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Cardiomyocyte apoptosis. Prevalence of cardiomyocyte apoptosis was determined by TUNEL (A) and annexin V (B). LV slices, obtained from five transgenic mice overexpressing bGH (Acro), five Acro treated with GH receptor antagonist (Acro-Peg), and five littermate control (Wt), aged 3 and 9 months, were counterstained with Sytox orange to visualize all cell nuclei in the myocardial section. Results represent the mean SD of data obtained in five animals for each group and represent the number of cells simultaneously stained with-sarcomeric actin (-actin) and TUNEL (or annexin V, as appropriate) divided by total cells stained with Sytox. Five Acro and five Wt aged 3 months were treated with doxorubicin for 24 h (C). Apoptosis was evaluated by TUNEL and expressed as mean SD of cells stained with-sarcomeric actin and TUNEL simultaneously divided by total cells stained with Sytox.-Sacromeric actin was used to visualize cardiomyocytes apoptosis in all experiments (see below).
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GH has antiapoptotic effects in cardiac or noncardiac cell lines; however, increased apoptosis has been found in myocardial samples of patients with acromegaly. The aim of this study was to investigate cardiac apoptosis and underlying molecular mechanisms in transgenic mice overexpressing bovine GH [acromegalic mice (Acro)] aged 3 or 9 months. Card...
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... was 0.29 0.014 and 0.25 0.008% in 3-and 9-month-old Wt, respectively, and 0.13 0.03 and 0.52 0.009% in young and elder Acro, respectively (P 0.0001) when measured by TUNEL (Fig. 2A); the trend was indis- tinguishable (0.32 0.02 and 0.25 0.016% in 3-and 9 month-old Wt, respectively, and 0.11 0.015 and 0.44 0.012% in young and old Acro, respectively (P 0.0001) FIG. 5. Myocardial level of pro-and antiapoptotic proteins in 3-month-old mice. A representative Western blot is shown of changes in the expression of ...
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... Bax, Bad, and Bcl-XL indistinguishable from that of Wt. Data are expressed as A.U., which represent the ratio between the intensity of the band of interest and the intensity of the band corresponding to the control protein and represent the mean SD of measurements obtained in five animals of each group. when apoptosis was evaluated by annexin V (Fig. 2B). Over- all, 3-month-old Acro had 55% lower apoptosis than Wt (P 0.001), whereas 9-month-old Acro had about 80% higher apoptosis degree than littermate controls (P 0.0001). Im- portantly, young Acro had a degree of cardiac apoptosis that was indistinguishable, after treatment with a GH receptor an- tagonist, from that of littermate ...
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... after treatment with a GH receptor an- tagonist, from that of littermate controls, suggesting a GH- dependent mechanism in the low degree of apoptosis observed in young Acro. On the contrary, treatment with a GH receptor antagonist had no effect in 9-month-old Acro, suggesting GH- independent mechanisms of increased apoptosis in elder ages (Fig. 2, A and B). . Cytochrome c, caspase-9, and caspase-3 expression in 9-month-old animals. A representative Western blot is shown of cytosolic and mitochondrial extracts of LVs obtained from 9-month-old transgenic mice (Acro), controls (Wt), or Acro treated with a GH receptor antagonist (Acro-Peg) (A). The expression of cytosolic proteins was ...
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... (0.05-0.5-2 mg/kg body weight). Acro treated with doxorubicin at 0.05 or 0.5 mg/kg body weight had about 50 and 35% lower apoptosis, respectively, than Wt (P 0.005). Higher doses of doxorubicin were associated with higher degree of apoptosis without a difference be- tween Acro and Wt (8.5 0.71 and 9.1 0.72%, respectively, P value not significant; Fig. ...
Citations
... The histological sections were prepared and stained as per the instructions (KeyGen Biotech Co Ltd, Nanjing, China), and the TUNEL-positive cells were counted using a quantitative digital analysis system (NIH Image 1.6; National Institutes of Health, Bethesda, MA, USA) and Image Pro Plus 6.0. The DNA fragmentation test was performed using the Annexin V-Fluos Staining Kit and activated caspase-3 antibody (Roche Diagnostics, Mannheim, Germany) according to the previous methods [28,29]. ...
Pentraxin 3 (PTX3) is synthesized locally and released into the circulation, reflecting local inflammation in the cardiovascular system. Therefore, we conducted a study to explore the effect of PTX3 in spontaneously hypertensive heart failure (SHHF) rats. Sprague Dawley (SD) and SHHF rats were treated with recombinant PTX3 protein, and the blood pressure (BP) and echocardiographic parameters were collected. Radioimmunoassay, enzyme immunoassay and enzyme-linked immunosorbent assay (ELISA) were applied to detect plasma levels of atrial/B-type natriuretic peptide (ANP/BNP) and PTX3. The pathological changes in the myocardial tissues were observed by hematoxylin and eosin (HE) and Masson stainings. The mRNA and protein expressions were detected by quantitative real-time reverse-transcription polymerase chain reaction (qPCR) and western blotting. Cardiomyocyte apoptosis was evaluated by TUNEL staining and DNA fragmentation test. Increased plasma concentrations of PTX3 were found in SHHF rats compared with SD rats, which was further enhanced by recombinant PTX3 protein. After injection with recombinant PTX3 protein, the heart function was improved in SHHF rats with the decreased systolic and diastolic BP, and the reduced plasma levels of ANP and BNP. Moreover, PTX3 improved the myocardial damage and interstitial fibrosis in SHHF rats with reduced cardiomyocyte apoptosis and decreased mRNA expressions of pro-inflammatory factors in myocardial tissues. PTX3 could decrease the BP and plasma levels of ANP and BNP in SHHF rats, as well as improve the inflammation, cardiomyocyte apoptosis, and pathological changes of myocardial tissues, suggesting it may be a useful intervention in the treatment of SHHF.
... Additionally, this result may suggest that GH replacement during the first 6 months might be insufficient to block the pro-apoptotic mechanisms induced in GHD patients presenting the chronic endogenous GH deficiency conditions. On the other hand, the opposite results were reported by a number of authors, who demonstrated that expression of BAX gene together with other pro-apoptotic factors, such as BAD or caspases, were down-regulated in an in vitro model of T cell lymphoma over-expressing GH [12] and in cardiomyocytes [46] or colonocytes [47] of transgenic mice overexpressing GH gene as well as in myocytes [48] and neurons [49] of GH-treated animals. Unfortunately, at this stage of our research, it is impossible to define the exact mechanism of the observed BAX gene up-regulation during GH treatment. ...
Growth hormone (GH) modulates hematopoietic cell homeostasis and is associated with apoptosis control, but with limited mechanistic insights. Aim of the study was to determine whether GH therapeutic supplementation (GH-TS) could affect apoptosis of CD34+ cells enriched in hematopoietic progenitor cells of GH deficient (GHD) children. CD34+ cells from peripheral blood of 40 GHD children were collected before and in 3rd and 6th month of GH-TS and compared to 60 controls adjusted for bone age, sex, and pubertal development. Next, apoptosis assessment via different molecular techniques was performed. Finally, to comprehensively characterize apoptosis process, global gene expression profile was determined using genome-wide RNA microarray technology. Results showed that GH-TS significantly reduced spontaneous apoptosis in CD34+ cells (p < 0.01) and results obtained using different methods to detect early and late apoptosis in analyzed cells population were consistent. GH-TS was also associated with significant downregulation of several members of TNF-alpha superfamily and other genes associated with apoptosis and stress response. Moreover, the significant overexpression of cyto-protective and cell cycle-associated genes was detected. These findings suggest that recombinant human GH has a direct anti-apoptotic activity in hematopoietic CD34+ cells derived from GHD subjects in course of GH-TS.
... Apoptosis was evaluated by methods assessing either early or late events. Specifically, phosphatidylserine exposition in the outer leaflet of the plasma membrane was determined in the initial apoptotic phase, using the Annexin V-FITC Apoptosis Detection kit (Sigma-Aldrich, St. Louis, MO, USA) [34]; DNA fragmentation was determined in the final stage of apoptosis, by both the cell death detection ELISA plus kit (Roche Applied Science, Penzberg, Germany) and the terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) assay (Roche Applied Science, Penzberg, Germany) [34,35]. For the Annexin V-FITC assay, pituitary cells, treated as described in the study design section, were collected and resuspended in 500 μl binding buffer containing 5 μl fluorescein isothiocyanate (FITC) conjugated annexin V and 10 μl propidium iodide solutions, according to manufacturer's instructions. ...
Polychlorinated biphenyls (PCBs) can disrupt the endocrine function, promote neoplasms and regulate apoptosis in some tissues; however, it is unknown whether PCBs can affect the apoptosis of pituitary cells. The study evaluated the effect of PCBs on the apoptosis of normal pituitary cells and the underlying mechanisms. Primary cell cultures obtained from mouse pituitary glands were exposed to Aroclor 1254 or selected dioxin-like (PCB 77, PCB 126) or non-dioxin-like (PCB 153, PCB 180) congeners. Apoptosis was evaluated by Annexin V staining, DNA fragmentation, and TUNEL assay. Both the expression and activity of caspases were analyzed. Selective thyroid hormone receptor (TR) or aryl-hydrocarbon receptor (AhR) or CYP1A1 antagonist were used to explore the mechanisms underlying PCBs action. Our results showed that Aroclor 1254 induced the apoptosis of pituitary cells as well as the final caspase-3 level and activity through the extrinsic pathway, as shown by the increased caspase-8 level and activity. On the other hand, the intrinsic pathway evaluated by measuring caspase-9 expression was silent. The selected non-dioxin-like congeners either increased (PCB 180) or reduced (PCB 153) pituitary cell apoptosis, affecting the extrinsic pathway (PCB 180), or both the extrinsic and intrinsic pathways (PCB 153), respectively. In contrast, the dioxin-like congeners (PCB 77 and PCB 126) did not affect apoptosis. The anti-apoptotic phenotype of PCB 153 was counteracted by a TR or a CYP1A1 antagonist, whereas the pro-apoptotic effect of PCB 180 was counteracted by an AhR antagonist. The induced apoptosis of Aroclor 1254 or PCB 180 was associated with a reduction of cell proliferation, whereas the decreased apoptosis due to PCB 153 increased cell proliferation by 30%. In conclusion, our data suggest that non-dioxin-like PCBs may modulate apoptosis and the proliferation rate of pituitary cells that have either pro- or anti-apoptotic effects depending on the specific congeners. However, the impact of PCBs on the process of pituitary tumorigenesis remains to be elucidated.
... In addition to the observed metabolic alterations resulting from chronic elevations in GH and IGF-1 concentrations which can lead to the development pro-apoptotic conditions in vivo [13,14], case studies have reported that a prolonged history of rhGH abuse can lead to the development of cardiomyopathy and heart failure [44,45], conditions which are associated with deregulated apoptosis [46]. Significant increases in cardiomyocyte apoptosis has been reported in myocardial biopsies from patients with acromegaly, apparently contributing to cell loss and functional abnormalities in acromegalic cardiomyopathy [46,47]. Indeed, the degree of cardiac apoptosis was found to exhibit a significant positive relationship with both serum IGF-1 concentrations and the reported duration of acromegalic disease [47]. ...
... Significant increases in cardiomyocyte apoptosis has been reported in myocardial biopsies from patients with acromegaly, apparently contributing to cell loss and functional abnormalities in acromegalic cardiomyopathy [46,47]. Indeed, the degree of cardiac apoptosis was found to exhibit a significant positive relationship with both serum IGF-1 concentrations and the reported duration of acromegalic disease [47]. In light of these observations, the finding that the anti-apoptotic effects exerted by rhGH on Bak protein levels does not persist, at least up until 15 days following the cessation of treatment could have significant health consequences for individuals who are administering rhGH at supra-physiological concentrations. ...
The purpose of this study was to determine whether recombinant human growth hormone (rhGH) would show any significant effects on the expression of apoptosis regulating proteins in peripheral blood mononuclear cells (PBMCs). Additionally, the potential for post-transcriptional regulation of gene expression by miRNA was assessed in two cellular compartments, the cytosol and the mitochondria. Ten male subjects were subcutaneously injected with either rhGH (1 mg) or saline (0.9%) for seven consecutive days in a double-blinded fashion. Blood sampling was undertaken prior to treatment administration and over a period of three weeks following treatment cessation. Bcl-2 and Bak gene and protein expression levels were measured in PBMCs, while attention was also directed to the expression of miR-181a and miR-125b, known translational inhibitors of Bcl-2 and Bak respectively. Results showed that rhGH significantly decreased Bak protein concentrations compared to placebo samples for up to 8 days post treatment. While cytosolic miRNA expression was not found to be significantly affected by rhGH, measurement of the expression of miR-125b in mitochondrial fractions showed a significant down-regulation eight days post-rhGH administration. These findings suggest that rhGH induces short-term anti-apoptotic effects which may be partially mediated through a novel pathway that alters the concentration of mitochondrially-associated miRNAs.
... Transgenic mice overexpressing a coding sequence of the bGH gene under the control of the metallothionein promoter in the C57BL/6J ϫ CBA genetic background have been described (14). The identity of bGH transgenic (Acro) mice was confirmed by PCR analysis as previously reported (15). ...
... Groups of Lean, Obe, or Acro animals were treated with pegvisomant (Pfizer), a specific antagonist of GH receptor (0.1 mg/daily, sc, for 15 d) as previously reported (15) or with pifithrin (Sigma-Aldrich), an inhibitor of p53 function (0.1 mg daily, ip, for 10 d) (17). Effectiveness of pegvisomant was evaluated by measuring serum IGF-I concentrations before and at the end of treatment; effectiveness of pifithrin was evaluated by Western blot, measuring the expression of p21 protein, which is regulated downstream by p53 (17). ...
... Total tissue extracts were obtained by homogenizing each organ sample in lysis buffer [Tris HCl (pH 7.5), 50 mM; NaCl, 150 mM; sodium deoxycholate, 0.25%; Nonidet P-40 1%; protease inhibitor] as previously reported (15). After incubation on ice for 30 minutes and subsequent centrifugation, supernatants were stored at Ϫ80°C. ...
Insulin resistance is a key marker of both obesity and GH excess. The purpose of the study was to assess the role of growth hormone (GH) on p53-mediated insulin resistance of male mice with obesity due to high-fat diet. C57BL/6JxCBA male mice fed on high-fat diet (Obe) were studied; male mice fed on normal diet (Lean) or transgenic mice for bovine GH under the same genetic background (Acro) served as controls. The convergence of p53 and GH pathways was evaluated by Western blot. Obe mice had insulin resistance, which was sustained by a selective increased expression of p53 in adipose tissue. Normal insulin sensitivity was restored and adipose p53 expression normalized when the GH pathway was blocked. Only the adipose p53 expression was sensitive to the GH blockage, which occurred through the p38 pathway. Adipose tissue of Obe mice had a coordinate overexpression of SOCS1-3 and STAT1, 3 and 5b, not different from that of Acro mice, suggesting an increased sensitivity of adipose tissue to GH. On the opposite, Lean mice were unaffected by changes of GH action.GH seems to be necessary for the increased adipose p53 expression and for insulin resistance of obese mice.
... Acromegalic patients often have kidney problems [23] and cardiovascular diseases [24]. Likewise, GH transgenic mice develop progressive glomerulosclerosis [25] and cardiovascular diseases [26][27][28]. In addition, GH transgenic mice show liver inflammation and liver tumors at old ages [29][30][31]. ...
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.
... On the other hand, the stimulation of apoptosis by long-term growth hormone exposure is at variance with the commonly reported protective role of the growth hormone /IGF-I axis against cell death [51]. However, increases in myocyte apoptosis are associated with high levels of growth hormone in patients with acromegaly [52] and in 9-month-old transgenic mice overexpressing growth hormone [53]. Furthermore, coho salmon implanted with growth hormone for 2 weeks showed stimulated Na-dependent proline absorption in the intestine [4], which may reflect increased permeability of the apoptotic intestinal epithelia in seawater. ...
The in-vitro effects of prolactin on the permeability of trout gill epithelia [16, 43] may also be associated with direct stimulation of cell proliferation by prolactin, as proposed above for the medaka esophagus. Prolactin has also been shown to induce cell proliferation in jejunal explants from fetal rat [44], and several studies have shown direct effects of prolactin on cell proliferation throughout vertebrates [21]. In teleosts, prolactin induces proliferative responses in cultured salmonid leukocytes [25, 26], and promotes osteoblastic activities in goldfish scales in vitro [27]. However, the epithelium appears to be the major target, as in human keratinocytes and prostate epithelial cells [3, 45, 46], and it is likely that a primary function of prolactin in teleost osmoregulation is direct stimulation of cell proliferation in osmoregulatory epithelia. On the other hand, prolactin has also been shown to stimulate apoptosis in newt spermatogonia and rat luteal tissues [47, 48]. In addition, we suggested that the inhibitory effect of prolactin on osteoclastic activity in goldfish scales is mediated in part through osteoclast apoptosis [27]. Further studies of intracellular signaling pathway will elucidate how prolactin regulates these cell turnover. At any rate, one of prolactin’s primary functions may be control of cell proliferation/apoptosis. Indeed, the prolactin receptors belong to the large superfamily of class 1 “cytokine” receptors.
There are few reports on the in vitro actions of growth hormone on teleost osmoregulatory organs, although growth hormone appears to be an important hormone for seawater adaptation. Direct regulatory roles of growth hormone on the gill Na⁺,K⁺-ATPase and heat-shock protein 70 in climbing perch and silver sea bream have been described [15, 17, 18]. Our experiment reveals direct effects of growth hormone on esophageal cell turnover. Induction of cell proliferation was observed 1 day after addition of 10 ng/ml growth hormone, whereas epithelial apoptosis was stimulated after 8 days. The direct action of growth hormone on cell proliferation may occur through locally produced insulin-like growth factor I (IGF-I), since IGF-I has been suggested to mediate the direct proliferative effects of growth hormone in mammalian gastrointestinal tracts [49, 50]. On the other hand, the stimulation of apoptosis by long-term growth hormone exposure is at variance with the commonly reported protective role of the growth hormone /IGF-I axis against cell death [51]. However, increases in myocyte apoptosis are associated with high levels of growth hormone in patients with acromegaly [52] and in 9-month-old transgenic mice overexpressing growth hormone [53]. Furthermore, coho salmon implanted with growth hormone for 2 weeks showed stimulated Na-dependent proline absorption in the intestine [4], which may reflect increased permeability of the apoptotic intestinal epithelia in seawater.
... Two observers blinded to the experimental protocols carried out all measurements independently. The DNA fragmentation test was performed using the Annexin-V-Fluos Staining Kit and activated Caspase III antibody (Roche Diagnostics, Mannheim, Germany) with DAPI nuclear contrast, as validated by Cittadini et al. (20) and Bogazzi et al. (23). ...
Insulin resistance is a recently identified mechanism involved in the pathophysiology of chronic heart failure (CHF). We investigated the effects of two insulin-sensitizing drugs (metformin and rosiglitazone) in a genetic model of spontaneously hypertensive, insulin-resistant rats (SHHF). Thirty SHHF rats were randomized into three treatment groups as follows: 1) metformin (100 mg/kg per day), 2) rosiglitazone (2 mg/kg per day), and 3) no drug. Ten Sprague-Dawley rats served as normal controls. At the end of the treatment period (12 months), the cardiac phenotype was characterized by histology, echocardiography, and isolated perfused heart studies. Metformin attenuated left ventricular (LV) remodeling, as shown by reduced LV volumes, wall stress, perivascular fibrosis, and cardiac lipid accumulation. Metformin improved both systolic and diastolic indices as well as myocardial mechanical efficiency, as shown by improved ability to convert metabolic energy into mechanical work. Metformin induced a marked activation of AMP-activated protein kinase, endothelial nitric oxide synthase, and vascular endothelial growth factor and reduced tumor necrosis factor-α expression and myocyte apoptosis. Rosiglitazone did not affect LV remodeling, increased perivascular fibrosis, and promoted further cardiac lipid accumulation. In conclusion, long-term treatment with metformin, but not with rosiglitazone, prevents the development of severe CHF in the SHHF model by a wide-spectrum interaction that involves molecular, structural, functional, and metabolic-energetic mechanisms.
... Indeed, this activity was reported to gauge caspase 3 activity [39]; these authors reported that the increased procaspase 3 protein observed in okadaic acid-induced apoptosis in PCa cells, reflected increased mRNA stabilization and paralleled PARP cleavage. There have been earlier reports of difficulties in detecting the active form of various caspases in LNCaP and other cells and of using changes in caspase levels as indicators of apoptosis [40,41]. The findings reported here are in apparent conflict with some earlier reports, mostly of modest mitogenic and anti-apoptotic effects of leptin, in AI PC3 and DU145 and lack of effect in AS LNCaP cells (Introduction Section). ...
Prostate cancer (PCa) progression is often associated with transactivation of the androgen receptor (AR) by endogenous hormones/growth factors. One such factor affecting growth, proliferation, and apoptostis (pro-/anti-) in various cancers is the adipokine leptin. This research studied leptin-induced signaling and apoptosis in androgen sensitive (LNCaP, PC3/AR) and insensitive (PC3, DU145) PCa cell lines.
Signaling was studied by immunoblotting in cells overexpressing leptin receptors (LRb), Janus kinase 2 (JAK2), and kinase negative-HER2-YFP cDNAs. Apoptosis was measured by immunoblotting of apoptotic proteins and by Hoechst staining of condensed DNA.
Leptin rapidly induced activation of JAK2, STAT3, and MAPK (ERK1/2) signaling cascades; it may also induce HER2 transactivation via leptin-induced phospho-JAK2. Leptin was then shown to exert clear pro-apoptotic effects, increasing levels of caspase 3, cleavage of its substrate, poly (ADP-ribose) polymerase (PARP) to cleaved PARP(89) , levels of CK 18, a cytoskeletal protein formed during apoptosis, and DNA condensation. Kinase inhibitors indicated that leptin-induced apoptosis is probably mediated by balanced activation of JAK2/STAT3, p38 MAPK, and PKC pathways in PCa cells. A human leptin mutein LRb antagonist, L39A/D40A/F41A, fully inhibited leptin-induced phosphorylation of JAK2, ERK1/2, and Akt/PKB, and partially abrogated effects on apoptotic proteins. In LNCaP and PC3/AR cells, leptin increased AR protein levels in correlation with raised apoptotic markers. Thus, AR may mediate, at least partly, the leptin-induced apoptotic response.
Leptin can clearly induce apoptosis in human PCa cell lines. These findings could lead to development of new leptin agonists with enhanced pro-apoptotic effects and targeted for use in human PCa.
... The association between GH hypersecretion/deficiency and cardiovascular health has been studied employing genetic mouse models of acromegaly (144,145) and GH deficiency (146); however, the impact of an intact GH/ IGF-I axis in cardiac morphology and function is still not well established. The GHRϪ/Ϫ mouse has provided additional insight into the role of the GH/IGF-I axis in cardiac structure and function. ...
Disruption of the GH receptor (GHR) gene eliminates GH-induced intracellular signaling and, thus, its biological actions. Therefore, the GHR gene disrupted mouse (GHR-/-) has been and is a valuable tool for helping to define various parameters of GH physiology. Since its creation in 1995, this mouse strain has been used by our laboratory and others for numerous studies ranging from growth to aging. Some of the most notable discoveries are their extreme insulin sensitivity in the presence of obesity. Also, the animals have an extended lifespan, which has generated a large number of investigations into the roles of GH and IGF-I in the aging process. This review summarizes the many results derived from the GHR-/- mice. We have attempted to present the findings in the context of current knowledge regarding GH action and, where applicable, to discuss how these mice compare to GH insensitivity syndrome in humans.
