Muscle Conditional Medium Reduces

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Molecular Sciences Article

Muscle Conditional Medium Reduces Intramuscular Adipocyte Differentiation and Lipid Accumulation through Regulating Insulin Signaling Haiyin Han, Wei Wei, Weiwei Chu, Kaiqing Liu, Ye Tian, Zaohang Jiang and Jie Chen * College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; [email protected] (H.H.); [email protected] (W.W.); [email protected] (W.C.); [email protected] (K.L.); [email protected] (Y.T.); [email protected] (Z.J.) * Corresponding: [email protected]; Tel./Fax: +86-25-8439-9269 Received: 17 July 2017; Accepted: 14 August 2017; Published: 20 August 2017

Abstract: Due to the paracrine effects of skeletal muscle, the lipid metabolism of porcine intramuscular (i.m.) preadipocytes was different from that of subcutaneous (s.c.) preadipocytes. To investigate the development of i.m. preadipocytes in vivo, the s.c. preadipocytes were cultured with muscle conditional cultured medium (MCM) for approximating extracellular micro-environment of the i.m. preadipocytes. Insulin signaling plays a fundamental role in porcine adipocyte differentiation. The expression levels of insulin receptor (INSR) and insulin-like growth factor 1 receptor (IGF-1R) in i.m. Preadipocytes were higher than that in s.c. preadipocytes. The effects of MCM on adipocyte differentiation, lipid metabolism and insulin signaling transdution were verified. MCM induced the apoptosis of s.c. preadipocytes but not of s.c. adipocytes. Moreover, MCM inhibited adipocyte differentiation at pre-differentiation and early stages of differentiation, while the expression levels of INSR and IGF-1R were increased. Furthermore, MCM treatment increased adipocyte lipolysis and fatty acid oxidation through induction of genes involved in lipolysis, thermogenesis, and fatty acid oxidation in mitochondria. Consistent with the above, treatment of s.c. adipocytes with MCM upregulated mitochondrial biogenesis. Taken together, MCM can approximate the muscle micro-environment and reduce intramuscular adipocyte differentiation and lipid accumulation via regulating insulin signaling. Keywords: intramuscular preadipocytes; extracellular micro-environment; insulin receptor; insulin-like growth factor 1 receptor; porcine; adipogenesis

1. Introduction The deposition of fat in muscle, recognized by the intramuscular fat (IMF), is an important meat quality. Development of adipocytes located within muscles has been studied. A wide variety of evidence indicated significant differences in both metabolic and secretory functions between intramuscular adipocytes and subcutaneous adipocytes [1,2]. Consequently, intramuscular adipocytes had lower lipogenic enzyme activities, smaller cell sizes, and lower lipid content than subcutaneous adipocytes [3]. However, the mechanism underlying regional differences in adipogenesis still remains unknown. The differentiation of preadipocytes to mature adipocytes includes activation of adipogenic gene expression and induction of insulin sensitivity [4]. Previous studies reported that insulin is an obligate hormone for preadipocyte differentiation [5–8]. Insulin signaling is initiated by binding of insulin to the insulin receptor (INSR), a receptor tyrosine kinase [9]. During adipocyte differentiation, the number of INSR increases approximately 25-fold [10]. Before the appearance of responsiveness of insulin receptor (INSR) to insulin during later stages of adipocyte differentiation, insulin mediates Int. J. Mol. Sci. 2017, 18, 1799; doi:10.3390/ijms18081799

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its early adipogenic effect via insulin-like growth factor-1 receptors (IGF-1R), which is initiated by activation of the tyrosine kinase at the cell surface [10]. Once triggered, the IGF-1R phosphorylates insulin receptor substrates-1 (IRS-1), a key cytosolic protein substrate, which subsequently activates downstream signal transduction pathways with impact on adipogenic gene transcription [11,12]. Meanwhile, insulin receptor was required for the full differentiation response because of the impaired adipocyte differentiation that was associated with decreased insulin receptor expression [13]. All of these findings suggested the molecular mechanism of insulin action on adipocyte differentiation, but the effects of insulin signaling on porcine intramuscular adipogenesis are not well understand. In this study, we turned our attention to the most proximal steps of insulin signaling for further study, including the INSR/IGF-1R and the major direct substrate, IRS-1. Intramuscular adipocytes and subcutaneous adipocytes development occurs in the different extracellular micro-environment. Systemic blood circulation provides transport capabilities for endocrine hormones, cytokines (adipokines and myokines) and nutrition for adipose tissue development. However, the development of intramuscular adipocytes are exclusively affected by the paracrine activity of skeletal muscle fibers due to their particular location in close vicinity to muscle fibers. For example, myokines interleukin-15 (IL-15), myostatin (MSTN) and irisin inhibit adipocyte differentiation and fat deposition via a skeletal muscle to fat signaling pathway [14–16]. In these in vitro studies, it just verified the role of one purified cytokine present in the micro-environment. It is possible to approximate the tissue micro-environment using commercially available products. However, in all cases, it is unlikely that the material used represents the specific combination of factors that exist within the tissue micro-environment. To address these challenges, we extracted the interstitial fluid from skeletal muscle, which can be applied as muscle conditional cultured medium (MCM) for approximating the extracellular micro-environment of i.m. preadipocytes in vivo. The subcutaneous preadipocytes were cultured in the MCM to study intramuscular adipose tissue development, including adipocyte differentiation and adipose tissue expansion. In the present study, the mechanism of insulin signaling in porcine adipocyte differentiation was verified. Meanwhile, the effects of extracellular micro-environment on adipocyte development were detected using skeletal muscle interstitial fluid that was applied as MCM. Furthermore, the molecular mechanism of MCM action on adipogenesis by modulating insulin signal transduction was investigated. The goal of this study was to understand the intramuscular adipogenesis with emphasis on cell micro-environment interactions that are pivotal in regulating adipocyte formation. 2. Results 2.1. Cellular Differentiation and Identification of Porcine Intramuscular and Subcutaneous Preadipocytes The intramuscular (i.m.) and subcutaneous (s.c.) preadipocytes which isolated using the ceiling culture method were resuspended in culture medium with an irregular triangular appearance when freshly inoculated. After three days of culture in growth medium, both i.m. and s.c. preadipocytes showed fibroblast-like morphology (Figure 1A). Both of them could redifferentiate into mature adipocytes with lipid droplets following by adipogenic stimulation at confluence (Figure 1B). Pref-1 is an adipocyte-specific protein, with a special expression in preadipocytes, inhibiting adipose differentiation in the early differentiation stage [17,18]. The fibroblast-like cells were identified by immunofluorescence staining of Pref-1. As shown in Figure 1C, positive Pref-1 reactions demonstrated that the isolated cells were preadipocytes with high purity. In conclusion, the date above confirmed that the fibroblast-like cells were porcine i.m. and s.c. preadipocytes, respectively.

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Figure dedifferentiated Figure1.1.Identification Identificationofofporcine porcinei.m. i.m. and ands.c. s.c.preadipocytes. preadipocytes.TheThe dedifferentiatedporcine porcine intramuscular (i.m.) and subcutaneous (s.c.) preadipocytes showed fibroblast-like morphology (A); intramuscular (i.m.) and subcutaneous (s.c.) preadipocytes showed fibroblast-like morphology (A); The i.m. and s.c.s.c. preadipocytes were induced in in adipogenic medium at at confluence. Nine days later, The i.m. and preadipocytes were induced adipogenic medium confluence. Nine days later, thethe i.m. and s.c.s.c. preadipocytes were re-differentiated to to mature adipocytes with lipid droplets (B); i.m. and preadipocytes were re-differentiated mature adipocytes with lipid droplets (B); The isolated i.m. and s.c. preadipocytes were seeded in 12-well plates, and the cells of day 3 were The isolated i.m. and s.c. preadipocytes were seeded in 12-well plates, and the cells of day 3 were detected byby Pref-1 immunofluorescent staining (C). Scale bar = 100 μm. detected Pref-1 immunofluorescent staining (C). Scale bar = 100 µm.

2.2. Inhibition Differentiation of of Porcine Adipocyte byby Downregulating thethe Expression 2.2. Inhibition Differentiation Porcine Adipocyte Downregulating ExpressionofofIGF-1R IGF-1RororINSR INSR Insulin Insulininitiates initiatesitsitspleiotropic pleiotropiceffects effectsononcellular cellulargrowth growthand andmetabolism metabolismbybybinding bindingtotoitsits specific cell-surface receptor, INSR and IGF-1R. To elucidate the function of INSR and inin specific cell-surface receptor, INSR and IGF-1R. To elucidate the function of INSR andIGF-1R IGF-1R determining adipocyte differentiation, s.c. preadipocytes were transfected with INSR and IGF-1R determining adipocyte differentiation, s.c. preadipocytes were transfected with INSR and IGF-1R siRNAs, 2424 h, h,the were induced to todifferentiate siRNAs,respectively. respectively.After After thes.c. s.c.preadipocytes preadipocytes were induced differentiatefollowing following adipogenic stimulation. adipogenic stimulation. Knockdown efficiency waswas examined by qPCR and immunoblotting for INSR and IGF-1R. The Knockdown efficiency examined by qPCR and immunoblotting for INSR and IGF-1R. mRNA expression levels of INSR and IGF-1R were significantly reduced by 64% and 45% in siRNA The mRNA expression levels of INSR and IGF-1R were significantly reduced by 64% and 45% in transfected cells, respectively (Figure 2A). The protein expression level of IGF-1R also reduced siRNA transfected cells, respectively (Figure 2A). The protein expression level was of IGF-1R was also significantly (Figure 2B). However, the protein of INSR could notcould be examined due to the reduced significantly (Figure 2B). However, theexpression protein expression of INSR not be examined due pig INSR antibody was not available. to the pig INSR antibody was not available. Knockdown Knockdownof ofINSR INSRusing usingsiRNA siRNAdecreased decreasedadipocyte adipocytedifferentiation differentiationshowing showingbybylower lower cytoplasm triglycerides content and the lipid droplets in mature adipocytes compared with cytoplasm triglycerides content and the lipid droplets in mature adipocytes compared withthe the control (NC) (Figure 2C,D). Similar results were obtained from IGF-1R knockdown (Figure 2C,D). control (NC) (Figure 2C,D). Similar results were obtained from IGF-1R knockdown (Figure 2C,D). These siRNAinterference interferencenot not only suppressed expression of INSR and Theseresults resultsindicated indicated that that siRNA only suppressed the the expression of INSR and IGF-1R IGF-1R but also blocked hormone-induced adipocyte differentiation. but also blocked hormone-induced adipocyte differentiation. 2.3. The Differences of of Insulin Signaling between Porcine i.m. Preadipocytes and s.c.s.c. Preadipocytes 2.3. The Differences Insulin Signaling between Porcine i.m. Preadipocytes and Preadipocytes The expression levels ofof INSR and IGF-1R between i.m. and s.c.s.c. preadipcytes were compared toto The expression levels INSR and IGF-1R between i.m. and preadipcytes were compared analyze the expressionlevels levelsofofINSR INSRand andIGF-1R IGF-1R analyze theregional regionaldifferences differencesin inadipogenesi. adipogenesi. The The mRNA expression inin i.m. i.m. preadipocytes significantly thaninthose in s.c. preadipocytes (Figure 3A). preadipocytes werewere both both significantly higherhigher than those s.c. preadipocytes (Figure 3A). Accordingly, Accordingly, the proteinpattern expression pattern of IGF-1R between i.m. and s.c. preadipocytes wasthe the protein expression of IGF-1R between i.m. and s.c. preadipocytes was consistent with consistent with the (Figure 3B). However, theofprotein expression of INSR could mRNA (Figure 3B).mRNA However, the protein expression INSR could not be examined due tonot thebe pig examined due to was the pig antibody was not available. INSR antibody notINSR available.

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Figure 2. Downregulation of insulin receptor (INSR) IGF-1 receptor (IGF-1R) by siRNA siRNA interference Figure 2. Downregulation of insulin receptor (INSR) or IGF-1 receptor (IGF-1R) by siRNA interference Figure 2. Downregulation of insulin receptor (INSR) ororIGF-1 receptor (IGF-1R) by interference decreased porcine adipocyte differentiation. The siRNAs targeting INSR and IGF-1R (si-INSR, decreased porcine adipocyte differentiation. The siRNAs targeting INSR and IGF-1R (si-INSR, si-IGF-1R) decreased porcine adipocyte differentiation. The siRNAs targeting INSR and IGF-1R (si-INSR, si-IGF-1R) si-IGF-1R) were transfected into porcine s.c. preadipocytes. The INSR/IGF-1R mRNA (A) (nlevels = 6) were transfected into porcine s.c. preadipocytes. The INSR/IGF-1R mRNA (A) (n = 6) and protein levels were transfected into porcine s.c. preadipocytes. The INSR/IGF-1R mRNA (A) (n = 6) and protein and protein levels (B) (n = 3) were verified after transfected with siNC and si-INSR/si-IGF-1R at 24 (B) (B) (n =(n3)=were verified after transfected with siNC andand si-INSR/si-IGF-1R at 24 48 h, 3) were verified after transfected with siNC si-INSR/si-IGF-1R at and 24 and 48respectively; h, respectively; and 48s.c. h, s.c. respectively; Transfected s.c. preadipocytes were induced todays; differentiate forintracellular nine days; Transfected preadipocytes were induced to to differentiate for for ninenine TheThe intracellular Transfected preadipocytes were induced differentiate days; The intracellular accumulation was determined by cytoplasm triglyceride content (C) (n = 4)(D) and accumulation was determined by cytoplasm triglyceride content (C) (n = 4) and Oil Red O staining (nOil accumulation was determined by cytoplasm triglyceride content (C) (n = 4) and Oil Red O staining (D) (n Red O staining (D) (n = 3). The mRNA expression differences were normalized to Ribosomal protein = 3).= The mRNA expression differences were normalized to Ribosomal protein lateral stalk subunit P0 P0 3). The mRNA expression differences were normalized to Ribosomal protein lateral stalk subunit lateral stalk subunit P0 β-actin (RPLP0) mRNA level. β-actin bands as loading. an loading. internal control for (RPLP0) mRNA level. TheThe β-actin bands served as an internal control forserved protein ** p** < p0.01, (RPLP0) mRNA level. bands served asThe an internal control for protein < 0.01, protein loading. ** p < 0.01, Scale bar = 200 µm. Scale barbar = 200 μm.μm. Scale = 200

Figure 3. INSR andand IGF-1R expression pattern in i.m. andand s.c.s.c. preadipocytes. TheThe comparison of INSR expression pattern in i.m. i.m. preadipocytes. Figure 3. INSR IGF-1R expression pattern in comparison of INSR andand IGF-1R mRNA expression levels in i.m. andand s.c.s.c. preadipocytes (A)(A) (n =(n protein expression IGF-1R mRNA expression levels in preadipocytes expression and IGF-1R mRNA expression levels in i.m. i.m. and s.c. preadipocytes (A) (n3);= = The 3); The protein level of of IGF-1R inin i.m. andand s.c.preadipocytes preadipocytes was detected by by Western blot andand subsequently level and s.c. was detected by Western blot and subsequently quantified. level ofIGF-1R IGF-1R in i.m. s.c. preadipocytes was detected Western blot subsequently quantified. (B) (n = 3). The mRNA expression differences were normalized to RPLP0 mRNA (B) (n = 3). The mRNA expression normalized RPLP0 mRNA level.mRNA Thelevel. β-actin quantified. (B) (n = 3). The mRNAdifferences expression were differences were to normalized to RPLP0 level. Thebands β-actin bands as an internal control forloading. protein loading. ** p**< p0.01. served asserved anserved internal for protein ** ploading. < 0.01. The β-actin bands ascontrol an internal control for protein < 0.01.

2.4.2.4. MCM Identification andand Its Its Effect on on Adipocyte Differentiation MCM Identification Effect on Adipocyte Adipocyte Differentiation Effect Differentiation TheThe protein factors in in thethe muscle conditional medium (MCM) were identified by by label freefree factors the muscle conditional protein factors muscle conditional medium (MCM) were identified label quantitation (date not shown). There were so many kinds of proteins, including cytokines released quantitation (date not shown). shown). There were so many many kinds kinds of of proteins, proteins, including including cytokines cytokines released released quantitation from muscle tissue, adipose tissue and other endocrine organ. The s.c.s.c. preadipocytes were cultured muscle tissue, adipose tissue and other endocrine organ. The s.c. preadipocytes were cultured from muscle tissue, adipose tissue and other endocrine organ. The preadipocytes were cultured with MCM at different concentrations to to verify thethe effect ofonof MCM onadipocyte porcine adipocyte with MCM different concentrations verify effect MCM on porcine adipocyte with MCM at at different concentrations to verify the effect of MCM porcine differentiation. differentiation. During the differentiation, the s.c. preadipocytes were simply incubated in in differentiation. During the differentiation, the s.c. preadipocytes were simply incubated −1 −1 adipogenic medium supplemented with MCM at at 0, 0, 10, 10, 40, 40, andand 120120 μg·mL protein adipogenic medium supplemented with MCM μg·mLtotal total protein

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5 of 15 the s.c. preadipocytes were simply incubated in adipogenic medium − 1 supplemented with MCM at 0, 10, 40, and 120 µg·mL total protein concentration. Nine days later, concentration. Nine days later, intracellular oil droplets were stained with Oil Red O. As indicated in intracellular oil droplets were stained with Oil Red O. As indicated in Figure 4, MCM suppressed FigureInt. 4,J.MCM lipid accumulation with significant effects at 120 μg·mL−1 total5 of protein Mol. Sci. suppressed 2017,with 18, 1799 15 lipid accumulation significant effects at 120 µg·mL−1 total protein concentration. The inhibition concentration. The inhibition effect of MCM on adipocyte differentiation was consistent with the effect of MCM on adipocyte differentiation was consistent with the development of i.m. preadipocytes −1 was concentration. days later, intracellular oilamount droplets of were stained withμg·mL Oil Red O. Asused indicated in development of i.m.Nine preadipocytes in vivo. − The MCM at 120 for further in vivo. The 4, amount of MCM atlipid 120 µg ·mL 1 waswith used for further study. −1 total protein Figure MCM suppressed accumulation significant effects at 120 μg·mL study.

concentration. The inhibition effect of MCM on adipocyte differentiation was consistent with the development of i.m. preadipocytes in vivo. The amount of MCM at 120 μg·mL−1 was used for further study.

Figure 4. The inhibitory effect of muscle conditional medium (MCM) on s.c. preadipocytes Figure 4. The inhibitory effect of muscle conditional medium (MCM) on s.c. preadipocytes differentiation. Confluenced s.c. preadipocytes were incubated in adipogenic medium supplemented differentiation. Confluenced s.c. preadipocytes were incubated in adipogenic medium supplemented Figure 4. Theamount inhibitory effectatofthemuscle conditional medium (MCM) on(ns.c. with an increasing of MCM indicated total protein concentration = 3).preadipocytes After nine days, with an increasing amount of MCM at the indicated total protein concentration (n = 3). After nine differentiation. Confluenced s.c.Red preadipocytes were incubated adipocytes were staining with Oil O. Scale bar = 200 µm. in adipogenic medium supplemented days, adipocytes were staining Oil at Red Scale bar = 200 μm. concentration (n = 3). After nine with an increasing amount with of MCM theO.indicated total protein days, adipocytes were staining with Oil Red O. Scale bar = 200 μm.

2.5. MCM MCM Induced Induced Apoptosis Apoptosis of of Porcine Porcine Preadipocytes Preadipocytes 2.5.

−1 2.5. MCM Induced Apoptosis and of Porcine Preadipocytes Porcine s.c. s.c.s.c. adipocytes werewere respectively treatedtreated with MCM 120 µgat ·mL Porcine s.c. preadipocytes preadipocytes and adipocytes respectively withatMCM 120 total protein concentration for three days andadipocytes then stained with fluorescein isothiocyanate (FITC)-labled −1 total Porcine s.c. concentration preadipocytes and s.c. respectively treated with MCM at 120 μg·mL protein for three days andwere then stained with fluorescein isothiocyanate −1 annexin V (Annexin V-FITC and propidium iodine (PI) to detect cell apoptosis. Flow cytometric μg·mL total protein concentration for three days and then stained with fluorescein isothiocyanate (FITC)-labled annexin V (Annexin V-FITC and propidium iodine (PI) to detect cell apoptosis. Flow (FITC)-labled annexin V (Annexin V-FITC and propidium iodine (PI) preadipocytes to detect cell apoptosis. Flow detection indicated MCM induced significantly apoptosis in s.c. when compared cytometric detectionthat indicated that MCM induced significantly apoptosis in s.c. preadipocytes when cytometric detection indicated that MCM induced significantly apoptosis in s.c. preadipocytes when with the control group (Figure 5A). The early and late apoptosis percentage was observed from 6.5% compared with the control group (Figure 5A). The early and late apoptosis percentage was observed compared with 2.8% the control group (Figure 5A). The early (Figure and late apoptosis percentage was observed to 15.1% (p < 0.01), to 6.4% (p < 0.05), respectively 5A). However, the apoptosis effects from 6.5% to 15.1% (p