and C.L.; writingoriginal draft, J.G., B.F., J.C., V.B., and C.L.; writingreview and editing, J.G., T.E., C.B., C.R., Schisandrin A R.L.G., B.F., O.L., J.C., V.B., and C.L. were higher in adipose tissue of SIV-infected macaques than in control macaques, indicating adipose tissue senescence. Tat and Nef induced higher senescence in ASCs, Schisandrin A characterized by higher levels of senescence-associated beta-galactosidase activity, p16 expression, and p53 activation vs. control cells. Treatment with Tat and Nef also induced oxidative stress and mitochondrial dysfunction. Prevention of oxidative stress (using N-acetyl-cysteine) reduced senescence in ASCs. Adipocytes having differentiated from Nef-treated ASCs displayed alterations in adipogenesis with lower levels of triglyceride accumulation and adipocyte marker expression and secretion, and insulin resistance. Conclusion: HIV/SIV promotes adipose tissue senescence, which in turn may alter adipocyte function and contribute to insulin resistance. (CEA, Fontenay-aux-Roses, France; CEA Permit Number A 92-032-02). The CEA animal facilities comply with the Standards for Human Care and Use of Laboratory of the Office for Laboratory Animal Welfare (OLAW, USA, assurance number #A5826-01) and with the European Directive Schisandrin A (2010/63, recommendation No. 9). The study was authorized by the local animal care and use committee (no. 44: Reference: 2015102713323361.02, APAFIS#2453) and the French Ministry of Research (= 0.0009) and of adipose tissue localization (= 0.05) for p16 expression. Thus, according to our results, the greater expression of p16 in VAT, suggests that VAT displays a higher aging phenotype. Moreover, p16 level and p53 activation in SCAT or VAT did not correlate with viral load, suggesting that the level of senescence was not linked to the severity of SIV infection. These results indicate that adipose tissue was more senescent in infected macaques and strongly suggest that SIV per se accentuates the aging of adipose tissue. Open in a separate window Figure 1 SIV infection of macaques was associated with higher expression of p16 and greater activation of p53 in the adipose tissue. Whole-tissue proteins were extracted from subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) from chronically infected macaques and controls and then analyzed by immunoblotting. (A) Representative immunoblots of p16, phosphorylated-p53, p53, and tubulin (loading control) are shown. (B) Densitometry analyses against tubulin as loading control were performed for p16 and p53 activation, and expressed as a mean SEM. Experiments were performed using SCAT and VAT from macaques from three control uninfected macaques (Ctrl) and four SIV-infected macaques (SIV+). * 0.05, ** 0.01 vs. noninfected macaques. 3.2. Tat- and Nef-Induced Cell Senescence in ASCs 3.2.1. Treatment of ASCs with Tat and Nef Resulted in a Lower Proliferative Capacity and Higher Levels of Senescence Markers Next, we looked at whether the HIV proteins Tat and Nef could induce senescence in ASCs. To this end, we first determined the impact of up to 30 days of exposure to Tat or Nef on cell proliferation in vitro. We found that Tat and Nef lowered the ASCs proliferation rate. This effect was seen after 15 days, and the low proliferation rate fell further with each cell passage (Figure 2A), when compared with nontreated cells. After 20 days of treatment, the cumulative PDL was significantly lower in ASCs treated with Tat or Nef than in nontreated cells. On day 15, the two Mouse monoclonal to CHUK HIV proteins enhanced senescence in ASCs, as characterized by a higher senescent cell Schisandrin A count (based on the SA–galactosidase activity). The percentage of senescent cells was 15.6 1.3% and 19.3 2.1% for Tat- and Nef-treated cells respectively, vs. 10.4 1.1% for control cells (Figure 2B). Furthermore, treatment with the HIV proteins was associated with greater lysosome accumulation (Figure 2C). Lastly, the expression of the cell cycle arrest proteins p16 and the level of p53 activation were higher after 15 days of Tat and Nef treatment, relative to controls (Figure 2D). Tat- or Nef-treated ASCs displayed signs of SASP, with greater secretion of the pro-inflammatory cytokines IL-6 and IL-8 (Figure 3A,B). Taken as a whole, these data indicated that treatment with the HIV protein Tat or Nef induced the cellular senescence in ASCs. In general, Nef had a greater effect than Tat on the induction of senescence and the secretion of inflammatory proteins. Open in a separate window Figure 2 Trans-activator of transcription (Tat) and negative-regulating factor (Nef) proteins induce cell senescence in adipose stem cells (ASCs). ASCs, isolated from different Schisandrin A abdominal SCAT healthy donors, were cultured with the HIV proteins Tat or Nef for 30 days. (A) Calculation of the cumulative population doubling level (PDL) is described in the.This effect was seen after 15 days, and the low proliferation rate fell further with each cell passage (Figure 2A), when compared with nontreated cells. of SIV-infected macaques and in human adipose stem cells (ASCs) exposed to Tat or Nef for up to 30 days. Results: p16 expression and p53 activation were higher in adipose tissue of SIV-infected macaques than in control macaques, indicating adipose tissue senescence. Tat and Nef induced higher senescence in ASCs, characterized by higher levels of senescence-associated beta-galactosidase activity, p16 expression, and p53 activation vs. control cells. Treatment with Tat and Nef also induced oxidative stress and mitochondrial dysfunction. Prevention of oxidative stress (using N-acetyl-cysteine) reduced senescence in ASCs. Adipocytes having differentiated from Nef-treated ASCs displayed alterations in adipogenesis with lower levels of triglyceride accumulation and adipocyte marker expression and secretion, and insulin resistance. Conclusion: HIV/SIV promotes adipose tissue senescence, which in turn may alter adipocyte function and contribute to insulin resistance. (CEA, Fontenay-aux-Roses, France; CEA Permit Number A 92-032-02). The CEA animal facilities comply with the Standards for Human Care and Use of Laboratory of the Office for Laboratory Animal Welfare (OLAW, USA, assurance number #A5826-01) and with the European Directive (2010/63, recommendation No. 9). The study was authorized by the local animal care and use committee (no. 44: Reference: 2015102713323361.02, APAFIS#2453) and the French Ministry of Research (= 0.0009) and of adipose tissue localization (= 0.05) for p16 expression. Thus, according to our results, the greater expression of p16 in VAT, suggests that VAT displays a higher aging phenotype. Moreover, p16 level and p53 activation in SCAT or VAT did not correlate with viral load, suggesting that the level of senescence was not linked to the severity of SIV infection. These results indicate that adipose tissue was more senescent in infected macaques and strongly suggest that SIV per se accentuates the aging of adipose tissue. Open in a separate window Figure 1 SIV infection of macaques was associated with higher expression of p16 and greater activation of p53 in the adipose tissue. Whole-tissue proteins were extracted from subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) from chronically infected macaques and controls and then analyzed by immunoblotting. (A) Representative immunoblots of p16, phosphorylated-p53, p53, and tubulin (loading control) are shown. (B) Densitometry analyses against tubulin as loading control were performed for p16 and p53 activation, and expressed as a mean SEM. Experiments were performed using SCAT and VAT from macaques from three control uninfected macaques (Ctrl) and four SIV-infected macaques (SIV+). * 0.05, ** 0.01 vs. noninfected macaques. 3.2. Tat- and Nef-Induced Cell Senescence in ASCs 3.2.1. Treatment of ASCs with Tat and Nef Resulted in a Lower Proliferative Capacity and Higher Levels of Senescence Markers Next, we looked at whether the HIV proteins Tat and Nef could induce senescence in ASCs. To this end, we first determined the impact of up to 30 days of exposure to Tat or Nef on cell proliferation in vitro. We found that Tat and Nef lowered the ASCs proliferation rate. This effect was seen after 15 days, and the low proliferation rate fell further with each cell passage (Figure 2A), when compared with nontreated cells. After 20 days of treatment, the cumulative PDL was significantly low in ASCs treated with Tat or Nef than in nontreated cells. On time 15, both HIV protein improved senescence in ASCs, as seen as a an increased senescent cell count number (predicated on the SA–galactosidase activity). The percentage of senescent cells was 15.6 1.3% and 19.3 2.1% for Tat- and Nef-treated cells respectively, vs. 10.4 1.1% for control cells (Amount 2B). Furthermore, treatment using the HIV protein was connected with better lysosome deposition (Amount 2C). Finally, the appearance from the cell routine arrest protein p16 and the amount of p53 activation had been higher after 15 times of Tat and Nef treatment, in accordance with controls (Amount 2D). Tat- or Nef-treated ASCs shown signals of SASP, with better secretion from the pro-inflammatory cytokines IL-6 and IL-8 (Amount 3A,B). As a whole, these data indicated that treatment using the HIV proteins Tat or Nef induced the mobile senescence in ASCs. Generally, Nef had a larger impact than Tat over the induction of senescence as well as the secretion of inflammatory proteins. Open up in another window Amount 2 Trans-activator of transcription (Tat) and negative-regulating aspect (Nef) protein induce cell senescence in adipose stem cells.
