Expression and functional activity of PPARgamma in pancreatic beta cells

Abstract

Rosiglitazone is an agonist of peroxisome proliferator activated receptor-gamma (PPARgamma) and ameliorates insulin resistance in type II diabetes. In addition, it may also promote increased pancreatic beta-cell viability, although it is not known whether this effect is mediated by a direct action on the beta cell. We have investigated this possibility. Semiquantitative real-time reverse transcription-polymerase chain reaction analysis (Taqman) revealed that freshly isolated rat islets and the clonal beta-cell line, BRIN-BD11, express PPARgamma, as well as PPARalpha and PPARdelta. The levels of expression of PPARgamma were estimated by reference to adipose tissue and were found to represent approximately 60% (islets) and 30% (BRIN-BD11) of that found in freshly isolated visceral adipose tissue. Western blotting confirmed the presence of immunoreactive PPARgamma in rat (and human) islets and in BRIN-BD11 cells. Transfection of BRIN-BD11 cells with a PPARgamma-sensitive luciferase reporter construct was used to evaluate the functional competence of the endogenous PPARgamma. Luciferase activity was modestly increased by the putative endogenous ligand, 15-deoxy-Delta12,14 prostaglandin J2 (15dPGJ2). Rosiglitazone also caused activation of the luciferase reporter construct but this effect required concentrations of the drug (50-100 microm) that are beyond the expected therapeutic range. This suggests that PPARgamma is relatively insensitive to activation by rosiglitazone in BRIN-BD11 cells. Exposure of BRIN-BD11 cells to the lipotoxic effector, palmitate, caused a marked loss of viability. This was attenuated by treatment of the cells with either actinomycin D or cycloheximide suggesting that a pathway of programmed cell death was involved. Rosiglitazone failed to protect BRIN-BD11 cells from the toxic actions of palmitate at concentrations up to 50 microm. Similar results were obtained with a range of other PPARgamma agonists. Taken together, the present data suggest that, at least under in vitro conditions, thiazolidinediones do not exert direct protective effects against fatty acid-mediated cytotoxicity in pancreatic beta cells.

Figure 1

Semiquantitative analysis of the expression of PPAR isoforms in rat islets and BRIN-BD11 cells. Panels a–c: RNA was extracted from rat islets (islet) BRIN-BD11 cells (Brin), rat liver and rat adipose tissue and amplified by semiquantitative RT–PCR (Taqman). Standard curves were constructed from liver (a, b) and adipose (c) and used to monitor the expression of PPAR isoforms (PPARα – panel a; PPARδ – panel b; PPARγ – panel c) in islet and BRIN-BD11 cells. The levels of expression of each PPAR isoform are expressed relative to that found in liver or adipose (defined as 1 in each case). Results are presented as relative expression±s.e.m. (n=3). Panel d: Proteins were extracted from adipose tissue (10 μg; lane 1), BRIN-BD11 cells (10 μg; lane 2), rat islets (5 μg; lanes 3–5), human islets (5 μg; lanes 6, 7) or HEK293 cells expressing PPARγ (5 μg; lane 8 – positive control) and separated by electrophoresis prior to transfer to PVDF membranes. The membranes were then probed with anti-PPARγ serum and immunoreactive bands were detected by chemiluminescence.

Figure 2

Effects of rosiglitazone on PPRE-luciferase reporter activity in transfected BRIN-BD11 or HEK 293T cells. BRIN-BD11 cells (squares) were transfected with PPRE-luciferase and HEK-293T cells (circles) were cotransfected with PPRE-luciferase and PPARγ prior to exposure to rosiglitazone and measurement of reporter activity 48 h later. Each point represents the mean value from triplicate determinations±s.e.m. from a representative experiment that was repeated three times with similar results. ^*P<0.01 relative to cells incubated in the absence of rosiglitazone.

Figure 3

Effects of 15dPGJ₂ on PPRE-luciferase reporter activity in transfected BRIN-BD11 cells. BRIN-BD11 cells were transfected with a PPRE-luciferase construct prior to exposure to 15dPGJ₂ and measurement of luciferase reporter activity 48 h later. Each point represents the mean value from triplicate determinations±s.e.m. from a representative experiment that was repeated three times with similar results. ^*P<0.05 relative to cells incubated in the absence of 15dPGJ₂.

Figure 4

Effects of high concentrations of rosiglitazone on PPRE-luciferase reporter activity in transfected BRIN-BD11. BRIN-BD11 cells were transfected with a PPRE-luciferase construct prior to exposure to rosiglitazone and measurement of luciferase reporter activity 48 h later. Each point represents the mean value±s.e.m. from triplicate determinations from a representative experiment that was repeated three times with similar results. ^*P<0.01 relative to cells incubated in the absence of rosiglitazone.

Figure 5

Effects of high concentrations of rosiglitazone on PPRE-luciferase reporter activity in transfected HEK-293T cells. HEK-293T cells were transfected with PPRE-luciferase alone (open bars) or PPRE-luciferase, together with PPARγ (grey bars) prior to exposure to rosiglitazone and measurement of luciferase reporter activity 48h later. Each point represents the mean value±s.e.m. from triplicate observations from a representative experiment that was repeated three times with similar results. ^*P<0.01 relative to the equivalent cells incubated in the presence of 10 μM rosiglitazone.

Figure 6

BRIN-BD11 cells were cultured in the absence (squares) or presence (circles) of 1 μM rosiglitazone for 24 h. After this time, increasing concentrations of palmitate were added (complexed to bovine serum albumin) and incubation continued for a further 18 h. At the end of this period, the cells were harvested and their viability was determined by vital dye staining. Each point represents the mean value±s.e.m. from quadruplicate measurements from a representative experiment that was repeated three times with similar results.

Figure 7

Effect of inhibition of transcription or translation on palmitate-induced cell death. BRIN-BD11 cells were incubated under control conditions or in the presence of 0.25 mM palmitate (bound to 1% fatty acid-free albumin) and either 2 μg ml⁻¹ actinomycin-D or 10 μg ml⁻¹ cycloheximide in cell culture medium containing 5.5 mM glucose. Cell death was determined after 18 h by vital dye staining. Results represent the mean level of viability±s.e.m. from triplicate determinations in a representative experiment that was repeated three times with similar results. ^*Significantly less than palmitate alone (P<0.01).

Figure 8

Effects of various PPAR ligands on palmitate-induced cell death in BRIN-BD11 cells. BRIN-BD11 cells were pretreated for 24 h under control conditions (Ctrl) or in the presence of rosiglitazone (Rosi, 1 μM), troglitazone (Trog, 1 μM), Pioglitazone (Pio, 1 μM), SB219994 (SB21, 2 nM), 15dPGJ₂ (PGJ2, 3 μM), compound F (Cpd F, 5 μM) or Wy14643 (Wy14, 20 μM). After this time, 0.25 mM palmitate was added (complexed to bovine serum albumin) and incubation continued for a further 18 h. At the end of this period, the cells were harvested and their viability was determined by vital dye staining. The reduction in cell viability was determined under each incubation condition and is expressed relative to that seen in the presence of palmitate alone.