Interleukin-4 Improves Metabolic Abnormalities in Leptin-Deficient and High-Fat Diet Mice

International Journal of Molecular Sciences

Article Interleukin-4 Improves Metabolic Abnormalities in Leptin-Deﬁcient and High-Fat Diet Mice

Shih-Yi Lin 1,2 , Ching-Ping Yang 3 , Ya-Yu Wang 2,4, Chiao-Wan Hsiao 5, Wen-Ying Chen 6, Su-Lan Liao 3, Yu-Li Lo 7, Yih-Hsin Chang 5, Chen-Jee Hong 8 and Chun-Jung Chen 3,9,10,*

1 Center for Geriatrics and Gerontology, Taichung Veterans General Hospital, Taichung City 407, Taiwan; [email protected] 2 Institute of Clinical Medicine, National Yang-Ming University, Taipei City 112, Taiwan; [email protected] 3 Department of Medical Research, Taichung Veterans General Hospital, Taichung City 407, Taiwan; [email protected] (C.-P.Y.); [email protected] (S.-L.L.) 4 Department of Family Medicine, Taichung Veterans General Hospital, Taichung City 407, Taiwan 5 Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei City 112, Taiwan; [email protected] (C.-W.H.); [email protected] (Y.-H.C.) 6 Department of Veterinary Medicine, National Chung-Hsing University, Taichung City 402, Taiwan; [email protected] 7 Department and Institute of Pharmacology, National Yang-Ming University, Taipei City 112, Taiwan; [email protected] 8 Institute of Brain Science, National Yang-Ming University, Taipei City 112, Taiwan; [email protected] 9 Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung City 404, Taiwan 10 Ph.D. Program in Translational Medicine, College of Life Sciences, National Chung Hsing University, Taichung City 402, Taiwan * Correspondence: [email protected]; Tel.: +886-4-2359-2525 (ext. 4022)

Received: 2 June 2020; Accepted: 22 June 2020; Published: 23 June 2020

Abstract: Obesity is a metabolic disorder that results from complex interactions between genetic predisposition and dietary factors. Interleukin-4 (IL-4), besides its role in immunity, has metabolic effects on insulin efficacy. We studied the effects of IL-4 on metabolic abnormalities in a mice model of obesity involving leptin deficiency and leptin resistance. Leptin-deficient 145E and leptin-resistant high-fat diet (HFD) mice showed lower levels of circulating IL-4. 145E and HFD mice showed a number of abnormalities: Obesity, hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, liver injury, and adiposity with concurrent inflammation, decreases in Akt, signal transducer and activator of transcription 3 (STAT3), and STAT6 phosphorylation in the hypothalamus, liver, and epididymal fat. Independent of leptin-deficient obesity and dietary obesity, a course of 8-week IL-4 supplementation improved obesity and impairment in Akt, STAT3, and STAT6 signaling. Amelioration of cytokine expression, despite variable extents, was closely linked with the actions of IL-4. Additionally, the browning of white adipocytes by IL-4 was found in epididymal white adipose tissues and 3T3-L1 preadipocytes. Chronic exercise, weight management, and probiotics are recommended to overweight patients and IL-4 signaling is associated with clinical improvement. Thus, IL-4 could be a metabolic regulator and antiobesity candidate for the treatment of obesity and its complications.

Keywords: adiposity; inﬂammation; IL-4; leptin; obesity

Int. J. Mol. Sci. 2020, 21, 4451; doi:10.3390/ijms21124451 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4451 2 of 19

1. Introduction Obesity is a public health problem worldwide. Obesity is a risk factor that increases the prevalence and severity of several chronic disorders, predisposing subjects to a number of disorders, like cardiovascular diseases, hypertension, stroke, mood disorders, cancers, and type 2 diabetes [1–3]. With body weight management, obese subjects show marked improvements in obesity-related complications, in both human and rodent studies [4–7]. These phenomena underscore the importance of identifying strategies to avoid overweight, with the aim of combating obesity and its complications. Energy balance is maintained through counter-regulations between food intake and energy expenditure via the communications of nutrients and hormones with distinct neuronal subpopulations and targeted tissues in the central nervous system (CNS) and the periphery. In the CNS, the critical neuronal populations are the anorexigenic proopiomelanocortin (POMC)- and orexigenic agouti-related protein (AgRP)-/neuropeptide Y (NPY)-expressing neurons, located in the arcuate nucleus of the hypothalamus. Regarding hormones, gut-derived ghrelin activates AgRP and NYP neurons, and inhibits the POMC neurons. In contrast, adipocyte-originating leptin has opposite effects [8–10]. When food nutrients exceed physiological requirements, the metabolized lipids are deposed at the intra-abdominal adipose tissues, particularly the white adipose tissue (WAT). The consequent adipocytic hyperplasia and hypertrophy increase the release of free fatty acids, proinflammatory cytokines, and adipokines into the blood stream, and lead to chronic and low-grade inflammations. The result is the impairment of insulin actions and glucose utility in the liver, skeletal muscles, and adipose tissues with a series of metabolic abnormalities, and the eventual development of obesity-associated complications [11–17]. Several studies have highlighted the crucial role of adipose tissues in metabolic changes, and their targeting for the prevention of obesity-induced complications. Chronic exercise, weight management, and probiotics have all been implicated in improving obesity-related metabolic abnormalities. Interleukin-4 (IL-4) signaling is known to be closely linked to such beneficial effects [18–21]. Th2 cytokine IL-4 directs macrophages towards an anti-inflammatory phenotype and inhibits proinflammatory cytokine expression through receptor engagement and subsequent activation of the Janus kinase (Jak)/signal transducer and activator of transcription 6 (STAT6) axis [22]. In addition, deficiency of the insulin receptor substrate (IRS) impairs IL-4-induced M2 macrophage polarization, indicating a possible crosstalk between IL-4 and the IRS/PI3K/Akt axis [23]. An association has been described between the IL-4/IL-4R genotype and type 2 diabetes as well as between IL-4 genotypes and high-density lipoprotein cholesterol [24,25]. IL-4 boosts insulin action in hepatocytes in vitro, and promotes myogenesis in myoblasts, resulting in greater insulin efficacy [26,27]. For adipocytes, IL-4 inhibits adipogenesis, promotes lipolysis, and restores insulin sensitivity against lipid-provoked insulin resistance [28–30]. Disruption of STAT6 impairs insulin actions in mice, and IL-4 administered intraperitoneally improves glucose and lipid metabolism in streptozotocin and high-fat diet (HFD) mice [15,31]. The above findings suggest the potential of IL-4 in combating obesity and its complications. Leptin is a key hormone in regulating food intake and body weight homeostasis through the leptin/STAT3 signaling pathways [32]. Congenital leptin deficiency causes hyperphagia, early severe obesity, and metabolic disorder [33], and on the other hand, hyperleptinemia and leptin resistance can also be associated with common obesity [32]. In experimental studies, leptin-deficient ob/ob mice, leptin receptor-deficient db/db mice, and HFD-induced obese mice are common models used for the study of obesity [34]. Besides, rats harboring a deletion of the 14th amino acid Ile, and mice carrying a substitution of the 145th amino acid from Val to Glu in the leptin protein also show signs of obesity, hyperglycemia, hyperinsulinemia, and insulin resistance [35–37]. Evidence further indicates a promoting effect of leptin on IL-4 secretion [38]. In lean mice, the insulin-sensitive state of adipose tissue is proposed to preserve local IL-4 secretion by eosinophils and maintenance of an anti-inflammatory milieu. Whereas, adipose eosinophils and IL-4 expression were reduced in mice fed HFD or in mice with genetic obesity secondary to leptin deficiency, and there was an inverse relationship between adipose eosinophil numbers and mouse Int. J. Mol. Sci. 2020, 21, 4451 3 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 19 weightnumbers [39 and]. Particularly, mouse weight it has [39]. been Particularly, reported that it has adipose been reported tissue macrophages that adipose from tissue obese macrophages mice can exhibitfrom obese an increased mice can expression exhibit an of increased the IL-4 receptor, expression and of thus the sensitivity IL‐4 receptor to IL-4, and [40 ].thus Currently,it sensitivity remains to IL‐4 unclear[40]. Currently, whether it IL-4 remains has beneficial unclear whether effects against IL‐4 has leptin beneficial deficiency- effects and against leptin leptin resistance-provoked deficiency‐ and metabolicleptin resistance abnormality‐provoked and obesity. metabolic To extend abnormality the knowledge and obesity. of biological To extend implications the knowledge of IL-4 on of obesitybiological and implica its complications,tions of IL‐4 we on studied obesity theand metabolic its complications, effects of IL-4we studied in two micethe metabolic models of effects obesity: of LeptinIL‐4 in deficiencytwo mice models and HFD. of obesity: Leptin deficiency and HFD.

2.2. ResultsResults

2.1.2.1. ObeseObese MiceMice HadHad aa LowerLower ProductionProduction ofof IL-4IL-4 ToTo initiateinitiate aa studystudy surroundingsurrounding IL-4IL‐4 eeffectsﬀects onon metabolicmetabolic changes,changes, wewe thereforetherefore determineddetermined thethe endogenousendogenous levelslevels ofof IL-4IL‐4 inin bothboth leanlean andand obeseobese mice.mice. ObeseObese mice,mice, bothboth 145E145E (Figure(Figure1 1A)A) and and HFD HFD (Figure(Figure1 1B),B), had had lower lower circulating circulating levels levels of of IL-4 IL‐4 than than lean lean mice. mice. Independent Independent ofof beingbeing leanleanor orobese, obese, exogenousexogenous IL-4IL‐4 supplementationsupplementation increased the circulating level in thethe bloodstreambloodstream (Figure1 1A,B).A,B). Therefore,Therefore, obeseobese micemice hadhad aa lowerlower levellevel ofof endogenousendogenous IL-4 IL‐4 in in their their blood blood circulation. circulation.

Figure 1. Obese mice showed reduced IL-4 expressions. (A) Wild-type C57BL/6 (WT) and leptin-deficient Figure 1. Obese mice showed reduced IL‐4 expressions. (A) Wild‐type C57BL/6 (WT) and 145E mice were fed the normal diet (ND) for 8 weeks. Simultaneously, normal saline and IL-4 leptin‐deficient 145E mice were fed the normal diet (ND) for 8 weeks. Simultaneously, normal saline (1 µg/mouse) were intraperitoneally administrated twice a week. Blood samples were collected and and IL‐4 (1 μg/mouse) were intraperitoneally administrated twice a week. Blood samples were subjected to ELISA for measuring IL-4 levels. * p < 0.05 vs. WT/saline group and # p < 0.05 vs. collected and subjected to ELISA for measuring IL‐4 levels. * p < 0.05 vs. WT/saline group and # p < 145E/saline group, n = 6. (B) C57BL/6 mice were fed with the normal diet (ND) or high-fat diet (HFD) for 0.05 vs. 145E/saline group, n = 6. (B) C57BL/6 mice were fed with the normal diet (ND) or high‐fat 12 weeks. Simultaneously, normal saline and IL-4 (1 µg/mouse) were intraperitoneally administrated diet (HFD) for 12 weeks. Simultaneously, normal saline and IL‐4 (1 μg/mouse) were twice a week for the last 8 weeks. Blood samples were collected and subjected to ELISA for the intraperitoneally administrated twice a week for the last 8 weeks. Blood samples were collected and measurement of IL-4 levels. * p < 0.05 vs. ND/saline group and # p < 0.05 vs. HFD/saline group, n = 6. subjected to ELISA for the measurement of IL‐4 levels. * p < 0.05 vs. ND/saline group and # p < 0.05 2.2. IL-4vs. HFD/ Amelioratedsaline group, Metabolic n = 6. Alterations in Leptin-Deficient Mice Within 8 weeks of feeding, 145E mice showed increases in food intake (Figure2A), body weight 2.2. IL-4 Ameliorated Metabolic Alterations in Leptin-Deficient Mice (Figure2B), feeding e fficiency (Figure2C), liver mass (Figure2D), epididymal fat mass (Figure2E), fastingWithin glucose 8 (Figureweeks 2 ofF), feeding, and insulin 145E (Figure mice2 G), showed as well increases as impaired in foodglucose intake tolerance (Figure (Figure 2A),2 H,I). body IL-4weight had (Figure little eff ect2B), on feeding WT lean efficiency mice, while (Figure it ameliorated 2C), liver the mass aforementioned (Figure 2D), metabolic epididymal alterations fat mass in 145E(Figure obese 2E), mice fasting (Figure glucose2). That (Figure is, IL-4 2 displaysF), and improvement insulin (Figure e ff ects2G), against as well metabolic as impaired alterations glucose in leptin-deficienttolerance (Figure 145E 2 obeseH,I). mice.IL‐4 had little effect on WT lean mice, while it ameliorated the aforementioned metabolic alterations in 145E obese mice (Figure 2). That is, IL‐4 displays improvement effects against metabolic alterations in leptin‐deficient 145E obese mice.

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FigureFigure 2.2. IL-4IL‐4 amelioratedameliorated metabolic changes in the leptin-deficientleptin‐deficient mice. Wild type C57BLC57BL/6/6 (WT) andand leptin-deficientleptin‐deficient 145E micemice werewere fedfed thethe normalnormal dietdiet (ND)(ND) forfor 88 weeks.weeks. Simultaneously,Simultaneously, normalnormal salinesaline andand IL-4IL‐4 (1(1 µμg/mouse)g/mouse) werewere intraperitoneallyintraperitoneally administratedadministrated twicetwice aa week.week. TheThe averageaverage foodfood intakeintake ((AA),), body body weight weight gain gain (B (),B feeding), feeding effi efficacycacy (C ),(C liver), liver mass mass (D), ( andD), and epididymal epididymal fat mass fat mass (E) were (E) measured.were measured. Blood Blood samples samples were collected were collected from 8-h from fasting 8‐h fasting mice and mice fasting and fastingglucose glucose (F) and (insulinF) and (insulinG) levels (G measured.) levels measured. The 8-h The fasting 8‐h mice fasting were mice intraperitoneally were intraperitoneally injected with injected a glucose with a solution glucose (2solution g/kg). Blood (2 g/kg). samples Blood were samples collected were from collected the tail veins from at the the tail indicated veins times at the after indicated treatments times and after the levelstreatments of glucose and werethe levels measured of glucose (H). AUC were of the measured glucose–time (H). AUC curves of was the calculated glucose–time (I). * curvesp < 0.05 was vs. WTcalculated/saline group(I). * p and< 0.05 # p vs.< 0.05WT/ vs.saline 145E group/saline and group, # p < n0.05= 6. vs. 145E/saline group, n = 6. 2.3. IL-4 Reduced Hypothalamic Changes in Leptin-Deficient Mice 2.3. IL-4 Reduced Hypothalamic Changes in Leptin-Deficient Mice In the hypothalamus of 145E mice, we found increased levels in the mRNA of orexigenic AgRP In the hypothalamus of 145E mice, we found increased levels in the mRNA of orexigenic AgRP (Figure3A) and NPY (Figure3B), and decreased levels in the mRNA of anorexigenic POMC (Figure3C). (Figure 3A) and NPY (Figure 3B), and decreased levels in the mRNA of anorexigenic POMC (Figure These observed changes were reduced by IL-4. Parallel increases in 145E mice and decreases by IL-4 were 3C). These observed changes were reduced by IL‐4. Parallel increases in 145E mice and decreases by found in the circulating levels of leptin (Figure3D) and ghrelin (Figure3E) as well as mRNA levels of IL‐4 were found in the circulating levels of leptin (Figure 3D) and ghrelin (Figure 3E) as well as tumor necrosis factor-α (TNF-α) (Figure3F) and interleukin-1 β (IL-1β) (Figure3G) in hypothalamic mRNA levels of tumor necrosis factor‐α (TNF‐α) (Figure 3F) and interleukin‐1β (IL‐1β) (Figure 3G) tissues. In addition, levels of protein phosphorylation in Akt, STAT3, and STAT6 decreased in the in hypothalamic tissues. In addition, levels of protein phosphorylation in Akt, STAT3, and STAT6 hypothalamic tissues of 145E mice but were reversed by IL-4 (Figure3H). Findings indicated that decreased in the hypothalamic tissues of 145E mice but were reversed by IL‐4 (Figure 3H). Findings the promotion of orexigenic and reduction of anorexigenic signaling in the hypothalamus of 145E indicated that the promotion of orexigenic and reduction of anorexigenic signaling in the mice were accompanied by increased mRNA expression of TNF-α/IL-1β cytokines and impaired hypothalamus of 145E mice were accompanied by increased mRNA expression of TNF‐α/IL‐1β phosphorylation of Akt, STAT3, and STAT6. These changes were ameliorated by IL-4. cytokines and impaired phosphorylation of Akt, STAT3, and STAT6. These changes were ameliorated by IL‐4.

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FigureFigure 3. IL-43. IL‐ ameliorated4 ameliorated hypothalamic hypothalamic changes changes in in leptin-deﬁcient leptin‐deficient mice. mice. Wild-type Wild‐type C57BL C57BL/6/6 (WT) (WT) and leptin-deﬁcientand leptin‐deficient 145E mice 145E were mice fed were the normalfed the dietnormal (ND) diet for (ND) 8 weeks. for 8 Simultaneously, weeks. Simultaneously, normal salinenormal and IL-4saline (1 µ gand/mouse) IL‐4 (1 were μg/mouse) intraperitoneally were intraperitoneally administrated administrated twice a week. twice Total a RNAsweek. wereTotal extractedRNAs were from theextracted hypothalamic from th tissuese hypothalamic and subjected tissues to qRT-PCR and subjected for the to measurement qRT‐PCR for ofthe AgRP measurement (A), NPY (ofB ),AgRP POMC (C),(A TNF-), NPYα ( (FB),), and POMC IL-1 (βC),(G TNF) mRNA‐α (F), expression. and IL‐1β (G Blood) mRNA samples expression. were collected Blood samples and subjected were collected to ELISA forand the measurement subjected to ELISA of leptin for ( D the) and measurement ghrelin (E) levels. of leptin Proteins (D) and were ghrelin extracted (E) levels. from the Prot hypothalamiceins were tissuesextracted and subjectedfrom the hypothalamic to Western blot tissues analysis and with subjected the indicated to Western antibodies. blot analysis Representative with the indicated blots and quantitativeantibodies. data Representative are shown blots (H). *andp < quantitative0.05 vs. WT data/saline are group shown and (H).# *p p< <0.05 0.05 vs.vs. WT/ 145Es/alinesaline group group, n =and6. # p < 0.05 vs. 145E/saline group, n = 6.

2.4.2.4. IL-4 IL- Reduced4 Reduced Hepatic Hepatic Changes Changes in in Leptin-DeficientLeptin-Deficient Mice ChangesChanges of of hepatic hepatic metabolism metabolism in in obesityobesity areare signs of metabolic metabolic abnormalities abnormalities [36]. [36 ].Serum Serum GPT GPT (Figure(Figure4A), 4A), GOT GOT (Figure (Figure4B), 4B), total total cholesterol cholesterol (Figure (Figure4 C),4C), and and triglyceride triglyceride (Figure(Figure4 4D)D) levelslevels were foundfound to beto higher be higher in 145E in 145E mice mice than than in WT in mice. WT mice. Hepatic Hepatic tissues tissues of 145E of mice 145E displayed mice displayed an elevated an accumulationelevated accumulation of triglycerides of triglycerides (Figure4E), mRNA (Figure expressions 4E), mRNA of expressions phosphoenolpyruvate of phosphoenolpyruvate carboxykinase (PEPCK)carboxykinase (Figure 4 (PEPCK)F) and TNF- (Figureα (Figure 4F) and4G), TNF as well‐α (Figure as reduced 4G), protein as well phosphorylation as reduced protein of Akt, STAT3,phosphorylation and STAT6 (Figure of Akt,4 H). STAT3, IL-4 amelioratedand STAT6 (Figureall these 4H abnormal). IL‐4 ameliorated changes (Figure all these4). The abnormal findings suggestedchanges a (Figure reversal 4). e ffTheect of findings IL-4 on suggested hepatic metabolic a reversal abnormalities effect of IL and‐4 on decreased hepatic Akt, metabolic STAT3, andabnormalities STAT6 signaling and decreased in 145E obese Akt, STAT3, mice. and STAT6 signaling in 145E obese mice.

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FigureFigure 4. 4.IL-4 IL‐ ameliorated4 ameliorated hepatic hepatic changes changes in in leptin-deﬁcient leptin‐deficient mice. Wild Wild-type‐type C57BL/6 C57BL/ 6 (WT) (WT) and and leptin-deﬁcientleptin‐deficient 145E 145E mice mice were were fed fed the the normal normal dietdiet (ND)(ND) for 8 weeks. weeks. Simultaneously, Simultaneously, normal normal saline saline andand IL-4 IL (1‐4µ g (1/ mouse) μg/mouse) were were intraperitoneally intraperitoneally administrated administrated twice twice a week. a week.Blood samplesBlood samples were collected were andcollected GPT (A and), GOT GPT (B (),A total), GOT cholesterol (B), total (C cholesterol), and triglycerides (C), and triglycerides (D) measured. (D) Hepatic measured. tissues Hepatic were collectedtissues andwere subjectedcollected and to measurementsubjected to measurement of triglycerides of triglycerides (E). Total RNAs(E). Total were RNAs extracted were extracted from the hepaticfrom tissuesthe hepa andtic tissues subjected and to subjected qRT-PCR to for qRT the‐PCR determination for the determination of PEPCK of (F )PEPCK and TNF- (F) αand(G TNF) mRNA‐α expressions.(G) mRNA Proteins expressions. were Proteins extracted were from extracted the hepatic from tissuesthe hepatic and subjectedtissues and to subjected Western blotto Western analysis withblot indicated analysis antibodies.with indicated Representative antibodies. Representative blots and quantitative blots and quantitative data are shown data are (H ).shown * p < (0.05H). * vs. WTp/ saline< 0.05 vs. group WT/ andsaline # p group< 0.05 and vs. # 145E p < 0.05/saline vs. 145E/ group,salinen = 6.group, n = 6.

2.5.2.5. IL-4 IL- Reduced4 Reduced Adipocytic Adipocytic Changes Changes in in Leptin-Deﬁcient Leptin-Deficient Mice AdiposeAdipose tissues tissues are are sites sites for for lipid lipid deposition deposition and and metabolism metabolism [29,30]. [29,30]. 145E 145E obese obese mice mice were were foundfound to have to have higher higher circulating circulating levels levels of freeof free fatty fatty acids acids and and these these increments increments were were reduced reduced by by IL-4 (FigureIL‐4 5 (FigureA). In the5A). epididymal In the epididymal fats of 145E fats mice, of 145E mRNA mice, levels mRNA were levels elevated were with elevated TNF- withα (Figure TNF‐5αB), IL-1(Figureβ (Figure 5B),5 C),IL‐1β and (Figure IL-6 (Figure 5C), and5D); IL the‐6 (Figure protein 5D) level; the was protein increased level was with increased cluster of with di ﬀ clustererentiation of 68differentiation (CD68) (Figure 685 E),(CD68) while (Figure protein 5E), phosphorylation while protein phosphorylation was lowered was with lowered Akt, STAT3, with Akt, and STAT3, STAT6 (Figureand 5 STAT6E). All these (Figure abnormal 5E). All changes these abnormal in mRNA, changes protein level, in mRNA and, protein protein phosphorylation level, and protein were phosphorylation were reduced by IL‐4 (Figure 5B–F). Intriguingly, the protein level of CD206 was reduced by IL-4 (Figure5B–F). Intriguingly, the protein level of CD206 was increased in the presence increased in the presence of IL‐4 (Figure 5E). The results are consistent with the higher secretion of of IL-4 (Figure5E). The results are consistent with the higher secretion of free fatty acid and higher free fatty acid and higher cytokine mRNA expression, along with impaired Akt, STAT3, and STAT6 cytokine mRNA expression, along with impaired Akt, STAT3, and STAT6 signaling in epididymal fats signaling in epididymal fats of 145E mice. These changes were reduced by IL‐4. of 145E mice. These changes were reduced by IL-4.

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FigureFigure 5. IL-45. IL‐ ameliorated4 ameliorated adipocytic adipocytic changes changes inin leptin-deﬁcientleptin‐deficient mice. Wild Wild-type‐type C57BL/6 C57BL/ 6(WT) (WT) and and leptin-deﬁcientleptin‐deficient 145E 145E mice mice were were fed fed the the normal normal diet diet (ND) (ND) for for 8 weeks. 8 weeks. Simultaneously, Simultaneously, normal normal saline saline and IL-4and (1 µ ILg/‐mouse)4 (1 μg/mouse) were intraperitoneally were intraperitoneally administrated administrated twice a week. twice Blood a week. samples Blood were samples collected were and freecollected fatty acids and (A free) measured. fatty acids Total (A) RNAs measure wered. Total extracted RNAs from were the extracted epididymal from fat the tissues epididymal and mRNA fat expressionstissues and measured mRNA expressions with qRT-PCR measured for TNF- withα ( B qRT), IL-1‐PCRβ ( C for), andTNF IL-6‐α (B (),D ). IL Proteins‐1β (C), and were IL extracted‐6 (D). fromProteins the epididymal were extracted fat tissuesfrom the and epididymal subjected fat to tissues Western and blotsubjected analysis to Western with indicated blot analysis antibodies. with Representativeindicated an blotstibodies and. quantitativeRepresentative data blots are shown and quantitative (E,F). * p < 0.05 data vs. are WT shown/saline (E group,F). * andp < # 0.05p < vs.0.05 vs.WT/ 145Esaline/saline group group, andn #= p6. < 0.05 vs. 145E/saline group, n = 6.

2.6.2.6. IL-4 IL Reduced-4 Reduced Metabolic Metabolic Changes Changes in in the the HFD HFD MiceMice AlthoughAlthough 145E 145E mice mice release release leptin leptin into the into blood the blood circulation, circulation, the mutant the mutant leptin loses leptin its loses biological its activitybiological [36]. activity To explore [36]. whether To explore IL-4 whether0s effects IL are‐4′s common effects are to common obese populations, to obese populations, HFD obese HFD mice wereobese analyzed mice were for analyzed comparison. for comparison. In these mice, In these IL-4 mice, showed IL‐4 showed similar similar effects effects regarding regarding food food intake (Figureintake6A), (Figur bodye 6A), weight body gain weight (Figure gain 6(FigureB), feeding 6B), feeding e fficiency efficiency (Figure (Figure6C), liver6C), liver mass mass (Figure (Figure6D), epididymal6D), epididymal fat mass fat (Figure mass 6(FigureE), fasting 6E), glucose fasting (Figureglucose6 (FigureF), insulin 6F), (Figure insulin6 G),(Figure and glucose6G), and tolerance glucose (Figuretolerance6H and (Figure 6I). In6H the and case 6I). of In HFD the case obese of mice,HFD obese when mice, compared when with compared the ND with lean the mice, ND lean they mice, they showed elevated mRNA expressions of AgRP (Figure 7A) and NPY (Figure 7B) in the showed elevated mRNA expressions of AgRP (Figure7A) and NPY (Figure7B) in the hypothalamus hypothalamus while mRNA expression of POMC remained unchanged. In these HFD mice, we while mRNA expression of POMC remained unchanged. In these HFD mice, we only found an only found an increased level of circulating leptin (Figure 7D) but not ghrelin (Figure 7E). increased level of circulating leptin (Figure7D) but not ghrelin (Figure7E). Additionally, in their Additionally, in their hypothalamic tissues, we found a moderate elevation in the expression of hypothalamic tissues, we found a moderate elevation in the expression of TNF-α mRNA (Figure7F) TNF‐α mRNA (Figure 7F) but unchanged expression of IL‐1β mRNA (Figure 7G). Compared with β butND unchanged mice, these expression HFD mice of IL-1 had mRNA decreased (Figure protein7G). phosphorylation Compared with NDof Akt, mice, STAT3, these HFD and mice STAT6 had decreased(Figure protein7H) in the phosphorylation hypothalamic of tissues. Akt, STAT3, Intriguingly, and STAT6 IL‐4 displayed (Figure7H) ameliorative in the hypothalamic effects only tissues. on Intriguingly,leptin (Figure IL-4 7 displayedD) and protein ameliorative phosphorylation effects only (Figure on leptin 7H). (Figure Regarding7D) and the proteinchanges phosphorylation in the hepatic (Figure(Figure7H). 8) Regarding and adipocytic the changes (Figure in9) thetissues, hepatic IL‐4 (Figure and HFD8) and mice adipocytic displayed (Figure the same9) tissues, effects IL-4as those and HFD mice displayed the same effects as those in IL-4 and leptin-deficient mice. The findings are

Int. J. Mol. Sci. 2020, 21, 4451 8 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 19 inconsistent IL‐4 and with leptin the‐deficient HFD and mice. 145E The mice findings sharing are most consistent metabolic with alterations, the HFD except and 145E some mice parts sharing of the mosthypothalamic metabolic signaling alterations pathways., except some parts of the hypothalamic signaling pathways.

Figure 6. 6. IL-4IL‐4 ameliorated ameliorated metabolic metabolic changes changes in leptin-resistant in leptin‐resistant mice. mice. C57BL C57BL/6/6 mice were mice fed were the fed normal the ndietormal (ND) diet or (ND) high-fat or diethigh (HFD)‐fat diet for (HFD) 12 weeks. for 12 Simultaneously, weeks. Simultaneously, normal saline normal and saline IL-4 (1 andµg/ mouse) IL‐4 (1 μg/mouse)were intraperitoneally were intraperitoneally administrated administrated twice a week twice for a week the last for 8 the weeks. last 8 Theweeks. average The average food intake food intake(A), body (A), weightbody weight gain ( Bgain), feeding (B), feeding eﬃcacy efficacy (C), liver (C), massliver (massD), and (D), epididymal and epididymal fat mass fat mass (E) were (E) weremeasured. measured. Blood Blood samples samples were collectedwere collected from 8-h from fasting 8‐h fasting mice and mice fasting and fasting glucose glucose (F) and ( insulinF) and insulin(G) levels (G) measured. levels measured. The 8-h The fasting 8‐h mice fasting were mice intraperitoneally were intraperitoneally injected withinjected a glucose with a solution glucose solution(2 g/kg). Blood(2 g/kg). samples Blood were samples collected were from collected the tail from veins the at the tail indicated veins at times the indicated after treatments times after and treatmentslevels of glucose and levels measured of glucose (H). measured AUC of the (H). glucose–time AUC of the glucose curves– wastime calculated curves was (I calculated). * p < 0.05 (I). vs. * pND < 0.05/saline vs. groupND/saline and #groupp < 0.05 and vs. # p HFD < 0.05/saline vs. HFD/ group,salinen = group,6. n = 6.

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FigureFigure 7. 7.IL-4 IL‐ ameliorated4 ameliorated hypothalamic hypothalamic changes changes in in leptin-resistant leptin‐resistant mice. mice. C57BL/6 C57BL/ 6 mice mice were were fed fed thethe normal normal diet diet (ND) (ND) or or high-fat high‐fat dietdiet (HFD)(HFD) for for 12 12 weeks. weeks. Simultaneously, Simultaneously, normal normal saline saline and andIL‐4 IL-4(1 (1 µμg/mouse)g/mouse) werewere intraperitoneallyintraperitoneally administrated administrated twice twice a aweek week for for the the last last 8 8weeks. weeks. Total Total RNA RNA were were extractedextracted from from the the hypothalamic hypothalamic tissues tissues and and mRNAmRNA expressions measured measured with with qRT qRT-PCR‐PCR for for AgRP AgRP (A),(A NPY), NPY (B ),(B POMC), POMC (C (),C), TNF- TNFα‐α(F (),F), and and IL-1 IL‐1ββ ((GG).). BloodBlood samples were were collected collected and and leptin leptin (D (D) and) and ghrelinghrelin (E )(E levels) levels measured measured with with ELISA. ELISA. Proteins Proteins werewere extractedextracted from from the the hypothalamic hypothalamic tissues tissues and and subjectedsubjected to to Western Western blot blot analysis analysis with with indicated indicated antibodies.antibodies. Represen Representativetative blots blots and and quantitative quantitative datadata are are shown shown (H ().H *).p *

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FigureFigure 8. IL-4 8. IL ameliorated‐4 ameliorated hepatic hepatic changes changes in leptin-resistant in leptin‐resistant mice. mice. C57BL C57BL/6/6 mice mice were were fed the fed normal the dietnormal (ND) ordiet high-fat (ND) or diet high (HFD)‐fat d foriet (HFD) 12 weeks. for 12 Simultaneously, weeks. Simultaneously, normal saline normal and saline IL-4 and(1 µ g IL/mouse)‐4 (1 wereμg/mouse) intraperitoneally were intraperitoneally administrated administrated twice a week twice for the a lastweek 8 weeks.for the last Blood 8 weeks. samples Blood were samples collected andwere GPT collected (A), GOT and (B GPT), total (A), cholesterol GOT (B), total(C), and cholesterol triglyceride (C), and (D) triglyceride levels measured. (D) levels Hepatic measured. tissues wereHepatic collected tissues and were subjected collected to and measurement subjected to of measurement triglycerides of (E triglycerides). Total RNAs (E). were Total extracted RNAs were from theextracted hepatic tissuesfrom the and hepatic mRNA tissues expression and mRNA measured expression with measured qRT-PCR with for PEPCK qRT‐PCR (F) for and PEPCK TNF-α (F()G ). Proteinsand TNF were‐α extracted (G). Proteins from were the hepatic extracted tissues from and the subjected hepatic tissues to Western and subjected blot analysis to Western with indicated blot analysis with indicated antibodies. Representative blots and quantitative data are shown (H). * p < antibodies. Representative blots and quantitative data are shown (H). * p < 0.05 vs. ND/saline group 0.05 vs. ND/saline group and # p < 0.05 vs. HFD/saline group, n = 6. and # p < 0.05 vs. HFD/saline group, n = 6.

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FigureFigure 9. 9.IL-4 IL‐4 ameliorated ameliorated adipocytic changes changes in in leptin leptin-resistant‐resistant mice. mice. C57BL/6 C57BL mice/6 micewere werefed the fed then normalormal d dietiet (ND) (ND) or or high high-fat‐fat diet diet (HFD) (HFD) for for 12 12weeks. weeks. Simultaneously, Simultaneously, normal normal saline saline and and IL‐4 IL-4 (1 (1 µμg/mouse)g/mouse) werewere intraperitoneallyintraperitoneally administrated administrated twice twice a aweek week for for the the last last 8 8weeks. weeks. Blood Blood samples samples werewere collected collected and and subjected subjected to to the the measurement measurement ofof freefree fatty acids ( (AA).). Total Total RNA RNA were were extracted extracted fromfrom the the epididymal epididymal fat tissues fat tissues and and mRNA mRNA expression expression measured measured with with qRT-PCR qRT‐PCR for TNF- for TNFα (B‐α), IL-1(B), β (C),IL and‐1β IL-6 (C), and (D). IL Proteins‐6 (D). wereProteins extracted were extracted from the from epididymal the epididymal fat tissues fat and tissues subjected and subjec to Westernted to blotWestern analysis blot with analysis indicated with antibodies. indicated Representativeantibodies. Representative blots and quantitative blots and quantitative data are shown data (areE,F ). * p

2.7.2.7. IL-4 IL Promoted-4 Promoted White White Adipocyte Adipocyte Browning Browning WhiteWhite adipose adipose tissue tissue is is a a storage storage site site ofof surplussurplus energy,energy, and brown adipose adipose tissue tissue consumes consumes energyenergy to togenerate generate heat. heat. The The conversion conversion of of white white adipose to to brown brown adipose adipose tissues tissues is is inversely inversely correlatedcorrelated with with adiposity adiposity and and metabolic metabolic abnormality abnormality [ [11,16,17].11,16,17]. To To ex exploreplore the the effects effects of of IL IL-4‐4 on on metabolicmetabolic regulation, regulation, we determined we determined the expression the expression of molecules of molecules regulating regulating white adipocyte white adipocyte browning in epididymalbrowning in fats. epididymal IL-4 caused fats. elevated IL‐4 caused levels elevated of PR levels domain of containingPR domain 16containing (PRDM16), 16 (PRDM16) peroxisome, proliferator-activatedperoxisome proliferator receptor‐activated gamma receptor coactivator gamma 1coactivatorα (PGC-1 α1α), (PGC and uncoupling‐1α), and uncoupling protein 1 p (UCP-1)rotein proteins1 (UCP compared‐1) proteins with compared controls inwith both controls 145E (Figure in both 10 145EA) and (Figure HFD (Figure10A) and 10 B)HFD mice. (Figure In the 10inB) vitro mice.cell model,In the 3T3-L1 in vitro preadipocytes cell model, 3T3 diff‐erentiatedL1 preadipocytes into mature differentiated adipocytes into (Figure mature 10 adipocytesC). During (Figure differentiation, 10C). IL-4During increased differentiation, mRNA expression IL‐4 increased of PRDM16 mRNA (Figure expression 10D), PGC-1 of PRDM16α (Figure (Figure 10E), 10 andD), UCP-1 PGC‐1α (Figure (Figure 10F). 10E), and UCP‐1 (Figure 10F). The findings are consistent with the promoting effect of IL‐4 on white The findings are consistent with the promoting effect of IL-4 on white adipocyte browning. adipocyte browning.

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FigureFigure 10. IL-4 10. promotedIL‐4 promoted white adipocyte white adipoc browning.yte browning. (A) Wild-type (A) WildC57BL‐type/6 (WT) C57BL/6 and leptin-deﬁcient (WT) and 145Eleptin mice‐deficient were fed145E the mice normal were fed diet the (ND) normal for d 8iet weeks. (ND) for Simultaneously, 8 weeks. Simultaneously, normal salinenormal and saline IL-4 (1 µandg/mouse) IL‐4 (1 wereμg/mouse) intraperitoneally were intraperitoneally administrated administrated twice a week. twice Proteinsa week. Proteins were extracted were extracted from the epididymalfrom the epididymal fat tissues andfat tissues subjected and tosubjected Western to blot Western analysis blot with analysis indicated with indicated antibodies. antibodies. * p < 0.05 * p vs. WT

3. Discussion3. Discussion LeptinLeptin is is a a pivotal pivotal hormone hormone regulating regulating food food intake and and body body weight weight by by coordinating coordinating communicationscommunications between between the the hypothalamus hypothalamus and and the the periphery. periphery. Leptin Leptin deficiency deﬁciency is a is result a result of a of a geneticgenetic mutationmutation predisposingpredisposing subjects to to severe severe obesity, obesity, while while leptin leptin resistance resistance is islinked linked to todietary dietary obesityobesity [32 [32–38–,38,41].41]. In In this this study, study, we we found found that that obese obese 145E 145E and and HFD HFD mice mice had had lower lower circulating circulating levels of IL-4levels than of IL wild-type‐4 than wild and‐type lean and mice. lean A course mice. A of course 8-week of IL-4 8‐week supplementation IL‐4 supplementation improves improves common phenotypescommon phenotypes of obesity and of impairedobesity and signaling impaired pathways signaling of pathways Akt, STAT3, of Akt, and STAT3, STAT6 inand the STAT6 hypothalamic, in the hypothalamic, hepatic, and epididymal fat tissues. Despite the inconsistent results across studies, hepatic, and epididymal fat tissues. Despite the inconsistent results across studies, amelioration of amelioration of cytokine expression and promotion of white adipocyte browning have been shown cytokine expression and promotion of white adipocyte browning have been shown to have a close link to have a close link with IL‐4. Since obesity is associated with lower circulating levels of IL‐4, our with IL-4. Since obesity is associated with lower circulating levels of IL-4, our current ﬁndings further current findings further highlighted IL‐4 being an alternative candidate in combating obesity and highlighted IL-4 being an alternative candidate in combating obesity and its complications. its complications. ConsistentConsistent with with a previousa previous report report [ 36[36]],, both bothNPY NPY andand AgRP mRNA mRNA levels levels in in the the hypothalamus hypothalamus werewere significantly significantly increased increased in in 145E 145E mice, mice, while while POMC POMC mRNA mRNA tended tended to to be be lower lower in in145E 145E micemice thanthan in wild-typein wild mice.‐type miceBesides,. Besides, the changes the changes in hypothalamic in hypothalamic orexigenic orexigeni/anorexigenicc/anorexigenic peptides peptides and cytokines and

Int. J. Mol. Sci. 2020, 21, 4451 13 of 19 along with circulating ghrelin in the leptin-deficient 145E mice were more marked than in HFD mice. It has been reported that exposure to HFD-induced obesity can cause dysregulation of the expression of NPY and AgRP in the hypothalamus [42,43]. However, the hypothalamic anorexigenic POMC can also be induced as an adaptive mechanism against obesity [44,45]. Therefore, in the HFD obese animals, although later eating less, they still consumed more calories, and gained more weight and adipose tissue mass. Leptin is primarily secreted by white adipose tissues and its circulating level is proportional to body fat mass. The interactions of leptin with its longest receptor isoform trigger STAT3 signaling in the hypothalamus, liver, and adipose tissues to coordinate food intake, energy expenditure, and lipid or glucose metabolism. Its actions are compromised by defective mutation, receptor mutation, resistance, and inflammation [9,14,35,36,41]. We previously identified and created a model with leptin mutation, the 145E mice, by replacing Val by Glu in the codon 145. These mice showed metabolic abnormalities and developed obese phenotypes. Intraperitoneal injection with wild-type leptin reduced food intake [36]. Here, we further demonstrated in 145E mice that hyperphagia, hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, hepatic steatosis, and adiposity were all accompanied by elevated cytokine expressions and lowered phosphorylation of Akt, STAT3, and STAT6 in the hypothalamus, liver, and epididymal fat. Such changes in the tissues towards proinflammation and impairment of Akt, STAT3, and STAT6 signaling were all duplicated in dietary obesity. As leptin can mediate the immune response [46], it was speculated that proinflammatory stress in various tissues of 145E mice might be related to their leptin deficiency status. Consistent with an earlier report [47], in HFD obese mice, we found a lesser trend of increased expression of cytokine mRNA in the hypothalamus than in peripheral organs, although several studies in HFD rodent models have indicated not only peripheral inflammation but also increased inflammatory signaling in the hypothalamus, which was linked to the development of central leptin resistance and obesity [48]. Taken altogether, in the current study, it was shown that leptin deficiency and leptin resistance shared some but not all metabolic changes in the development of obesity. IL-4 is a Th2 cytokine that can promote M2 macrophage polarization in adipose tissues [12,23], and it has been proposed that IL-4 signaling could be a potential target to sustain insulin sensitivity in obesity [40]. A large amount of literature documents the effect of IL-4 signaling to block immune-mediated inflammation in both peripheral tissues and the brain [49,50]. In leptin-deficient obese mice, the expression of hypothalamic orexigenic/anorexigenic peptides and inflammatory cytokines as well as signaling pathways of Akt, STAT3, and STAT6 in the hypothalamus, body weight, and glucose dysregulation after IL-4 administration was restored towards those in wild-type mice along with circulating ghrelin. On the contrary, there seemed to be less effects of IL-4 on hypothalamic orexigenic/anorexigenic peptides and inflammatory cytokines expression, except preserved higher phosphorylation levels of Akt, STAT3, and STAT6 in the hypothalamus in HFD obese mice, although their weight was also lowered. In fact, a previous study reported that central administration of IL-4 exacerbated hypothalamic inflammation and weight gain during high-fat feeding [47]. These findings indicated the central sensitivity of IL-4 in leptin-deficient and -resistant obese animals might be different. The above issues warrant further investigation. It was speculated that body weight loss as well as restoration of metabolic phenotypes in leptin-deficient and HFD obese mice after IL-4 administration might come from other effects on peripheral organs (e.g., liver and adipose tissues, and pancreatic islets). Disruption of STAT6, downstream of IL-4, inhibits insulin action in mice, and IL-4 enhances insulin efficacy on adipocytes, hepatocytes, myoblasts, and diabetic mice [15,26–31]. Akt, a crucial member of insulin action, is also a candidate target of IL-4 signaling [22,23]. Exogenous IL-4 preserved higher phosphorylation levels of Akt, STAT3, and STAT6 in the liver and epididymal fat of both 145E and HFD obese mice. Since typical leptin/STAT3 and IL-4/STAT6 are closely linked to insulin/Akt in metabolic regulation, our present findings are consistent with IL-4 being able to affect the STAT3 and STAT6 cascades and Akt under certain situations while it is ineffective in WT and lean mice. In addition, IL-4 protects pancreatic beta cells from injury and the Th2 cytokine IL-13 from interfering in gluconeogenic enzyme expression via STAT3 [51,52]. In 145E and HFD mice, elevation in the mRNA levels of the hepatic gluconeogenic Int. J. Mol. Sci. 2020, 21, 4451 14 of 19 enzyme PEPCK was reduced by IL-4. On top of the improved insulin/Akt action, the direct effect of IL-4/STAT3 on the gluconeogenic enzyme might also lower the glucose output. White adipose tissues are central to metabolic regulation through the release of adipokines. It is known that obesity is associated with chronic low-grade inflammation of the adipose tissue and increased M1 macrophages. However, a recent paper reported that locally proliferating macrophages in obese adipose were not classically activated (M1, CD68 positive), but rather they exhibited an alternatively activated (M2, CD206 positive) immune phenotype with increased gene expression of the IL-4 receptor [40]. Obese adipose tissues showed an increased CD68 content, while it had minor effect on the CD206 content. IL-4 not only decreased the increment of CD68 but also elevated CD206, indicating the suppression of M1 macrophages and promotion of M2 macrophages. Based on this, adipose sensitivity to IL-4 stimulation can be enhanced by increased phosphorylation of STAT6, lipolysis in fat cells, and insulin sensitivity [31]. Currently, white adipocyte browning is an emerging strategy to improve metabolic abnormalities. PDM16 is a zinc-finger nuclear protein that controls transcriptional programs at the differentiating brown adipocytes and mitochondrial biogenesis involving PGC-1α and UCP-1. The processes of adipocyte browning are accompanied by STAT6 hyperphosphorylation [16]. The leptin/STAT3 signaling pathways coordinate and promote the browning of white adipocytes [11,17]. In the epididymal white adipose tissues and 3T3-L1 cells, IL-4 elevated the mRNA expression and protein levels of PRDM16, PGC-1α, and UCP-1. As IL-4 increased STAT3 and STAT6 phosphorylation, adipocyte browning was consequently achieved. In WT and lean mice, generally, there seemed little effects of IL-4 on weight, intake, glucose tolerance, leptin, ghrelin, and inflammatory cytokines as shown in obese mice. It was possible that chronic IL-4 administration in lean animals may elicit compensatory responses in the body with a final neutral metabolic influence. Besides, even in control animals, many factors, such as fasting and feeding duration, stress, and environment, can all transiently affect the expression of circulating leptin, ghrelin, glucose levels, and inflammatory cytokines, and thus a more careful standardization is necessary to avoid such variations [53]. Nevertheless, the regulatory effects of IL-4 on metabolism in animals with a non-obese status need further investigation. In clinical practice, chronic exercise, weight management, and probiotics are recommended to overweight patients. IL-4 signaling is one parameter associated with clinical improvement [18–21]. Through this study, we provided experimental evidence showing that IL-4 induced improvements of metabolic abnormalities in a mice model of obesity with leptin deficiency and HFD. The models of 145E and HFD obese mice showed lower levels of circulating IL-4. The metabolic abnormalities in 145E and HFD obese mice were accompanied by inflammation and reduced Akt, STAT3, and STAT6 signaling in the hypothalamus, liver, and epididymal fat. IL-4-mediated improvements were found in parallel with the amelioration of inflammation and the promotion of Akt, STAT3, and STAT6 phosphorylation. Additionally, the browning of white adipocyte by IL-4 was found in epididymal white adipose tissues and 3T3-L1 preadipocytes. Despite some limitations of our experiments and some inconsistency with the literature, our results suggested that the Th2 cytokine IL-4 is a metabolic regulator and antiobesity candidate for the treatment of obesity and its complications. It should be noted that IL-4 can exert systemic effects around inflammation. Therefore, before IL-4 can be translated into clinical applications, a deeper investigative insight into its metabolic actions is still required.

4. Materials and Methods

4.1. Animals and Treatments Adult male leptin-deﬁcient Leptin145E/145E (145E) mice [36] and species-matched wild-type (WT) C57BL/6 mice were housed in a controlled animal facility and handled according to procedures approved by the Animal Experimental Committee of Taichung Veterans General Hospital (IACUC No. La-1081629, 17 April, 2019). Mice of WT (n = 24) and 145E (n = 24) strains, aged 8 weeks old, were fed with a normal diet (ND, LabDiet 5001, 13% energy provided by fat) for 8 weeks. Simultaneously, normal Int. J. Mol. Sci. 2020, 21, 4451 15 of 19 saline and IL-4 (1 µg/mouse) were intraperitoneally administrated twice a week (see previously reported protocols for details [31]). For diet-induced obesity, C57BL/6 mice (8-week-old) were fed with ND (n = 24) or HFD (n = 24) (TestDiet, 58Y1, 61% energy provided by fat) for 12 weeks. Normal saline and IL-4 (1 µg/mouse) were intraperitoneally administrated twice a week during the last 8 weeks.

4.2. Cell Culture and Diﬀerentiation

The 3T3-L1 preadipocytes were maintained in Dulbecco0s modified eagle medium (DMEM) containing 10% calf serum. For adipogenesis, postconfluent cells (2 days old) were maintained in DMEM by adding 0.5 mM 3-isobutyl-1-methyxantine (IBMX) IBMX, 1 µM dexamethasone, 10 µg/mL insulin, and 10% fetal bovine serum (FBS) for 2 days. Thereafter, the cultured media was switched to DMEM containing 5 µg/mL insulin and 10% FBS and changed every 2 days. Cells were analyzed 8 days later. IL-4 (10 ng/mL) and vehicle were added to the cells during the 2-day induction period. Treatments and Oil Red O staining were performed in accordance with previously reported methods with slight modifications [30].

4.3. Glucose Tolerance Test For the intraperitoneal glucose tolerance test (IPGTT) (n = 6/group), mice were fasted for 8 h and intraperitoneally given glucose solution (2 g/kg body weight). Blood samples were collected from the tail veins at speciﬁed time points, and glucose levels were determined using a hand-held Accucheck glucometer (Roche Diagnostics, Indianapolis, IN, USA). The total area under the curve (AUC) for IPGTT was calculated using the trapezoidal (trapezium) rule.

4.4. Blood Sample Analyses Mice (n = 6/group) were anesthetized with isoflurane (2–4%), blood samples were withdrawn from the left femoral artery, and the serum was kept at 80 C until analyses. Serum levels of insulin (Shibayagi, − ◦ Gunma, Japan), leptin, ghrelin, and medium level of IL-4 (R&D Systems, Minneapolis, MN, USA) were measured using the Enzyme-Linked Immunosorbent Assay (ELISA) kit, according to the manufacturer0s instructions. Serum levels of total cholesterol (Cholesterol E, Wako, Osaka, Japan), free fatty acids (NEFA C, Wako, Osaka, Japan), triglycerides (Stanbio Laboratory, San Antonin, TX, USA), and GPT/GOT were determined using the Alanine Aminotransferase Activity Assay Kit (ALT/GPT)/Aspartate Aminotransferase Activity Assay Kit (AST/GOT), MyBioSource, San Diego, CA, USA.

4.5. RNA Isolation and Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) Hypothalami, epididymal fats, liver (n = 6/group), and 3T3-L1 cells were isolated and stored in RNAlater solution (Ambion, Austin, TX, USA). RNAs were extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Levels of mRNA were analyzed with SYBR green-based qRT-PCR (Applied Biosystems, Foster City, CA, USA), and the internal control was β-actin. Primers used for ampliﬁcations were as follows: AgRP 50-CGGAGGTGCTAGATCCACAGAA-30 and 50-AGGACTCGTGCAGCCTTACAC-30; NPY 50-AGAGATCCAGCCCTGAGACA-30 and 50-TTTCATTTCCCATCACCACA-30; POMC 50-GTTACGGTGGCTTCATGACCTC-30 and 50-CGCGTTCTTGATGATGGCGTTC-30; TNF-α 50-TCTTCTCATTCCTGCTTGTGG-30 and 50-GGTCTGGGCCATAGAACTGA-30; IL-1β 50-TTCATCTTTGAAGAAGAGCCCAT-30 and 50-TCGGAGCCTGTAGTGCAGTT-30; PEPCK 50-GAACACACCCTCGGTCAACA-30 and 50-CAAGGTCATCCAGGGCAGCCTC-30; IL-6 50-TGATGGATGCTACCAAACTGG-30 and 50-TTCATGTACTCCAGGTAGCTATGG-30; PRDM16 50-GATGGGAGATGCTGACGGAT-30 and 50-TGATCTGACACATGGCGAGG-30, PGC-1α 50-TCCTCTGACCCCAGACTCAC-30 and 50-TAGAGTCTTGGAGCTCCT-30; UCP-1 50-GAAAGGGACCCCTAATC-30 and 50-GGGACGTCATCTGCCAGTA-30 and β-actin 50-CCTCTATGCCAACACAGTGCTGTCT-30 and 50-GCTCAGGAGGAGCAATGATCTTGA-30. Int. J. Mol. Sci. 2020, 21, 4451 16 of 19

4.6. Western Blot Analysis Hypothalami, epididymal fats, and liver tissues (n = 6/group) were isolated, and proteins extracted using the commercial Tissue Protein Extraction Reagents (T-PER, Pierce Biotechnology, Rockford, IL, USA). Equal amounts of proteins were resolved by SDS-PAGE, before being transferred to the polyvinylidine ﬂuoride membrane. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies, horseradish peroxidase-conjugated IgG, and enhanced chemiluminescence Western blotting reagents. The visualized signals were quantitated by a densitor meter. Primary antibodies used were: Akt, phospho-Akt, signal transducers and activators of transcription 3 (STAT3), phospho-STAT3, STAT6, phospho-STAT6, PRDM16, PGC-1α, UCP-1, CD68, CD206, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

4.7. Statistical Analyses Statistical results were expressed as mean standard deviation. The one-way analysis of variance ± (ANOVA) was performed to compare inter-group differences. Then, Dunnett0s test or Tukey post-hoc test was performed for the purpose of comparison. Statistically significant differences were set at p < 0.05.

Author Contributions: S.-Y.L. and C.-J.C. conceived and designed the experiments; C.-P.Y., Y.-Y.W., C.-W.H., W.-Y.C. and S.-L.L. performed the experiments; Y.-L.L., Y.-H.C. and C.-J.H. analyzed the data; S.-Y.L. wrote the paper; C.-J.C. edited the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants from the Taichung Veterans General Hospital (TCVGH-1088202C) and Veterans General Hospitals and University System of Taiwan Joint Research Program (VGHUST104-G7-8-2 and VGHUST105-G7-8-2), Taiwan. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

AgRP Agouti-Related Protein AUC Area Under Curve CNS Central Nervous System DMEM Dulbecco0s Modiﬁed Eagle Medium ELISA Enzyme-Linked Immunosorbent Assay GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase HFD High-Fat Diet IGPTT Intraperitoneal Glucose Tolerance Test IL-1β Interleukin-1β IL-4 Interleukin-4 IRS Insulin Receptor Substrate Jak Janus Kinase NPY Neuropeptide Y PEPCK Phosphoenolpyruvate Carboxykinase PGC1α Peroxisome Prolioferator-Activated Receptor Gamma Coactivator 1α POMC Pro-Opiomelanocortin PRDM16 PR-Domain Containing 16 RT-PCR Reverse Transcriptase-Polymerase Chain Reaction STAT3 Signal Transducer and Activator of Transcription 3 TNF-α Tumor Necrosis Factor-α UCP-1 Uncoupling Protein 1 WAT White Adipose Tissue Int. J. Mol. Sci. 2020, 21, 4451 17 of 19

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