Kinome Profiling Reveals Abnormal Activity of Kinases in Skeletal Muscle From Adults With Obesity and Insulin Resistance

Abstract

Context Obesity-related insulin resistance (OIR) is one of the main contributors to type 2 diabetes and other metabolic diseases. Protein kinases are implicated in insulin signaling and glucose metabolism. Molecular mechanisms underlying OIR involving global kinase activities remain incompletely understood. Objective To investigate abnormal kinase activity associated with OIR in human skeletal muscle. Design Utilization of stable isotopic labeling-based quantitative proteomics combined with affinity-based active enzyme probes to profile in vivo kinase activity in skeletal muscle from lean control (Lean) and OIR participants. Participants A total of 16 nondiabetic adults, 8 Lean and 8 with OIR, underwent hyperinsulinemic-euglycemic clamp with muscle biopsy. Results We identified the first active kinome, comprising 54 active protein kinases, in human skeletal muscle. The activities of 23 kinases were different in OIR muscle compared with Lean muscle (11 hyper- and 12 hypo-active), while their protein abundance was the same between the 2 groups. The activities of multiple kinases involved in adenosine monophosphate–activated protein kinase (AMPK) and p38 signaling were lower in OIR compared with Lean. On the contrary, multiple kinases in the c-Jun N-terminal kinase (JNK) signaling pathway exhibited higher activity in OIR vs Lean. The kinase-substrate–prediction based on experimental data further confirmed a potential downregulation of insulin signaling (eg, inhibited phosphorylation of insulin receptor substrate-1 and AKT1/2). Conclusions These findings provide a global view of the kinome activity in OIR and Lean muscle, pinpoint novel specific impairment in kinase activities in signaling pathways important for skeletal muscle insulin resistance, and may provide potential drug targets (ie, abnormal kinase activities) to prevent and/or reverse skeletal muscle insulin resistance in humans.

Profiling the Skeletal Muscle Proteome in Patients on Atypical Antipsychotics and Mood Stabilizers

“…Three mg of proteins from each biopsy and 300 μg “heavy” Stable Isotope Labeling by Amino Acid (SILAC) labeled protein standards were mixed. The “heavy” SILAC labeled protein standards were from the stock we prepared with SILAC incorporation rate >95%, as described in [ 34 ]. The protein mixture underwent reduction (10 mM dithiothreitol (DTT) incubation for 30 min at 55 °C) and alkylation (50 mM iodoacetamide (IAA) incubation for 30 min at room temperature in dark).…”

“…To minimize the experimental variation during sample preparation and HPLC-ESI-MS/MS data acquisition, we developed and validated a modified Super-SILAC approach (we now call it Universal-SILAC), in which SILAC labeled protein lysates were spiked-in to each experimental sample and were used as a universal standard for quantification purpose [ 34 , 39 ]. The normalized peak area for non-labeled peptides was calculated by normalizing the peak area of a peptide (PAi) against the sum of the peak area of the isotope-labeled peptide, which were identified in the same sample [ 34 , 39 ]. Please note that traditional SILAC/spike-in Super-SILAC requires both light peptides and corresponding heavy-labeled peptides to be identified for quantification.…”

“…Our Universal-SILAC approach does not require both light peptides and corresponding heavy-labeled peptides to be identified for quantification, therefore it provides more quantitative information than the traditional SILAC/Super-SILAC quantification. Please see our publication [ 34 , 39 ] for more details. The Universal-SILAC strategy can be easily applied to proteome quantification [ 34 ]: …”

Metformin Increases Protein Phosphatase 2A Activity in Primary Human Skeletal Muscle Cells Derived from Lean Healthy Participants

“…Eligible subjects underwent in-patient clinical tests: a skeletal muscle biopsy followed by a 2-hour hyperinsulinemic-euglycemic clamp to assess insulin sensitivity. Biopsies were immediately blotted free of blood and cleaned of connective tissue (~30 sec), submerged in ice-cold DPBS for primary skeletal muscle cell culturing as described in references [ 15 – 18 ]. The primary cells were differentiated into myotubes and treated with/out metformin.…”

“…The hyperinsulinemic-euglycemic clamp is considered to be the gold standard for measuring insulin sensitivity in vivo as previously described [ 18 – 20 ]. The study began at approximately 8 am (time −60 min) after an overnight fast.…”