Metformin and Glucose Concentration as Limiting Factors in Retinal Pigment Epithelial Cell Viability and Proliferation

Abstract

Metformin is a well-established drug for the treatment of type 2 diabetes; however, the mechanism of action has not been well described and many aspects of how it truly acts are still unknown. Moreover, regarding in vitro experiments, the glycaemic status when metformin is used is generally not considered, which, added to the suprapharmacological drug concentrations that are commonly employed in research, has resulted in gaps of its mechanism of action. The aim of this study was to determine how glucose and metformin concentrations influence cell culture. Considering that diabetic retinopathy is one of the most common complications of diabetes, a retinal pigment epithelial cell line was selected, and cell viability and proliferation rates were measured at different glucose and metformin concentrations. As expected, glucose concentration by itself positively influenced cell proliferation rates. When the metformin was considered, results were conditioned, as well, by metformin concentration. This conditioning resulted in cell death when high concentrations of metformin were used under physiological concentrations of glucose, while this did not happen when clinically relevant concentrations of metformin were used independently of glucose status. Our study shows the importance of in vitro cell growth conditions when drug effects such as metformin's are being analysed.

Keywords: cell proliferation; glucose; metformin; type 2 diabetes.

Figure 1

Effect of glucose concentration in the hTERT RPE-1 cell culture. (a) Cell viability of the hTERT RPE-1 cell line after 72 h of cell culture and under different concentrations of glucose (N = 3). (b) Microscopy images (magnification: 50×) of hTERT RPE-1 under different glucose concentrations. (c) Glucose time course for both conditions of glucose of the cell culture supernatant (N = 3). PhG: physiological glucose; HG: high glucose; C-VE: negative control.

Figure 2

hTERT RPE-1 cell viability time course comparing the different concentrations of metformin (1 M, 10 mM,1 mM, 50 µM) under both conditions of glucose (N = 3). PhG: physiological glucose; HG: high glucose; C-VE: negative control. ($: p = 0.020; &: p = 0.012; 10 mM PhG vs. 10 mM HG).

Figure 3

Continuous cell proliferation rate registry by iCelligence under two concentrations of metformin (10 mM, 50 µM) (a) and physiological (b) and high concentrations of glucose (N = 3). Cell culture of hTERT RPE-1 (magnification: 50×) under the two concentrations of glucose and for (c) 10 mM of metformin and (d) 50 µM of metformin. PhG: physiological glucose; HG: high glucose; C-VE: negative control.

Figure 4

Glucose consumption time course. (a) Cell supernatant glucose consumption time course for the two concentrations of glucose. Cell supernatant glucose concentration at 6 h, 24 h and 48 h of (b) high and (c) physiological concentration culture media for the different concentrations of metformin (C-VE, 10 mM, 50 µM) (N = 3). PhG: physiological glucose; HG: high glucose; C-VE: negative control. * p < 0.05 (10 mM vs. C-VE).