. Conversely, GH transgenic mice have excess GH action and die prematurely. We have studied bovine (b) GH transgenic mice (n = 9) and their wild type (WT) littermates (n = 8) longitudinally and have determined several age-related changes. Compared to WT mice, bGH mice lost fat mass, became hypoglycemic and had lower insulin levels at older ages despite being hyperinsulinemic when young. To examine plasma protein differences in bGH mice relative to controls, samples at 2, 4, 8, 12 and 16 months of age were analyzed by two-dimensional gel electrophoresis followed by identification using mass spectrometry. We found several differences in plasma proteins of bGH mice compared to controls, including increased apolipoprotein E (five isoforms), haptoglobin (four isoforms) and mannose-binding protein-C (one out of three isoforms), and decreased transthyretin (six isoforms). In addition, clusterin (two out of six isoforms) and haptoglobin (four isoforms) were up-regulated in bGH mice as a function of age. Finally, alpha-2 macroglobulin (seven isoforms) was altered in an isoform-specific manner with two isoforms increased and two decreased in bGH mouse plasma compared to controls. In conclusion, identification of these proteins suggests that bGH mice exhibit an increased inflammatory state with an adverse lipid profile, possibly contributing to their diminished life expectancy. Also, these newly discovered plasma proteins may be indicative or ‘biomarkers’ of a shortened lifespan.
doi:10.1007/s11248-011-9499-5
PMCID: PMC3176978  PMID: 21365322
Proteomics; Growth hormone; Plasma; Two-dimensional gel electrophoresis; Aging; Inflammation
16.  Plasma biomarkers of mouse aging 
Age  2010;33(3):291-307.
Normal aging is accompanied by a series of physiological changes such as gray hair, cataracts, reduced immunity, and increased susceptibility to disease. To identify novel biomarkers of normal aging, we analyzed plasma proteins of male mice longitudinally from 2 to 19 months of age. Plasma proteins were analyzed by two-dimensional gel electrophoresis and identified using mass spectrometry (MS), MS/MS and liquid chromatography MS/MS. We found that many plasma proteins exist as multiple isoforms with different masses and/or charges. Thirty-nine protein spots (corresponding to six distinct proteins) have been identified, 13 of which exhibited significant changes with age. For example, several proteins increased significantly during aging including one isoform of transthyretin, two isoforms of haptoglobin, and three isoforms of immunoglobulin kappa chain. Conversely, several proteins decreased significantly during aging including peroxiredoxin-2, serum amyloid protein A-1, and five isoforms of albumin. Identification of these proteins provides new biomarkers of normal aging in mice. If validated in humans, these biomarkers may facilitate therapeutic interventions to identify premature aging, delay aging, and/or improve healthspan of the elderly.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-010-9179-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-010-9179-z
PMCID: PMC3168609  PMID: 20842533
Mouse aging; Biomarkers; Proteomics; Two-dimensional gel electrophoresis; Plasma
17.  Morphology of ovaries in laron dwarf mice, with low circulating plasma levels of insulin-like growth factor-1 (IGF-1), and in bovine GH-transgenic mice, with high circulating plasma levels of IGF-1 
Background
It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals, and one of the signs of aging is progressive gonadal dysfunction.
Methods
To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on ovaries, we analyzed ovaries isolated from 2-year-old growth hormone receptor knockout (GHR-KO) Laron dwarf mice, with low circulating plasma levels of IGF-1, and 6-month-old bovine growth hormone transgenic (bGHTg) mice, with high circulating plasma levels of IGF-1. The ages of the Laron dwarf mutants employed in our studies were selected based on their overall survival (up to ~ 4 years for Laron dwarf mice and ~ 1 year for bGHTg mice).
Results
Morphological analysis of the ovaries of mice that reached ~50% of their maximal life span revealed a lower biological age for the ovaries isolated from 2-year-old Laron dwarf mice than their normal-lifespan wild type littermates. By contrast, the ovarian morphology of increased in size 6 month old bGHTg mice was generally normal.
Conclusion
Ovaries isolated from 2-year-old Laron dwarf mice exhibit a lower biological age compared with ovaries from normal WT littermates at the same age. At the same time, no morphological features of accelerated aging were found in 0.5-year-old bGHTg mice compared with ovaries from normal the same age-matched WT littermates.
doi:10.1186/1757-2215-5-18
PMCID: PMC3583234  PMID: 22747742
Murine ovary; Laron dwarf mouse; Bovine growth hormone transgenic mouse; Growth hormone; Insulin-like growth factor-1; Aging
18.  Growth hormone and adipose tissue: beyond the adipocyte 
The last two decades have seen resurgence in the interest in, and research on, adipose tissue. In part, the increased interest stems from an alarming increase in obesity rates worldwide. However, an understanding that this once simple tissue is significantly more intricate and interactive than previously realized has fostered additional attention. While few would argue that growth hormone (GH) radically alters adipose tissue, a better appreciation of the newer complexities requires that GH's influence on this tissue be reexamined. Therefore, the objective of this review is to describe the more recent understanding of adipose tissue and how GH may influence and contribute to these newer complexities with special focus on the available data from mice with altered GH action.
doi:10.1016/j.ghir.2011.03.002
PMCID: PMC3112270  PMID: 21470887
growth hormone; body composition; obesity; aging; adipose tissue; gender and age differences
19.  Activation of the GH/IGF-1 axis by CJC-1295, a long acting GHRH analog, results in serum protein profile changes in normal adult subjects 
Objective
To identify biomarkers of growth hormone (GH) and insulin-like growth factor 1 (IGF-1) action in human serum.
Background
The search for new markers of GH activity has received extensive attention given that the current biomarkers (IGF-1, IGFBP-3 and collagen peptides) show substantial variability in the population, and are not reliably predictive of either the physiologic effects of GH therapy or the detection of GH abuse by athletes. GH releasing hormone (GHRH) is a polypeptide synthesized in the hypothalamus that binds to receptors on pituitary somatotropes to promote the synthesis and release of GH. Serum GH and IGF-1 levels have been shown to increase with administration of GHRH or CJC-1295, a long acting GHRH analog.
Design
Sera from 11 healthy young adult men before and one week after CJC-1295 injection were analyzed by two-dimensional gel electrophoresis for proteomic changes. Serum proteins displaying significant changes before and after treatment were subsequently identified using mass spectrometry. In addition, correlations between these proteins and GH or IGF-1 levels were evaluated.
Results
Two protein spots that displayed decreased intensities after treatment were identified as an apolipoprotein A1 isoform and a transthyretin isoform. Three protein spots upregulated by CJC-1295 treatment included beta-hemoglobin, a C-terminal fragment of albumin, and a mix of an immunoglobulin fragment and another C-terminal albumin fragment. A linear relationship was found between the spot containing immunoglobulin and albumin fragments and IGF-1 levels.
Conclusions
Although the molecular mechanisms linking the identified proteins to GH and IGF-1 biological activity remain to be clarified, the results suggest that they represent potential biomarkers of GH and/or IGF-1 action.
doi:10.1016/j.ghir.2009.03.001
PMCID: PMC2787983  PMID: 19386527
serum proteomics; biomarkers; growth hormone; IGF-1; GHRH analog; apolipoprotein A1; transthyretin; albumin; hemoglobin
20.  Perspective: Proteomic approach to detect biomarkers of human growth hormone 
Several serum biomarkers for recombinant human growth hormone (rhGH) have been established, however, none alone or in combination have generate a specific, sensitive, and reproducible ‘kit’ for the detection of rhGH abuse. Thus, the search for additional GH specific biomarkers continues. In this review, we focus on the use of proteomics in general and 2-dimensional electrophoresis (2-DE) in particular for the discovery of new GH induced serum biomarkers. Also, we review some of the protocols involved in 2DE. Finally, the possibility of tissues other than blood for biomarker discovery is discussed.
doi:10.1016/j.ghir.2009.04.018
PMCID: PMC2760539  PMID: 19501004
proteomics; two-dimensional gel electrophoresis; growth hormone; doping; biomarker; blood; urine; skin
21.  Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects 
PLoS ONE  2012;7(2):e31857.
Background
Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.
Methodology/Principal Findings
The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.
Conclusions/Significance
Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.
doi:10.1371/journal.pone.0031857
PMCID: PMC3285196  PMID: 22384088
22.  Plasma Protein Biomarkers Correlated with the Development of Diet-Induced Type 2 Diabetes in Mice 
Clinical proteomics  2010;6(1-2):6-17.
Introduction
Early detection, assessment of disease progression, and application of an appropriate therapeutic intervention are all important for the care of patients with type 2 diabetes. Currently, however, there is no simple test for early detection of type 2 diabetes. Established diagnostic tests for the disease including oral glucose tolerance, fasting blood glucose, and hemoglobin A1c are relatively late markers where the disease has already progressed. Since blood is in direct contact with many tissues, we hypothesized that pathological tissue changes are likely to be reflected in proteomic profiles of plasma.
Methods
Mice were reared either on regular chow or a high-fat diet at weaning and several physiological responses (i.e., weight, fasting plasma glucose and insulin, and glucose tolerance) were monitored at regular time intervals. Plasma was collected at regular intervals for proteomic analysis by two-dimensional gel electrophoresis and subsequent mass spectrometry.
Results
Onset of hyperinsulinemia with corresponding glucose intolerance was observed in 2 weeks and fasting blood glucose levels rose significantly after 4 weeks on the high-fat diet. Many proteins were found to exist in multiple forms (isoforms). Levels of some isoforms including plasma retinol binding protein, transthyretin, Apolipoprotein A1, and kininogen showed significant changes as early as 4 weeks which coincided with the very early development of glucose intolerance.
Conclusions
These results show that a proteomic approach to study the development of type 2 diabetes may uncover unknown early post-translationally modified diagnostic and/or therapeutic protein targets.
doi:10.1007/s12014-009-9040-5
PMCID: PMC2899708  PMID: 20625478
Diabetes; Biomarkers; Mice; Protein isoforms; Two-dimensional gel electrophoresis; Plasma
23.  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
24.  Two-Year Body Composition Analyses of Long-Lived GHR Null Mice 
Growth hormone receptor gene–disrupted (GHR−/−) mice exhibit increased life span and adipose tissue mass. Although this obese phenotype has been reported extensively for young adult male GHR−/− mice, data for females and for other ages in either gender are lacking. Thus, the purpose of this study was to evaluate body composition longitudinally in both male and female GHR−/− mice. Results show that GHR−/− mice have a greater percent fat mass with no significant difference in absolute fat mass throughout life. Lean mass shows an opposite trend with percent lean mass not significantly different between genotypes but absolute mass reduced in GHR−/− mice. Differences in body composition are more pronounced in male than in female mice, and both genders of GHR−/− mice show specific enlargement of the subcutaneous adipose depot. Along with previously published data, these results suggest a consistent and intriguing protective effect of excess fat mass in the subcutaneous region.
doi:10.1093/gerona/glp175
PMCID: PMC2796884  PMID: 19901018
Body composition; Growth hormone; Obesity; Adipose depots; Gender differences
25.  Endocrine Regulation of Heat Shock Protein mRNA Levels in Long-lived Dwarf Mice 
Heat shock proteins (HSPs) maintain proteostasis and may protect against age-associated pathology caused by protein malfolding. In C. elegans, the lifespan extension and thermotolerance in mutants with impaired insulin/IGF signals depends partly on HSP elevation. Less is known about the role of HSPs in the increased lifespan of mice with defects in GH/IGF-I pathways. We measured HSP mRNAs in liver, kidney, heart, lung, muscle and cerebral cortex from long-lived Pit1(dw/dw) Snell dwarf mice. We found many significant differences in HSP mRNA levels between dwarf and control mice, but these effects were complex and organ-specific. We noted 15 instances where HSP mRNAs were lower in Pit1(dw/dw) liver, kidney, or heart tissues, and 14/15 of these were also seen in Ghr(-/-) mice, which lack GH receptor. In contrast, of 12 examples where HSP mRNAs were higher in Snell liver, kidney, or heart, none were altered in Ghr(-/-) mice. Four liver mRNAs were depressed in both Pit1(dw/dw) and Ghr(-/-) mice, and each of these was elevated by GH injection in Ames (Prop1(df/df)) dwarf mice, consistent with the hypothesis that these declines depended on GH and/or IGF-I. Contributions of chaperones to longevity in mice may be more complex than those inferred from C. elegans.
doi:10.1016/j.mad.2009.03.004
PMCID: PMC2718793  PMID: 19428459

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