Caloric restriction results in decreased expression of peroxisome proliferator-activated receptor superfamily in muscle of normal and long-lived growth hormone receptor/binding protein knockout mice

Resistance to growth hormone, reduced insulin-like growth factor 1 (IGF1) action, and enhanced insulin sensitivity are likely mediators of extended life span and delayed aging process in growth hormone receptor/binding protein knockout (GHR-KO) mice. Fat metabolism and genes involved in fatty acid oxidation are strongly involved in insulin action. Using real-time polymerase chain reaction and western blot we have examined expression of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptor (RXR) genes in the skeletal muscle of normal and GHR-KO mice subjected to 30% caloric restriction. The results indicate that caloric restriction decreased the expression of PPARgamma, PPARalpha, and PPARbeta/delta which would lead to down-regulation of fat metabolism. This suggested metabolic change clearly does not affect whole-body insulin action. These findings suggest that whole-animal insulin sensitivity is not regulated through skeletal muscle insulin action.     

Aging-related characteristics of growth hormone receptor/binding protein gene-disrupted mice

Since generation of the growth hormone receptor/binding protein (GHR/BP) gene-disrupted mouse nearly 10 years ago, use of this mouse model has become widespread in the elucidation of the physiological roles of GH and insulin-like growth factor-1 (IGF-1). In particular, it serves as a useful model to study mechanisms of aging. This review highlights the evidence demonstrating that the loss of GH signaling leads to lifespan extension in mice, and presents the multiple characteristics of this mouse line that suggest the life extension is due to alteration of the aging process.     

Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40-50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.     

Protection against diabetes-induced nephropathy in growth hormone receptor/binding protein gene-disrupted mice

To further investigate the role of GH in diabetic nephropathy, experimental diabetes was induced with streptozotocin (STZ) in mice in which the GH receptor/binding protein gene was disrupted. Body weight, blood glucose, and renal histology and morphometry were studied 10 weeks after diabetes induction in wild-type (+/+) mice and in mice heterozygous (+/-) and homozygous (-/-) for the disruption. Equivalent levels of hyperglycemia developed in all diabetic groups. Normal weight gain was absent in +/+ and +/- diabetic groups, and -/- diabetics lost weight during the study. Diabetic +/+ and +/- groups both showed evidence of glomerulosclerosis, increases in glomerular volume, and increases in the ratio of mesangial area to total glomerular area, whereas diabetic -/- mice showed none of these pathological changes. These results extend our previous findings of protection against diabetes-associated kidney damage in transgenic mice expressing a GH antagonist. Taken together, the results argue for an important role of GH in the development of diabetes induced end-organ damage.     

Inhibition of toll-like receptor 2 expression improves insulin sensitivity and signaling in muscle and white adipose tissue of mice fed a high-fat diet

The aims of the present study were to investigate the expression of toll-like receptor 2 (TLR2) in muscle and white adipose tissue (WAT) of diet-induced obesity (DIO) mice, and also the effects of its inhibition, with the use of TLR2 antisense oligonucleotide (ASON), on insulin sensitivity and signaling. The expression of TLR2 was increased in muscle and WAT of DIO mice, compared with those that received standard chow. Inhibition of TLR2 in DIO mice, by TLR2 ASON, improved insulin sensitivity and signaling in muscle and WAT. In addition, data show that the inhibition of TLR2 expression prevents the activation of IKBKB, MAPK8, and serine phosphorylation of IRS1 in DIO mice, suggesting that TLR2 is a key modulator of the crosstalk between inflammatory and metabolic pathways. We, therefore, suggest that a selective interference with TLR2 presents an attractive opportunity for the treatment of insulin resistance in obesity and type 2 diabetes.     

Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue.

Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine beta-hydroxylase (Dbh-/-), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 microg/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mRNA) and protein expression were 5-fold higher in BAT from control (Dbh+/-) compared with Dbh-/- mice. Leptin produced a 4-fold increase in UCP1 mRNA levels in Dbh+/- mice but had no effect on UCP1 expression in Dbh-/-. The beta3-adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to beta-adrenergic stimulation. Similarly, exogenous leptin reduced leptin mRNA in WAT from Dbh+/- but not Dbh-/- mice. In separate experiments, leptin produced comparable reductions in food intake in both Dbh+/- and Dbh-/- mice, illustrating that norepinephrine is not required for leptin's effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mRNA in response to exogenous leptin but increased UCP1 mRNA in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor.     

Insulin-like growth factor I, growth hormone and insulin in white adipose tissue

Maturation of adipose tissue results from both the expansion of mature adipocytes and the formation of new adipocytes from adipocyte precursor cells. A variety of hormones related to adipogenesis have been identified recently. Both growth hormone (GH) and insulin-like growth factor I (IGF-I) are of major significance in adipocyte differentiation. IGF-I has been suggested to be a major regulator of cell proliferation, differentiation and metabolism, thus regulating, among other biological processes, adipose tissue growth and differentiation of pre-adipocytes into adipocytes. GH exerts its effects by increasing the pool of adipocyte precursor cells capable of differentiating into mature adipocytes. In addition, GH seems to have the potential to reduce the volume of mature adipocytes, thus inhibiting the expansion of adipose tissue and reducing body fat. This chapter gives an overview of studies that have investigated the roles of insulin, GH and IGF-I in adipogenesis.     

Effects of caloric restriction and growth hormone resistance on the expression level of peroxisome proliferator-activated receptors superfamily in liver of normal and long-lived growth hormone receptor/binding protein knockout mice

Growth hormone receptor/binding protein knockout (GHR-KO) mice live approximately 40% longer than their normal siblings do. These mice have dramatically reduced plasma levels of insulin-like growth factor 1 (IGF1) and enhanced insulin sensitivity. We examined the expression level of peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors family genes in the livers of normal and GHR-KO mice fed ad libitum or subjected to long-term 30% caloric restriction (CR). The levels of PPARgamma and PPARalpha messenger RNA and proteins and the levels of retinoid X receptors messenger RNA were elevated in long-lived GHR-KO mice as compared to normal mice with no major effect of CR in either genotype. These findings suggest that enhanced insulin sensitivity of GHR-KO mice may be related to the altered actions of PPARs family members in the liver. The results also indicate that CR may increase insulin sensitivity through a different mechanism.     

Nicotine induces uncoupling protein 1 in white adipose tissue of obese mice.

OBJECTIVE: To test the hypothesis that nicotine not only activates uncoupling protein1 (UCP1) in brown adipose tissue (BAT), but also induces UCP1 in white adipose tissue (WAT), which contributes to the mitigation of obesity in obese mice. DESIGN: Weights of the whole body, the gastrocnemius muscle, interscapular BAT and subcutaneous and retroperitoneal WAT, food intake and the mRNA and protein of UCP1 in these tissues were measured and immunohistochemistry using antiserum against UCP1 was also performed in obese yellow KK mice treated with nicotine for 6 months and control mice treated with physiological saline. RESULTS: Obese mice treated with nicotine for 6 months, compared with those injected with saline, weighed significantly less (P < 0.01) and had smaller subcutaneous and retroperitoneal WAT pads (P < 0.01), while obese mice that received nicotine ate less (P < 0.05) than those injected with saline. In mice treated with nicotine, the mRNA and protein of UCP1 was detected not only in BAT, but also in subcutaneous and retroperitoneal WATs. Immunohistochemically, the BAT of obese mice contained large lipid droplets and appeared rather WAT-like, but changed to typical brown adipocytes after nicotine treatment. The fat pads of nicotine-treated mice contained many multilocular cells that were positive for UCP1. CONCLUSION: Nicotine not only activates UCP1 in BAT, but also induces UCP1 in WAT and decreases food intake, which contributes to the mitigation of obesity.     

Increased visceral adipose tissue is associated with increased circulating insulin and decreased sex hormone binding globulin levels in massively obese adolescent girls.

The current study was designed to examine the relationship between body fat distribution, as evaluated by anthropometry and magnetic resonance imaging (MRI), and circulating insulin, sex hormone and SHBG levels in obese adolescent girls. Twenty-nine obese adolescent girls, aged 12.6-16.9 years with a mean BMI of 30.51+/-1.86 participated in this study. All girls had breast stage B4-5 and pubic hair stage P4-5. Percent obesity and BMI as indices of being overweight were calculated; the waist-to-hip ratio (WHR) and the waist-to-thigh ratio (WTR) were calculated to obtain two anthropometric indices for the pattern of body fat distribution. The areas of visceral (VAT) and subcutaneous adipose tissue (SAT) were evaluated by MRI at the L4-L5 level. Serum concentrations of total T, DHEAS, 17beta-estradiol, progesterone and SHBG were measured. Plasma glucose and insulin concentrations were evaluated during an oral glucose tolerance test. WHR was the only anthropometric parameter that was significantly associated with the area of VAT. Insulin level showed correlation with both WHR and the area of VAT; no correlation was found between insulin levels and WTR. Both WHR and VAT were negatively correlated with serum DHEAS level and positively correlated with T level. There were strong negative correlations between serum SHBG level and the area of VAT and WHR. Inverse correlation was found between serum SHBG level and insulin. Serum 17beta-estradiol and progesterone levels showed no significant correlation with all the patterns of body fat distribution. SAT was not significantly correlated with both anthropometric parameters and any of the sex hormones evaluated. We can draw two main conclusions. Firstly, in massively obese adolescent girls, the WHR seems to be a good indicator for the accumulation of VAT, and abdominal obesity, rather than adiposity per se, appears to be related to biochemical complications. Secondly, increased upper body adiposity and, in particular, the intra-abdominal fat area are associated with increased insulin levels in massively obese adolescent girls. The associated reductions in SHBG and DHEAS levels represent an early general risk factor for the development of metabolic and cardiovascular diseases in this population, as previously described for obese adult women.     

Intracerebroventricular administration of neuropeptide Y to normal rats increases obese gene expression in white adipose tissue

Summary The aim of this work was to determine the possible inter-relationship between neuropeptide Y (NPY, a hypothalamic stimulator of feeding) and adipose tissue expression of the ob protein (a novel potent inhibitor of feeding). Such a relationship could be of importance in the maintenance of normal body weight. To this end, normal rats were intracerebro-ventricularly (i.c.v.) infused for 6 days with NPY. NPY infusion resulted in hyperphagia and a marked increase in adipose tissue ob mRNA levels. The effect of NPY on ob expression persisted when hyperphagia was prevented by pair-feeding, and was reversed following cessation of NPY infusion. Basal and glucose-stimulated insulinaemia were increased by i. c. v. NPY infusion compared to control values, regardless of whether animals were ad libitum-fed or pair-fed. Cessation of NPY infusion was accompanied by normalisation of insulinaemia. These changes in insulinaemia produced by i. c. v. NPY infusion paralleled the observed changes in ob expression. When normal rats were made hyperinsulinaemic-euglycaemic for 24 h, such hyperinsulinaemia also resulted in increased ob mRNA levels in white adipose tissue. This suggested that NPY-induced hyperinsulinaemia could be responsible for the upregulation of ob mRNA levels of NPY-infused rats. It is concluded that central (i. c. v.) NPY infusion increases adipose tissue ob expression, a functional relationship that is linked, at least in part, via NPY-induced hyperinsulinaemia.Abbreviations NPY Neuropeptide Y - i. c. v. intracerebroventricular     

Localization of the growth hormone receptor/binding protein in skin

Acromegaly is characterized by coarsening of facial features, acanthosis nigricans, hypertrichosis and oily skin. To determine the site through which GH exerts these effects, we have used immunohistochemistry to localize the GH receptor/binding protein (BP) in rat, rabbit and human skin. Three monoclonal antibodies (MAb 1, 43, 263) were immunoreactive in identical locations, whereas no immunoreactivity was evident when control monoclonal antibodies (MAb 50.8 and MAb 7 (rat] were used. Skin from neonatal and adult animals was used to determine whether GH receptor/BP expression was developmentally regulated. Immunoreactivity of the GH receptor/BP in the three species was consistently localized in the stratum basale and stratum spinosum. Intermittent staining was observed in the stratum granulosum. Scattered basal epidermal cells often displayed more intense immunoreactivity. This distribution was observed at all maturational stages examined. Intense GH receptor/BP immunoreactivity was observed in all histological layers of the lower one-third of hair follicles and in hair matrix cells of the dermal papillae. Immunoreactivity was also detected in the outer epithelial root sheath of the upper two-thirds of hair follicles, in sebaceous glands and in fibroblasts of the connective tissue sheath surrounding the follicle. GH receptor/BP immunoreactivity was also present in the secretory duct and myoepithelial cells of human eccrine sweat glands. Fibroblasts, Schwann cells of peripheral nerve fascicles, skeletal muscle cells and adipocytes of the dermis were also immunoreactive as were medial smooth muscle and endothelial cells of arteries. These results provide evidence that GH acts locally on the epidermis and epidermal appendages concordant with our recent localization of GH receptor/BP to epithelial cell types of the gastrointestinal and reproductive systems.     

Growth hormone regulates leptin gene expression in bovine adipose tissue: correlation with adipose IGF-1 expression

Leptin, the product of the ob gene, is secreted from white adipocytes and regulates food intake and whole-body energy metabolism. In rodents and humans, leptin gene expression is under complex endocrine and metabolic control, and is strongly influenced by energy balance. Growth hormone (GH) has myriad effects on adipose tissue metabolism. The primary aim of this study was to determine the ability of GH to regulate leptin mRNA expression in bovine adipose tissue in vitro and in vivo. Incubation of subcutaneous adipose tissue explants for 24 h with GH alone had no effect on bovine leptin gene expression, whereas high concentrations of insulin or dexamethasone (DEX) potently stimulated bovine leptin mRNA abundance. GH, in combination with high concentrations of insulin, DEX, or both, attenuated the ability of insulin or DEX to stimulate leptin expression in vitro. These data indicate that GH can indirectly regulate leptin expression in vitro by altering the adipose tissue response to insulin or DEX. We extended these studies to examine the ability of GH to regulate leptin expression in vivo, using young castrate male cattle treated with no hormone (control) or GH (200 micrograms/kg body weight per day) for 3 days. GH increased plasma GH and insulin concentrations, but not those of cortisol or non-esterified fatty acid (NEFA) concentrations. GH treatment increased adipose tissue leptin and IGF-1 mRNA concentrations (n=9, P>0.001). In addition, leptin abundance was highly correlated with adipose tissue IGF-1 mRNA in GH-treated animals (P>0.001). The timing of GH-induced changes in leptin gene expression preceded measurable GH effects on adiposity.     

Effect of circulating growth hormone on muscle IGF-I protein concentration in female mice with growth hormone receptor gene disruption

Growth hormone (GH) is a potent secretague for circulating insulin-like growth factor-I (IGF-I). The purpose of this study was to examine the effect of circulating GH on muscle IGF-I protein expression using GH transgenic animal models. Three different models were used: mice that overexpress bovine GH (bGH; n = 10), mice without a functional GH receptor (GHR-/-; n = 10), and wildtype mice (n = 10). All mice were 16-week old females and each group differed in their basic phenotypic characteristics. Immediately after euthanization the triceps surae muscle group (soleus, plantaris, and gastrocnemius muscles) was removed. IGF-I was extracted from the muscle with an acid–ethanol solution (12.5% 2N hydrochloric acid and 87.5% ethanol, pH 1.5) followed by neutralization with Tris-base and subsequently quantified using a radioimmunoassay. Analysis revealed that bGH mice had significantly greater muscle IGF-I protein expression compared to GHR-/- and wildtype mice. No difference in IGF-I protein concentration was found between GHR-/- and wildtype animals. This study found that overexpression of GH leading to high circulating GH concentrations increase muscle IGF-I protein expression. However, the absence of a functional GHR did not affect muscle IGF-I protein expression compared to wildtype despite high circulating levels of GH and low circulating levels of IGF-I. In conclusion, it appears that at rest high circulating levels of GH augment muscle IGF-I protein expression only in the presence of an intact GHR but that the absence of a functional GH receptor does not affect basal levels of muscle IGF-I protein in female mice.     

Differential expression of renal growth hormone receptor and its binding protein in experimental diabetes mellitus.

Growth hormone (GH) may have a role in the development of diabetic nephropathy. The effect of experimental diabetes on renal expression of the growth hormone receptor gene products, including the receptor itself (GHR) and its binding protein (GHBP) was examined. Adult female rats received i.v. streptozotocin and were killed at 7, 30, 90 and 180 days after the induction of diabetes. Diabetic animals had a pronounced increase in kidney weight and progressive albuminuria. In renal cortex, no change was seen in GHR mRNA levels throughout the observation period of 6 months, while a significant increase in cortical GHBP mRNA levels was observed after 1 month of diabetes and sustained for the rest of the study period. Immunohistochemical analysis of kidney sections revealed a stronger staining for GHBP at the cortical and inner medullary areas in the diabetic animals. These data indicate that although the GHR and GHBP mRNAs originate from the same gene, their renal levels are differentially regulated during the development of experimental diabetic kidney disease, suggesting a functional role for GHBP.     

Sex differences in the effect of high-fat diet feeding on rat white adipose tissue mitochondrial function and insulin sensitivity

Obesity-induced mitochondrial dysfunction in white adipose tissue (WAT) leads to a dysregulation of adipokine secretion, which is involved in insulin resistance development. Taking into account the sex differences previously found both in mitochondrial function and for the insulin sensitivity profile in different tissues, the aim of this study was to investigate whether a sex-dependent effect of a long-term high-fat diet (HFD) feeding exists on WAT mitochondrial function. Indeed, HFD effects on the levels of the key components of the insulin and adiponectin signaling pathways, and the consequences of these effects on the systemic profile of insulin sensitivity were also studied. Wistar rats of both sexes were fed a standard diet or an HFD. Serum markers of insulin sensitivity, protein, and mRNA levels of the main elements of the insulin and adiponectin signaling pathways, and the markers of mitochondrial function and biogenesis, were measured. Our results indicate that different physiological strategies are adopted by male and female rats in response to HFD. In this regard, HFD induced mitochondrial proliferation in males and mitochondrial differentiation in females, as well as a greater retroperitoneal WAT expandability capacity, which allows them to preserve a better insulin sensitivity profile than male rats for both control and HFD groups. Moreover, female WAT showed a decrease in adiponectin and insulin signaling pathway element levels. This sexual dimorphism suggests that there are different strategies for retroperitoneal WAT to maintain the energetic and metabolic homeostasis in response to HFD feeding.