Protein kinase C-gamma activation in the early streptozotocin diabetic rat lens.
OBJECTIVE
The purpose of this study is to demonstrate the early activation of the protein kinase C-gamma (PKC-gamma) pathway in the streptozotocin (STZ)-induced diabetic rat lens.


METHODS
Twelve-week-old male and female Sprague-Dawley rats were injected with 80 mg/kg (body weight) of STZ (N-[methylnitrosocarbamoyl]-D-glucosamine) intraperitoneally. Very high glucose (VHG) diabetes was defined as a nonfasting blood glucose level of at least 450 mg/dl, confirmed by daily monitoring with Accu-Check Advantage test strips, and occurred about 2 weeks after STZ administration. All assayed lenses were from VHG or age-matched control rats, harvested within 24 hr of VHG detection. PKC-gamma activation was measured by enzyme activity assay and by Western blotting to show autophosphorylation on Thr514. Cellular insulin-like growth factor-1 (IGF-1), PKC-gamma phosphorylation of Cx43 on Ser368, and activation of phospholipase C-gamma 1 (PLC-gamma 1), extracellular signal-regulated kinase (ERK1/2), and caspase-3 were determined by Western blotting. Endogenous diacylglycerol (DAG) levels were measured with a DAG assay kit. Lens gap junction activity was determined by the microinjection/Lucifer yellow dye transfer assay. Electron microscopy was applied to affirm fiber cell damage in the VHG diabetic lenses.


RESULTS
In the lenses of VHG diabetic rats, PKC-gamma enzyme was activated. PKC-gamma could be further activated by 400 nM phorbol-12-myristate-13-acetate (PMA), but the PKC-gamma protein levels remained constant. No elevation of IGF-1 level was observed. Western blots showed that activation of PKC-gamma may be due to activation of PLC-gamma 1, which synthesized endogenous DAG, a native PKC activator. The level of PKC-gamma -catalyzed phosphorylation of Cx43 on Ser368 and resulting inhibition of lens gap junction dye transfer activity was increased in the VHG diabetic lenses. At this early time period, the diabetic lens showed no activation of either caspase-3 or ERK1/2. Only a single fiber cell layer deep within the cortex (approximately 90 cell layers from capsule surface) showed vacuoles and damaged cell connections.


CONCLUSIONS
Early activation of PLC-gamma 1 and elevated DAG were observed within VHG diabetic lenses. These were correlated with activation of PKC-gamma, phosphorylation of Cx43 on Ser368, and inhibition of dye transfer. Abnormal signaling from PKC-gamma to Cx43 in the epithelial cells/early fiber cells, observed within VHG diabetic lenses, may be responsible for fiber cell damage deeper in the lens cortex.
Lin
Dingbo
D
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Harris
Richie
R
Stutzman
Rachael
R
Zampighi
Guido A
GA
Davidson
Harriett
H
Takemoto
Dolores J
DJ
D000818:Animals; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D018031:Connexin 43 / Q000378:metabolism; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D017629:Gap Junctions; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018031:Connexin 43; D004075:Diglycerides; D007527:Isoenzymes; D007334:Insulin-Like Growth Factor I; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C463939:protein kinase A Cgamma; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D053148:Caspase 3; D013755:Tetradecanoylphorbol Acetate