Different sites of actions make different responses to thiazolidinediones between mouse and rat models of fatty liver.

Therapeutic approach for NAFLD is limited and there are no approved drugs. Pioglitazone (PGZ), a thiazolidinedione (TZD) that acts via peroxisome proliferator activated receptor gamma (PPARγ) is the only agent that has shown consistent benefit and efficacy in clinical trials. However, the mechanism of its therapeutic effect on NAFLD remains unclear. The poor understanding may be due to problems with mouse, a species most used for animal experiments. TZDs exacerbate fatty liver in mouse models while they improve it in rat models like in human patients. Therefore, we compared the effects of TZDs including PGZ and rosiglitazone (RGZ) in ob/ob mice and Lepmkyo/Lepmkyo rats, models of leptin-deficient obesity, and A-ZIP/F-1 mice and seipin knockout (SKO) rats, models of generalized lipodystrophy. Pparg mRNA expression was markedly upregulated in fatty livers of mouse models while it was unchanged in rat models. TZDs exacerbated fatty liver in ob/ob and A-ZIP/F-1 mice, improved it in Lepmkyo/Lepmkyo rats and showed no effect in SKO rats. Gene expression analyses of Pparg and its target gene, Fsp27 revealed that PPARγ in the adipose tissue is the exclusive therapeutic target of TZDs in rats but PPARγ in the liver in addition to the adipose tissue is also a major site of actions for TZDs in mice. Although the response to TZDs in mice is the complete opposite of that in human patients, no report has pointed out the problem with TZD studies using mouse models so far. The present study might provide useful suggestions in research on TZDs.

Impaired Leptin Signalling in Obesity: Is Leptin a New Thermolipokine?

Leptin is a principal adipose-derived hormone mostly implicated in the regulation of energy balance through the activation of anorexigenic neuronal pathways. Comprehensive studies have established that the maintenance of certain concentrations of circulating leptin is essential to avoid an imbalance in nutrient intake. Indeed, genetic modifications of the leptin/leptin receptor axis and the obesogenic environment may induce changes in leptin levels or action in a manner that accelerates metabolic dysfunctions, resulting in a hyperphagic status and adipose tissue expansion. As a result, a vicious cycle begins wherein hyperleptinaemia and leptin resistance occur, in turn leading to increased food intake and fat enlargement, which is followed by leptin overproduction. In addition, in the context of obesity, a defective thermoregulatory response is associated with impaired leptin signalling overall within the ventromedial nucleus of the hypothalamus. These recent findings highlight the role of leptin in the regulation of adaptive thermogenesis, thus suggesting leptin to be potentially considered as a new thermolipokine. This review provides new insight into the link between obesity, hyperleptinaemia, leptin resistance and leptin deficiency, focusing on the ability to restore leptin sensitiveness by way of enhanced thermogenic responses and highlighting novel anti-obesity therapeutic strategies.

TGF-ß1 downregulation in the hypothalamus of obese mice through acute exercise.

Obesity and aging lead to abnormal transforming growth factor-ß1 (TGF-ß1) signaling in the hypothalamus, triggering the imbalance on glucose metabolism and energy homeostasis. Here, we determine the effect of acute exercise on TGF-ß1 expression in the hypothalamus of two models of obesity in mice. The bioinformatics analysis was performed to evaluate the correlation between hypothalamic Tgf-ß1 messenger RNA (mRNA) and genes related to thermogenesis in the brown adipose tissue (BAT) by using a large panel of isogenic BXD mice. Thereafter, leptin-deficient (ob/ob) mice and obese C57BL/6 mice fed on a high-fat diet (HFD) were submitted to the acute exercise protocol. Transcriptomic analysis by using BXD mouse reference population database revealed that hypothalamic Tgf-ß1 mRNA is negatively correlated with genes related to thermogenesis in brown adipose tissue of BXD mice, such as peroxisome proliferator-activated receptor gamma coactivator and is positively correlated with respiratory exchange ratio. In agreement with these results, leptin-deficient (ob/ob) and HFD-fed mice displayed high levels of Tgf-ß1 mRNA in the hypothalamus and reduction of Pgc1α mRNA in BAT. Interestingly, an acute exercise session reduced TGF-ß1 expression in the hypothalamus, increased Pgc1α mRNA in the BAT and reduced food consumption in obese mice. Our results demonstrated that acute physical exercise suppressed hypothalamic TGF-ß1 expression, increasing Pgc1α mRNA in BAT in obese mice.

Role for fatty acid amide hydrolase (FAAH) in the leptin-mediated effects on feeding and energy balance.

Endocannabinoid signaling regulates feeding and metabolic processes and has been linked to obesity development. Several hormonal signals, such as glucocorticoids and ghrelin, regulate feeding and metabolism by engaging the endocannabinoid system. Similarly, studies have suggested that leptin interacts with the endocannabinoid system, yet the mechanism and functional relevance of this interaction remain elusive. Therefore, we explored the interaction between leptin and endocannabinoid signaling with a focus on fatty acid amide hydrolase (FAAH), the primary degradative enzyme for the endocannabinoid N-arachidonoylethanolamine (anandamide; AEA). Mice deficient in leptin exhibited elevated hypothalamic AEA levels and reductions in FAAH activity while leptin administration to WT mice reduced AEA content and increased FAAH activity. Following high fat diet exposure, mice developed resistance to the effects of leptin administration on hypothalamic AEA content and FAAH activity. At a functional level, pharmacological inhibition of FAAH was sufficient to prevent leptin-mediated effects on body weight and food intake. Using a novel knock-in mouse model recapitulating a common human polymorphism (FAAH C385A; rs324420), which reduces FAAH activity, we investigated whether human genetic variance in FAAH affects leptin sensitivity. While WT (CC) mice were sensitive to leptin-induced reductions in food intake and body weight gain, low-expressing FAAH (AA) mice were unresponsive. These data demonstrate that FAAH activity is required for leptin's hypophagic effects and, at a translational level, suggest that a genetic variant in the FAAH gene contributes to differences in leptin sensitivity in human populations.

Leptin Replacement Reestablishes Brain Insulin Action in the Hypothalamus in Congenital Leptin Deficiency.

OBJECTIVE: Human obesity is associated with impaired central insulin signaling, and in very rare cases, severe obesity can be caused by congenital leptin deficiency. In such patients, leptin replacement results in substantial weight loss and improvement in peripheral metabolism. RESEARCH DESIGN AND METHODS: In a leptin-deficient patient, we investigated the impact of leptin substitution on central insulin action, as quantified by changes in neuronal activity after intranasal insulin application. This was assessed before and during the first year of metreleptin substitution. RESULTS: After only 1 year, treatment with metreleptin reestablishes brain insulin sensitivity, particularly in the hypothalamus and, to a lesser degree, in the prefrontal cortex. Results are depicted in comparison with a control group. In our patient, brain activation changes were accompanied by substantial weight loss, reduced visceral adipose tissue, reduced intrahepatic lipid content, and improved whole-body insulin sensitivity. CONCLUSIONS: Leptin replacement and weight loss improved homeostatic insulin action in the patient in question.

Melatonin Efficacy in Obese Leptin-Deficient Mice Heart.

Cardiomyocytes are particularly sensitive to oxidative damage due to the link between mitochondria and sarcoplasmic reticulum necessary for calcium flux and contraction. Melatonin, important indoleamine secreted by the pineal gland during darkness, also has important cardioprotective properties. We designed the present study to define morphological and ultrastructural changes in cardiomyocytes and mainly in mitochondria of an animal model of obesity (ob/ob mice), when treated orally or not with melatonin at 100 mg/kg/day for 8 weeks (from 5 up to 13 week of life). We observed that ob/ob mice mitochondria in sub-sarcolemmal and inter-myofibrillar compartments are often devoid of cristae with an abnormally large size, which are called mega-mitochondria. Moreover, in ob/ob mice the hypertrophic cardiomyocytes expressed high level of 4hydroxy-2-nonenal (4HNE), a marker of lipid peroxidation but scarce degree of mitofusin2, indicative of mitochondrial sufferance. Melatonin oral supplementation in ob/ob mice restores mitochondrial cristae, enhances mitofusin2 expression and minimizes 4HNE and p62/SQSTM1, an index of aberrant autophagic flux. At pericardial fat level, adipose tissue depot strictly associated with myocardium infarction, melatonin reduces adipocyte hypertrophy and inversely regulates 4HNE and adiponectin expressions. In summary, melatonin might represent a safe dietary adjuvant to hamper cardiac mitochondria remodeling and the hypoxic status that occur in pre-diabetic obese mice at 13 weeks of life.

Does leptin cause an increase in blood pressure in animals and humans?

PURPOSE OF REVIEW: Cardiovascular diseases (CVDs) are the number one cause of death globally. The risk for the development of CVDs is significantly increased in obesity. Leptin, the product of white adipose tissue, appears to contribute to the development of CVDs in obesity. Here, we discuss the premise that leptin engages the sympathetic nervous system and contributes to elevated blood pressure (BP) developing in obesity. RECENT FINDINGS: The long-term regulation of BP is dependent on the activity of the autonomic nervous system and specifically the sympathetic nervous system. Sympathetic nerve activity is significantly increased in obese rodents and humans. Leptin increases sympathetic nerve activity in rodents and humans; however, leptin only consistently increases BP chronically in rodents. The ability of leptin to increase BP in rodents is via both hypothalamic and extrahypothalamic regions. In leptin-deficient and leptin receptor-deficient humans, leptin appears to be the key reason for decreased systolic BP. However, in other research conducted in humans, chronic administration of leptin does not elevate BP. SUMMARY: Further research into the role of leptin in the development of CVDs, especially in humans, needs to be conducted.

Modeling the impact of growth and leptin deficits on the neuronal regulation of blood pressure.

The risk of hypertension is increased by intrauterine growth restriction (IUGR) and preterm birth. In the search for modifiable etiologies for this life-threatening cardiovascular morbidity, a number of pathways have been investigated, including excessive glucocorticoid exposure, nutritional deficiency and aberration in sex hormone levels. As a neurotrophic hormone that is intimately involved in the cardiovascular regulation and whose levels are influenced by glucocorticoids, nutritional status and sex hormones, leptin has emerged as a putative etiologic and thus a therapeutic agent. As a product of maternal and late fetal adipocytes and the placenta, circulating leptin typically surges late in gestation and declines after delivery until the infant consumes sufficient leptin-containing breast milk or accrues sufficient leptin-secreting adipose tissue to reestablish the circulating levels. The leptin deficiency seen in IUGR infants is a multifactorial manifestation of placental insufficiency, exaggerated glucocorticoid exposure and fetal adipose deficit. The preterm infant suffers from the same cascade of events, including separation from the placenta, antenatal steroid exposure and persistently underdeveloped adipose depots. Preterm infants remain leptin deficient beyond term gestation, rendering them susceptible to neurodevelopmental impairment and subsequent cardiovascular dysregulation. This pathologic pathway is efficiently modeled by placing neonatal mice into atypically large litters, thereby recapitulating the perinatal growth restriction-adult hypertension phenotype. In this model, neonatal leptin supplementation restores the physiologic leptin surge, attenuates the leptin-triggered sympathetic activation in adulthood and prevents leptin- or stress-evoked hypertension. Further pathway interrogation and clinical translation are needed to fully test the therapeutic potential of perinatal leptin supplementation.

The Acute-Phase Protein Orosomucoid Regulates Food Intake and Energy Homeostasis via Leptin Receptor Signaling Pathway.

The acute-phase protein orosomucoid (ORM) exhibits a variety of activities in vitro and in vivo, notably modulation of immunity and transportation of drugs. We found in this study that mice lacking ORM1 displayed aberrant energy homeostasis characterized by increased body weight and fat mass. Further investigation found that ORM, predominantly ORM1, is significantly elevated in sera, liver, and adipose tissues from the mice with high-fat diet (HFD)-induced obesity and db/db mice that develop obesity spontaneously due to mutation in the leptin receptor (LepR). Intravenous or intraperitoneal administration of exogenous ORM decreased food intake in C57BL/6, HFD, and leptin-deficient ob/ob mice, which was absent in db/db mice and was significantly reduced in mice with arcuate nucleus (ARC) LepR knockdown, whereas enforced expression of ORM1 in ARC significantly decreased food intake, body weight, and serum insulin level. Furthermore, we found that ORM is able to bind directly to LepR and activate the receptor-mediated JAK2-STAT3 signaling in hypothalamus tissue and GT1-7 cells, which was derived from hypothalamic tumor. These data indicated that ORM could function through LepR to regulate food intake and energy homeostasis in response to nutrition status. Modulating the expression of ORM is a novel strategy for the management of obesity and related metabolic disorders.

Neonatal growth restriction-related leptin deficiency enhances leptin-triggered sympathetic activation and central angiotensin II receptor-dependent stress-evoked hypertension.

BACKGROUND: Neonatal growth restriction (nGR) leads to leptin deficiency and increases the risk of hypertension. Previous studies have shown nGR-related hypertension is normalized by neonatal leptin (nLep) and exacerbated by psychological stress. With recent studies linking leptin and angiotensin signaling, we hypothesized that nGR-induced nLep deficiency increases adult leptin sensitivity; leading to leptin- or stress-induced hypertension, through a pathway involving central angiotensin II type 1 receptors. METHODS: We randomized mice with incipient nGR, by virtue of their presence in large litters, to vehicle or physiologic nLep supplementation (80 ng/g/d). Adult caloric intake and arterial pressure were monitored at baseline, during intracerebroventricular losartan infusion and during systemic leptin administration. RESULTS: nGR increased leptin-triggered renal sympathetic activation and hypertension with increased leptin receptor expression in the arcuate nucleus of the hypothalamus; all of those nGR-associated phenotypes were normalized by nLep. nGR mice also had stress-related hyperphagia and hypertension, but only the stress hypertension was blocked by central losartan infusion. CONCLUSION: nGR leads to stress hypertension through a pathway that involves central angiotensin II receptors, and nGR-associated leptin deficiency increases leptin-triggered hypertension in adulthood. These data suggest potential roles for preservation of neonatal growth and nLep supplementation in the prevention of nGR-related hypertension.

Ipragliflozin Improves Hepatic Steatosis in Obese Mice and Liver Dysfunction in Type 2 Diabetic Patients Irrespective of Body Weight Reduction.

Type 2 diabetes mellitus (T2DM) is associated with a high incidence of non-alcoholic fatty liver disease (NAFLD) related to obesity and insulin resistance. Currently, medical interventions for NAFLD have focused on diet control and exercise to reduce body weight, and there is a requirement for effective pharmacological therapies. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote the urinary excretion of glucose by blocking its reabsorption in renal proximal tubules. SGLT2 inhibitors lower blood glucose independent of insulin action and are expected to reduce body weight because of urinary calorie loss. Here we show that an SGLT2 inhibitor ipragliflozin improves hepatic steatosis in high-fat diet-induced and leptin-deficient (ob/ob) obese mice irrespective of body weight reduction. In the obese mice, ipragliflozin-induced hyperphagia occurred to increase energy intake, attenuating body weight reduction with increased epididymal fat mass. There is an inverse correlation between weights of liver and epididymal fat in ipragliflozin-treated obese mice, suggesting that ipragliflozin treatment promotes normotopic fat accumulation in the epididymal fat and prevents ectopic fat accumulation in the liver. Despite increased adiposity, ipragliflozin ameliorates obesity-associated inflammation and insulin resistance in epididymal fat. Clinically, ipragliflozin improves liver dysfunction in patients with T2DM irrespective of body weight reduction. These findings provide new insight into the effects of SGLT2 inhibitors on energy homeostasis and fat accumulation and indicate their potential therapeutic efficacy in T2DM-associated hepatic steatosis.

Localizing Effects of Leptin on Upper Airway and Respiratory Control during Sleep.

STUDY OBJECTIVES: Obesity hypoventilation and obstructive sleep apnea are common complications of obesity linked to defects in respiratory pump and upper airway neural control. Leptin-deficient ob/ob mice have impaired ventilatory control and inspiratory flow limitation during sleep, which are both reversed with leptin. We aimed to localize central nervous system (CNS) site(s) of leptin action on respiratory and upper airway neuroventilatory control. METHODS: We localized the effect of leptin to medulla versus hypothalamus by administering intracerbroventricular leptin (10 µg/2 µL) versus vehicle to the lateral (n = 14) versus fourth ventricle (n = 11) of ob/ob mice followed by polysomnographic recording. Analyses were stratified for effects on respiratory (nonflow-limited breaths) and upper airway (inspiratory flow limitation) functions. CNS loci were identified by (1) leptin-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and (2) projections of respiratory and upper airway motoneurons with a retrograde transsynaptic tracer (pseudorabies virus). RESULTS: Both routes of leptin administration increased minute ventilation during nonflow-limited breathing in sleep. Phrenic motoneurons were synaptically coupled to the nucleus of the solitary tract, which also showed STAT3 phosphorylation, but not to the hypothalamus. Inspiratory flow limitation and obstructive hypopneas were attenuated by leptin administration to the lateral but not to the fourth cerebral ventricle. Upper airway motoneurons were synaptically coupled with the dorsomedial hypothalamus, which exhibited STAT3 phosphorylation. CONCLUSIONS: Leptin relieves upper airway obstruction in sleep apnea by activating the forebrain, possibly in the dorsomedial hypothalamus. In contrast, leptin upregulates ventilatory control through hindbrain sites of action, possibly in the nucleus of the solitary tract.

The Effects of Leptin Replacement on Neural Plasticity.

Leptin, an adipokine synthesized and secreted mainly by the adipose tissue, has multiple effects on the regulation of food intake, energy expenditure, and metabolism. Its recently-approved analogue, metreleptin, has been evaluated in clinical trials for the treatment of patients with leptin deficiency due to mutations in the leptin gene, lipodystrophy syndromes, and hypothalamic amenorrhea. In such patients, leptin replacement therapy has led to changes in brain structure and function in intra- and extrahypothalamic areas, including the hippocampus. Furthermore, in one of those patients, improvements in neurocognitive development have been observed. In addition to this evidence linking leptin to neural plasticity and function, observational studies evaluating leptin-sufficient humans have also demonstrated direct correlation between blood leptin levels and brain volume and inverse associations between circulating leptin and risk for the development of dementia. This review summarizes the evidence in the literature on the role of leptin in neural plasticity (in leptin-deficient and in leptin-sufficient individuals) and its effects on synaptic activity, glutamate receptor trafficking, neuronal morphology, neuronal development and survival, and microglial function.

Dietary component isorhamnetin is a PPARγ antagonist and ameliorates metabolic disorders induced by diet or leptin deficiency.

Studies on peroxisome proliferator-activated receptor (PPAR)-γ ligands have been focused on agonists. However, PPARγ activation may induce obesity and nonalcoholic fatty liver disease (NAFLD), one of the most challenging medical conditions. Here, we identified that isorhamnetin, a naturally occurring compound in fruits and vegetables and the metabolite of quercetin, is a novel antagonist of PPARγ. Isorhamnetin treatment inhibited the adipocyte differentiation induced by the PPARγ agonist rosiglitazone, reduced obesity development and ameliorated hepatic steatosis induced by both high-fat diet treatment and leptin deficiency. Our results suggest that dietary supplement of isorhamnetin may be beneficial to prevent obesity and steatosis and PPARγ antagonists may be useful to treat hepatic steatosis.

Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production.

Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E-deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.

Beneficial Effects of Supplementation of the Rare Sugar "D-allulose" Against Hepatic Steatosis and Severe Obesity in Lep(ob)/Lep(ob) Mice.

A rare sugar, D-allulose (also called D-psicose), has recently been applied as a food supplement in view of controlling diabetes and obesity in Japan. D-allulose has been proven to have unique effects against hyperglycemia and hyperlipidemia in a number of studies using several species of rats and mice. However, the antiobesity effects of D-allulose have not yet been assessed in Lep(ob)/Lep(ob) (ob/ob) mice. Therefore, this study explored the dietary supplemental effects of this sugar in leptin-deficient ob/ob mice. Consequently, the subchronic ingestion of D-allulose in ob/ob mice for 15 wk significantly decreased the body and liver weights, and the loss of body weight was involved in the reduction of the total fat mass, including abdominal visceral fat, and not fat-free body mass, including muscle. Furthermore, D-allulose improved hepatic steatosis, as evaluated using hepatic histological studies and MRI. In the normal mice, none of these parameters were influenced by the single or long-term ingestion of D-allulose. These results indicate that dietary supplementation of D-allulose especially influences postprandial hyperglycemia and obesity-related hepatic steatosis, without exercise therapy or dietary restriction. Therefore, D-allulose may be useful as a supplement for preventing and improving obesity and obesity-related disorders.

Leptin signaling as a therapeutic target of obesity.

INTRODUCTION: Leptin is a hormone with a key role in food intake and body weight homeostasis. Congenital leptin deficiency (CLD) is a rare disease that causes hyperphagia and early severe obesity. However, common obesity conditions are associated with hyperleptinemia and leptin resistance. AREAS COVERED: The main signaling pathways activated by leptin as well as the mechanisms underlying the regulatory actions of leptin on food intake and on lipid and glucose metabolism are reviewed. The potential mechanisms involving leptin resistance and the main regulatory hormonal and nutritional factors controlling leptin production/functions are also analyzed. The pathophysiology of leptin in human obesity, and especially the trials analyzing effects of leptin replacement therapy in patients with CLD or in subjects with common obesity and in post-obese weight-reduced subjects are also summarized. EXPERT OPINION: The use of drugs or specific bioactive food components with anti-inflammatory properties to reduce the inflammatory state associated with obesity, especially at the hypothalamus, may help to overcome leptin resistance. Research should also be focused on investigating dietary strategies, food supplements or drugs capable of avoiding or reversing the leptin fall during weight management, in order to promote sustained body weight lowering and weight loss maintenance.

Hypothalamic obesity.

Hypothalamic obesity represents a rare diagnosis applicable to only a small subset of obese patients. It is important to identify, diagnose, and treat these patients. This article reviews the physiology of the hypothalamus, focusing on its role in regulation of hunger, feeding, and metabolism. The causes of hypothalamic obesity are discussed including genetic, anatomic, and iatrogenic etiologies. The complex hormonal environment leading to obesity is explored for each etiology and treatment strategies are discussed. Reproductive consequences are also reviewed.

Monoclonal antibody targeting of fibroblast growth factor receptor 1c ameliorates obesity and glucose intolerance via central mechanisms.

We have generated a novel monoclonal antibody targeting human FGFR1c (R1c mAb) that caused profound body weight and body fat loss in diet-induced obese mice due to decreased food intake (with energy expenditure unaltered), in turn improving glucose control. R1c mAb also caused weight loss in leptin-deficient ob/ob mice, leptin receptor-mutant db/db mice, and in mice lacking either the melanocortin 4 receptor or the melanin-concentrating hormone receptor 1. In addition, R1c mAb did not change hypothalamic mRNA expression levels of Agrp, Cart, Pomc, Npy, Crh, Mch, or Orexin, suggesting that R1c mAb could cause food intake inhibition and body weight loss via other mechanisms in the brain. Interestingly, peripherally administered R1c mAb accumulated in the median eminence, adjacent arcuate nucleus and in the circumventricular organs where it activated the early response gene c-Fos. As a plausible mechanism and coinciding with the initiation of food intake suppression, R1c mAb induced hypothalamic expression levels of the cytokines Monocyte chemoattractant protein 1 and 3 and ERK1/2 and p70 S6 kinase 1 activation.