骨骼肌細胞中神經醯胺影響胰島素訊息傳遞之機轉

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(1) . 國立臺灣師範大學生命科學系碩士論文. 骨骼肌細胞中神經醯胺影響胰島素訊息傳遞之機轉 Mechanisms of Decreased Insulin Signaling by Ceramides in Skeletal Muscle Cells. 研究生：謝章亭 Chang-Ting Hsieh. 指導教授：林炎壽博士 Yen-Shou Lin, Ph.D.. 中華民國一百年十二月 .

(2) . 致謝時光飛逝，轉眼間我踏入師大這個大環境已經兩年半的時間，每一段歷程對我來說彷彿都歷歷在目，從一開始進入實驗室的陌生以及對未來的不確定感到後來能夠為自己的實驗負責甚至能夠有餘力幫忙實驗室的其他成員，這樣的轉變對我來說是相當難能可貴的。雖然在這段不算長也不算短的實驗歷程中也經歷了許多大風大浪，但都在自己的堅持以及實驗室成員互相幫忙下成功的克服，因此對於在這段時間曾經幫助過我的人更是滿懷感激。首先要感謝在我碩士班期間給予我相當大指導的林炎壽老師，無論是在實驗上的討論或是設計上都給予我適當的指導以及發揮的空間，使我有信心去面對各種挑戰；另外也要感謝實驗室的大學姐仁華學姐，在我實驗屢屢遇到困難時能夠利用實驗上經歷與見解給予我許多建議，使問題能夠迎刃而解；當然的和我同時進入實驗室的柏強，一同在實驗室奮鬥了兩年半，如今我們終於能夠帶著滿滿的收穫笑著離開師大，甚感欣慰；而我也要感謝來參與我口試的梁美智老師以及李冠群老師在論文上面給予我許多的建議與指導。最後，感謝師大的所有師長及各位學長姐們，有了大家的幫助，讓我能夠在師大度過這段充實的時光，而我相信這一切的一切都將成為我人生中一段美好回憶。. .

(3) . Contents Abbreviation list ....................................................................................... 1 Abstract (Chinese) ...................................................................................... 2 Abstract (English) ....................................................................................... 4 Introduction ............................................................................................... 6 Materials and Methods ............................................................................ 13 Results ..................................................................................................... 20 Discussion ............................................................................................... 24 References ............................................................................................... 28 Figures ..................................................................................................... 36 Appendix. Antibodies used in study ....................................................... 54. .

(4)   . Abbreviation list. Abbreviation. Full name. PKB. Akt/protein kinase B. GSK3. glycogen synthase kinase-3. NEFA. non-esterified fatty acid. IMTG. intramuscular triacylglycerol. CEMs. caveolin enriched membrane domains. mTOR. The mammalian target of rapamycin. mTORC1. mTOR complex 1. mTORC2. mTOR complex 2. Rheb. Ras homolog enriched in brain. GAP. GTPase activating protein. S6K. ribosomal S6 kinase. 4E-BP. 4E-binding protein. 1 .

(5)   . 摘要肥胖是引發胰島素不敏感以及第二型糖尿病的高度危險因子，而目前已經被證實，當血液中脂肪酸的濃度提高時會造成胰島素所誘導的葡萄糖吸收能力受到阻斷。神經醯胺 (ceramide)，為一脂肪酸的衍生物，會透過降低 AKT/protein kinase B (PKB)和 glycogen synthase kinase-3 (GSK3)活性而影響胰島素的訊息傳遞，然而神經醯胺對於上游分子，例如 IRS1 以及 PI3K 則沒有影響。本研究目的主要是進一步探討神經醯胺在胰島素訊息傳遞的詳細作用機轉。胰島素刺激的 C2C12 肌肉細胞中，100 μM C2-神經醯胺會造成 AKT 在絲胺酸 473 (Serine 473)位點磷酸化程度降低，這一點同文獻所報導。另一方面，在訊息傳遞的上游分子中，例如 IRS1 在酪胺酸 (Tyrosine)位點磷酸化程度以及 IRS1 總蛋白表現量上則沒有太大的影響。而在同樣的處理條件中，我們發現 S6K 的磷酸化程度卻有顯著的增加。我們利用 mTORC1 的抑制劑 Rapamycin 處理，進一步證實 C2-神經醯胺所造成 AKT 在絲胺酸 473 (serine 473)位點磷酸化程度降低和增加的 S6K 的磷酸化可能有因果關係。所以根據目前已建立的胰島素訊息傳遞模式中，S6K 上游分子如 TSC1/TSC2，Rheb，mTORC1 均有可能為 C2-神經醯胺的直接作用標的。我們利用 shRNA 減少這些蛋白質在細胞中的表現以進一步檢測 C2-神經醯胺對於胰島素訊息傳遞破壞作用是否因 2 .

(6)   . 此受到影響，發現在 C2C12 肌肉細胞中減弱了 Rheb 分子表現量時確實會對 C2-神經醯胺所造成 Akt473 磷酸化程度下降的作用產生干擾，因此我們找出 C2-神經醯胺的作用應該位於 PI3K 的下游，但 Rheb 分子/或上游。此一發現對於 C2-神經醯胺在肌肉細胞所造成之胰島素不敏感的現象，可謂訂出了 C2-神經醯胺的標的分子，而詳細的作用層級及其作用機轉將有助於藥物標的的開發。關鍵字: 肌肉細胞、胰島素不敏感、 C2-神經醯胺. 3 .

(7)   . Abstract Obesity is a high risk factor to develop insulin resistance and type II diabetes. Chronic elevation of free fatty acid levels in plasma has been found to be closely associated with impaired insulin-mediated glucose uptake. Ceramide, a fatty acid derived lipid, was confirmed as a negative regulator of insulin signaling pathway by reducing Akt/protein kinase B (PKB) and glycogen synthase kinase-3 (GSK3) activity. However, there are no effects on the upstream signaling molecules, such as IRS1 and PI3K. In this study, we intend to investigate the detail molecular mechanism of C2-ceramide on insulin signaling. We showed that 100 µM C2-ceramide caused a decrease of Akt Ser473 phosphorylation in insulin-stimulated C2C12 myotubes, which is in consistent with the results on the literatures. While, the phosphorylation of the upstream signaling molecules, IRS1 tyrosine residue, and IRS1 total abundance were not changed. Interestingly, an increased phosphorylation of S6K was observed in the same condition. Utilizing rapamycin, an mTORC1 inhibitor, we further demonstrated that the decreased Akt Ser473 phosphorylation might be mediated through the activated S6K. According to current model of insulin signaling pathway, the upstream molecules of S6K such as TSC1/2, Rheb and mTORC1 could have been the targets of C2-ceramide. Utilizing shRNA to individually knockdown these molecules in order to examine the effect of loss-of-function on C2-ceramide impaired insulin signaling pathway, we found that Rheb knockdown in C2C12 myoblasts can block the decreased Akt Ser473 phosphorylation induced by C2-ceramide. Taken together, we explored a 4 .

(8)   . possible novel pathway of C2-ceramide impaired insulin signaling through activated S6K to interfere with Akt Ser473 phosphorylation. The affected signaling molecules had also been pinpointed to upstream of Rheb but downstream of PI3K. Further investigation on mapping and detail mechanism will provide great therapeutically strategy on treatment of type II diabetes. Key word: C2C12 myocytes, insulin resistance, C2-ceramide. 5 .

(9)   . Introduction. Insulin resistance Insulin resistance is a phenomenon in which biological functions on its target tissues in responded to insulin stimulation was decreased. When insulin resistance occurred, the body compensates for this state by producing additional amounts of insulin. This results in hyperinsulinemia in the blood. Over time, this process causes an imbalance in glucose metabolism. It is also one of the features of the metabolic syndrome associated with obesity (Reaven, 2003).. The relationship between fatty acid and insulin resistance Skeletal muscle plays an essential role in the regulation of whole-body glucose homeostasis because it takes great part of body weight and is a major tissue regulated by insulin. Therefore, insulin resistance in skeletal muscle is closely related to the development of type II diabetes. Elevation of plasma free fatty acids (fFAs) has been shown to impair insulin action and gradually develop into type II diabetes (Paolisso et al., 1995, Schmitz-Peiffer et al., 1999, Ragheb et al., 2009). Much effort is being marshaled to investigate how fFAs induce insulin resistance in skeletal muscle. Non-esterified fatty acid (NEFA) sustainedly oversupplied to skeletal muscle led to accumulation of intramuscular triacylglycerol (IMTGs) and fatty acid derivate metabolites, like diacylglycerol (DAG) and ceramide (Chavez et al., 2003). In addition, 6 .

(10)   . it is reported that lipids, both unsaturated fFAs and saturated fFAs, can impair insulin actions such as inhibition of insulin-stimulated phosphorylation of Akt and decrease of glucose uptake and glycogen synthesis in skeletal muscle (Schmitz-Peiffer et al., 1999).. Ceramides Ceramides consist of a long chain sphingosine base conjugated with fatty acid via amine bond and a polar head group. Ceramide have also different compositions and synthesis in different compartments or membranes of the cell through a variety of mechanisms. Like other lipid second messengers in signal transduction, they are produced rapidly and transiently in response to specific stimulation on target proteins (Morales et al., 2007). In general, ceramides are the minor components of the membrane and they are concentrated in the lateral liquid ordered microdomains (Stancevic and Kolesnick, 2010). Although the structures of fatty acid derived metabolites ceramide and diacylglycerol are similarities, their occurrence, location, and behavior in membranes are different (Hannun and Obeid, 2008). The roles of ceramides in the regulation of insulin action, apoptosis, cell differentiation, transformation and proliferation have been demonstrated. Many disease states such as cancer, diabetes, neuropathies, Alzheimer's disease, Parkinson's disease, and atherosclerosis caused by ceramide treatment were also reported (Gangoiti et al., 2010). In the variety of ceramide analog, short-chain ceramides, like C2-ceramide and C6-ceramide, were water soluble and membrane permeable which can be delivered into cell easily (Kitatani et 7 .

(11)   . al., 2008).. Other biological function of ceramides and its relevant molecule, palmitate in skeletal muscle Fatty acid over-supplied in skeletal muscle has been demonstrated to result in not only insulin resistance, but also cause mitochondrial dysfunction and induce apoptosis (Wang et al., 2009, Rizvi et al., 2011). It was shown that muscle cells preincubated with saturated FFA, palmitate, could result in accumulation of ceramide, and diacylglycerol. Meanwhile, such incubation also reduced insulin-stimulated phosphorylation of GSK3 and Akt (Chavez et al., 2003). In addition, palmitate induced apoptosis by activate caspase-9 and caspase-3. Therefore, ceramide could play an important role in palmitate-induced apoptosis (Zha et al., 1996, Li et al., 1997, Cai et al., 2002, Peterson et al., 2008).. The mTORC1 and mTORC2 pathway The mammalian target of rapamycin (mTOR) is a conserve Ser/Thr protein kinase that forms two compositions and functionally distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 which is consisted of mTOR, mLST8, PRAS40 and raptor is acutely sensitive to immunosuppressant drug rapamycin. It plays a crucial role in regulating several cellular processes, like cell growth and proliferation, protein synthesis, ribosome biosynthesis, 8 .

(12)   . transcription, and autophagy (Wullschleger et al., 2006, Polak and Hall, 2009, Gan et al., 2011). The small GTPase Rheb (Ras homolog enriched in brain) is a direct upstream activator of mTORC1 and is negative regulated by the protein complex of tuberous sclerosis complex 1/2 (TSC1/TSC2). Within the TSC1 and TSC2 protein complex, TSC1gene encodes the protein hamartin (130KD) and TSC2 gene encodes the protein tuberin (198KD). Harmartin have the function to mediate binding with tuberin and form a functional and stable heterodimer, this protein complex can act as a tumor suppressor to regulate cells growth proliferation (van Slegtenhorst et al., 1997, Plank et al., 1998, van Slegtenhorst et al., 1998, Hodges et al., 2001, Cai et al., 2006). Tuberin is a GTPase activating protein (GAP) for Rheb. TSC1/TSC2 complex induced Rheb-GTP to convert to Rheb-GDP and affected mTORC1 activity (Inoki et al., 2003, Zhang et al., 2003). The active form of GTP-bound Rheb can stimulate mTORC1 and regulate its downstream substrate, such as ribosomal S6 kinases (S6K) and the eukaryotic initiation factor 4E-binding protein (4E-BP) (Long et al., 2005, Cai et al., 2006, Huang et al., 2008). Phosphorylation of S6K and 4E-BP by mTORC1 lead to S6K activation and 4E-BP inhibition, thereby promoting the protein synthesis. The other mTOR protein complex mTORC2 which is comprised of mTOR, mLST8, mSIN1, and rictor is resistant to rapamycin, but prolong the rapamycin treatment also can disrupt the composition of mTORC2 (Frias et al., 2006, Jacinto et al., 2006, Yang et al., 2006, Rosner and Hengstschlager, 2008, Julien et al., 2010, Gan et al., 2011). The mTORC2 activity has been demonstrated to 9 .

(13)   . phosphorylate the hydrophobic sites of downstream target Akt at Ser473. Moreover, mTORC2 can also regulate cytoskeleton reorganization. (Jacinto et al., 2004, Sarbassov et al., 2004, Gan et al., 2011). Previously studies have been shown that the product of TSC1 gene, harmartin, binds and co-localizes with hypophosphorylated the product of TSC2 gene, tuberin, at the membrane where the tuberin utilizes its GAP activity to inhibit downstream molecules, Rheb. In respond to growth factor, it promotes activation of mTOR in C2C12 myotubes through phosphorylation, and distribution of TSC1/TSC2 complex from the membrane to the cytosol. Some studies have shown that phosphorylation of ser 939 of tuberin is the primary site for 14-3-3 protein binding although phosphorylation of ser 981 and 1130 can also be recognized by 14-3-3 protein. Phosphorylation of these sites does not change its GAP activity toward downstream target Rheb but promote tuberin translocation from membrane to cytosol where interact with 14-3-3 protein and segregate away from membrane bound activation partner, hamartin, relieved its inhibit effect on Rheb (Cai et al., 2006, Miyazaki et al., 2010) .. Mechanisms of ceramide impaired insulin signaling pathway In fatty acid derivated lipids, ceramide is one of the most active metabolite to negative regulate insulin actions and signaling (Blouin et al., 2010). C2-Ceramide can reduce insulin-stimulated glycogen synthesis and glucose uptake by decreasing phosphorylation of Akt and glycogen synthase kinase-3 (GSK3) (Schmitz-Peiffer et al., 1999). However there 10 .

(14)   . are no effects on their upstream molecules such as IRS1, PI3K (JeBailey et al., 2007). Two mechanisms of how ceramide inhibited insulinstimulated Akt Ser473 phosphorylation have been proposed. In one of these mechanisms, to be convinced that Akt is the primary important target of ceramide, some studies showed that ceramide can active PKCζ on caveolin enriched membrane domains (CEMs) and phosphorylates PH domain of Akt on threonine 34 residue, through preventing Akt translocation and recruitment to plasma membrane (Bourbon et al., 2000, Powell et al., 2003, Blouin et al., 2010). In the other mechanism, C2-ceramide decreased phosphorylation of Akt through the direct activation of cytosolic serine/threonine phosphatase PP2A which is responsible for dephosphorylating Akt and prevent insulin signaling pathway (Dobrowsky et al., 1993, Resjo et al., 2002, Julien et al., 2010). However, in addition to these mechanisms, an interesting phenomenon was observed when C2C12 myotubes were treated with C2-cermide in our experiments. An increased phosphorylation of S6K was always accompanied with a decrease of Akt phosphorylation in abovementioned experiments. When insulin or insulin-like growth factor-1 (IGF-1) binding to its receptor, it can phosphorylate IRS-1 on tyrosine residue and induce the insulin signaling pathway. Activation of mTORC1 and its downstream target S6K has been found to negative regulate the degree of Akt phosphorylation in respond to insulin or IGF-1.This serves as a feedback loop to promote phosphorylation of insulin receptor substrate-1 (IRS-1) at serine residue and it can lead to IRS-1 instable and inability of insulin or 11 .

(15)   . IGF-1 activated PI3K, Akt (Harrington et al., 2004, Shah et al., 2004, Um et al., 2004, Shah and Hunter, 2006, Huang et al., 2008). Recently, mTORC1 can regulate mTORC2 signaling through the activated S6K1 phosphorylates rictor on threonine 1135. A rictor mutant which lacks this phosphorylation site increases phosphorylation of Akt (Julien et al., 2010). Therefore we proposed that the impairment of C2-ceramide on the insulinstimulated Akt phosphorylation could be mediated through mTOCR1 pathway. There are four specific aims listed below. (1) To determine the effect of C2-ceramide on insulin signaling transduction; (2) To explore the effect of C2-ceramide on caspase-3 activity; (3) To determine whether C2-ceramide can regulate mTORC2 signaling mediated through S6K activation; and (4) To study whether C2-ceramide can act on upstream molecules of S6K, likeTSC1/TSC2, Rheb or mTORC1. Our data suggest that signaling molecules between PI3K and Rheb play a critical role in ceramide-impaired insulin signaling pathway.. 12 .

(16)   . Materials and methods. Materials The materials/ reagents were purchased from companies indicated in the parentheses. Dulbecco’s modification Eagle’s medium (DMEM), horse serum (Gibco-BRL-Life Technologies, Grand Island, NY, USA), fetal Bovine Serum (Thermo Scientific, South Logan, Utah, USA); Monoclonal phospho-specific anti S6K, Rictor Thr1135 and anti caspase-3 antibodies (Cell Signaling Technology, Danvers, MA, USA); Polyclonal anti IRS1 Ser307 and monoclonal anti-phosphotyrosine, antibodies (Upstate Biotechnology, Lake Placid, NY, USA); Polyclonal anti IRS1, monoclonal anti pro-caspase-3, anti S6K antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Monoclonal anti-TSC2, anti-Akt and phospho-specific anti Akt Ser473 antibodies (Epitomics, CA, USA); Horseradish peroxidase anti-rabbit, anti-mouse secondary antibodies (Jackson, West Grove, PA, USA); C2-ceramide (Calbiochem, Nottingham, UK); Palmitate, anisomycin, and bovine serum albumin (BSA, fatty acid free) (Sigma Chemical Co, St. Louis, MO, USA). A table of antibodies source, catalog numbers, titer, and molecular weight is also summarized in appendix.. Cell culture and transfection Mice C2C12 myoblasts was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). HEK 293T and C2C12 13 .

(17)   . myoblasts were maintained in DMEM with 10% fetal bovine serum. For muscle cells differentiation, C2C12 myoblasts were seeded in 6 cm plates and cultivated in the same medium until 95% confluence initially. C2C12 myoblasts were then cultivated in DMEM containing 2% horse serum. Medium was changed every other days until formation of C2C12 myotubes which typically takes 4~5 days to be observed. HEK293T cells were plated in 6 cm dishes, grown in 10% FBS for 24 h until 60-70% confluence initially before transient transfection using Lipofectamine™ 2000 reagent or calcium phosphate approach. For lipofectamine 2000 transfection, 6 µg DNA and 15 µl Lipofectamine™ 2000 was placed into 150 µl medium individually, mixed gently then incubated 20-30 min at room temperature. Add the DNALipofectamine™ 2000 complexes directly to each 6 cm dishes and incubated for additional 36-48 h. In approach of using calcium phosphate precipitation on a 10 cm dish, 500 µl, 2×HBSS (38 mM HEPE, 274 mM NaCl, 11 mM Dextrose, 10 mM KCl, 1.41 mM Na2HPO4) was aliquoted into a 15 ml microfuge tube (250 µl 2×HBSS if using 6 cm dishes). Prepare another microfuge tube containing 60 µl CaCl2, 10 µg DNA, replenish distilled water enough to achieve the total volume to 500 µl (30 µl CaCl2, 5 µg DNA and 250 µl total volume if using 6 cm dishes). Add DNA/CaCl2 complex into the 2×HBSS and mixed gently. This mixture was added to cells drop by drop and incubated for additional 36-48 h.. Lipid preincubation Palmitate and C2-ceramide was respectively dissolved in 100% 14 .

(18)   . ethanol and DMSO conjugated with 1~2% BSA by vortex. It’s now named as palmitate or C2-ceramide mixture. C2C12 myotubes were washed by no serum DMEM once, followed by 0.75 mM, 1.2 mM palmitate or 12.5, 25, 50, 100 µM C2-ceramide mixture treatment for 16 and 2 h, respectively. In control groups, cells were incubated with no serum DMEM containing vehicle solvents plus 1~2% BSA. As time is up, cells were further treated 100 nM insulin for 15 min before harvesting.. Preparation of whole cell lysate and immunoblotting Cells were lysed in lysis buffer (20 mM Tris base, PH 7.9, 20 mM NaCl, 1 mM EDTA, 5 mM EGTA, 20 mM β-glycerolphosphate, 1 mM DTT, 1 mM PMSF in isopropanol, protease inhibitor, 25 nM Calyculin A, 0.5% TritonX-100). On overage one 6 cm plate was lysed in around 160 μl lysis buffer. Lysates were centrifuged at 13,500 rpm for 10 min. Aliquots of the supernatants containing equal amounts of protein, measured by Bradford assay (Bio-Rad, Hercules, CA, USA), were loaded onto gels for SDS-PAGE separation. For immunoblotting, proteins were separated on appropriate percentage of SDS-PAGE and transferred onto the PVDF membrane (Millipore, UK). Membranes were probed with primary antibodies as indicates on figures. Blots were visualized using horseradish peroxide-conjugated secondary antibody followed by chemiluminescence as outlined by the manufacturer (Thermo Scientific).. Substrate preparation, immunoprecipitation and kinase assay 15 .

(19)   . To prepare substrates, FLAG-tagged hAKT WT and PH domain deletion were constructed and expressed in competent E.coli BL21 (DE3) pLysS. One colony was picked to cultivate at 37°C for overnight then transfer 5 ml into 500 ml fresh LB medium until the OD600 of grow reach 0.5~1(usually take 3 h). After adding inducer 100 µM IPTG (isopropyl -1-thio-β-D-galactopyranoside), the medium was shaked at 37°C for additional 4~5 h. The culture was collected to a 500 ml bottle and centrifugation 4000 rpm at 4°C for 15 min. The pellet was stored at -80°C until use. Resuspend pellet in mTORC2 lysis buffer (2 mM EDTA, 0.1% β-mercatoethanol, 0.2 mM pMSF, 5 mM benzamidine). Sonicate 5~6 times, 15 sec for each time. After sonication, the lysates were spinned at 13000 rpm for 30 min at 4°C. The supernatant was incubated with FLAG-agarose beads for overnight at 4°C. Immunocomplex were washed four times by mTORC2 lysis buffer and two times by mTORC2 lysis buffer containing 0.5 M NaCl. The immunocomplex with mTORC2 kinase buffer (10 mM HEPES, PH 7.4, 25 mM β-glycerolphosphate) were separated to several eppendorfs and washed one time with mTORC2 kinase buffer. The substrate was eluted by adding 0.3 µg/µl FLAG peptide and the supernatant was collected using spin columns. The substrate was snap frozen using liquid nitrogen and stored at -80°C until use. Lysates were centrifuged at 13500 rpm for 10 min. Aliquots of the supernatants containing equal amounts of protein, measured by Bradford assay (Bio-Rad), were added to 15 µl of settled pre-incubated anti-rictor-protein A-agarose beads and incubated at 4°C for 2 h. Beads were washed four times with 1ml of lysis buffer, two times with 1ml of 16 .

(20)   . lysis buffer containing 0.5 M NaCl, and again with 1ml of kinase buffer. The kinase assay was performed in 25 µl of reaction buffer (FLAG-tagged substrates, 10 mM MgCl2, and 100 µM ATP) for 30 min at 30°C on a thermomixer. The reaction was stopped by addition SDS sample buffer to 1x concentration followed.. Short hairpin RNAs (shRNAs) and viral infection Short hairpin RNAs (shRNAs) targeting luciferase, TSC2, and Rheb were purchased from National RNAi Core Facility in Academia Sinica. The functional clones and correspond target sequence of TSC2 and Rheb were Luciferase, 5’ CCGGCGCTGAGTACTTCGAAATGTCCTCGAGGACATTTCGAAGTA CTCAGCGTTTTT Rheb C1, 5’ GCCCGTCATCCTTGAAGATAAACTCGAGTTTATCTTCAAG GATGACGGGTTTTTG; Rheb E1, 5’ CCGGCAGACATACTCCATAGATATTCTCGAGAATATCTATGGAGTAT GTCTGTTTTTG’ TSC2 G1, 5’ CCGGCCCTTATATCACTAAGGGTTTCTCGAGAAACCCTTAGTGATAT AAGGGTTTTTG’ HEK 293T cells were used to perform the viral package. Briefly, Luciferase, Rheb, or TSC2 shRNA along with packaging vectors: (pCMV5-∆R8.91and pCMV5-pMD.G) were co-transfected into HEK 293T 17 .

(21)   . cells by using calcium phosphate approach. Viruses were collected after 24, 48 and 72 h transfection. Virus were concentrated by centrifugation at 25000 rpm, 4°C for 1 h and the pellet were suspended by 1×PBS then stored at -80°C until use. Stable pools of cells were generated by lentivirus infection of C2C12 cells in growth medium containing 4 µg/ml polybrene and 3 µg/ml puromycin.. Subcellular fractionation: In the initial stage to establish this methodology, HEK 293T were plated in 6 cm plates and transfected with FLAG tagged nPKCθ. These cells were treated with 200 nM PMA for different timing. Cells were lysed in lysis buffer (250 mM sucrose, 20 mM Tris base, PH 7.9 , 1 mM EDTA, 0.4 mM EGTA, 20 mM β-glycerolphosphate, 1 mM DTT, 1 mM PMSF in isopropanol, protease inhibitor, 25 nM Calyculin A). The samples were ultracentrifuged at 140,000g for 1 h at 4°C by using table top ultracentrifuge (Beckman TLX-120, TLA 100 rotor). The supernatant were collected as cytosolic fraction. The pellet was further stroke several times with lysis buffer containing 0.2% MEGA10. The mixtures were ultracentrifuged at 140,000 g for 1 h at 4°C again. The supernatant was collected as membrane fraction. A portion of cytosolic and membrane fractions were loaded on to SDS-PAGE and perform Western-blot analysis.. Statistical Analysis Data are presented as mean ±S.E. Treatment effects were evaluated 18 .

(22)   . using a two-tailed Student’s t test. A p value < 0.05 was considered to be statistically significant.. 19 .

(23)   . Results. Impairment of C2-ceramide on insulin signaling pathway C2C12 myotubes were treated with various dosages of C2-ceramide (12.5 µM to 100 µM). At low dosage range shown in Fig 1, 12.5 µM and 25 µM have no noticeable effects on Akt Ser473. Only with high concentration of C2-ceramide such as 50 µM and 100 µM can cause markedly reduction in insulin-induced Akt Ser 473 phosphorylation. On the other hand, no matter which concentration of C2-ceramide was used, insulin stimulated tyrosine phosphorylation of IRS1 was not altered. When the effect of C2-cermide was compared with the one of palmitate, the survey of signaling molecules was shown in Fig 2. Both palmitate and ceramide induced a decrease of insulin-stimulated Akt Ser473 phosphorylation. However, phosphorylation of Tyr residues in IRS1 is decreased in palmitate-treated cells but not in ceramide-treated cells. In order to further verify the effect of these lipids on the upstream molecules of insulin signaling pathway, the negatively regulative residues, Ser307 of IRS1, a site phosphorylated by PKC and demonstrated to inhibit IRS1 activity were examined. As shown in Fig 2B, these sites were increased in palmitate treated groups and unchanged in ceramide treated groups. More signaling molecules including MAPK and S6K were examined. Within these results, the increase of S6K phosphorylation in ceramide treated cells is particular obvious in all the signaling molecules examined. A statistical quantization from three independent experiments 20 .

(24)   . is demonstrated in Fig 2C.. Caspase-3 is induced by C2-ceramide treatment Several evidences have been reported that accumulation of fatty acid or fatty acid derivates, like palmitate and ceramide in skeletal muscle could induce apoptosis through activate caspase-9 and caspase-3 (Turpin et al., 2006). Therefore, we examined the effects of palmitate and ceramide under the designed experimental condition in Fig 3. Treatment of high concentration of palmitate (1.2 mM) for 16 h could promote the cleavage form of caspase-3 significantly (lane 4 versus lane 2). On the other hands, C2C12 myotubes treated with 100 µM C2-ceramide for 2 h modestly increase the casepase-3 cleavage.. The decreased of Akt Ser473 phosphoylation might mediate through the activated S6K As experiments progress, we keep noticing that increased phosphorylation of S6K Thr389 was always accompanied by the decrease of Akt Ser473 phosphorylation. In addition, it is recently shown that mTORC2 promotes Akt phosphorylation could be negatively regulated by mTORC1 signaling (Julien et al., 2010). Therefore, we explored whether C2-ceramide-decreased Akt Ser473 phosphorylation induced by insulin mediated through such a mTORC1/mTORC2 crosstalk. Rapamycin, an mTORC1 inhibitor, was employed to verify the relationship of cause/ effect. As shown in Fig 4A, treatment of C2-ceramide caused decrease of 21 .

(25)   . insulin-stimulated Akt phsphorylation at Ser473 by which it accompanied with increase of S6K Thr389 phosphorylation (lane 3 versus lane 4). When C2C12 myoblasts were pretreated with 1 µM rapamycin for 30 min, the result demonstrated that rapamycin could relieve the inhibitory effect of C2-ceramide on insulin-stimulated phosphorylation of Akt Ser473 (lane 4 versus lane 6). This indicated S6K could play a role in the pathway of C2-ceramide- impaired insulin signaling pathway. A quantitative result from three experiments have been plotted and statistically presented in Fig 4B.. Mechanism of how C2-ceramide affects insulin-stimulated Akt Ser473 phosphorylation is mapped to be in the pathway between PI3K and Rheb molecule. According to the current model of pathway involved in mTORC1 signaling, we further examine whether C2-ceramide act on upstream molecules of S6K, such as mTORC1, Rheb and TSC1/TSC2 complex. First of all, two distinct Rheb shRNA targeting mouse Rheb sequences (Rheb C1 and Rheb E1) were chosen and found that both shRNA were functional (Fig 5A). As shown in Fig 5B, the result demonstrated that knockdown of Rheb protein can also relieve the decreased of insulin-stimulated Akt Ser473 phosphorylation caused by C2-ceramide (Fig 5B, lane 12 versus lane 8, 10). A statistic result was plotted in Fig 5C. This demonstrated that Rheb, an upstream molecule of mTORC1, plays a role in the mechanism of C2-ceramide impaired insulin signaling pathway. 22 .

(26)   . Next, we examined whether more upstream molecules, TSC1/TSC2, play a role in the abovementioned mechanism. Several studies have demonstrated that endogenous TSC2 knockdown can enhance mTORC1 activity to activate S6K, by which it could cause a feedback loop to inhibit Akt signaling (Huang et al., 2008). As shown in Fig 6, TSC2 knockdown does not change insulin-stimulated Akt Ser473 phosphorylation (lane 11 versus lane 9 and lane 7). Comparing to luciferase knockdown in C2C12 myoblasts, TSC2 knockdown does not altered phosphorylation of Akt Ser473 in C2-ceramide treatment (lane 12 versus lane 10). Three experiments were quantitated and statistically plotted as Fig 6B.. 23 .

(27)   . Discussion Previous studies have shown that the decrease of Akt phosphorylation caused by C2-ceramide could mediate through PKCζ and PP2A (Bourbon et al., 2000, Powell et al., 2003, Blouin et al., 2010). In present studies, we proposed a novel mechanism of how C2-ceramide impaired insulin signaling. C2C12 cells treated with 100 µM C2-ceramide could cause phosphorylation of Akt Ser473 decreased and always accompanied by the increase of S6K Thr389 phosphorylation. Utilizing rapamycin to block mTORC1 activated S6K, we demonstrated that the decrease of Akt Ser473 phosphorylation might be mediated through the S6K. Utilizing shRNA to stably knockdown Rheb, the results suggested that the signaling molecule(s) between PI3K and Rheb is (are) involved in C2-ceramide-impaired insulin signaling pathway. Hence, it is plausible that C2-ceramide might mediate more than one pathway to affect insulin signaling. Ceramide has several important physiological functions to regulate cellular homeostatsis. In addition to paticipitating the metabolism and membrane structure formation, it also can be an important siganling molecule to regulate intracelular signaling transduction. However, there are still dramatic differences between short-chain ceramide and natural ceramide in terms of physical properties. Nature or long-chain ceramide is not water soluble and they have difficulty to pass the lipid bilayer into cytoplasm. Comparing to nature ceramide, short-chain ceramide like C2-ceramide or C6-ceramide are water soluble and membrane permeable. C2-ceramide can disorder membrane stucture and lead to lipid packing 24 .

(28)   . disrupt. For this reason, C2-ceramide have different lateral distribution in the membrane. On the other hand, C2-ceramide precents its amohiphilic nature, can leave to plasma membrane and translocate to cytosol or other membrane of organelles inside the cell. Therefore, C2-ceramide can react with its target protein more rapidly and effectly in the cells (van Blitterswijk et al., 2003). Perhaps due to its mutiple effects on cell biology, it’s reasonable that C2-ceramide could lead to insulin signaling through different mechanisms. Previos studies have shown that ceramide can induce apoptosis through inhibiting Akt signaling then consequently activating caspase-9 and caspase-3. In our experiments, C2C12 myoblasts treated with C2-ceramide only for 2 h. Thus, it may be about to enter the early apoptotic stage. The decrease of Akt Ser473 phosphorylation caused by C2-ceramide seems to have nothing to do with caspase activation at that time point compared to the one seen in palmitate-treated myoblasts. According to this result, we hypothesized that the decrease of Akt Ser473 phosphorylation induced by C2-ceramide was not caused by apoptosis. According to Fig 6, TSC2 knockdown does not alter insulinstimulated Akt Ser473 phosphorylation. There are some possible explanations to interpret it. First of all, since the TSC2 knockdown in C2C12 myoblasts were not complete, the feedback inhibition of Akt Ser473 phosphorylation caused by mTORC1activated S6K as reported on literatures was not observed. Therefore, C2-ceramide may still have the ability to inhibit the Akt phosphorylation through the remnant TSC2. To clearly confirm the proper role of TSC2 could play, we will propose to 25 .

(29)   . utilize TSC2 knockout MEF transfect with TSC2 protein to observe the effect of C2-ceramide on Akt Ser473 phosphorylation. Alternatively, the distribution of TSC2 after C2-ceramide treatment will be examined. We initially hypothesize that C2-ceramide may suppress the insulin signaling pathway by directly affecting mTORC2 kinase activity or the composition of mTORC2 complex. We utilized mTORC2 kinase assay and pGEX-FLAG-hAKT and pGEX-FLAG-∆PH-hAKT as the substrates to test our hypothesis. Due to the technique problems, we temporarily can not progress further. On the other hand, immunoprecipitation with anti-rictor antibody from lysate of C2C12 cells, we found that the abundance of mTOR associated with rictor does not altered in C2-ceramide treatment (data not show). Therefore, the hypothesis of the defect between PI3K and Rheb after C2-ceramide treatment is prevailing. How can mTORC1 cross talk mTORC2? We found the decrease of insulin-stimulated Akt Ser473 phosphorylation caused by C2-ceramide could result from the C2-ceramide-induced S6K activation. As previously studies have published that mTORC1-activated S6K1 phosphorylates rictor on threonine 1135 and regulates mTORC2 signaling (Julien et al., 2010). It is possible that mTORC1 could cross talk with mTORC2 signaling in current C2-ceramide treatment condition. Either employe rapamycin or Rheb shRNA, the inhibitory effect of C2-ceramide on insulin-stimulated Akt Ser473 phosphorylation could be relieved. Based on these results, C2-ceramide inhibit phosphorylation of Akt Ser473 could be indeed mediated through the activation of the Rheb/mTORC1/S6K pathway to negatively regulate mTORC2 signaling transduction. 26 .

(30)   . In current content, we hypothesized the novel pathway of C2-ceramide could mediate through the molecule between PI3K and Rheb to inhibit Akt Ser473 phosphorylation. We will further explore the molecule which C2-ceramide directly acts on to impair insulin pathway. Completely elucidating this pathophysiological state might eventually provide a clue for interference of metabolite-related diseases such as diabetes and metabolite syndrome. .. 27 .

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(38)   . Nat Cell Biol 5:578-581. . 35 .

(39)   . Figure 1. . . 36 .

(40)   . Figure 1. The effect of different C2-ceramide dosage on insulin signaling transduction. C2C12 myotubes was treated with various dosage of C2-ceramide, 12.5 µM, 25 µM, 50 µM, 100 µM for 2 h followed by 100 nM insulin for 15 min before being harvested. Cell lysates were processed as “Materials and Methods” and Western blots were peformed using with different antibodies as indicated. Actin was included as a loading control. The representative of one experiment which has been repeated two times. . 37 .

(41)   . Figure 2.. A. . B. . 38 .

(42)   . C. D. 39 .

(43)   . Figure 2. The effect of both C2-ceramide and palmitate on insulin signaling molecules. (A) and (B), C2C12 myotubes were preincubated various dosage of palmitate for 16 h or C2-ceramide for 2 h, followed by 100 nM insulin for 15 min before being harvested. Cell lysates were processed as “Materials and Methods” and Western blots were peformed using different antibodies as indicated. Anisomycin was included as a positive control for antibody of phospho-Ser307 IRS1. The representative of one experiment which has been repeated three times. (C) and (D), key signaling molecules such as phosphorylation of Akt Ser 473, S6K Thr389 were quantited and using freeware NIH Image J. Y-axis represents persentage of insulin stimulaton which was set 100% A.U., arbituary unit. Data are shown as mean ± S.E (n＝3). * compared with the group of 100 nM insulin treatment.. . 40 .

(44)   . Figure 3..                      . . 41 .

(45)   . Figure 3. The effect of both C2-ceramide and palmitate on caspase-3 activity. C2C12 myotubes were preincubated with various dosage of palmitate for 16 h or C2-ceramide for 2 h, followed by 100 nM insulin for 15 min before being harvested. Cell lysates were processed as “Materials and Method” and Western blots were peformed using caspase-3 and pro-caspase 3 antibodies. Cyclohexamide (CHX) was included as a positive control for caspase-3 antibody. These blots are representative of one experiment repeat two times.. . 42 .

(46)   . Figure 4.. A. 43 .

(47)   . B. C. 44 .

(48)   . Figure 4. The decreased Akt Ser473 could mediate through the activated S6K. (A) C2C12 myotubes were preincubated with 1 µM rapamycin for 30 min, then 100 µM C2-ceramide for 2 h, follwed by 100 nM insulin treatment for 15 min before being harvested. Cell lysates were processed as “Materials and Methods” and Western blots were peformed using different antibodies as indicated. The representative of one experiment which has been repeated three times. (B) and (C), Akt Ser473, S6K Thr389 were quantited and using freeware NIH Image J. Y-axis represents persentage of insulin stimulaton which was set 100% A.U., arbituary unit. Data are shown as mean ± S.E (n＝3). * compared with the group of 100 nM insulin treatment.. . 45 .

(49)   . Figure 5. A. B. 46 .

(50)   . C. D. . 47 .

(51)   . Figure 5. The decreased phosphorylation of Akt Ser473 could mediate through up regulation of the upstream molecules of S6K, Rheb. (A) Wild type, luciferase, and Rheb shRNA (two clones). C2C12 myoblasts were examined for the efficiency of Rheb protein knockdown. (B) The lysate of C2C12 myoblast after treatment indicated on the figure were processed as “Materials and methods” and Western blots were performed using different antibodies as indicated. The representative of one experiment which has been repeated three times. (C) and (D), Akt Ser473, S6K Thr389 were quantited and using freeware NIH Image J. Y-axis represents persentage of insulin stimulaton which was set 100% A.U., arbituary unit. Data are shown as mean ± S.E (n＝3). * compared with the group of 100 nM insulin treatment.. . 48 .

(52)   . Figure 6. A. 49 .

(53)   . C. 50 .

(54)   . Figure 6. The phosphorylation of Akt Ser473 might or might not mediate through the upstream molecules of Rheb, TSC2. (A) C2C12 myoblast from wild type, luciferase, or TSC2 shRNA treatment were treated with C2-ceramidefor 2 h followed by 100 nM insulin treatment for 15min before being harvested. Cell lysates were processed as “Materials and Methods” and Western blots were performed using different antibodies as indicated. The representative of one experiment which has been repeated five times. (B) and (C), Akt Ser473, S6K Thr389 were quantited and using freeware NIH Image J. Y-axis represents persentage of insulin stimulaton which was set 100% A.U., arbituary unit. Data are shown as mean ± S.E (n＝3). * compared with the group of 100 nM insulin treatment.. . 51 .

(55)   . Figure 7.. . 52 .

(56)   . Figure 7. Propose mechanism of C2-ceramide decreased insulin-stimulated Akt Ser473 phosphorylation. C2C12 myoblasts treated with C2-ceramide for 2 h make a decrease of insulin-stimulated Akt Ser473 phosphorylation and enhanced phosphorylation of S6K Thr389 simultaneously. The decreased of AKT Ser473 phosphorylation can mediate through the activated Rheb/mTORC1/S6K pathway or direct act on their upstream molecule, TSC2 to phosphorylate rictor on threonine 1135 to regulate mTORC2 signaling.. 53 .

(57)   . Appendix. Antibodies used in study Antibody. Brand. Source. Catalog. Titer. Number pS473 AKT. Epitomics. Rabbit. 2118-S. 1:1000. AKT. Epitomics. Rabbit. 1085-S. 1:8000. pT389 S6K. Cell Signaling Rabbit. 9205. 1:1000. Epitomics. Rabbit. 1175-1. 1:2000. S6K. Santa Cruz. Rabbit. sc-230. 0.2µg/λ. Cleaved Caspase-3. Cell Signaling Rabbit. 9661. 1:1000. Pro-caspase-3. Santa Cruz. Rabbit. sc-7148. 0.2µg/λ. pTyr IRS1. Milipore. mouse. 05-321X 1mg/ml. pS307 IRS1. Milipore. Rabbit. 07-247. 1:600. IRS1. Santa Cruz. Rabbit. Sc-560. 1:400. pT202/204 MAPK. Epitomics. Rabbit. 2219-1. 1:1000. MAPK. Cell Signaling Rabbit. 9212. 1:1000. mTOR. -. Rabbit. -. 0.2µg/λ. Rictor. -. Rabbit. -. 0.1µg/λ. Raptor. -. Rabbit. -. 0.4µg/λ. pT1135 Rictor. Cell Signaling Rabbit. 3806. 1:1000. TSC2/Tuberin. Epitomics. rabbit. 1613-S. 1:1000. Sigma. mouse. A5441. 1:2000. (C-term) Actin. 54 .

(58)