PEGylated AdipoRon derivatives improve glucose and lipid metabolism under insulinopenic and high fat diet conditions

3. IntroductionSedentary lifestyle(Biswas A. Oh P.I. Faulkner G.E. Bajaj R.R. Silver M.A. Mitchell M.S. Alter D.A. Sedentary time and its association with risk for disease incidence, mortality, and hospitalization in adults: a systematic review and meta-analysis.) and high-fat diet(Dietary fat intake does affect obesity!., Emerging paradigms for understanding fatness and diabetes risk.) result in increased adiposity, associated with insulin resistance, hyperglycemia and an increased risk of type 2 diabetes. The pathogenesis of this disease is further augmented by glucolipotoxicity-induced apoptosis of pancreatic β-cells and inadequate production or secretion of insulin.(Lipotoxicity of beta-cells in obesity and in other causes of fatty acid spillover., beta-cell dysfunction and failure in type 2 diabetes: potential mechanisms.) The adipocyte-derived protein hormone, adiponectin, counteracts fatty acid and cytokine-induced apoptosis in the pancreatic β-cell line INS-1,(Rakatzi I. Mueller H. Ritzeler O. Tennagels N. Eckel J. Adiponectin counteracts cytokine- and fatty acid-induced apoptosis in the pancreatic beta-cell line INS-1.) and protects MIN6 cells and mouse islets from serum-starvation-induced apoptosis.(Wijesekara N. Krishnamurthy M. Bhattacharjee A. Suhail A. Sweeney G. Wheeler M.B. Adiponectin-induced ERK and Akt Phosphorylation Protects against Pancreatic Beta Cell Apoptosis and Increases Insulin Gene Expression and Secretion.) Adiponectin levels are reduced as obesity progresses and adipocytes become dysfunctional.(beta-cell dysfunction and failure in type 2 diabetes: potential mechanisms., Dahlman I. Elsen M. Tennagels N. Korn M. Brockmann B. Sell H. Eckel J. Arner P. Functional annotation of the human fat cell secretome.) Interestingly, adiponectin was shown to maintain β-cell mass and glucose homeostasis in ob/ob mice when a 2-3 fold overexpression of adiponectin was induced.(Kim J.-Y. van de Wall E. Laplante M. Azzara A. Trujillo M.E. Hofmann S.M. Schraw T. Durand J.L. Li H. Li G. Jelicks L.A. Mehler M.F. Hui D.Y. Deshaies Y. Shulman G.I. Schwartz G.J. Scherer P.E. Obesity-associated improvements in metabolic profile through expansion of adipose tissue.) Adiponectin is therefore regarded as a promising antidiabetic adipokine.Adiponectin receptor1 (AdipoR1) and Adiponectin receptor2 (AdipoR2) are the main receptors of adiponectin through which molecular and cellular actions take place, with downstream effectors including ceramidase and sphingolipids, AMP-activated protein kinase (AMPK), Peroxisome proliferator-activated receptor (PPAR) alpha (PPARα, PPAR gamma (PPARγ) and additional components.(Yamauchi T. Kamon J. Ito Y. Tsuchida A. Yokomizo T. Kita S. Sugiyama T. Miyagishi M. Hara K. Tsunoda M. Murakami K. Ohteki T. Uchida S. Takekawa S. Waki H. Tsuno N.H. Shibata Y. Terauchi Y. Froguel P. Tobe K. Koyasu S. Taira K. Kitamura T. Shimizu T. Nagai R. Kadowaki T. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects., Adiponectin Receptor as a Key Player in Healthy Longevity and Obesity-Related Diseases.) Overexpression and siRNA knockdown of AdipoR1 and AdipoR2 revealed their important roles in cellular binding of adiponectin, as well as the downstream signaling events involving AMPK and PPARα. Many studies point to metabolically beneficial effects of both AdipoR1/2. We previously defined the very potent ceramide lowering effects induced by adiponectin signaling through its receptors.(Holland W.L. Miller R.A. Wang Z.V. Sun K. Barth B.M. Bui H.H. Davis K.E. Bikman B.T. Halberg N. Rutkowski J.M. Wade M.R. Tenorio V.M. Kuo M.-S. Brozinick J.T. Zhang B.B. Birnbaum M.J. Summers S.A. Scherer P.E. Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin.) These effects on sphingolipid concentrations can explain most physiological and cell-signaling effects, such as the insulin sensitizing, anti-inflammatory and anti-apoptotic actions attributed to adiponectin. Evidence is emerging for the pleiotropic roles of ceramides and the other sphingolipids in the development of insulin resistance and other metabolic disorders.(

Chavez, Jose A., and Scott A. Summers. A Ceramide-Centric View of Insulin Resistance. Cell Metabolism 15: 585-594.

) More recent data showed that adiponectin was essential to maintain minimal lipid homeostasis under insulinopenic conditions to improve local lipid metabolism in the islets,(Ye R. Holland W.L. Gordillo R. Wang M. Wang Q.A. Shao M. Morley T.S. Gupta R.K. Stahl A. Scherer P.E. Adiponectin is essential for lipid homeostasis and survival under insulin deficiency and promotes β-cell regeneration.) and the process acts as an important anti-lipotoxic phenomenon to boost β-cell regeneration primarily mediated by adiponectin’s action, at least in part, on the β-cells directly.(Ye R. Wang M. Wang Q.A. Scherer P.E. Adiponectin-Mediated Antilipotoxic Effects in Regenerating Pancreatic Islets.) This is consistent with the direct action of adiponectin on β-cells as both AdipoR1 and AdipoR2 are abundantly expressed in islet β-cells.(Kharroubi I. Rasschaert J. Eizirik D.L. Cnop M. Expression of adiponectin receptors in pancreatic β cells., Segerstolpe A. Palasantza A. Eliasson P. Andersson E.M. Andreasson A.C. Sun X. Picelli S. Sabirsh A. Clausen M. Bjursell M.K. Smith D.M. Kasper M. Ammala C. Sandberg R. Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes.)In addition to its pleiotropic protective effects on obesity-related metabolic disorders, adiponectin elicits a central role in the cold-induced browning of subcutaneous white adipose tissue (scWAT), contributing to reduced obesity in mice.(Hui X. Gu P. Zhang J. Nie T. Pan Y. Wu D. Feng T. Zhong C. Wang Y. Lam K.S. Xu A. Adiponectin Enhances Cold-Induced Browning of Subcutaneous Adipose Tissue via Promoting M2 Macrophage Proliferation., Cohen P. Levy J.D. Zhang Y. Frontini A. Kolodin D.P. Svensson K.J. Lo J.C. Zeng X. Ye L. Khandekar M.J. Wu J. Gunawardana S.C. Banks A.S. Camporez J.P. Jurczak M.J. Kajimura S. Piston D.W. Mathis D. Cinti S. Shulman G.I. Seale P. Spiegelman B.M. Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch.) Upon chronic cold exposure, adiponectin is sequestered toward the stromal vascular fraction (SVF) of scWAT, where adiponectin is predominately tethered to M2 macrophages in a T-cadherin/Akt-dependent manner. This results in the proliferation and accumulation of M2 macrophages in the scWAT. This accumulation is accompanied by upregulated expression of tyrosine hydroxylase for production of catecholamine, a key factor required for browning of WAT.(Hui X. Gu P. Zhang J. Nie T. Pan Y. Wu D. Feng T. Zhong C. Wang Y. Lam K.S. Xu A. Adiponectin Enhances Cold-Induced Browning of Subcutaneous Adipose Tissue via Promoting M2 Macrophage Proliferation.) However, a more recent study indicated that M2 macrophages do not synthesize relevant amounts of catecholamines, deeming them unlikely to have a direct role in adaptive thermogenesis.(Fischer K. Ruiz H.H. Jhun K. Finan B. Oberlin D.J. van der Heide V. Kalinovich A.V. Petrovic N. Wolf Y. Clemmensen C. Shin A.C. Divanovic S. Brombacher F. Glasmacher E. Keipert S. Jastroch M. Nagler J. Schramm K.W. Medrikova D. Collden G. Woods S.C. Herzig S. Homann D. Jung S. Nedergaard J. Cannon B. Tschop M.H. Muller T.D. Buettner C. Alternatively activated macrophages do not synthesize catecholamines or contribute to adipose tissue adaptive thermogenesis.) Regardless of whether M2 macrophages are relevant local thermogenic efferent during cold-induced browning in scWAT, hypoadiponectinemia was shown to result in a significant reduction in energy expenditure.(Adiponectin and energy homeostasis.)Various factors prevent the use of adiponectin as an attractive treatment for obesity-related disorders. Injection of recombinant adiponectin, though therapeutically effective, is not attractive due to its relatively high plasma concentrations, short half-life and its complex quaternary structure, which requires proper post-translational modifications for multimerization.(Pajvani U.B. Hawkins M. Combs T.P. Rajala M.W. Doebber T. Berger J.P. Wagner J.A. Wu M. Knopps A. Xiang A.H. Utzschneider K.M. Kahn S.E. Olefsky J.M. Buchanan T.A. Scherer P.E. Complex Distribution, Not Absolute Amount of Adiponectin, Correlates with Thiazolidinedione-mediated Improvement in Insulin Sensitivity.) Interestingly, recent efforts by the Kadowaki and colleagues have identified an orally active low molecular weight compound known as AdipoRon that activates AdipoR1 and AdipoR2 as well as the downstream AMPK and PPARγ pathways in liver and skeletal muscles.(Okada-Iwabu M. Yamauchi T. Iwabu M. Honma T. Hamagami K.-i. Matsuda K. Yamaguchi M. Tanabe H. Kimura-Someya T. Shirouzu M. Ogata H. Tokuyama K. Ueki K. Nagano T. Tanaka A. Yokoyama S. Kadowaki T. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity.) Several studies have investigated the metabolic actions of AdipoRon as well as the associated protective roles against liver inflammation and fibrosis,(Sha M. Gao Y. Deng C. Wan Y. Zhuang Y. Hu X. Wang Y. Therapeutic effects of AdipoRon on liver inflammation and fibrosis induced by CCl4 in mice., Adiponectin receptor agonist AdipoRon relieves endotoxin-induced acute hepatitis in mice.) diabetic nephropathy and kidney disease,(Choi S.R. Lim J.H. Kim M.Y. Kim E.N. Kim Y. Choi B.S. Kim Y.S. Kim H.W. Lim K.M. Kim M.J. Park C.W. Adiponectin receptor agonist AdipoRon decreased ceramide, and lipotoxicity, and ameliorated diabetic nephropathy., Kim Y. Lim J.H. Kim M.Y. Kim E.N. Yoon H.E. Shin S.J. Choi B.S. Kim Y.S. Chang Y.S. Park C.W. The Adiponectin Receptor Agonist AdipoRon Ameliorates Diabetic Nephropathy in a Model of Type 2 Diabetes.) atherosclerosis, myocardial ischemia/reperfusion,(Zhang Y. Zhao J. Li R. Lau W.B. Yuan Y.X. Liang B. Li R. Gao E.H. Koch W.J. Ma X.L. Wang Y.J. AdipoRon, the first orally active adiponectin receptor activator, attenuates postischemic myocardial apoptosis through both AMPK-mediated and AMPK-independent signalings.) cardiac hypertrophy,(Hu X. Ou-Yang Q. Wang L. Li T. Xie X. Liu J. AdipoRon prevents l-thyroxine or isoproterenol-induced cardiac hypertrophy through regulating the AMPK-related pathway.) spinal cord injury,(Yu J. Zheng J. Lu J. Sun Z. Wang Z. Zhang J. AdipoRon Protects Against Secondary Brain Injury After Intracerebral Hemorrhage via Alleviating Mitochondrial Dysfunction: Possible Involvement of AdipoR1-AMPK-PGC1alpha Pathway.) corticosterone-induced depression,(Nicolas S. Debayle D. Bechade C. Maroteaux L. Gay A.S. Bayer P. Heurteaux C. Guyon A. Chabry J. Adiporon, an adiponectin receptor agonist acts as an antidepressant and metabolic regulator in a mouse model of depression.) cognitive dysfunction of Alzheimer’s disease,(Liu B. Liu J. Wang J.G. Liu C.L. Yan H.J. AdipoRon improves cognitive dysfunction of Alzheimer's disease and rescues impaired neural stem cell proliferation through AdipoR1/AMPK pathway.) and bone,(Sapio L. Nigro E. Ragone A. Salzillo A. Illiano M. Spina A. Polito R. Daniele A. Naviglio S. AdipoRon Affects Cell Cycle Progression and Inhibits Proliferation in Human Osteosarcoma Cells.) ovarian,(Ramzan A.A. Bitler B.G. Hicks D. Barner K. Qamar L. Behbakht K. Powell T. Jansson T. Wilson H. Adiponectin receptor agonist AdipoRon induces apoptotic cell death and suppresses proliferation in human ovarian cancer cells.) and pancreatic cancer.(Messaggio F. Mendonsa A.M. Castellanos J. Nagathihalli N.S. Gorden L. Merchant N.B. VanSaun M.N. Adiponectin receptor agonists inhibit leptin induced pSTAT3 and in vivo pancreatic tumor growth.)Here, we explored whether AdipoRon, upon exposure to fatty acids, can act as an exogenous modulator of β-cell responses by activating AdipoR-elicited cellular ceramidase activity.(Holland W.L. Miller R.A. Wang Z.V. Sun K. Barth B.M. Bui H.H. Davis K.E. Bikman B.T. Halberg N. Rutkowski J.M. Wade M.R. Tenorio V.M. Kuo M.-S. Brozinick J.T. Zhang B.B. Birnbaum M.J. Summers S.A. Scherer P.E. Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin., Vasiliauskaite-Brooks I. Sounier R. Rochaix P. Bellot G. Fortier M. Hoh F. De Colibus L. Bechara C. Saied E.M. Arenz C. Leyrat C. Granier S. Structural insights into adiponectin receptors suggest ceramidase activity.) Additionally, we employed a cell-based assay to determine how structural modifications may generate more potent AdipoRon derivatives that can thwart the toxic effect of palmitate on the pancreatic β-cell line INS-1. This led us to investigate the favorable effects of an AdipoRon derivative, namely, AdipoRonPEG5, on glucose and lipid homeostasis in INS-1 cells, STZ-induced insulinopenic adiponectin knockout mice and HFD-induced obese mouse models. In addition, we examined the hepatic AdipoR1/AdipoR2 expression and the downstream pathway activation in type 2 diabetic mice. The data suggests that AdipoRonPEG5 ameliorates gluco- and lipotoxicity by activating the AMPK pathway through AdipoR1, and by upregulating ceramidase expression.4. Materials and Methods MaterialsPalmitic acid (PA) was supplied by Sigma-Aldrich (P5585). The recombinant C-terminal globular domain of adiponectin (gAcrp30) was a product from Novus Biologicals (NBP199295). Bovine serum albumin (BSA, fatty-acid free) was obtained from Sigma-Aldrich (A8806). Propidium iodide (PI) for discrimination of necrotic from apoptotic cells was product of Invitrogen (P3566). RPMI Phenol Red-free 1640 medium and penicillin/streptomycin were provided by Gibco (11835055) and Invitrogen (15140163), respectively. AdipoRon and its analogues including AdipoRonPEG5 were synthesized as described in the Supplemental Information. MiceAll mice including the adiponectin knockout mouse (Nawrocki A.R. Rajala M.W. Tomas E. Pajvani U.B. Saha A.K. Trumbauer M.E. Pang Z. Chen A.S. Ruderman N.B. Chen H. Rossetti L. Scherer P.E. Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists.) were bred in the C57BL/6 genetic background. Mice were fed on regular (LabDiet #5058) or high fat (60%, Research #D12492). High-fat diet was started from 6 weeks. Mice were maintained in 12-h dark/light cycles, with ad libitum access to diet and water. All protocols for mouse use and euthanasia were reviewed and approved by the Institutional Animal Care and Use Committee. logP prediction and solubility measurements of AdipoRon and AdipoRonPEG5We computed octanol-water partition coefficients (logP) using Molinspiration, which was developed by fitting calculated logP with experimental logP for a training set of more than twelve thousand, mostly drug-like molecules (https://www.molinspiration.com). We quantified the solubilities of AdiponRons by LC-MS/MS. Briefly, water (HPLC grade) was added in 50-100 μL increments to a glass vial containing either AdipoRon (0.97 mg) or AdipoRonPEG5 (1.69 mg), vortexed until the compound started to dissolve (AdipoRon, 3100 mL; AdipoRonPEG5, 400 mL). The suspension was then stirred overnight away from light. The solution was transferred to Teflon tubes and centrifuged at 15,000 rpm at room temperature for 10 min. We then collected the supernatant (aqueous saturated solution) for dilution and analysis.

For standards, 98 microliters water were added to an Eppendorf tube and combined with 200 microliters of methanol containing 0.15% formic acid and 75 ng/ml Initial Standard (IS, n-Benzeylbenzamide in MeOH. IS final conc. = 50 ng/ml). Samples were spiked with 2 microliter of IS. The samples were vortexed 15 sec, and spun 2 x 13,200 rpm in a standard microcentrifuge. The supernatant was then analyzed by LC-MS/MS on a QTrap 4000 system (AB Sciex) with the following settings: Ion Source/Gas Parameters: CUR = 25, CAD = medium, IS = 5000, TEM = 500, GS1 = 70, GS2 = 70. Buffer A: Water + 0.1% formic acid + 2 mM NH4OAC; Buffer B: methanol + 0.1% formic acid + 2 mM NH4OAC; flow rate 1.5 ml/min; Agilent C18 XDB column, 5 micron packing 50 X 4.6 mm size. HPLC elution gradient: 0 - 0.5 min 97% A, 0.5 - 2.0 min gradient to 100% B, 2.0 - 3.5 min 100% B, 3.5 - 3.6 min gradient to 97% A, 3.6 - 4.1 97% A. Compound transition 429.156 to 174.1 for AdipoRon, and 665.289 to 135.2 for AdipoRonPEG5.

 Pharmacokinetics and liver bioavailability

Two groups of male C57BL/6J mice (21 mice/group) at 8 weeks of age were dosed IP with 20 mg/kg AdipoRon or AdipoRonPEG5, 0.2 mL/mouse formulated as 10% DMSO/10% Cremophor/20% PEG400/60% PBS. Whole blood was harvested at seven time points post IP dosing, 10 min, 30 min, 90 min, 180 min, 360 min, 960 min and 1440 min. Three mice were sacrificed at each time point. Plasma was processed from whole blood by centrifugation of the ACD treated blood for 10' at 10,000 rpm in a standard centrifuge. Additionally, liver was harvested. The tissues were weighed and snap frozen in liquid nitrogen.

To quantify plasma drug concentration and PK, we prepared standards by adding 98 microliters blank plasma (BioIVT) to an eppendorf. These were spiked with 2 microliter of initial standard. For QCs, we added 98.8 microliters blank plasma to Eppendorf, spiked with 1.2 microliters of initial standard. For samples, we mixed 100 microliters plasma with 200 microliters of MeOH containing 0.15% formic acid and 75 ng/ml IS (IS final conc. = 50 ng/ml). The samples were vortexed 15 sec, incubated at room temp for 10' and spun 2x 13,200 rpm in a standard microcentrifuge. The supernatant was then analyzed by LC-MS/MS.

To measure liver drug concentration and bioavailability, we prepared standards by adding 98 microliters blank liver homogenate to an Eppendorf, spiked with 2 microliters of initial standard. For QCs, we added 98.8 microliters blank liver homogenate to an Eppendorf, spiked with 1.2 microliters of initial standard. For samples, we mixed 100 microliters liver homogenate with 200 microliters of MeOH containing 0.15% formic acid and 75 ng/ml IS (IS final conc. = 50 ng/ml). The samples were vortexed 15 sec, incubated at room temp for 10' and spun 2x 13,200 rpm in a standard microcentrifuge. The supernatant was then analyzed by LC-MS/MS.

 Genotyping PCRApproximately 3 mm of mouse tail tip was incubated in 80 μL 50 mM NaOH at 95 °C for 1.5 h. 8 μL 1 M Tris–HCl (pH 8.0) was added for neutralization. After vortexing and a short spin down, 0.5–1 μL of supernatant was used as PCR template. Primer sequences for genotyping PCR are listed in Table S1. The PCR program was: 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s, 62 °C for 30 s, and 72 °C for 30 s, and ended with 72 °C for 3 min. qPCRTotal RNA was extracted with the RNeasy Mini kit (Qiagen #74106) for pancreas or Trizol (Invitrogen #15596018) for liver and adipose tissue. cDNA was synthesized with iScript cDNA Synthesis Kit (Bio-Rad #170-8891). Quantitative real-time PCR(qPCR) was performed with the Powerup SYBR Green PCR master mix (Applied Biosystems # A25742) on Quantistudio 6 Flex Real-Time PCR System (Applied Biosystems # 4485694). Primer sequences for qPCR are listed in Table S2. Cell culture

The rat insulinoma cell line INS-1 (passages 20–30) was grown in monolayer culture in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 500 U/ml penicillin, 50 μg/ml streptomycin, 2 mM glutamine and 50 μM 2-mercaptoethanol in 100 mm petri dishes in an atmosphere of 5% CO2 at 37°C. Subconfluent cells were maintained in continuous passages by trypsinization of cultures

7 days after plating. The medium was changed every 42 h. Cell number was determined after cell dissociation with trypsin/EDTA at 37 °C. INS-1 cells were routinely seeded at 60× 103 cells/well of a 96-well plate for cell death detection experiments at 60-70% confluence.

 In vitro beta cell stress by palmitic acid (PA) and protection with AdipoRons (PA) / BSA complex solution.

A 7.1 mM palmitic acid stock solution was prepared in 95% ethanol by incubating at 37°C. In parallel, 2% fatty acid-free BSA was solubilized in sterile nanopure water at 37°C. The palmitic acid/BSA complex solution was then prepared by incubating the components in a molar ratio of 5:1 at 37°C in a shaking water bath for 1 h and was used directly for INS-1 stimulation experiments. Control cultures were carried out in the presence of BSA/ethanol in RPMI without palmitic acid.

Preparation of INS-1 cells for sphingolipid measurement upon treatment with AdipoRon analogs.

INS-1 cells were seeded at 2 × 106/well in a 6-well plate. Cells were treated as follows for 24 h: (i) Control: No FBS or PA (0.25 mM); (ii) Positive Control: 10% FBS + PA (0.25 mM); (iii) Adiponectin (10 nM) + PA (0.25 mM); (iv) AdipoRonPEG6HTL (10 nM) + PA (0.25 mM); (v) AdipoRonPEG6HTL (1 μM) + PA (0.25 mM); (vi) AdipoRonPEG5 (1 μM) + PA (0.25 mM); (vii) AdipoRonPEG5 (20 μM) + PA (0.25 mM). Thereafter, treatment was aspirated, and the cells were washed with cold PBS x 2, scraped off with 1-2 mL PBS and spun down at 2500 rpm for 5 min. The supernatant was aspirated, and the pallet was stored at 80 °C for sphingolipid analysis.

 Quantification of beta cell death by Hoechst 33342/PI staining

Following a 2-h pre-incubation under serum-free conditions INS-1 cells were kept for 24 h in Phenol Red-free FBS-free RPMI 0.5% BSA as a control or containing 0.25 mM PA-BSA in the presence or absence of treatment. Treatment includes 10% FBS, 20 nM adiponectin, 10 μM AdipoRon, 10 μM AdipoRonPEG5, or 1.0 μM AdipoRonPEG6HTL. Cells were double stained for 10 min at 4 °C with Hoechst 33342 and propidium iodide (PI) at 10 μg/mL and 1 μg/mL, respectively. Adding Hoechst 33342/PI to Phenol Red-free cell media ensures that any floating dead cells were not lost since aspirating the treatment media is no longer necessary. Images were acquired on a confocal laser scanning microscope (Zeiss LSM 510) in the green (Ex 488 nm, Em 500-530 nm) or red channel (Ex 543 nm, Em 565-615 nm) with a ×40/1.3 objective lens connected to a confocal scanner and a workstation. Hoechst 33342/PI double staining enables the differentiation of apoptotic cells from necrotic cells. Hoechst 33342 stains the condensed chromatin in apoptotic cells more brightly than the chromatin in normal cells. PI is a red-fluorescence dye that is only permeant to dead cells.

Quantification of beta cell death by PI staining was carried out on a fluorescence plate reader. Following a 2-h pre-incubation under serum-free conditions INS-1 cells (60k cells/well, 96-well plate (COSTAR ref # 3603)) were kept for 24 h in Phenol Red-free FBS-free RPMI 0.5% BSA as a control or containing 0.25 mM PA-BSA in the presence or absence of treatment. Cells were stained for 10 min at 4 °C with propidium iodide (PI) at 1 μg/mL (added to the existing media to avoid loss of dead cells). PI fluorescence intensity was measured with a plate reader (PHERAstar, BMG Labtech).

 Systemic testsFor oral glucose tolerance test (OGTT), mice were fasted for 4–6 h and subjected to an oral gavage of dextrose (2 mg/g body weight). Tail blood was collected at 0, 15, 60, and 120 min and prepared for serum and assayed for glucose and insulin. For insulin tolerance test (ITT), insulin (0.75 U/kg Humulin R; Eli Lilly, Indianapolis, IN, USA) was administered under fed condition. Serum glucose level was measured at 0, 15, 60, and 90 minutes time point. Triglyceride tolerance test (TGTT) was initiated by oral gavage of 20% Intralipid (10 μl/g BDW, l141-100mL, Sigma), and serum was collected at 0, 1, 1.5, 3, and 6 hr for triglyceride assay. Glucose, insulin and triacylglyceride levels were measured using an oxidase-peroxidase assay (Sigma P7119), insulin ELISA (Crystal Chem, Elk Grove village, IL, USA, #15596018) and Infinity Triglycerides Reagent (Thermo Fisher Scientific TR22421). Western blotProtein was extracted from kidney tissue by homogenization in PBS supplemented with 1 mM EDTA, 20 mM NaF, 2 mM Na3VO4, and protease inhibitor cocktail. 5× RIPA buffer was added to the homogenate for a final concentration of 10 mM Tris-HCl, 2 mM EDTA, 0.3% NP40, 0.3% deoxycholate, 0.1% SDS, and 140 mM NaCl, pH 7.4. The sample was centrifuged at 10,000 g for 5 minutes. 20–50 μg/lane of supernatant protein was separated by SDS-PAGE (NP0335BOX, Thermo Fisher) and transferred to nitrocellulose membrane. The blots were then incubated overnight at 4°C with primary antibodies (1:1000) in a 1% BSA TBST-blocking solution. Primary antibodies are listed in Table S3. Primary antibodies were detected using secondary antibodies labelled with infrared dyes emitting at 700 nm or 800 nm (1:5000, Li-Cor Bioscience 925-68073 and 926-32213, respectively) The Odyssey Infrared Imager was used to visualized Western blots with Li-Cor IRdye secondary antibodies. STZ treatment

Streptozotocin (STZ; Sigma S1030) was dissolved by 0.05 M citrate buffer (final; 20mg/ml). Following 6 hours fasting, mice received a single intraperitoneal injection of streptozotocin (200 mg/kg) to induce insulinopenic conditions.

 AdipoRon derivative treatment

6-weeks old wild type male mice were fed high fat diet for 4 weeks or 7 months. 5 mg/Kg of PBS, Adiporon, AdipoRonPEG5, AdipoRonPEG6HTL were injected twice a day for five days. After 5 five days of treatment, systemic test is performed and serum (Before and after treatment), subcutaneous, epididymal, liver, heart samples were harvested for qPCR and sphingolipid analysis.

 Cold exposure

5 mg/Kg of PBS and AdipoRonPEG5 were injected twice a day for five days at room temperature followed by housing in the cold chamber (4°C) for 7days. Body temperature was measured at 0, 1, 2, 3, 4 and 5-hour timepoint at the first day of cold exposure. 5 mg/Kg of PBS and AdipoRonPEG5 was injected once a day for 7 days after cold exposure followed by harvesting tissues for qPCR analysis.

 RNA-seq

RNA-sequence was performed by Novogene (Sacramento, CA, USA) by utilizing isolated RNA. After the QC procedures, mRNA from eukaryotic organisms is enriched from total RNA using oligo(dT) beads. For prokaryotic samples, rRNA is removed using a specialized kit that leaves the mRNA. The mRNA from either eukaryotic or prokaryotic sources then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added, with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation and AMPure XP beads is used to purify the cDNA. The final cDNA library is ready after a round of purification, terminal repair, Atailing, ligation of sequencing adapters, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to I ng/gl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM). Libraries are fed into

留言 (0)

沒有登入
gif