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314 DIFFERENTIAL ACTIVATION OF INTRACELLULAR SIGNALING BY HIGH GLUCOSE, METFORMIN, AND AICAR IN HUMAN MESANGIAL CELLS.
  1. E. Gosmanova1,
  2. G. Tigyi1,
  3. B. M. Wall1,2
  1. 1The University of Tennessee Health Science Center, Memphis, TN
  2. 2VAMC, Memphis, TN

Abstract

Introduction Hyperglycemia-induced activation of extracellular mitogen-activated protein kinase (ERK1,2) is considered to be one of the central mechanisms contributing to the development of diabetic nephropathy (DN). Metformin has been shown to provide nephroprotective action in patients with diabetes mellitus. However, intracellular mechanisms by which this drug exerts beneficial effects on DN remain to be determined.

Purpose To investigate the effects of hyperglycemia on activation of ERK1,2 and AMP-kinase (AMPK) in human mesangial cells. The effects of hyperglycemia were also studied in the presence of known activators of AMP-kinase (metformin and 5-amonoimidazole-4-carboxamide-1-b-D-ribofuranoside [AICAR]).

Methods Human mesangial cells were incubated in normal (5.5 mM) or high (25 mM) glucose in the presence or absence of 1 mM metformin or 1 mM AICAR for 24 hours. Activation of ERK1,2 and AMPK was assessed by detection of phosphorylated forms of ERK1,2 and AMPK by Western blotting.

Results High glucose induced activation of ERK1,2 by 60% (p = .001) and inhibited phosphorylation of AMPK by 17% (p = .04). In the presence of 5.5 mM glucose, metformin induced ERK1,2 phosphorylation by 51% (p = .03) and AICAR inhibited ERK1,2 phosphorylation by 57% (p = .02). While metformin did not alter high glucose-induced activation of ERK1,2, AICAR was able to inhibit ERK1,2 by 58% (p = .001). Both metformin and AICAR stimulated AMPK phosphorylation in the presence of 5.5 mM glucose by 2.1-fold (p = .07) and 12-fold (p = .04), respectively. Incubation of mesangial cells with metformin in the presence of 25 mM glucose increased AMPK phosphorylation 2.9-fold (p = .001) compared with high glucose alone. Similarly, AICAR activated AMPK 13-fold in the presence of high glucose (p = .03).

Conclusion High glucose down-regulates AMPK and induces activation of ERK1,2 in human mesangial cells. Both metformin and AICAR were able to prevent inhibition of AMPK induced by high glucose. In contrast, AICAR, but not metformin, led to inhibition of ERK1,2. These data suggest that the beneficial effects of metformin may involve additional signaling pathways.

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