High fat-diet and saturated fatty acid palmitate inhibits IGF-1 function in chondrocytes  S.A. Nazli, R.F. Loeser, S. Chubinskaya, J.S. Willey, R.R. Yammani  Osteoarthritis and Cartilage  Volume 25, Issue 9, Pages 1516-1521 (September 2017) DOI: 10.1016/j.joca.2017.05.011 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 High-fat diet inhibits IGF-1 mediated PG synthesis in cartilage explants. Femoral cartilage explants isolated from hip joints of mice that were fed a high fat-diet (HFD) and low fat-diet (LFD) (n = 10/group) were cultured and treated with 100 ng/ml of IGF-1 overnight. PG synthesis was measured using the [35S] sulfate incorporation method. Data indicate mean counts per minute normalized by μg DNA/replicate; error bars indicate 95% confidence interval. Data were analyzed via ANOVA (α = 0.05); the presented p values identify significant between-group differences determined by Tukey's post hoc test. Osteoarthritis and Cartilage 2017 25, 1516-1521DOI: (10.1016/j.joca.2017.05.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Palmitate induces ER stress in human chondrocytes. A) Human primary chondrocytes were stimulated overnight with 0–500 μM of the FFA palmitate and oleate and detection of CHOP was used as a measure of ER stress. B) In other experiments, cells were pretreated with or without 1 mM PBA for 30 min followed by overnight treatment with palmitate or oleate (500 μM) or C) 2 μM of TG. After incubation, cell lysates were immunoblotted with antibodies to C/EBP homologous protein (CHOP). Blots were stripped and reprobed with GAPDH as a loading control. Data are representative of three independent experiments performed with cells obtained from different biological donors. Osteoarthritis and Cartilage 2017 25, 1516-1521DOI: (10.1016/j.joca.2017.05.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Palmitate inhibits IGF-1 signaling. A. Chondrocytes were pretreated with or without 1 mM PBA or 500 μM of FFA (palmitate or oleate) for 24 h followed by 50 ng/ml IGF-1 for 30 min. B. Cells were pretreated with or with PBA or 2 μM of TG for 24 h followed by 50 ng/ml IGF-1 for 30 min. Cell lysates were immunoblotted with phosphospecific antibodies to IRS-1, AKT, and CHOP. In some experiments, blots were striped and reprobed with GAPDH for loading control. Data are representative of three independent experiments performed with cells obtained from different biological donors. Osteoarthritis and Cartilage 2017 25, 1516-1521DOI: (10.1016/j.joca.2017.05.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Palmitate inhibits IGF-1-stimulated PG synthesis. (A) Normal human chondrocytes were pretreated with or without PBA or FFA (palmitate or oleate) overnight, followed by 24-hour stimulation with 50 ng/ml IGF-1. (B) Cells were stimulated with or without PBA or 2 μM of TG overnight, followed by 24 h treatment with 100 ng/ml. PG synthesis was measured using [35S] sulfate incorporation and DNA was analyzed with a PicoGreen assay. Data indicate mean counts per minute normalized by μg DNA/replicate; error bars represents mean ± standard deviation of n = 3 independent experiments performed with cells obtained from different donors. Data were analyzed via ANOVA (α = 0.05); the presented p values identify significant between-group differences determined by Tukey's post hoc test. Osteoarthritis and Cartilage 2017 25, 1516-1521DOI: (10.1016/j.joca.2017.05.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Palmitate induces JNK phosphorylation. Chondrocytes were pretreated with or without 1 mM PBA for 30 min followed by treatment with 500 μM of palmitate or oleate (A) or 2 μM TG (B) for 24 h. In other experiments, cells were pretreated with JNKi for 30 min followed by treatment with 500 μM of palmitate or oleate (C & E) or 2 μM TG (D &F) for 24 h followed by 50 ng/ml IGF-1 for 30 min. After incubation, cell lysates were immunoblotted with total or phosphospecific antibodies to JNK, IRS-1 and AKT, Immunoblots presented are representative of three independent experiments performed with cells obtained from different biological donors. Osteoarthritis and Cartilage 2017 25, 1516-1521DOI: (10.1016/j.joca.2017.05.011) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions