This study, designed to further investigate the direct effects of Pioglitazone on pancreatic islets, demonstrates that the effect of this TZD on islet apoptosis rate and gene expression profile depends on the glucose concentration.
Parton et al.  had investigated gene expression profiles of rat islets treated with a PPAR-γ agonist (GW-347845) only at 3 mM glucose and found that 49 out of 9,563 genes exhibited altered expression levels. A substantial increase in the number of genes affected was observed only when islets overexpressing PPAR-γ were treated with GW-347845. Our current finding, in which 101 out of 13,059 genes were affected by Pioglitazone in islets maintained at 5.6 mM glucose, suggests that gene expression in β-cells is not very sensitive to TZDs in euglycemic conditions and in cells with physiological expression of PPAR-γ. A further similarity between both studies was the lack of modulation of the genes coding for glucokinase and UCP2, previously suggested as PPAR-γ targets [29, 30].
At both glucose concentrations Pioglitazone increased the expression of Scd2 and Fabp4, lipogenic genes which are PPAR-γ targets in adipocytes [31, 32] and Srebf1, a transcription factor involved in lipogenesis in adipocytes . In the opposite metabolic direction, Pioglitazone increased the expression of Insig1 (at 5.6 mM), a protein that participates in the inhibition of cholesterol synthesis  and of Lipe (at 23 mM), which codes for hormone-sensitive lipase, responsible for hydrolysis of triglycerides . Thus, in conditions of physiological expression of PPAR-γ, Pioglitazone stimulates expression of genes that limit lipid accumulation in islets, but we cannot rule out that these changes are a response to a primary activation of lipogenic pathways, which is more concordant with the role of PPAR-γ in other tissues.
The transcriptional response to Pioglitazone was strikingly different under conditions of elevated glucose, an effect not previously described. Concomitant treatment of islets with Pioglitazone and 23 mM glucose increased by 12-fold the number of modulated genes. Little is known about the mechanisms involved in glucose regulation of β-cell gene expression. It has been suggested that glucose metabolism alters gene expression directly at the transcriptional level by interfering with proteins bound to the preinitiation complex or by modifying the concentrations of transcription factors . Although further studies are necessary to determine the contribution of enhanced RNA stability or direct transcriptional activation on the observed response, it can be hypothesized that at high concentrations, glucose interferes with ligand-dependent PPAR-γ transcriptional activity, directly modulating the expression of genes that are not affected when islets are exposed solely to the PPAR-γ ligand. However, since the transcriptional response to Pioglitazone was determined after 24 h in the absence of protein synthesis inhibitors, it is likely that part of the observed gene regulation is not directly due to ligand binding to PPAR-γ, but resulted from changes in expression of other transcriptional regulators targeted by the combination of glucose and ligand. It is also worth mention that both high glucose concentrations  and PPAR ligands  promote chromatin remodeling and transcription, which could also contribute to the observed finding.
At 48 h, Pioglitazone elicited a reduction in islets apoptosis rate at 5.6 mM glucose. The transient nature of this response makes it difficult to state that this TZD exerts a protective effect on pancreatic islets, but one can conclude that in such experimental condition, Pioglitazone is not deleterious, a result in contrast to that obtained at supraphysiological glucose concentration, at which an increased apoptosis rate was found after 48 and 72 h. This pro-apoptotic tendency was also revealed by the gene expression analysis after 24 h, since genes related to cell cycle and cell death were significantly modulated. Because genes related to lipid metabolism were also modulated at 5.6 mM, this single pathway does not seem to be related to the apoptotic fate of islets. Additional data that reinforce a pro-apoptotic trend of islets exposed to Pioglitazone at 23 mM glucose are upregulation of antioxidant defense genes such as Hmox1 and Sod2, believed to be activated, respectively, in response to cellular stress  and in ß-cell defense , and a lower Bcl2 RNA expression after 48 and 72 h of exposure. Although several studies have already demonstrated that TZD´s may counteract direct fatty acid induced deleterious effects on β-cell function [11, 41–44], few studies addressed the direct effects of TZD´s in conditions mimicking hyperglycemia. Zeender et al. had found that Pioglitazone decreased the apoptosis rate of human islets exposed to 33.3 mM glucose after 48 h (5 μM) and 96 h (1 μM) while Saitoh et al. did not observe any effect of Pioglitazone on the apoptotic rate of MIN-6 cells exposed to 25 mM glucose  and Ohtani et al. found that troglitazone (10-4 to 10-6 M) induced apoptosis on HIT-15 cell exposed to 7 mM glucose for 72 h . Reasons for the discrepancies between the studies might be the use of different experimental conditions such as glitazones and glucose concentrations, different cell lines or islets from different species, but the controversial results point out the need for further research on this issue.
It is probable that in vivo the indirect beneficial effects of TZDs on islets compensate for the potential deleterious direct effects in the presence of high glucose, given that previous studies have already demonstrated restoration of islet function in rodents [7, 8] and humans [4–6]. Furthermore, besides hyperglycemia, other conditions not addressed in this study participate in the loss of β-cell observed in type 2 diabetes, and there are reports supporting a protective effect of TZDs in presence of lipotoxicity [44, 46, 47], deposition of islet amyloid  and endoplasmic reticulum stress . In agreement with the present study, however, a former in vitro study has already found that troglitazone protective effects on islets were less pronounced at high glucose .
In summary, our data demonstrate for the first time that the effect of Pioglitazone on pancreatic islet gene expression profile and apoptosis rate depends on the glucose concentration, reinforcing the necessity of additional studies designed to evaluate TZDs effects on the preservation of β-cell function in situations where glucotoxicity might be more relevant than lipotoxicity.