Figure 5.

MCFA helps the β-cells recover from chronic palmitate induced dysfunction. (a) Representative immunoblotting of INS1E cells after chronic treatment with increased concentrations of palmitate or MCFA C8:C10 mixture with antibodies against Nrf2 and mitochondrial respiratory chain proteins. (b) Assessment of apoptotic and ER stress signaling after chronic treatment with palmitate (C16:0) or MCFA C8:C10 mixture after immunoblotting with antibodies against PARP (Poly(ADP-Ribose) Polymerase), CHOP, Cox4 (Cytochrome C Oxidase Subunit 4) and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) as loading controls. (c) Mitochondrial membrane potential (MMP) in response to 16.7 mM glucose in INS-1E cells chronically treated with vehicle control (Ctrl) or the MCFA C8:C10 mixture, using the fluorescent lipophilic cation IP10. Changes in MMP are expressed as the ratio of the fluorescence in mitochondria divided by the cytosolic fluorescence (Fmito/Fcyto) measured in the same cells. At the end of the recording, the protonophore, FCCP (2 μM) was used to dissipate MMP. Data shown are the means ± SEM from three independent experiments done in sextuplet in 96-welled plates, ** p < 0.01. (d) Expression of CHOP and Glut2 by qPCR in INS1E cells after chronic treatment with palmitate and recovery in control medium or MCFA-enriched medium. (e) Western blot analysis of transcription factors regulating β-cell function (MafA, NeuroD1, PDX1) or mitochondrial proteins (NDUFA9, DLD, Cox4) after treatment as above; GAPDH was used as a loading control. (f) Analysis of β-cell function after impairment with chronic palmitate and recovery as above by insulin secretion under basal (2 mM glucose) and stimulated (16.7 mM glucose + 0.1 µM Ex-4) conditions. Data represent means + SEM of three independent experiments; * p < 0.05, ** p < 0.01 relative to control cells and # p < 0.05 relative to palmitate control cells.