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1.  Age-Related and Depot-Specific Changes in White Adipose Tissue of Growth Hormone Receptor-Null Mice 
Growth hormone receptor-null (GHR−/−) mice are dwarf, insulin sensitive, and long-lived in spite of increased adiposity. However, their adiposity is not uniform, with select white adipose tissue (WAT) depots enlarged. To study WAT depot–specific effects on insulin sensitivity and life span, we analyzed individual WAT depots of 12- and 24-month-old GHR− /− and wild-type (WT) mice, as well as their plasma levels of selected hormones. Adipocyte sizes and plasma insulin, leptin, and adiponectin levels decreased with age in both GHR− /− and WT mice. Two-dimensional gel electrophoresis proteomes of WAT depots were similar among groups, but several proteins involved in endocytosis and/or cytoskeletal organization (Ehd2, S100A10, actin), anticoagulation (S100A10, annexin A5), and age-related conditions (alpha2-macroglobulin, apolipoprotein A-I, transthyretin) showed significant differences between genotypes. Because Ehd2 may regulate endocytosis of Glut4, we measured Glut4 levels in the WAT depots of GHR− /− and WT mice. Inguinal WAT of 12-month-old GHR− /− mice displayed lower levels of Glut4 than WT. Overall, the protein changes detected in this study offer new insights into possible mechanisms contributing to enhanced insulin sensitivity and extended life span in GHR− /− mice.
doi:10.1093/gerona/glt110
PMCID: PMC3859361  PMID: 23873966
Aging; Growth hormone receptor; Adipose tissue depots; Endocytosis; Glut4.
2.  Growth hormone signaling is necessary for lifespan extension by dietary methionine 
Aging Cell  2014;13(6):1019-1027.
Growth hormone significantly impacts lifespan in mammals. Mouse longevity is extended when growth hormone (GH) signaling is interrupted but markedly shortened with high-plasma hormone levels. Methionine metabolism is enhanced in growth hormone deficiency, for example, in the Ames dwarf, but suppressed in GH transgenic mice. Methionine intake affects also lifespan, and thus, GH mutant mice and respective wild-type littermates were fed 0.16%, 0.43%, or 1.3% methionine to evaluate the interaction between hormone status and methionine. All wild-type and GH transgenic mice lived longer when fed 0.16% methionine but not when fed higher levels. In contrast, animals without growth hormone signaling due to hormone deficiency or resistance did not respond to altered levels of methionine in terms of lifespan, body weight, or food consumption. Taken together, our results suggest that the presence of growth hormone is necessary to sense dietary methionine changes, thus strongly linking growth and lifespan to amino acid availability.
doi:10.1111/acel.12269
PMCID: PMC4244257  PMID: 25234161
dwarf; lifespan; growth hormone; methionine; mice
3.  Specific suppression of insulin sensitivity in growth hormone receptor gene-disrupted (GHR-KO) mice attenuates phenotypic features of slow aging 
Aging Cell  2014;13(6):981-1000.
In addition to their extended lifespans, slow-aging growth hormone receptor/binding protein gene-disrupted (knockout) (GHR-KO) mice are hypoinsulinemic and highly sensitive to the action of insulin. It has been proposed that this insulin sensitivity is important for their longevity and increased healthspan. We tested whether this insulin sensitivity of the GHR-KO mouse is necessary for its retarded aging by abrogating that sensitivity with a transgenic alteration that improves development and secretory function of pancreatic β-cells by expressing Igf-1 under the rat insulin promoter 1 (RIP::IGF-1). The RIP::IGF-1 transgene increased circulating insulin content in GHR-KO mice, and thusly fully normalized their insulin sensitivity, without affecting the proliferation of any non-β-cell cell types. Multiple (nonsurvivorship) longevity-associated physiological and endocrinological characteristics of these mice (namely beneficial blood glucose regulatory control, altered metabolism, and preservation of memory capabilities) were partially or completely normalized, thus supporting the causal role of insulin sensitivity for the decelerated senescence of GHR-KO mice. We conclude that a delayed onset and/or decreased pace of aging can be hormonally regulated.
doi:10.1111/acel.12262
PMCID: PMC4326932  PMID: 25244225
endocrinology and metabolism; growth hormone hormonal signaling; insulin sensitivity; longevity regulation; (neuro)endocrinology of senescence
4.  Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment 
Biochemical and biophysical research communications  2013;441(1):10.1016/j.bbrc.2013.10.023.
Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.
doi:10.1016/j.bbrc.2013.10.023
PMCID: PMC3855845  PMID: 24134847
Growth hormone receptor; melanoma; growth hormone; prolactin receptor; cancer
5.  The effect of low and high plasma levels of insulin-like growth factor-1 (IGF-1) on the morphology of major organs: studies of Laron dwarf and bovine growth hormone transgenic (bGHTg) mice 
Histology and histopathology  2013;28(10):1325-1336.
Summary
It is well known that somatotrophic/insulin signaling affects lifespan in experimental animals. To study the effects of insulin-like growth factor-1 (IGF-1) plasma level on the morphology of major organs, we analyzed lung, heart, liver, kidney, bone marrow, and spleen 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 two mutant strains employed in our studies were selected based on their overall ~50% survival (Laron dwarf mice live up to ~4 years and bGHTg mice up to ~1 year). Morphological analysis of the organs of long-living 2-year-old Laron dwarf mice revealed a lower biological age for their organs compared with normal littermates, with more brown adipose tissue (BAT) surrounding the main body organs, lower levels of steatosis in liver, and a lower incidence of leukocyte infiltration in different organs. By contrast, the organs of 6-month-old, short-living bGHTg mice displayed several abnormalities in liver and kidney and a reduced content of BAT around vital organs.
PMCID: PMC3988530  PMID: 23613169
Aging; Brown adipose tissue; Insulin-like growth factor-1 (IGF-1); Growth hormone (GH); Laron dwarf mice
6.  Selective inner retinal dysfunction in growth hormone transgenic mice 
Objective
The discovery of locally produced growth hormone (GH) and its receptor in the retina of rodents raises the possibility that GH might modulate retinal function. To test this hypothesis, we determined the retinal electroretinogram (ERG) of bovine GH (bGH) transgenic mice.
Design
ERGs were recorded from 11 wild type (WT) and 9 bGH mice, at 2 months of age in response to a series of light flashes at increasing intensity. Three ERG components were assessed for their amplitude and timing: a-wave, b-wave and oscillatory potentials (OPs). OPs were isolated with a 75–300 Hz digital filter. Retina layer sizes, nuclei number and vascularisation were assessed by respectively staining cross sections with DAPI and Bandeiraea simplicifolia.
Results
OPs were selectively affected in the bGH mouse compared to WT. When OP amplitude values were normalized to the a-wave amplitude (to account for inter-animal variability in WT and bGH groups), OP2, OP3, and OP4 showed amplitude reductions (of 65%, 72%, and 68%, respectively) in the bGH mouse compared to the WT. This was accompanied by a prolongation of the implicit time for the peak of OP3 (28.1 vs 31.1 ms, WT vs bGH) and OP4 (37.8 vs 41.6 ms), while the implicit time of a- and b-waves were unaffected. Fast Fourier transform analysis revealed that the OPs’ dominant frequency was significantly reduced (P < 0.05) in the bGH mice (100 Hz) compared to WT (108 Hz). There was no significant change in retinal histology except for a significant increase in the axial length of the eye in bGH mice.
Conclusions
Mice expressing bGH display a selective inner retinal defect as demonstrated using ERG recordings. The specific OP defect observed in these mice is similar to the ERG results obtained in patients with diabetic retinopathy and in related animal models.
doi:10.1016/j.ghir.2011.05.008
PMCID: PMC4151295  PMID: 21705251
growth hormone; retina; electroretinography; growth factors; diabetic retinopathy; retinopathy of prematurity
7.  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
8.  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
9.  The GH/IGF-1 axis in ageing and longevity 
Nature reviews. Endocrinology  2013;9(6):366-376.
Secretion of growth hormone (GH), and consequently that of insulin-like growth factor 1 (IGF-1), declines over time until only low levels can be detected in individuals aged ≥60 years. This phenomenon, which is known as the ‘somatopause’, has led to recombinant human GH being widely promoted and abused as an antiageing drug, despite lack of evidence of efficacy. By contrast, several mutations that decrease the tone of the GH/IGF-1 axis are associated with extended longevity in mice. In humans, corresponding or similar mutations have been identified, but whether these mutations alter longevity has yet to be established. The powerful effect of reduced GH activity on lifespan extension in mice has generated the hypothesis that pharmaceutically inhibiting, rather than increasing, GH action might delay ageing. Moreover, mice as well as humans with reduced activity of the GH/IGF-1 axis are protected from cancer and diabetes mellitus, two major ageing-related morbidities. Here, we review data on mouse strains with alterations in the GH/IGF-1 axis and their effects on lifespan. The outcome of corresponding or similar mutations in humans is described, as well as the potential mechanisms underlying increased longevity and the therapeutic benefits and risks of medical disruption of the GH/IGF-1 axis in humans.
doi:10.1038/nrendo.2013.67
PMCID: PMC4074016  PMID: 23591370
10.  Serum proteome changes in acromegalic patients following transsphenoidal surgery: novel biomarkers of disease activity 
Context
Transsphenoidal adenomectomy is the primary treatment for acromegaly. However, assessment of the therapeutical outcome remains problematic since the existing biomarkers of disease activity frequently show discordant results.
Objective
To discover novel serum biomarkers of disease activity in acromegalic patients before and after surgery.
Design
Serum samples of eight newly diagnosed acromegaly patients before and after transsphenoidal surgery were analyzed for proteomic changes by two-dimensional gel electrophoresis. Protein spots displaying statistically significant changes, pre- versus post-surgery, were identified by mass spectrometry (MS), tandem MS (MS/MS), and western blot analysis.
Results
Six protein spots displaying decreased intensities after surgery were identified as transthyretin (two isoforms), haptoglobin a2, b-hemoglobin, and apolipoprotein A-1 (two isoforms). One protein spot, identified as complement C4B precursor, was increased after the surgery.
Conclusions
Seven serum protein spots were differentially expressed following surgery in acromegalic patients. The identified proteins represent potential novel biomarkers to assess the effectiveness of surgical treatment in acromegalic individuals. Future studies will validate the use of the identified proteins as biomarkers of disease activity after medical treatment of acromegaly.
doi:10.1530/EJE-10-0754
PMCID: PMC4074021  PMID: 21059862
11.  Proteomic analysis allows for early detection of potential markers of metabolic impairment in very young obese children 
Background
Early diagnosis of initial metabolic derangements in young obese children could influence their management; however, this impairment is frequently not overt, but subtle and undetectable by routinely used clinical assays. Our aim was to evaluate the ability of serum proteomic analysis to detect these incipient metabolic alterations in comparison to standard clinical methods and to identify new candidate biomarkers.
Methods
A cross-sectional study of fasting serum samples from twenty-two prepubertal, Caucasian obese (OB; 9.22 ± 1.93 years; 3.43 ± 1.08 BMI-SDS) and twenty-one lean controls (C; 8.50 ± 1.98 years; -0.48 ± 0.81 BMI-SDS) and a prospective study of fasting serum samples from twenty prepubertal, Caucasian obese children (11 insulin resistant [IR]) before (4.77 ± 1.30 BMI-SDS) and after weight reduction (2.57 ± 1.29 BMI-SDS) by conservative treatment in a reference hospital (Pros-OB) was performed. Proteomic analysis (two-dimension-eletrophoresis + mass spectrometry analysis) of serum and comparative evaluation of the sensitivity of routinely used assays in the clinics to detect the observed differences in protein expression level, as well as their relationship with anthropometric features, insulin resistance indexes, lipid profile and adipokine levels were carried out.
Results
Study of the intensity data from proteomic analysis showed a decrease of several isoforms of apolipoprotein-A1, apo-J/clusterin, vitamin D binding protein, transthyretin in OBvs. C, with some changes in these proteins being enhanced by IR and partially reversed after weight loss. Expression of low molecular weight isoforms of haptoglobin was increased in OB, enhanced in IR and again decreased after weight loss, being positively correlated with serum interleukin-6 and NAMPT/visfatin levels. After statistical correction for multiple comparisons, significance remained for a single isoform of low MW haptoglobin (OB vs. C and IR vs. non-IR) and Apo A1 (IR vs. non-IR). Assays routinely used in the clinical setting (ELISA/kinetic nephelometry), only partially confirmed the changes observed by proteomic analysis (ApoA1 and haptoglobin).
Conclusion
Proteomic analysis can allow for the identification of potential new candidate biomarkers as a complement to routinely used assays to detect initial changes in serum markers of inflammation and lipid metabolism impairment in young obese children.
doi:10.1186/1687-9856-2014-9
PMCID: PMC4063220  PMID: 24949022
Proteomic; Two dimension electrophoresis; Childhood obesity; Insulin resistance
12.  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.
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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