High Glucose Concentration Attenuates Insulin‐Stimulated Protein Kinase B Levels In Cultured Astrocytes

Abstract

BACKGROUNDGlucoregulatory abnormalities have been implicated to contribute to the pathophysiology of Alzheimer‐type dementia. The brain requires insulin for supply of energy critical for processing of memory and cognition. Insulin acts on the insulin receptor initiating signaling cascades that activate protein kinase B (Akt) through phosphorylation (p‐Akt) in cultured astrocytes. However, it is not yet clear whether the insulin induced production of p‐Akt could be attenuated by high glucose concentrations through stimulation of an inhibitory signaling pathway involving activation of protein kinase C (PKC).OBJECTIVEThe objective of our study is to examine the phosphorylation of Akt in cultured neonatal astrocytes following insulin stimulation and to determine if this response could be attenuated by culturing astrocytes in high glucose media via activation of PKC.METHODSNeonatal astrocyte cultures were derived from rat hippocampus and cultured for three passages in normal (5.5 mM) or high glucose (25 mM) media. Astrocyte cultures were stimulated with increasing concentrations of insulin (10−11 to 10−6 M) and Western immunoblotting analysis was used to determine protein levels of p‐Akt and insulin receptor. Protein levels of PKC isoforms (alpha, gamma, and delta) were determined in whole cell lysates from separate astrocyte cultures grown in normal or high glucose media.RESULTSInsulin‐induced a dose‐dependent increase in p‐Akt protein level in astrocytes cultured in normal glucose. However, insulin stimulation of astrocytes cultured in high glucose media resulted in an attenuated protein level of p‐Akt (100 nM insulin: 3.6 ± 1.0 vs. 8.9 ± 1.6 fold increase compared to control), requiring more insulin to increase the protein levels of p‐Akt (interaction effect: p = 0.024). The protein levels of insulin receptor in cultured astrocytes also increased in response to stimulation with insulin. Astrocytes cultured in high glucose media exhibited increased protein levels of PKC alpha (1.5 fold increase, p < 0.05) and gamma (2 fold increase, p < 0.05) but not of the PKC delta isoform compared to astrocytes cultured in normal glucose media.CONCLUSIONSInsulin‐dependent intracellular signaling involving phosphorylation of Akt is attenuated in astrocytes cultured in high glucose media. Astrocytes respond to increased environmental glucose with isoform specific increases in PKC protein levels which may have resulted in negative regulation of astrocyte insulin receptor signaling.Support or Funding InformationFunding from NIH (NHLBI) T35 HL072483, R01 HL033833 and HL105997 and the Veterans Administration Research Career Scientist Award.