Growth Hormone Receptor Knockout Research Articles

Metabolic Alterations Due to Caloric Restriction and Every Other Day Feeding in Normal and Growth Hormone Receptor Knockout Mice

Mutations causing decreased somatotrophic signaling are known to increase insulin sensitivity and extend life span in mammals. Caloric restriction and every other day (EOD) dietary regimens are associated with similar improvements to insulin signaling and longevity in normal mice; however, these interventions fail to increase insulin sensitivity or life span in growth hormone receptor knockout (GHRKO) mice. To investigate the interactions of the GHRKO mutation with caloric restriction and EOD dietary interventions, we measured changes in the metabolic parameters oxygen consumption (VO2) and respiratory quotient produced by either long-term caloric restriction or EOD in male GHRKO and normal mice. GHRKO mice had increased VO2, which was unaltered by diet. In normal mice, EOD diet caused a significant reduction in VO2 compared with ad libitum (AL) mice during fed and fasted conditions. In normal mice, caloric restriction increased both the range of VO2 and the difference in minimum VO2 between fed and fasted states, whereas EOD diet caused a relatively static VO2 pattern under fed and fasted states. No diet significantly altered the range of VO2 of GHRKO mice under fed conditions. This provides further evidence that longevity-conferring diets cause major metabolic changes in normal mice, but not in GHRKO mice.

Prolonged Rapamycin treatment led to beneficial metabolic switch

Prolonged Rapamycin treatment led to beneficial metabolic switch

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.

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.

Serum IGF-1 Is Insufficient to Restore Skeletal Size in the Total Absence of the Growth Hormone Receptor

States of growth hormone (GH) resistance, such those observed in Laron dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout [GHRKO]) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKO-HIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1, allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron syndrome does not achieve full skeletal growth.

Editorial:Molecular EndocrinologyArticles in the Spotlight for March 2013

Welcome to the March issue of Molecular Endocrinology. We think you will find these articles to be interesting and enlightening: In their article, “Impaired Fertility and FSH Synthesis in Gonadotrope-Specific Foxl2 Knockout Mice,” Tran et al describe the first gonadotrope-specific knockout model with a selective impairment in FSH synthesis and secretion. Their work establishes forkhead protein L2 (FOXL2) as the first cell-restricted transcription factor required for FSH -subunit (FSHb) expression in vivo. This important study advances our understanding of mechanisms of differential regulation of the gonadotropins. “The Endocrine Disrupting Chemical Tolylfluanid Alters Adipocyte Metabolism via Glucocorticoid Receptor Activation” by Neel et al finds that despite lacking structural similarity to known glucocorticoids, the fungicide tolylfluanid activates glucocorticoid signaling and alters insulin signal transduction through enhanced glucocorticoid receptor (GR) binding to a specific response element in the insulin receptor substrate-1 (IRS-1) gene. This effect mimics the action of low-dose corticosterone. Furthermore, tolylfluanid and corticosterone result in duration-dependent effects on insulin signaling. Therefore, tolylfluanid, an endocrine-disrupting chemical found on fruit crops in Europe that acts on GR may play a heretofore-unrecognized role in metabolic disorders. List et al’s “The Role of GH in Adipose Tissue: Lessons From Adipose-Specific GH Receptor Gene-Disrupted Mice” (freely available at http://mend.endojournals.org/) uses healthy obese mice in which the GH receptor (GHR) gene is disrupted to study the in vivo effects of GH directly on adipose tissue. Surprisingly, the adipose-specific GH receptor knockout shares few metabolic characteristics of a global GHR knockout. For example, the adipose-specific GHR knockout mice are obese with increased total body fat and increased adipocyte size, but resistin and adiponectin levels as well as glucose metabolism are not altered. Therefore, increased circulating adipokines in GHR global knockout mice are likely due to altered GH signaling in nonadipose tissue. Our thanks and congratulations go out to these and all of the authors of this issue’s excellent research. Donald B. DeFranco, PhD. Editor-in-Chief, Molecular Endocrinology

Effect of Growth Hormone and Calorie Restriction on the Expression of Antioxidant Enzymes in the Liver and Kidney of Growth Hormone Receptor Knockout Mice.

Caloric restriction (CR) can delay aging and prolong life span and these actions may be related to reduced oxidative damage. Mice with disrupted growth hormone (GH) receptor/binding protein knockout (GHRKO) live significantly longer than their normal siblings. Therefore, it is of interest to examine the effects of chronic CR on hepatic and renal antioxidant enzymes as well as lipid peroxidation (LP) as an oxidative stress marker in GHRKO mice. Female GHRKO and normal mice were either fed ad libitum (AL) or subjected to 30% CR starting at 2 months of age and examined at the age of 9 months. In the liver, catalase (CAT) activity was significantly increased in GHRKO-AL as compared to normal control -AL animals. CR reduced CAT activity in both GHRKO and normal phenotypes. Cu/Zn superoxide dismutase (SOD1) activity was also higher in GHRKO-AL as compared to normal-AL mice. However, CR reduced SOD1 activity in GHRKO mutants. Glutathione peroxidase (GPx) activity was significantly decreased in GHRKO-AL mice and further reduced in GHRKO-CR group of animals. CR significantly increased LP in GHRKOs while its activity was not altered in GHRKO-AL group of mice. In the kidney, CAT activity was lower in GHRKO-AL as compared to normal-AL, however CR did not induce any significant effect in both phenotypes. Similarly, SOD1 levels were significantly lower in GHRKO than in normal mice. GPx expression was higher in GHRKO-AL as compared to control-AL. CR reduced GPx activity in GHRKO mice but increased it in controls as compared to their AL counterparts. There was no difference in LP expression between GHRKO-AL and normal-AL mice. However, CR significantly increased its levels in both phenotypes. Although these findings do not support the hypothesis that CR would increase the capacity of ROS defense mechanisms in GHRKO mice by increasing antioxidant enzymes levels, they do agree with some of the reported effects of CR on their expression. We suspect that GH resistance and CR may affect aging by different mechanisms and if CR delays aging in GHRKO animals it is not due to changes in the activity of antioxidant enzymes.

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.

Upregulation of the angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas receptor axis in the heart and the kidney of growth hormone receptor knock-out mice

ObjectiveGrowth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1–7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR−/−) mouse. DesignRAS components were evaluated in the heart and the kidney of GHR−/− and control mice by immunohistochemistry and Western blotting (n=12 for both groups). ResultsThe immunostaining of Ang-(1–7) was increased in both the heart and the kidney of GHR−/− mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR−/− mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues. ConclusionsThe shift within the RAS towards an exacerbation of the ACE2/Ang-(1–7)/Mas receptor axis observed in GHR−/− mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.

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.

BackgroundIt is well known that somatotrophic/insulin signaling affects lifespan in experimental animals, and one of the signs of aging is progressive gonadal dysfunction.MethodsTo 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).ResultsMorphological 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.ConclusionOvaries 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.

Abstract LB-190: Growth hormone signaling in mammary stem and progenitor cells

Abstract The role of growth hormone (GH) in development of the normal mammary gland has been convincingly demonstrated in studies that show dramatically impaired mammary development in spontaneous dwarf rats and growth hormone receptor knock-out (GHR-KO) mice. There is also evidence that GH signaling may have a role in breast cancer. Animals treated with GH develop hyperproliferative lesions in the mammary gland and have increased incidence of spontaneous mammary tumors. Conversely, animals deficient in GH signaling are resistant to carcinogen-induced cancers, including mammary carcinoma. In humans, there is an increased incidence of cancer in patients with acromegaly (syndrome caused by excess GH production by pituitary gland) and a decreased incidence in patients with Laron syndrome (GH deficiency). In this study, we show that GH may increase the risk of breast cancer by stimulating stem and progenitor cells to enter the cell cycle. Using immunohistochemistry and immunostaining coupled with flow cytometry analysis, we found that GHR is expressed in stem and progenitor cells in the normal breast epithelium. We have previously shown that cells with ALDH activity from the human mammary epithelium have functional properties of stem/progenitor cells. Two thirds of these ALDH positive cells express GHR. GHR positive mammary epithelial cells are capable to initiate mammosphere formation in suspension culture and can generate both luminal and myoepithelial cell lineages in vitro. GH treatment increased mammosphere formation in a dose dependent manner. Although the total number of progeny was increased in these conditions, cell dynamics analysis using PKH26 dye dilution showed that the ratios between stem cells, early and late progenitor cells were not changed. GH treatment reduced the number of cells in quiescent state (G0) and decreased p21 protein levels, a cell-cycle regulator previously associated with stem cells quiescence. Taken together, these results indicated that GH treatment did not change symmetry of self-renewal cell divisions but increased the number of cycling mammary stem/progenitor cells. We found that GH is present in normal human mammary epithelial cells both in vitro and in vivo. In vitro, GH is secreted by mammary epithelial cells upon progesterone stimulation. We propose a model in which GH, estrogen and progesterone establish a paracrine signaling loop that stimulates GHR positive/steroid receptor negative stem and progenitor cells to enter the cell cycle. We propose that increased levels or prolonged exposure steroid hormones and/or GH would lead eventually to an expanded undifferentiated population at higher risk for transformation through oncogenic hits. Consistent with this concept, analysis of 180 cases of clinical precursor lesions (DCIS) showed an expanded GHR+ cell population in 92% of cases, with expression in 50-100% of the cell population in more than half of cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-190. doi:1538-7445.AM2012-LB-190

Decreased thyroid follicle size in dwarf mice may suggest the role of growth hormone signaling in thyroid growth regulation

BackgroundAltered somatotrophic signaling is among the most important potential mechanisms of extended longevity. Ames dwarf (df/df) mice are homozygous for mutation at the Prop-1 gene, leading to a lack of growth hormone (GH), prolactin and thyroid stimulating hormone (TSH). Mice homozygous for targeted disruption of the growth hormone receptor/growth hormone binding protein gene are known as GH receptor knockout (GHRKO) mice or “Laron dwarf”. Both, df/df and GHRKO mice, are characterized by reduced body size, low plasma insulin and insulin-like growth factor-I (IGF-I), remarkably extended longevity, and severe (in df/df mice) or mild (in GHRKO mice) thyroid hypofunction. Recently, by crossing df/df and GHRKO mice, double-mutant Ames dwarf/GHRKO (df/KO) mice were created. Interestingly, these mice are smaller than Ames dwarfs or GHRKOs, and also have reduced insulin and IGF-I levels. The aim of the study was to investigate if and to what extent certain thyroid morphological parameters, such as inner follicular surface area, inner follicular perimeter, as well as the follicular epithelium thickness are changed in the examined dwarf mice.MethodsThis quantification was performed in thyroids collected from df/df, GHRKO and df/KO female mice, at approximately 5–6 months of age. We used a computerized plotting programme that combines a live microscopic image of the slide with an operator-generated overlay.ResultsInner follicular surface area and inner follicular perimeter were decreased in all examined kinds of dwarf mice as compared to normal animals. Furthermore, decreases in these two parameters were more pronounced in df/df and df/KO than in GHRKO mice. Concerning the follicular epithelium thickness, only a tendency towards decrease of this parameter was found in all three kinds of dwarf mice.ConclusionsParameters characterizing thyroid follicle size are decreased in all three examined models of dwarf mice, which may explain decreased thyroid hormone levels in both basal mutants (Ames dwarfs and GHRKOs). df/df mutation seems to predominate over GHRKO genetic intervention concerning their effects on thyroid growth. Beside TSH, also GH signaling seems to constitute a crucial element in the regulation of thyroid growth and, possibly, function.

GH Does Not Modulate the Early Fasting-Induced Release of Free Fatty Acids in Mice

Fasting results in the mobilization of adipose stores and the elevation of levels of free fatty acids (FFA). In humans, this process is driven by a release in GH. Little is known regarding the role of GH in modulating this process during early stages of fasting in the mouse. Confirmation of the role of GH in modulating FFA release in the fasting mouse is of particular importance given the frequent use of mouse models to study metabolic mechanisms. Here, we correlate the initial release of FFA throughout fasting in mice with pulsatile GH secretion. Observations illustrate the rapid release of FFA in response to food withdrawal. This does not correlate with a rise in GH secretion. Rather, we observed a striking loss in pulsatile secretion of GH throughout the first 6 h of fasting, suggesting that GH does not modulate the initial release of FFA in the mouse in response to fasting. This was confirmed in GH receptor knockout mice, in which we observed a robust fasting-induced rise in FFA. We further illustrate the dynamic relationship between the orexigenic and anorexigenic hormones ghrelin and leptin during fasting in the mouse. Our findings show an initial suppression of leptin and the eventual rise in circulating levels of acyl-ghrelin with fasting. However, altered acyl-ghrelin and leptin secretion occurs well after the rise in FFA and the suppression of GH secretion. Consequently, we conclude that although acyl-ghrelin and leptin may modulate the physiological response to drive food intake, these changes do not contribute to the initial loss of pulsatile GH secretion. Rather, it appears that the suppression of GH secretion in fasting may occur in response to an elevation in fasting levels of FFA or physiological stress. Observations highlight a divergent role for GH in modulating FFA release between man and mouse.

Post-transcriptional regulation of IGF1R by key microRNAs in long-lived mutant mice.

Long-lived mutant mice, both Ames dwarf and growth hormone receptor gene-disrupted or knockout 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, miR-669b, and miR-681, are involved in posttranscriptional regulation of genes pertinent to growth hormone/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, miR-669b, or miR-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 growth hormone signaling-deficient mutant mouse brain suggests reduced IGF1 signaling at the posttranscriptional level, for numerous gains of neuronal function in these long-lived mice.

Expression of Key Regulators of Mitochondrial Biogenesis in Growth Hormone Receptor Knockout (GHRKO) Mice is Enhanced but is Not Further Improved by Other Potential Life-Extending Interventions

Mitochondrial biogenesis is essential for cell viability. Growth hormone receptor knockout (GHRKO), calorie restriction, and surgical visceral fat removal constitute experimental interventions to delay aging and increase life span. We examined the expression of known regulators of mitochondriogenesis: peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin-1 (SIRT-1) and sirtuin-3 (SIRT-3), endothelial nitric oxide synthase (eNOS), nuclear respiratory factor-1, mitochondrial transcription factor A (TFAM), and mitofusin-2 (MFN-2) in the skeletal muscles and hearts of control and calorie-restricted female GHRKO mice and in the kidneys of male GHRKOs after visceral fat removal or sham surgery. Expression of PGC-1α in skeletal muscles, AMPK, SIRT-1, SIRT-3, eNOS, and MFN-2 in the heart and PGC-1α, AMPK, SIRT-3, eNOS, and MFN-2 in kidneys was increased in GHRKO mice but was not affected by calorie restriction or visceral fat removal. GHRKO mice have increased expression of key regulators of mitochondriogenesis, which is not improved further by calorie restriction or visceral fat removal.

Key regulators of mitochondrial biogenesis are increased in kidneys of growth hormone receptor knockout (GHRKO) mice

The growth hormone receptor knockout (GHRKO) mice are remarkably long-lived and highly insulin sensitive. Alterations in mitochondrial biogenesis are associated with aging and various metabolic derangements. We have previously demonstrated increased gene expression of key regulators of mitochondriogenesis in kidneys, hearts and skeletal muscles of GHRKO mice. The aim of the present study was to quantify the protein levels of the following regulators of mitochondriogenesis: peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), AMP-activated protein kinase α (AMPKα), phospho-AMPKα (p-AMPKα), sirtuin-3 (SIRT-3), endothelial nitric oxide synthase (eNOS), phospho-eNOS (p-eNOS), nuclear respiratory factor-1 (NRF-1) and mitofusin-2 (MFN-2) in skeletal muscles and kidneys of GHRKOs in comparison to normal mice. We also were interested in the effects of calorie restriction (CR) and visceral fat removal (VFR) on these parameters. Both CR and VFR improve insulin sensitivity and can extend life span. The renal levels of PGC-1α, AMPKα, p-AMPKα, SIRT-3, eNOS, p-eNOS and MFN-2 were increased in GHRKOs. In the GHRKO skeletal muscles, only MFN-2 was increased. Levels of the examined proteins were not affected by CR (except for PGC-1α and p-eNOS in skeletal muscles) or VFR. GHRKO mice have increased renal protein levels of key regulators of mitochondriogenesis, and this may contribute to increased longevity of these knockouts.

The Effects of Aging and Genotype on NMDA Receptor Expression in Growth Hormone Receptor Knockout (GHRKO) Mice

Caloric restriction enhances N-methyl-D-aspartate (NMDA) receptor binding and upregulates messenger RNA expression of the GluN1 subunit during aging. Old growth hormone receptor knockout mice resemble old calorically restricted rodents in enhanced life span and brain function, as compared with aged controls. This study examined whether aged growth hormone receptor knockout mice also show enhanced expression of NMDA receptors. Six or 23- to 24-month-old male normal-sized control or dwarf growth hormone receptor knockout mice were assayed for NMDA-displaceable [(3)H]glutamate binding (autoradiography) and GluN1 subunit messenger RNA (in situ hybridization). There was slight sparing of NMDA receptor binding densities within aged medial prefrontal and motor cortices, similar to caloric restriction, but there were greater age-related declines in GluN1 messenger RNA in growth hormone receptor knockout versus control mice. These results suggest that some of the functional improvements in aged mice with altered growth hormone signaling may be due to enhancement of NMDA receptors, but not through the upregulation of messenger RNA for the GluN1 subunit.

Deletion of growth hormone receptor gene but not visceral fat removal decreases expression of apoptosis-related genes in the kidney—potential mechanism of lifespan extension

Mice homozygous for the targeted disruption of the growth hormone (GH) receptor (Ghr) gene (GH receptor knockout; GHRKO; KO) are hypoinsulinemic, highly insulin sensitive, normoglycemic, and long-lived. Visceral fat removal (VFR) is a surgical intervention which improves insulin signaling in normal (N) mice and rats and extends longevity in rats. We have previously demonstrated decreased expression level of certain pro-apoptotic genes in skeletal muscles and suggested that this may contribute to the regulation of longevity in GHRKO mice. Alterations in apoptosis-related genes expression in the kidneys also may potentially lead to lifespan extension. In this context, we decided to examine the renal expression of the following genes: caspase-3, caspase-9, caspase-8, bax, bad, bcl-2, Smac/DIABLO, Apaf-1, p53, and cytochrome c1 (cyc1) in male GHRKO and N mice subjected to VFR or sham surgery, at approximately 6 months of age. The kidneys were collected 2 months after VFR. As a result, caspase-3, caspase-9, and bax expressions were decreased in KO mice as compared to N animals. Expressions of Smac/DIABLO, caspase-8, bcl-2, bad, and p53 did not differ between KOs and N mice. VFR did not change the expression of the examined genes in KO or N mice. In conclusion, endocrine abnormalities in GHRKO mice result in decreased expression of pro-apoptotic genes and VFR did not alter the examined genes expression in N and KO mice. These data are consistent with a model in which alterations of GH signaling and/or insulin sensitivity lead to increased lifespan mediated by decreased renal expression of pro-apoptotic genes.

Renal pro-apoptotic proteins are reduced by growth hormone resistance but not by visceral fat removal

Growth hormone (GH) receptor knockout (GHRKO) mice are highly insulin sensitive and long-lived. Surgical visceral fat removal (VFR) improves insulin signaling in normal mice and rats and extends longevity in rats. We have previously demonstrated decreased expression of certain pro-apoptotic genes in kidneys of GHRKO mice and suggested that this could contribute to the increased longevity of these animals. The aim of the present study was to examine the level of the following proteins: caspase-3, caspase-9, caspase-8, bax, bad, phospho-bad, bcl-2, Smac/DIABLO, Apaf-1, phospho-p53 (pp53) and cytochrome c in male GHRKO and normal (N) mice subjected to VFR or sham surgery, at approximately six months of age. The kidneys were collected two months after VFR. Caspase-3, caspase-8, bax, bad, Smac/DIABLO, Apaf-1 and pp53 levels were decreased in GHRKO mice as compared to N animals. VFR did not change the level of any of the examined proteins. The decreased renal levels of pro-apoptotic proteins could contribute to the extended life-span caused by targeted disruption of the GH receptor gene but are apparently not involved in mediating the effects of VFR.

Decreased expression level of apoptosis-related genes and/or proteins in skeletal muscles, but not in hearts, of growth hormone receptor knockout mice

The long-lived growth hormone (GH) receptor knockout (GHRKO; KO) mice are GH-resistant due to targeted disruption of the GH receptor (Ghr) gene. Apoptosis is a physiological process in which cells play an active role in their own death and is a normal component of the development and health of multicellular organisms. Aging is associated with the progressive loss of strength of skeletal and heart muscles. Calorie restriction (CR) is a well-known experimental model to delay aging and increase lifespan. The aim of the study was to examine the expression of the following apoptosis-related genes: caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 (cyc1) in the skeletal muscles and hearts of female normal and GHRKO mice, fed ad libitum or subjected to 40% CR for six months, starting at two months of age. Moreover, skeletal muscle caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 and cytochrome c (cyc) protein expression levels were assessed. Expression of caspase-3, caspase-9, bax and Smac/DIABLO genes and proteins was decreased in GHRKO's skeletal muscles. The Apaf-1 protein expression also was diminished in this tissue. In contrast, bcl-2 and pbad protein levels were increased in skeletal muscles in knockouts. No changes were demonstrated for the examined genes' expression in GHRKO's hearts except for the increased level of cyc1 mRNA. CR did not alter the expression of the examined genes and proteins in skeletal muscles of knockouts versus normal (N) mice. In heart homogenates, CR increased caspase-3 mRNA level as compared with ad libitum mice. Decreased expression of certain proapoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by CR.

GH modulates hepatic epidermal growth factor signaling in the mouse.

Epidermal growth factor (EGF) is a key regulator of cell survival and proliferation involved in the pathogenesis and progression of different types of cancer. The EGF receptor (EGFR) is activated by binding of the specific ligand but also by transactivation triggered by different growth factors including GH. Chronically, elevated GH levels have been associated with the progression of hepatocellular carcinoma. Considering EGF and GH involvement in cell proliferation and their signaling crosstalk, the objective of the present study was to analyze GH modulatory effects on EGF signaling in liver. For this purpose, GH receptor-knockout (GHR-KO) and GH-overexpressing transgenic mice were used. EGFR content was significantly decreased in GHR-KO mice. Consequently, EGF-induced phosphorylation of EGFR, AKT, ERK1/2, STAT3, and STAT5 was significantly decreased in these mice. In contrast, EGFR content as well as its basal tyrosine phosphorylation was increased in transgenic mice overexpressing GH. However, EGF stimulation caused similar levels of EGFR, AKT, and ERK1/2 phosphorylation in normal and transgenic mice, while EGF induction of STAT3 and STAT5 phosphorylation was inhibited in the transgenic mice. Desensitization of the STATs was related to decreased association of these proteins to the EGFR and increased association between STAT5 and the tyrosine phosphatase SH2-containing phosphatase-2. While GHR knockout is associated with diminished expression of the EGFR and a concomitant decrease in EGF signaling, GH overexpression results in EGFR overexpression with different effects depending on the signaling pathway analyzed: AKT and ERK1/2 pathways are induced by EGF, while STAT3 and STAT5 activation is heterologously desensitized.