Effects of Methylmercury and Theaflavin Digallate on Adipokines in Mature 3T3-L1 Adipocytes

Abstract

Diabetes is a contributor to morbidity across the globe and is often associated with obesity, metabolic syndrome and other inflammatory diseases associated with aging. In addition to genetic and lifestyle factors, environmental factors such as metals and persistent organic pollutants may increase the severity or lower the threshold of these conditions. In cell culture, methylmercury is toxic to adipocytes and may impact adipokine secretions. In this study, we determined the effects of different concentrations of theaflavin digallate on methylmercury exposed 3T3-L1 adipocytes in cell culture. Secretions of resistin, adiponectin and lipid peroxidation product, 4-hydroxynonenal (4-HNE) were monitored using ELISA assays. Cell morphology of methylmercury and theaflavin-3,3'-digallate treated adipocytes was assessed using Lipid (Oil Red O) staining. Exposure to methylmercury increased the levels of resistin and adiponectin as well as 4-HNE when compared to the control cells. Methylmercury treated cells resulted in smaller number of adipocytes and clumped lipid droplets. These results suggest that methylmercury induces reactive oxygen species leading to development of an inflammatory response. Theaflavin-3,3'-digallate reduced the impact of methylmercury by maintaining the adipocytes morphology and secretion patterns of adiponectin, resistin and 4-hydroxynonenal. With this experimental model system other anti-inflammatory and signaling agents could be tested at the biochemical level before eventually leading to studies in animal models.

Keywords: adipokines; adiponectin; diabetes; lipid peroxidation; methylmercury; resistin; theaflavin digallate.

Figure 1

Morphology changes caused due to different treatments to mature adipocytes on Day 28 using lipid staining. (a) shows the stained lipids in the control group (Population 1); (b) shows the stained lipids under MeHg exposure (Population 2); (c) shows the stained lipids exposed to MeHg and 3.14µM TF-3 (Population 3); (d) shows the stained lipids exposed to MeHg and 6.25 µM TF-3 (Population 4).

Figure 2

Adiponectin secretion in all eight populations from Day 18 to Day 28. Overall statistically significant difference was determined using the Kruskal–Wallis method and p-value was less than 0.0001. Differences are considered significant (*) if p-value is less than or equal to 0.00625, very significant (**) if less than or equal to 0.001 and extremely significant (***) if less than or equal to 0.0001.

Figure 3

Resistin secretion in all eight populations from Day 18 to Day 28. Overall statistically significant difference was determined using the Kruskal–Wallis method and p-value was less than 0.0001. “***” shows an extremely significant difference having a p-value less than or equal to 0.0001.

Figure 4

4-HNE secretion in all six populations from Day 18 to Day 28. Overall statistically significant difference was determined using the Kruskal–Wallis method and p-value was less than 0.0001. Differences are considered significant (*) if p-value is less than or equal to 0.00625, very significant (**) if less than or equal to 0.001 and extremely significant (***) if less than or equal to 0.0001.

Figure 5

Timeline of the exposure study.