The present study demonstrates for the first time that ER stress, which exists in obese adipose tissue, can downregulate resistin in vitro in mouse adipocytes. The effect of ER stress induction appeared to be primarily transcriptional, and three transcription factors were implicated in mediating the effects of ER stress on resistin levels. C/EBPα and PPARγ, which are known activators of resistin, are downregulated by treatment with tunicamycin and have diminished binding at the resistin gene. Knockdown of these factors mimicked the effects of ER stress induction. On the other hand, CHOP10 is a transcriptional repressor that is activated by ER stress in adipocytes and can interact in a dominant negative fashion with C/EBPα (37
). Knockdown of this protein was able to partially rescue the effects of tunicamycin on resistin expression, suggesting that CHOP10 may also be responsible for the changes in resistin mRNA. Thus, the effects of ER stress on resistin levels in mouse adipocytes appear to be a combination of decreased expression of activating transcription factors, including C/EBPα and PPARγ, and increased expression of repressors such as CHOP10 and possibly others.
A remaining question is how activation of the unfolded protein response leads to downregulation of C/EBPα and PPARγ. One possibility is that repressors of gene transcription such as CHOP10 and ATF3 that are activated as part of the ER stress response may be directly involved. For example, it is known that both C/EBPα and PPARγ genes are activated by C/EBPα binding to their promoters (30
), and therefore CHOP10 may decrease their expression via its dominant negative interactions with C/EBPα. Notably, however, CHOP10 knockdown was not able to fully rescue the effects of tunicamycin treatment on C/EBPα, indicating that other factors must be involved. Knockdown of ATF3, which is another transcriptional repressor (40
) that is activated by ER stress (26
), did not affect the ability of tunicamycin to reduce resistin levels (data not shown), suggesting that it does not play a role in this process.
The implication of these findings is that ER stress activation may provide an explanation for the decrease in resistin mRNA in adipose tissue of obese mice in the setting of elevated serum resistin levels. Indeed, this study demonstrates that decreased resistin expression co-exists with markers of ER stress activation such as increased BiP mRNA and phospho-eIF2α in adipose tissue from obese mice fed an HFD. Furthermore, C/EBPα mRNA and protein were decreased under these conditions, consistent with the role of this transcription factor as an important regulator of resistin (31
). In addition, the study demonstrates that in EWAT the levels of resistin protein reflect the downregulation in the mRNA, similar to what is observed for 3T3-L1 adipocytes in vitro. This raises the possibility that the discrepancy between adipose tissue and plasma resistin levels may not be occurring at the level of individual adipocytes but rather results from various global defects characteristic of obesity and insulin resistance. For example, it has been shown that the development of obesity is associated with an increase in fat cell number (41
), and therefore the net effect in obesity may be elevated resistin release into the circulation even if resistin secretion is decreased on a per-cell basis. In addition, a number of recent studies have demonstrated a negative correlation between renal function and resistin levels (43
), suggesting that resistin may be cleared through the kidney. Thus, in the setting of diabetic nephropathy, resistin clearance may be impaired leading to accumulation of the protein in the circulation. Another possibility is that resistin half-life in obesity may be increased because of oligomerization. A number of studies have shown that both mouse and human resistin can form oligomers, which can be detected in the circulation (45
) and have different biological actions compared with the monomer form (45
); and the propensity to oligomerize is concentration dependent (47
It has been previously hypothesized that the discrepancy between resistin mRNA and circulating protein levels may be because of the hyperinsulinemia associated with obesity and insulin resistance (15
). In vitro experiments have demonstrated that insulin treatment of mature adipocytes downregulates resistin expression (15
) although the mechanism has not been elucidated. However, there is evidence that insulin can potently decrease C/EBPα expression in differentiated 3T3-L1 cells leading to decreased C/EBPα binding at target DNA sequences (50
). This suggests that both ER stress and hyperinsulinemia may contribute to the decreased resistin mRNA levels in obesity by converging on C/EBPα. Moreover, insulin treatment of 3T3-L1 adipocytes did not lead to a discrepancy between resistin mRNA and protein secretion (data not shown), suggesting that at least in this model system both insulin and ER stress downregulated the protein levels along with the mRNA of resistin. It should be noted that the effects of insulin and ER stress in vivo may be different from those observed in vitro in cultured 3T3-L1 cells. However, 3T3-L1 cells, which are derived from immortalized mouse embryonic fibroblasts and can be differentiated into adipocytes with a hormonal cocktail, are generally considered a valid model for adipocyte function. Moreover, transplantation of 3T3-L1 cells into nude mice has been shown to result in formation of adipose tissue that is essentially identical to normal fat (51
), suggesting that this cell line is fully capable of reproducing the in vivo adipocyte phenotype.
The overall significance of the findings presented here is that ER stress, which develops in obese adipose tissue, can affect adipocyte function on many levels including dysregulation of adipokine production. It was previously shown that ER stress can impair insulin signaling both in vitro in adipocytes and in vivo in obese XBP+/−
mice, which are unable to respond properly to ER stress, and develop dramatically worse adipose tissue insulin resistance compared with wild-type controls (21
). Importantly, treatment of obese mice with chemical chaperones that alleviate ER stress improves signaling through the insulin receptor (52
), indicating that the effects of ER stress on adipose tissue insulin resistance may be reversible. Thus, targeting ER stress may constitute a feasible strategy for treating obesity and insulin resistance, although it will be critical to understand the various mechanisms by which ER stress affects adipose tissue, including its effects on adipokine expression and function.