The differential role of resistin on invasive liver cancer cells.

OBJECTIVES: To determine whether inhibition of kinase signaling will suppress resistin-induced liver cancer progression. Resistin is located in monocytes and macrophages of adipose tissue. This adipocytokine is an important link between obesity, inflammation, insulin resistance, and cancer risk. Pathways that resistin is known to be involved include but are not limited to mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinases (ERK). The ERK pathway promotes cellular proliferation, migration, survival of cancer cells, and tumor progression. The Akt pathway is known to be up-regulated in many cancers including liver cancer. METHODS: Using an in vitro model, HepG2 and SNU-449 liver cancer cells were exposed to resistin ± ERK, Akt, or both inhibitors. The following physiological parameters were assessed: cellular proliferation, ROS, lipogenesis, invasion, MMP, and lactate dehydrogenase activity. RESULTS: The inhibition of kinase signaling suppressed resistin-induced invasion and lactate dehydrogenase in both cell lines. In addition, in SNU-449 cells, resistin increased proliferation, ROS, and MMP-9 activity. Inhibition of PI3K and ERK decreased phosphorylated Akt and ERK, and pyruvate dehydrogenase. CONCLUSIONS: In this study, we describe the effect of Akt and ERK inhibitors to determine if inhibition suppresses resistin-induced liver cancer progression. Resistin promotes cellular proliferation, ROS, MMP, invasion and LDH activity in SNU-449 liver cancer cells which is differentially mediated by Akt and ERK signaling pathways.

Central s-resistin deficiency ameliorates hypothalamic inflammation and increases whole body insulin sensitivity.

S-resistin, a non-secretable resistin isoform, acts as an intracrine factor that regulates adipocyte maduration, inflammatory and insulin response in 3T3-L1 cells. However, its intracellular function in vivo is still unknown. In this study, we analyze the central role of s-resistin, decreasing its hypothalamic expression using an intracerebroventricular injection of lentiviral RNAi. The data present herein support an improvement in the hypothalamic leptin and insulin signaling pathway upon s-resistin downregulation. Furthermore, hypothalamic levels of pro-inflammatory markers decrease, meanwhile those of the anti-inflammatory cytokine IL-10 increases. Interestingly, peripheral NEFA decreases alike circulating leptin and resistin levels. These data demonstrate that hypothalamic s-resistin controls fuel mobilization and adipokines secretion. Importantly, central s-resistin downregulation improves systemic insulin sensitivity, as demonstrated after an IPGTT. Interestingly, our data also indicate that s-resistin downregulation could improve hypothalamic inflammation in aged Wistar rats. Altogether, our findings suggest that hypothalamic s-resistin seems to be a key regulator of the brain-fat axis which links inflammation with metabolic homeostasis.

Resistin Gene Expression is Downregulated in CD4(+) T Helper Lymphocytes and CD14(+) Monocytes in Rheumatoid Arthritis Responding to TNF-α Inhibition.

Rheumatoid arthritis (RA) is caused by complex interactions between immune cells and sustained by Th1 response cytokines. Resistin [resistance to insulin; (RETN)] is an inflammatory cytokine, first discovered in murine adipocytes. In man, RETN is mainly secreted by monocytes. The distinct role of RETN in the immune reaction is uncertain; however, RETN has pro-inflammatory, pro-fibrotic and possibly tolerogenic properties. The aim was to assess the reaction of RETN gene expression to TNF-α inhibition (I) in pathogenetic immune cell subsets in RA, in the context of Th1, inflammatory and regulatory cytokine gene expressions. Accordingly, we measured RETN, IFN-γ, TNF-ß, IL-1ß, TNF-α, TGF-ß and IL-10 gene expressions in CD14(+) monocytes, CD4(+) T helper (Th) lymphocytes (ly), CD8(+) T cytotoxic (Tc) ly and CD19(+) B ly in active RA before and 3 months after start of TNF-αI. Leucocyte subsets were separated by specific monoclonal antibody-covered beads, RNA extracted and levels of RETN, Th1 response, inflammatory and regulatory cytokine mRNAs measured by quantitative reverse transcription-polymerase chain reaction technique. We found that TNF-αI caused a significant downregulation of RETN gene expression in CD14(+) monocytes and CD4(+) Th ly and was unchanged in CD8(+) Tc ly and CD19(+) B ly. Both in active RA and during TNF-αI, RETN mRNA levels were significantly higher in CD14(+) monocytes than in all other examined cell types. In monocytes, fold change in RETN and TGF-ß gene expressions upon TNF-αI correlated significantly. Our findings indicate that RETN has pro-inflammatory as well as proresolving roles in active RA.

Antiresistin RNA Oligonucleotide Ameliorates Diet-Induced Nonalcoholic Fatty Liver Disease in Mice through Attenuating Proinflammatory Cytokines.

The aim of this study was to determine whether inhibition of resistin by a synthetic antiresistin RNA (oligonucleotide) oligo ameliorates metabolic and histological abnormalities in nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD) in mice. The antiresistin RNA oligo and a scrambled control oligo (25 mg/kg of body weight) were i.p. injected to HFD mice. Serum metabolic parameters and hepatic enzymes were measured after 4-week treatment. The treatment significantly reduced epididymal fat and attenuated the elevated serum resistin, cholesterol, triglycerides, glucose, and insulin with an improved glucose tolerance test. Antiresistin RNA oligo also normalized serum AST and ALT levels with improved pathohistology of NAFLD. Immunoblotting and qRT-PCR revealed that decreased protein and mRNA expression of resistin in fat and liver tissues of the treated mice were associated with reduction of adipose TNF-α and IL-6 expression and secretion into circulation. mRNA and protein expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and sterol regulatory element-binding protein-1c (SREBP-1c) were also significantly decreased in the treated mice. Our results suggest that resistin may exacerbate NAFLD in metabolic syndrome through upregulating inflammatory cytokines and hepatic PEPCK and SREBP-1c. Antiresistin RNA oligo ameliorated metabolic abnormalities and histopathology of NAFLD through attenuating proinflammatory cytokines.

Resistin and visfatin in steatotic and non-steatotic livers in the setting of partial hepatectomy under ischemia-reperfusion.

BACKGROUND & AIMS: This study examined whether the regulation of resistin and visfatin could reduce damage and improve regeneration in both steatotic and non-steatotic livers undergoing partial hepatectomy under ischemia-reperfusion, a procedure commonly applied in clinical practice to reduce bleeding. METHODS: Resistin and visfatin were pharmacologically modulated in lean and obese animals undergoing partial hepatectomy under ischemia-reperfusion. RESULTS: No evident role for these adipocytokines was observed in non-steatotic livers. However, obese animals undergoing liver surgery showed increased resistin in liver and plasma, without changes in adipose tissue, together with visfatin downregulation in liver and increment in plasma and adipose tissue. Endogenous resistin maintains low levels of visfatin in the liver by blocking its hepatic uptake from the circulation, thus regulating the visfatin detrimental effects on hepatic damage and regenerative failure. Indeed, the administration of anti-resistin antibodies increased hepatic accumulation of adipocyte-derived visfatin, exacerbating damage and regenerative failure. Interestingly, treatment with anti-visfatin antibodies protected steatotic livers, and similar results were obtained with the concomitant inhibition of resistin and visfatin. Thus, when visfatin was inhibited, the injurious effects of anti-resistin antibodies disappeared. Herein we show that upregulation of visfatin increased NAD levels in the remnant steatotic liver, whereas visfatin inhibition decreased them. These later observations suggest that visfatin may favour synthesis of NAD instead of DNA and induces alterations in amino acid metabolism-urea cycle and NO production, overall negatively affecting liver viability. CONCLUSIONS: Our results indicate the clinical potential of visfatin blocking-based therapies in steatotic livers undergoing partial hepatectomy with ischemia-reperfusion.

TNFα blockade for inflammatory rheumatic diseases is associated with a significant gain in android fat mass and has varying effects on adipokines: a 2-year prospective study.

PURPOSE: To evaluate the long-term consequences of TNFα inhibitors on body composition and fat distribution, as well as changes in serum adipokines in patients with rheumatoid arthritis (RA) or ankylosing spondylitis (AS). METHODS: Eight patients with RA and twelve with AS requiring a TNFα inhibitor were prospectively followed for 2 years. Body composition was evaluated by dual X-ray absorptiometry and included measurements of total fat mass, lean mass, fat in the gynoid and android regions, and visceral fat. Serum leptin, total and high molecular weight (HMW) adiponectin, resistin, and ghrelin were also assessed. RESULTS: There was a significant gain in body mass index (p = 0.05) and a tendency for weight (p = 0.07), android fat (p = 0.07), and visceral fat (p = 0.059) increase in patients with RA, while in AS, total fat mass significantly increased (p = 0.02) with a parallel weight gain (p = 0.07). When examining the whole population of patients, we observed after 2 years a significant increase in body weight (+1.9%; p = 0.003), body mass index (+2.5%; p = 0.004), total fat mass (+11.1%; p = 0.007), and fat in the android region (+18.3%; p = 0.02). There was a substantial, albeit nonsignificant gain in visceral fat (+24.3%; p = 0.088). Lean mass and gynoid fat were not modified. No major changes were observed for serum leptin, total adiponectin, and ghrelin, while HMW adiponectin and the HMW/total adiponectin ratio tended to decrease (-15.2%, p = 0.057 and -9.3%, p = 0.067, respectively). Resistin decreased significantly (-22.4%, p = 0.01). CONCLUSIONS: Long-term TNFα inhibition in RA and AS is associated with a significant gain in fat mass, with a shift to the android (visceral) region. This fat redistribution raises questions about its influence on the cardiovascular profile of patients receiving these treatments.

Resistin affects lipid metabolism during adipocyte maturation of 3T3-L1 cells.

Resistin, an adipose-tissue-specific secretory factor, aggravates metabolic syndrome through impairment of glucose metabolism. Previously, we demonstrated that resistin expression was induced in both 3T3-L1 cells and primary pre-adipocytes derived from Zucker obese rats during the process of differentiation and maturation (Ikeda Y, Hama S, Kajimoto K, Okuno T, Tsuchiya H & Kogure K (2011) Biol Pharm Bull 34, 865-870). However, the biological function of resistin in adipocytes is poorly understood. In the present study, we examined the effects of resistin knockdown on the biological features of 3T3-L1 cells. We found that lipid content was significantly decreased in 3T3-L1 cells transfected with anti-resistin small interfering RNA (siRNA) after adipocyte differentiation. While expression of peroxisome proliferator activated receptor γ and CCAAT/enhancer-binding protein α was not affected, protein expression and transcriptional activity levels of carbohydrate response element binding protein (ChREBP), which upregulates transcription of lipogenic genes, decreased after anti-resistin siRNA treatment. Moreover, gene expression of fatty acid synthase and acetyl-CoA carboxylase 2, which are known to be regulated by ChREBP, were also suppressed by resistin knockdown. In contrast, activity of the fatty acid ß-oxidation-regulating protein carnitine palmitoyltransferase 1 increased. These results suggest that resistin knockdown induces suppression of lipid production and activation of fatty acid ß-oxidation. Consequently, resistin may affect lipid metabolism during adipocyte maturation.

Oral Angiotensin-(1-7) prevented obesity and hepatic inflammation by inhibition of resistin/TLR4/MAPK/NF-κB in rats fed with high-fat diet.

Obesity is characterized by a pro-inflammatory state commonly associated with type 2 diabetes and fat-liver disease. In the last few years, different studies pointed out the role of Angiotensin (Ang)-(1-7) in the metabolic regulation. The aim of the present study was to evaluate the effect of oral-administration of Ang-(1-7) in metabolism and inflammatory state of high-fat feed rats. Twenty-four male Sprague Dawley rats were randomized into three groups: High Fat Diet (HFD); Standard Diet (ST); High Fat Diet+Angiotensin-(1-7) [HFD+Ang-(1-7)]. Glycemic profile was evaluated by glucose tolerance and insulin sensitivity tests, plasmatic glucose and insulin. Cholesterol, HDL and triglycerides analyses presented lipidic profile. RT-PCR evaluated mRNA expression to ACE, ACE2, resistin, TLR4, IL-6, TNF-α and NF-κB genes. The main results showed that oral Ang-(1-7) decreased body weight and abdominal fat-mass. In addition, HFD+Ang-(1-7) treated rats presented enhanced glucose tolerance, insulin-sensitivity and decreased plasma-insulin levels, as well as a significant decrease in circulating lipid levels. These alterations were accompanied by a marked decreased expression of resistin, TLR4, ACE and increased ACE2 expression in liver. Furthermore, Ang-(1-7) decreases phosphorylation of MAPK and increases NF-κB expression. These alterations diminished expression of interleukin-6 and TNF-α, ameliorate inflammatory state in liver. In summary, the present study showed that oral-treatment with Ang-(1-7) in high-fat feed rats improved metabolism down-regulating resistin/TLR4/NF-κB-pathway.

The effects of designed angiopoietin-1 variant on lipid droplet diameter, vascular endothelial cell density and metabolic parameters in diabetic db/db mice.

Metabolic syndrome consists of metabolic abnormality with central obesity, hypertriglyceridemia, insulin resistance and hypertension. Adipose tissue has been known as a primary site of insulin resistance and its adipocyte size may be correlated with the degree of insulin resistance. A designed angiopoietin-1, COMP-Angiopoietin-1 (COMP-Ang1), mitigated high-fat diet-induced insulin resistance in skeletal muscle. In this study, we examined effects of COMP-Ang1 on adipocyte droplet size, vascular endothelial cell density in adipose tissue and metabolic parameters in db/db mice by administering COMP-Ang1 or LacZ (as a control) adenovirus. Administration of COMP-Ang1 decreased fat droplet diameter in epididymal and abdominal visceral adipocyte and visceral fat content in db/db mice. The density of vascular endothelial cell in adipose tissue was increased in db/db mice after treatment with COMP-Ang1. Serum resistin and tumor necrosis factor-α level was lower after treatment with COMP-Ang1 in db/db mice. COMP-Ang1 caused a restoration of fasting glycemic control in db/db mice and decreased serum insulin level and insulin resistance measured by HOMA index. These findings indicate that COMP-Ang1 regulates adipocyte fat droplet diameter, vascular endothelial cell density and metabolic parameters in db/db mice.

Ellagic acid in pomegranate suppresses resistin secretion by a novel regulatory mechanism involving the degradation of intracellular resistin protein in adipocytes.

Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. Pomegranate is a rich source of compounds used to treat metabolic diseases including type 2 diabetes. In this study, we found that consumption of pomegranate fruit extract (PFE) predominantly reduced the serum resistin levels in ovariectomized mice, an animal model with elevated resistin levels in serum and upregulated resistin mRNA expression in white adipose tissue. Moreover, the PFE significantly reduced the secretion and intracellular protein levels of resistin in differentiated murine 3T3-L1 adipocytes, but it did not alter resistin mRNA expression. When de novo protein synthesis was inhibited by the protein synthesis inhibitor cycloheximide, the intracellular resistin protein levels were drastically reduced by the PFE, suggesting that the PFE promoted the degradation of resistin at the protein level. We also found that ellagic acid (EA), a main component of pomegranate, had the same effects on the secretion and intracellular protein level of resistin. These results suggest that EA in pomegranate suppresses resistin secretion by a novel mechanism involving the degradation of intracellular resistin protein in adipocytes.

Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-alpha in macrophages.

Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-alpha stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-alpha. Addition of mevalonate induced resistin protein expression similar to TNF-alpha stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA) completely attenuated the resistin protein expression induced by TNF-alpha and mevalonate. TNF-alpha induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-alpha. The gel shift and promoter activity assay showed that TNF-alpha increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-alpha. Recombinant resistin and TNF-alpha significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-alpha. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-alpha.

Dietary supplementation with chitosan derived from mushrooms changes adipocytokine profile in diet-induced obese mice, a phenomenon linked to its lipid-lowering action.

Recent data reported that chitosan reduces high-fat (HF) diet-induced obesity in mice without describing the metabolic consequences of such an effect. The aim of this study was to investigate the capacity of chitosan derived from edible mushrooms to modify adipocytokine levels and to assess the relevance of this effect on the development of fat mass, and on glucose and lipid metabolism in obese mice. Mice were fed a HF diet or a HF diet supplemented with 5% fungal chitosan for ten weeks. HF-induced hypertriglyceridaemia, fasting hyperinsulinaemia and fat accumulation in liver, muscle and white adipose tissue (WAT) were reduced after chitosan treatment. The higher lipid content in the caecum following treatment with chitosan suggested that this dietary fiber reduced lipid absorption. We postulated that the lower triglyceridaemia observed upon chitosan treatment could also be the result of the lower FIAF (fasting-induced adipose factor) expression observed in visceral adipose tissue. IL-6, resistin and leptin levels decreased in the serum after chitosan supplementation. We conclude that fungal chitosan counteracts some inflammatory disorders and metabolic alterations occurring in diet-induced obese mice since it decreases feed efficiency, fat mass, adipocytokine secretion and ectopic fat deposition in the liver and the muscle.

Psoriasis patients show signs of insulin resistance.

BACKGROUND: Recent observations suggest that psoriasis is a risk factor for the development of coronary artery calcification which in turn represents an indicator for atherosclerosis. OBJECTIVE: To clarify a possible pathogenetic link between psoriasis and atherosclerosis, we studied the metabolic state of patients with psoriasis. METHODS: Thirty-nine consecutive patients with moderate-to-severe plaque-type psoriasis were enrolled in the study. Detailed information was obtained on the patients' clinical picture and history of psoriasis, smoking habits and medication. The body mass index (BMI) of the patients was calculated. Laboratory investigations focused on values for inflammation, lipid profile and multiple cytokines. The intima-media thickness of the carotid artery was measured by ultrasound, and an oral glucose tolerance test was performed to calculate the homeostasis model assessment of insulin resistance (HOMA). RESULTS: Numerous well-recognized correlations such as between BMI and HOMA (P < 0.02) as well as BMI and vessel wall thickness (P < 0.05) were successfully reproduced, thus confirming consistency of our dataset. With regard to psoriasis, we observed a significant correlation between the Psoriasis Area and Severity Index (PASI) score and insulin secretion. Moreover, the PASI score was significantly correlated with serum resistin levels--a cytokine known to be increased in insulin resistance. CONCLUSIONS: Taken together, several measurements indicative of insulin resistance were found to be significantly correlated with the PASI score. The concept of insulin resistance as a consequence of chronic inflammation and possible pathogenetic cause for comorbidities known to be associated with psoriasis is supported by these data. Our findings validate further studies on larger cohorts as well as interventional studies.

Resistin-binding peptide antagonizes role of resistin on white adipose tissue.

AIM: To investigate the direct effects of resistin and resistin-binding peptide (RBP) on lipid metabolism and endocrine function in adipose tissue. METHODS: Rat white adipose tissue was cultured in vitro and incubated for 24 h with 30 ng/mL recombinant rat resistin protein (rResistin) or combined with RBP of varying concentrations(1x10(-12) mol/L, 1x10(-10) mol/L, 1x10(-8) mol/L). Free fatty acids (FFA) released into medium was measured by a colorimetric kit. The levels of protein secretion and mRNA expression of TNF-alpha and adiponectin were detected by ELISA kit and RT-PCR respectively. RESULTS: The levels of FFA released into medium were significantly increased after 24 h of exposure to rResistin, but significantly decreased after RBP was applied, although there was no difference between the 3 concentrations. The protein level and gene expression of TNF-alpha in adipose tissue were significantly increased after 24 h of exposure to rResistin, but only obviously decreased after incubated with 1x10(-8) mol/L RBP. The levels of protein secretion and mRNA expression of adiponectin in adipose tissue were significantly decreased after 24 h of exposure to rResistin, but increased after incubated with RBP with the higher concentrations. CONCLUSION: RBP can effectively antagonize the role of resistin on the lipid metabolism and endocrine function of adipose tissue.

A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation.

BACKGROUND: Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor. METHODS: Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide. RESULTS: Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation. CONCLUSION: RBP could effectively rescue the promoted differentiation of resistin overexpressed 3T3-L1 preadipocyte.