Involvement of PFKFB3/iPFK2 in Beneficial Effects of Metformin on 3T3-L1 Adipocytes and Temporal Effects of Pioglitazone on Adipocytes

Abstract

Metformin, as one of the first-line clinical anti-diabetic oral drugs for T2DM, has been shown to have beneficial effects including decreasing hepatic glucose production (HGP), improving insulin sensitivity, and suppressing inflammation, primarily in the liver though AMPK pathway. However, the effect of metformin on adipose tissue and adipocytes has not been fully investigated, as well as the role of PFKFB3/iPFK2, the gene that was shown to be protective against insulin resistance and inflammatory response in adipose tissue caused by overnutrition, in beneficial effects of metformin.

In this study, the involvement of PFKFB3/iPFK2 in metformin actions is investigated using 3T3-L1 adipocytes, on the aspect of anti-inflammatory effect and improvement in insulin sensitivity. In PFKFB3/iPFK2-Control (iPFK2-Ctrl) cells, metformin treatment was shown to have significant insulin-sensitizing effect, evidenced by an increase in p-Akt/Akt signaling, and some effect on anti-inflammatory response. In contrast, insulin sensitivity was not improved, but impaired under metformin treatment on PFKFB3/iPFK2-knockdown (iPFK2-KD) adipocytes. Also, metformin did not exhibit anti-inflammatory response in iPFK2-KD cells. Meanwhile, the phosphorylation of AMPK on control cells was increased with metformin treatment, but did not alter in iPFK2-KD cells. These results supported that metformin treatment on 3T3-L1 adipocytes could increase insulin sensitivity and possibly ameliorate inflammatory response, and a disruption of PFKFB3/iPFK2 in adipocytes impairs the insulin-sensitizing and anti-inflammatory effect of metformin, possibly through the AMPK pathway.

Other than drugs, circadian rhythm also takes an important role in regulation of adipocyte physiology. Therefore, it is pharmacologically meaningful to investigate the timing of drug delivery and the potential differential outcomes. In this study, 3T3-L1 adipocytes displayed an internal oscillation, evidenced by temporal variations in levels of core clock proteins and expression of core clock genes. Furthermore, 3T3-L1 adipocytes responded circadian time-differently when stimulated with LPS and insulin. Anti-inflammatory effect of pioglitazone at ZT19 was more effective than that at ZT7, while no significant insulin-sensitizing effect was seen at both time points.