Despite intense investigation, we have a very incomplete understanding of the specific roles played by PPARγ in many of the tissues where it is expressed. The identification of the pancreatic β cell as a site of PPARγ expression, combined with data demonstrating the effects of TZD administration on islet architecture and function, suggested to us that PPARγ could be important in the biology of the β-cell. We have critically analyzed this issue by using Cre-lox technology to target PPARγ in a β-cell-specific manner. Counter to expectations, we could detect no significant metabolic abnormality in βγKO mice relative to the metabolism of PPARγfl/fl
, or PPARγcre
controls. Our analysis included measurements of insulin and glucose in fasting and fed mice on both the lean (chow) and insulin-resistant (high-fat) diets. Glucose and insulin tolerance testing and levels of acute-phase insulin secretion similarly revealed no differences between βγKO and control mice. Although it is not possible to confirm that we have deleted PPARγ expression in every β cell, several lines of evidence indicate that we have eliminated its expression in the vast majority of cells. First, RT-PCR of β cells from the dispersed islets of βγKO mice reveals the absence of wild-type PPARγ mRNA. Second, PCR of whole islets (which contain numerous non-β cells, such as α and δ cells, endothelial cells, etc.) reveals a significant reduction in the expression of the floxed allele, while the 400-bp PCR product indicating the presence of the deleted allele is present only in βγKO islets. Third, the effect of troglitazone on insulin secretion is totally eliminated in the βγKO islets. Fourth, in other studies, the expression of the RIP-Cre transgene has been demonstrated to be virtually completely penetrant (24
The absence of metabolic consequences in βγKO mice does not exclude the β cell as an antidiabetic target of TZD drugs. To assess this possibility, we measured insulin secretion after TZD administration from isolated whole islets of control or βγKO mice. In control islets, troglitazone caused a roughly twofold induction of insulin secretion, even in the absence of glucose. This effect was totally eliminated in βγKO islets. When these studies were extended to the intact mouse, however, we found that the improved glucose homeostasis associated with TZD administration was preserved in βγKO animals. This finding demonstrates that PPARγ in β cells is unlikely to play a major role in the therapeutic response to TZDs. Even if there is a slight improvement in insulin secretion after TZD administration in vivo, this effect is likely masked by the peripheral insulin sensitization that dominates the clinical response to TZDs.
The most striking difference between control mice and βγKO mice was islet mass. Loss of PPARγ is associated with a hyperplastic response in the targeted β cells. PPARγ is known to regulate growth in a variety of cell types, including preadipocytes, myeloid leukemia cells, and epithelial tumor lines derived from breast, colon, and prostate. The fact that βγKO islets do not proliferate indefinitely suggests that other growth-regulating pathways eventually come into play, which may explain why tumor formation in the islet tissue of βγKO mice was not observed.
β-Cell hyperplasia is most commonly seen as a response to obesity and insulin resistance; it may be caused by a circulating growth factor or factors (14
). Despite several theories, no specific factor has been definitively identified. Recent evidence has suggested that insulin itself might be important as a mediator of β-cell hyperplasia (1
). All the known components of the insulin signaling pathway are present in β cells (16
) (reviewed in reference 23
). Loss of insulin receptor substrate 1 (IRS-1) results in hyperplastic, but dysfunctional, islets (3
), while loss of IRS-2 results in diabetes without compensatory β-cell hyperplasia (44
). The overexpression of a constitutively active form of Akt increases β-cell mass in transgenic mice (7
). Furthermore, loss of β-cell insulin receptors (βIRKO) is associated with reduced β-cell hyperplasia during normal aging (24
). Crossing βIRKO mice to animals with hepatic insulin resistance (due to the inactivation of insulin receptors in their livers) results in progeny that are insulin resistant but which do not appropriately expand their β-cell mass (R. N. Kulkarni, unpublished data). Given the obvious defect in β-cell hyperplasia after high-fat feeding in βγKO mice, it is tempting to speculate that the lack of PPARγ may cause local insulin resistance within β cells.
Other mechanisms may also contribute to our findings, of course. For example, excess lipid accumulation in the islet has been associated with the lipoapoptosis of β cells (43
). TZD treatment of islets from Zucker fatty rats causes reductions in cellular triglyceride levels as well as reduced lipoapoptosis (17
). It is possible, therefore, that for βγKO animals on the high-fat diet, the smaller islet mass than that of controls results from enhanced apoptosis in the setting of reduced PPARγ activity.
One of the more intriguing aspects of our study is the discordance between islet mass and function, especially for mice in the obese, insulin-resistant state. Specifically, the fact that glucose and insulin levels are identical in βγKO and PPARγfl/fl mice on a high-fat diet implies that there must be a compensatory gain in β-cell function in the βγKO mice. This observation is supported by the elevated insulin content on a per cell basis that was seen in βγKO islets (at least in the chow-fed mice). PPARγ can thus be identified as a factor that couples peripheral insulin resistance to changes in β-cell proliferation, perhaps by promoting lipid deposition in the islet. In this scenario, removal of PPARγ makes the β cell work more efficiently. However, obese βγKO mice were still glucose intolerant, indicating that the absence of PPARγ cannot compensate fully for the β-cell dysfunction seen in states of peripheral insulin resistance.