Acclimation of C₂C₁₂ myoblasts to physiological glucose concentrations for in vitro diabetes research

The interplay between hyper-glycemia and -lipidemia in diabetes mellitus (DM) is important in simulating diabetic conditions. However, cell culture media typically contain supraphysiological levels of glucose to stimulate cellular growth, which also desensitizes cells to elevated glucose levels. Moreover, creating hyperlipidemic conditions in vitro requires specialized carriers because unbound lipids form micelles when introduced to liquid media. This study sought to develop a novel method for simulating DM conditions in vitro.

We acclimated the C₂C₁₂ mouse myoblasts to culture medium with 5.6 mM glucose, which mimics physiological levels, and created a bovine serum albumin-palmitic acid conjugate for lipid transport to explore the effects of hyperlipidemia. We simulated diabetic conditions in vitro by using both hyper-glycemic and -lipidemic conditions and compared the results to that of only hyperglycemic or hyperlipidemic conditions.

Acclimated cells exposed to these hyper-glycemic (15 mM glucose) and/or -lipidemic (0.25 mM palmitate) conditions for 2 h showed increased mitochondrial fragmentation and membrane potential as well as elevated reactive oxygen species production compared to control cells. These findings suggest altered mitochondrial morphology and function, which have been confirmed using isolated rat flexor digitorum brevis myofibers. Hyper-glycemic and/or -lipidemic stimulations for 24 h significantly increased mitogen-activated protein kinase kinase MEK 1/2 protein expression, upregulated the early pro-apoptotic transcription factor C/EBP homologous protein (CHOP), and induced apoptosis.

Our results further support and confirm the utility of this method which will allow for subsequent investigations studying the effects of hyper-glycemia and/or -lipidemia in vitro.