Early in vivo detection of denervation-induced atrophy by luminescence transient nanothermometry.

Denervation induces skeletal muscle atrophy due to the loss of control and feedback with the nervous system. Unfortunately, muscle atrophy only becomes evident days after the denervation event when it could be irreversible. Alternative diagnosis tools for early detection of denervation-induced muscle atrophy are, thus, required. In this work, we demonstrate how the combination of transient thermometry, a technique already used for early diagnosis of tumors, and infrared-emitting nanothermometers makes possible the in vivo detection of the onset of muscle atrophy at short (<1 day) times after a denervation event. The physiological reasons behind these experimental results have been explored by performing three dimensional numerical simulations based on the Pennes' bioheat equation. It is concluded that the alterations in muscle thermal dynamics at the onset of muscle atrophy are consequence of the skin perfusion increment caused by the alteration of peripheral nervous autonomous system. This work demonstrates the potential of infrared luminescence thermometry for early detection of diseases of the nervous system opening the venue toward the development of new diagnosis tools.

Role of Glucocorticoid Signaling and HDAC4 Activation in Diaphragm and Gastrocnemius Proteolytic Activity in Septic Rats.

Sepsis increases glucocorticoid and decreases IGF-1, leading to skeletal muscle wasting and cachexia. Muscle atrophy mainly takes place in locomotor muscles rather than in respiratory ones. Our study aimed to elucidate the mechanism responsible for this difference in muscle proteolysis, focusing on local inflammation and IGF-1 as well as on their glucocorticoid response and HDAC4-myogenin activation. Sepsis was induced in adult male rats by lipopolysaccharide (LPS) injection (10 mg/kg), and 24 h afterwards, rats were euthanized. LPS increased TNFα and IL-10 expression in both muscles studied, the diaphragm and gastrocnemius, whereas IL-6 and SOCS3 mRNA increased only in diaphragm. In comparison with gastrocnemius, diaphragm showed a lower increase in proteolytic marker expression (atrogin-1 and LC3b) and in LC3b protein lipidation after LPS administration. LPS increased the expression of glucocorticoid induced factors, KLF15 and REDD1, and decreased that of IGF-1 in gastrocnemius but not in the diaphragm. In addition, an increase in HDAC4 and myogenin expression was induced by LPS in gastrocnemius, but not in the diaphragm. In conclusion, the lower activation of both glucocorticoid signaling and HDAC4-myogenin pathways by sepsis can be one of the causes of lower sepsis-induced proteolysis in the diaphragm compared to gastrocnemius.

IGF-1 and IGFBP-3 in Inflammatory Cachexia.

Inflammation induces a wide response of the neuroendocrine system, which leads to modifications in all the endocrine axes. The hypothalamic-growth hormone (GH)-insulin-like growth factor-1 (IGF-1) axis is deeply affected by inflammation, its response being characterized by GH resistance and a decrease in circulating levels of IGF-1. The endocrine and metabolic responses to inflammation allow the organism to survive. However, in chronic inflammatory conditions, the inhibition of the hypothalamic-GH-IGF-1 axis contributes to the catabolic process, with skeletal muscle atrophy and cachexia. Here, we review the changes in pituitary GH secretion, IGF-1, and IGF-1 binding protein-3 (IGFBP-3), as well as the mechanism that mediated those responses. The contribution of GH and IGF-1 to muscle wasting during inflammation has also been analyzed.

Olive leaf extract supplementation improves the vascular and metabolic alterations associated with aging in Wistar rats.

Olive leaves are rich in bioactive substances which exert anti-inflammatory, antioxidant, insulin-sensitizing and antihypertensive effects. The aim of this study was to analyze the possible beneficial effects of an olive leaf extract (OLE) rich in secoiridoids and phenolic compounds on the aging-induced metabolic and vascular alterations. Three experimental groups of rats were used: 3-month-old rats, 24-month-old rats and 24-month-old rats supplemented 21 days with OLE (100 mg/kg). Administration of OLE to aged rats decreased the weight of adrenal glands and prevented the aging-induced loss of body weight and muscle mass. In the serum, OLE reduced the circulating levels of LDL-cholesterol and IL-6 and increased the concentrations of leptin and adiponectin. In the liver OLE attenuated the decreased gene expression of SOD-1, GSR, GCK and GSK-3ß and reduced the aging-induced overexpression of NOX-4, Alox-5, iNOS and TNF-α. In aorta segments, OLE prevented endothelial dysfunction and vascular insulin resistance and improved vasoconstriction in response to KCl and NA. Improvement in vascular function was associated with the attenuation of the alterations in the gene expression of COX-2, IL-6, GPx, NOX-1 and IL-10. In conclusion, OLE exerts anti-inflammatory and antioxidant effects in aged rats and attenuates the alterations in vascular function associated with aging.

Hormones and Muscle Atrophy.

The endocrine system is an essential regulator of muscle metabolism in both health and disease. Hormones such as growth hormone (GH), insulin-like growth factor-I (IGF-I) and androgens are the main regulators of muscle metabolism in both health and disease; have profound influences on muscle, acting as anabolic factors; and are important regulators of muscle mass. On the contrary, glucocorticoids have direct catabolic effects and induce muscle protein loss. Muscle wasting is a systemic response to fasting and several diseases like cancer, sepsis, renal and cardiac failure and trauma. Muscle atrophy also occurs in specific muscles with denervation, immobilization or inactivity. All of these conditions are characterized by significant changes in the endocrine environment. The aim of this review was to describe the role of endocrine system on the development of muscle atrophy. Understanding hormonal regulation of the skeletal muscle in these conditions might facilitate the development of hormone-mediated therapies for muscle atrophy.

Formoterol treatment prevents the effects of endotoxin on muscle TNF/NF-kB, Akt/mTOR, and proteolytic pathways in a rat model. Role of IGF-I and miRNA 29b.

Inflammatory diseases are associated with muscle wasting as a result of an increase in proteolysis. The purpose of this study was to elucidate whether administration of a ß2 adrenergic agonist, formoterol, was able to prevent the acute effects of sepsis induced by liposaccharide (LPS) injection on rat gastrocnemius muscle and to evaluate the possible roles of corticosterone, IGF-I, miR-23a, and miR-29b. For this purpose, male Wistar rats were injected with LPS and/or formoterol. Formoterol treatment decreased LPS-induced increase in serum corticosterone, TNFα upregulation, and NF-κB(p65) and Forkhead box protein O1 activation in the gastrocnemius. Atrogin-1, muscle RING-finger protein-1, microtubule-associated protein-1 light chain 3b (LC3b), and the lipidation of LC3b-I to LC3b-II were increased by LPS, and formoterol blocked these effects. Serum IGF-I and its mRNA levels in the gastrocnemius were decreased, whereas mecano growth factor and IGF binding protein 3 mRNA levels were increased in the rats injected with LPS but not in the rats that received LPS and formoterol. Similarly, LPS decreased Akt and mammalian target of rapamycin phosphorylation, and formoterol blocked these decreases. Finally, miR-29b expression in the gastrocnemius was upregulated by endotoxin injection, whereas miR-23a was not significantly different. Formoterol treatment did not significantly modify LPS-induced increase in muscle miR-29b. Furthermore, in control rats formoterol increased the expression of this miRNA. We conclude that formoterol decreases endotoxin-induced inflammation and proteolysis in rat skeletal muscle. Those responses can be a direct effect of ß2 adrenergic receptor stimulation or/and of blocking the effects of LPS on corticosterone and IGF-I. Muscle miR-23a and -29b do not seem to play an important role in those responses.

Metabolic programming of sirtuin 1 (SIRT1) expression by moderate energy restriction during gestation in rats may be related to obesity susceptibility in later life.

In rats, 20% gestational energy restriction programmes offspring for higher food intake, which in adulthood results in higher body weight in males but not in females. Here, we aimed to assess whether the effects of moderate energy restriction during gestation and the sex-related outcomes on adult body weight may be related to the metabolic programming of sirtuin expression in different tissues. For this purpose, 25-d-old offspring of control and 20% energy-restricted (ER) rats (from days 1-12 of pregnancy) were studied. Body weight and the weight of white adipose tissue (WAT) depots and liver were recorded and mRNA expression of sirtuin 1 (SIRT1) and selected genes in the WAT, liver, muscle and hypothalamus were analysed. No differences were found in body weight or the weight of WAT and liver between the control and ER animals. A similar pattern of SIRT1 mRNA expression was found in the WAT, liver and skeletal muscle of ER animals, but in a sex-dependent manner: ER males showed lower SIRT1 mRNA levels than the controls, while no differences were found in females. A sex-different pattern was also observed in the hypothalamus. ER males, but not females, also showed lower mRNA levels of adipose TAG lipase (ATGL) and uncoupling protein 2 in WAT and of sterol response element binding protein 1c and stearoyl-CoA desaturase-1 in the liver. Both sexes of ER animals showed lower mRNA levels of 5' adenosine monophosphate-activated protein kinase and ATGL in the liver. In conclusion, moderate maternal energy restriction during gestation programmes a particular, sex-dependent gene expression profile of SIRT1 in different peripheral tissues, which may be related to obesity predisposition in adulthood; therefore SIRT1 expression emerges as a potential early biomarker of obesity susceptibility.

Sex-associated differences in the leptin and ghrelin systems related with the induction of hyperphagia under high-fat diet exposure in rats.

Leptin and ghrelin are known to be main hormones involved in the control of food intake, with opposing effects. Here we have explored whether changes in the leptin and ghrelin system are involved in the long-term effects of high-fat (HF) diet feeding in rats and whether sex-associated differences exist. Male and female Wistar rats were fed until the age of 6 months with a normal-fat (NF) or an HF-diet. Food intake and body weight were followed. Gastric and serum levels of leptin and ghrelin, and mRNA levels of leptin (in stomach and adipose tissue), ghrelin (in stomach), and NPY, POMC, and leptin and ghrelin receptors (OB-Rb and GHS-R) (in the hypothalamus) were measured. In both males and females, total caloric intake and body weight were greater under the HF-diet feeding. In females, circulating ghrelin levels and leptin mRNA expression in the stomach were higher under HF-diet. HF-diet feeding also resulted in higher hypothalamic NPY/POMC mRNA levels, more marked in females, and in lower OB-Rb mRNA levels, more marked in males. In addition, in females, serum ghrelin levels correlated positively with hypothalamic NPY mRNA levels, and these with caloric intake. In males, hypothalamic OB-Rb mRNA levels correlated positively with POMC mRNA levels and these correlated negatively with caloric intake and with body weight. These data reflect differences between sexes in the effects of HF-diet feeding on food intake control systems, suggesting an impairment of the anorexigenic leptin-POMC system in males and an over-stimulation of the orexigenic ghrelin-NPY system in females.

Adiponectin is involved in the protective effect of DHEA against metabolic risk in aged rats.

The aim of the present work was to analyze the effect of dehydroepiandrosterone (DHEA) on several metabolic risk factors, including cardiovascular health and insulin resistance, in aged rats submitted to a high-fat diet. For that, weaned rats were fed on a high-fat diet until 20 months of age. In the last 13 weeks of life, a group (n=11) received the diet supplemented with DHEA (0.5%, w/w), serving the rest (n=10) as controls. Body weight, body fat, serum lipids (triglycerides, total cholesterol and non-esterified fatty acids (NEFA)), HOMA index, n-6/n-3 polyunsaturated fatty acid (PUFA) ratios, serum adiponectin, leptin, resistin and TNF-alpha, as well as adiponectin expression in adipose tissue, were measured. A stepwise discriminant test was used to analyze these variables, and an index of overall metabolic risk was generated from them. DHEA treatment resulted in a significantly lower overall metabolic risk index, as generated by the discriminant test (P<0.01). The DHEA group had lower body fat and n-6/n-3 polyunsaturated fatty acid (PUFA) ratios than the control group (P<0.01), and the same trends were observed for serum cholesterol, triglycerides and HOMA index; in contrast, adiponectin expression in adipose tissue increased in DHEA-treated rats (P<0.05). The discriminant analysis revealed that adiponectin, both from serum and adipose tissue, was the most influencing factor, followed by n-6/n-3 ratios in adipose tissue, and by body fat. Our results then suggest that adiponectin is involved in the protective effect of DHEA against metabolic risk demonstrated in the present work.

Dehydroepiandrosterone prevents age-associated alterations, increasing insulin sensitivity.

The age decline in DHEA levels has been associated with the appearance of age-related disorders such as obesity and insulin resistance. The aim of this study was to analyse the effect of chronic administration (13 weeks) of DHEA (5 g/kg diet) to old female rats fed on a high-fat diet on body weight and adiposity, and concretely on the expression of the adipokines related to obesity and insulin resistance, such as leptin, adiponectin and resistin. DHEA treatment induced a decrease in body weight, adipose tissue mass and serum insulin, adiponectin and leptin levels. Adiponectin mRNA expression in visceral fat depots decreased with aging, but this reduction was attenuated by DHEA treatment. DHEA treatment also stimulated resistin gene expression in the ovaric and renal adipose depots, which is associated with an increase in its circulating levels. In conclusion, DHEA treatment decreases body weight and adiposity in old female rats fed a high-fat diet, leading to an improvement of the HOMA index for insulin sensitivity, with decreasing circulating insulin levels, and preventing the age-associated decline of visceral-adipose adiponectin expression.

Anti-tumor necrosis factor agent PEG-sTNFRI improves the growth hormone/insulin-like growth factor-I system in adjuvant-induced arthritic rats.

Adjuvant-induced arthritis is associated with body weight loss and decreased pituitary growth hormone (GH) and hepatic insulin-like growth factor-I (IGF-I) synthesis. Cytokines as tumor necrosis factor (TNF) mediate wasting associated with chronic inflammation. The aim of this study was to analyse whether the inhibition of TNF is able to revert the decrease in the body weight and the GH/IGF-I axis in arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with polyethylene glycol linked to soluble TNF receptor p55 (PEG-sTNFRI) (1 mg/kg, s.c.) or saline for 8 days. There was a significant decrease in pituitary GH mRNA (P<0.05), hepatic IGF-I mRNA (P<0.01) and serum concentrations of IGF-I (P<0.01) in arthritic rats. The 8-day administration of PEG-sTNFRI resulted in an increase in food intake (P<0.05) and body weight gain (P<0.01) in arthritic but not in control rats. There was an increase in pituitary GH mRNA after PEG-sTNFRI treatment both in control and in arthritic rats. There was a significant increase in IGF-I serum concentrations (P<0.05) and hepatic IGF-I mRNA expression (P<0.05) in control rats treated with PEG-sTNFRI, whereas the effect of this anti-TNF agent in arthritic rats was only statistically significant in hepatic IGF-I mRNA expression (P<0.05). These data suggest that TNF seems to be involved in the decrease in GH and IGF-I synthesis in arthritic rats.

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle.

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.

Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats.

Chronic arthritis induces hypermetabolism and cachexia. Ghrelin is a gastrointestinal hormone that has been proposed as a treatment to prevent cachexia. The aim of this work was to examine the effect of administration of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) to arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with GHRP-2 (100 microg/kg) or with saline for 8 days. Arthritis induced an increase in serum ghrelin (P < 0.01) and a decrease in serum concentrations of leptin (P < 0.01), whereas GHRP-2 administration increased serum concentrations of leptin. GHRP-2 increased food intake in control rats but not in arthritic rats. However, in arthritic rats GHRP-2 administration ameliorated the external symptoms of arthritis, as it decreased the arthritis score (10.4 +/- 0.8 vs. 13.42 +/- 0.47, P < 0.01) and the paw volume. In addition, circulating IL-6 and nitrites/nitrates were increased by arthritis, and GHRP-2 treatment decreased the serum IL-6 levels (P < 0.01). To elucidate whether GHRP-2 is able to modulate IL-6 release directly on immune cells, peritoneal macrophage cultures were incubated with GHRP-2 or ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor. Both GHRP-2 (10(-7) M) and ghrelin (10(-7) M) prevented endotoxin-induced IL-6 and decreased nitrite/nitrate release from peritoneal macrophages in vitro. These data suggest that GHRP-2 administration has an anti-inflammatory effect in arthritic rats that seems to be mediated by ghrelin receptors directly on immune cells.