Abstract
Obesity is tightly linked to hepatic steatosis and insulin resistance. One feature of this association is the paradox of selective insulin resistance: insulin fails to suppress hepatic gluconeogenesis but activates lipid synthesis in the liver. How lipid accumulation interferes selectively with some branches of hepatic insulin signaling is not well understood. Here we provide a resource, based on unbiased approaches and established in a simple cell culture system, to enable investigations of the phenomenon of selective insulin resistance. We analyzed the phosphoproteome of insulin-treated human hepatoma cells and identified sites in which palmitate selectively impairs insulin signaling. As an example, we show that palmitate interferes with insulin signaling to FoxO1, a key transcription factor regulating gluconeogenesis, and identify altered FoxO1 cellular compartmentalization as a contributing mechanism for selective insulin resistance. This model system, together with our comprehensive characterization of the proteome, phosphoproteome, and lipidome changes in response to palmitate treatment, provides a novel and useful resource for unraveling the mechanisms underlying selective insulin resistance.
Highlights
Obesity is often accompanied by liver steatosis and type 2 diabetes mellitus (T2D)1
Palmitate did not affect the level of SREBP1c transcripts, showing a selective effect of palmitate treatment on insulin signaling for gluconeogenesis, but not lipid synthesis
Because we found that palmitate pretreatment causes impairment of insulin’s effects on phosphorylation of FoxO1, we hypothesized that this impairment may be because of physical isolation of FoxO1 from kinases, rendering it insensitive to insulin signaling in the cytoplasm
Summary
T2D, type 2 diabetes mellitus; CAA, chloroacetamide alkylating reagent; DG, diacylglycerol; DMEM, T2D remain unclear, but a common feature is hepatic insulin resistance with impaired insulin signaling. HepG2 cells are immortalized and, are fully compatible with stable isotope labeling (SILAC)-based phosphoproteomic analyses by mass spectrometry [11,12,13] Using this model system, we show here that HepG2 cells treated with palmitate exhibit selective insulin resistance, and we characterize global changes by lipidomic, proteomic, and phosphoproteomic analyses. By further developing phosphoproteomic methodology with a protocol that omits peptide fractionation but includes multiple rounds of enrichment and mass spectrometry measurements for phosphopeptides, we quantified 18,000 sites in the phosphoproteome Several thousand of these sites are responsive to insulin, and we show how palmitate treatment interferes with some of these changes. As a proof-of-principle, we explored how palmitate interferes with a key node of insulin signaling regulating gluconeogenesis; we show that impaired FoxO1 signaling with palmitate treatment is associated with changes in its localization, suggesting a novel contributing mechanism for selective insulin resistance
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