HSG4112 induces insulin-synergistic and selective glucose uptake in muscle cells
The observed increase in basal metabolic rate hinted at heightened activity in muscle, which is responsible for most energy expenditure, and decrease in fat, which has lower metabolic activity. To explore this idea, we performed in vitro glucose uptake assays in 3T3-L1 adipocytes and C2C12 skeletal muscle cells to determine whether HSG4112 directly or indirectly influences energy metabolism and mediate transport of major energy source – glucose – into either or both types of cells. We tested (R )-HSG4112 and (S )-HSG4112 separately, in order to further examine the comparability between each stereoisomer’s effect in energy metabolism at cellular level. Metformin, the first-line medication for type 2 diabetes (T2DM), was used as the positive control for its well-known effect on facilitating glucose uptake in adipose tissue and skeletal muscle (Polianskyte-Prauseet al. , 2019).
In 3T3-L1 adipocytes, both (R )-HSG4112 and (S )-HSG4112 minimally increased glucose uptake; the highest concentration (10.0µM) of both compounds resulted in an average of 1.32- and 1.21-fold increase in glucose uptake relative to control, respectively, while metformin increased glucose uptake by 2.33-fold (Figure 6A ). In C2C12 skeletal muscle cell, (R )-HSG4112 and (S )-HSG4112 both significantly increased glucose uptake; here, the highest concentration led to fold-increase of 1.90 and 1.74, respectively, which were greater than 1.34-fold increase by metformin. While a dose-dependent increase in glucose uptake was observed in (S )-HSG4112, (R )-HSG4112 already reached saturation at the lowest concentration of 2.5µM, suggesting stronger direct effect of (R )-HSG4112 in glucose uptake in muscle cell (Figure 6B ).
We further tested whether HSG4112 improves glucose uptake synergistically, independently, or dependently with insulin, because insulin resistance is a key phenotype of obesity and T2DM. In adipocytes, both (R )-HSG4112 and (S )-HSG4112 did not further increase glucose uptake after insulin administration, while metformin expectedly led to synergistic two-fold increase (Figure 6C ). In muscle cells, both (R )-HSG4112 and (S )-HSG4112 significantly increased glucose uptake after insulin administration to 3.4- and 3.9- fold increase, respectively, in a dose-dependent manner. This was a significantly greater than metformin’s 2.1-fold increase, which is an ‘insulin-sensitizer’ that enhances glucose disposal to skeletal muscle when co-treated with insulin (Giannarelli et al. , 2003) (Figure 6D ). These results show that HSG4112 is a robust insulin-sensitizer for glucose uptake in muscle cells and not in adipocytes.