Proteomic profiling of metformin effects in 3T3‐L1 adipocytes by SILAC‐based quantification

Abstract

Metformin is a common and generally the first medication prescribed for treatment of type 2 diabetes. Its mechanism involves affecting pathways that regulate glucose and lipid metabolism in metabolic cells such as that of muscle and liver cells. In spite of various studies exploring its effects, the proteome changes in adipocytes in response to metformin remains poorly understood. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)‐based quantitative proteomic profiling to study the effects of metformin specifically on 3T3‐L1 adipocytes. We define proteins that exhibited altered levels with metformin treatment, 400 of them showing statistically significant changes in our study. Our results suggest that metformin affects not only the PPAR signaling pathway, as well as glucose and lipid metabolism, but also protein folding, endoplasmic reticulum stress, negative regulation of appetite, and one‐carbon folate metabolism in adipocytes. This proteomic investigation provides important insight into effects of metformin in adipocytes.

Comprehensive Characterization of Protein Turnover by Comparative SILAC Labeling Analysis in 3T3-L1

“…We observed that 3T3-L1 adipocytes accumulated lipid drops 5 days after initiation of differentiation; we thus performed protein turnover experiments 10 days after initiation of differentiation (Supplementary Figure 1A). For biological replicates, we employed a SILAC label-swap replication strategy where light SILAC-labeled adipocytes were changed to heavy SILAC containing Arg10 and Lys8 and heavy SILAC-labeled adipocytes were changed to light SILAC containing Arg and Lys for eight different time intervals: 6, 18, 30, 48, 60, 96, 120, and 144 h. This strategy has been shown to improve reproducibility of the experiments and help in the correction of any experimental errors. ,,− …”

“…We employed a SILAC labeling approach to determine protein half-lives from 0 to 144 h in 3T3-L1 adipocytes. To confirm reproducibility of the protein turnover in 3T3-L1 adipocytes, we applied a SILAC label-swap strategy. ,,− , It is reported that label-swapping analysis of SILAC cell culture enhances confidence in the quantification and helps in the monitoring of any experimental errors, for example, incomplete labeling of SILAC. − …”

“…They were maintained in a 5% CO 2 humidified atmosphere at 37 °C. To ensure complete SILAC labeling, cells were cultured for five passages in SILAC DMEM, as described previously. , …”

“…To ensure complete SILAC labeling, cells were cultured for five passages in SILAC DMEM, as described previously. 9,10 For differentiation of 3T3-L1 preadipocyte to adipocyte, cells were added 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 μM dexamethasone, and 1 μM insulin, as described in our previous study. 9,10 After 10 days of initiation of differentiation, light or heavy SILAC-labeled 3T3-L1 adipocytes were exposed to heavy or light SILAC DMEM for 6, 18, 30, 48, 60, 96, 120, and 144 h, respectively; cells were washed with phosphatebuffered saline (PBS) three times before changing SILAC DMEM at each time point.…”

“…9,10 For differentiation of 3T3-L1 preadipocyte to adipocyte, cells were added 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 μM dexamethasone, and 1 μM insulin, as described in our previous study. 9,10 After 10 days of initiation of differentiation, light or heavy SILAC-labeled 3T3-L1 adipocytes were exposed to heavy or light SILAC DMEM for 6, 18, 30, 48, 60, 96, 120, and 144 h, respectively; cells were washed with phosphatebuffered saline (PBS) three times before changing SILAC DMEM at each time point. For cell harvest and sample collection, cells were washed with PBS three times and then treated with trypsin at 37 °C, followed by centrifugation at 21,130 × g at 4 °C for 15 min.…”