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A novel 68-kDa adipocyte protein phosphorylated on tyrosine in response to insulin and osmotic shock
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Abstract:
Osmotic shock can cause insulin resistance in 3T3-L1 adipocytes by inhibiting insulin activation of glucose transport, p70S6 kinase, glycogen synthesis, and lipogenesis. By further investigating the relationship between insulin and hypertonic stress, we have discovered that osmotic shock enhanced by 10-fold the insulin-stimulated tyrosine phosphorylation of a 68-kDa protein. Phosphorylation by insulin was maximal after 1 min and was saturated with 50-100 nm insulin. The effect of sorbitol was completely reversible by 2.5 min. pp68 was a peripheral protein that was localized to the detergent insoluble fraction of the low density microsomes but was not associated with the cytoskeleton. Stimulation of the p42/44 and the p38 MAP kinase pathways by osmotic shock had no effect on pp68 phosphorylation. Treatment of adipocytes with the phosphotyrosine phosphatase inhibitor phenylarsine oxide also enhanced insulin-activated tyrosine phosphorylation of pp68 suggesting that osmotic shock may increase pp68 phosphorylation by inhibiting a phosphotyrosine phosphatase. Dissociation of pp68 from the low density microsomes with RNase A indicated that pp68 binds to RNA. Failure to immunoprecipitate pp68 using antibodies directed against known 60-70-kDa tyrosine-phosphorylated proteins suggest that pp68 may be a novel cellular target that lies downstream of the insulin receptor.
Insights
Osmotic shock induces insulin resistance by inhibiting insulin signaling. This study identifies a novel 68-kDa protein (pp68) whose phosphorylation is enhanced by hypertonic stress, potentially revealing a new mechanism in insulin resistance.
Area of Science:
- Cell Biology
- Metabolic Signaling
- Biochemistry
Background:
- Osmotic shock impairs insulin signaling in 3T3-L1 adipocytes, leading to insulin resistance.
- Hypertonic stress inhibits key insulin-activated pathways, including glucose transport and lipogenesis.
Purpose of the Study:
- To investigate the molecular mechanisms linking insulin signaling and hypertonic stress.
- To identify novel cellular targets affected by osmotic shock in adipocytes.
Main Methods:
- Utilized 3T3-L1 adipocytes subjected to osmotic shock (sorbitol).
- Measured insulin-stimulated tyrosine phosphorylation of cellular proteins.
- Investigated protein localization, association with cytoskeleton, and effects of MAP kinase pathways.
- Used phosphotyrosine phosphatase inhibitor and RNase A treatment.
Main Results:
- Osmotic shock enhanced insulin-stimulated tyrosine phosphorylation of a 68-kDa protein (pp68) 10-fold.
- pp68 phosphorylation was rapid, reversible, and saturated by insulin.
- pp68 is a peripheral protein associated with low-density microsomes and binds to RNA.
- Osmotic shock may increase pp68 phosphorylation by inhibiting a phosphotyrosine phosphatase.
Conclusions:
- pp68 represents a novel cellular target potentially involved in insulin resistance.
- Osmotic shock's effect on pp68 phosphorylation may occur via phosphotyrosine phosphatase inhibition.
- pp68's interaction with RNA suggests a role in post-transcriptional regulation within insulin signaling.