Quantitative In Vivo Proteomics of Metformin Response in Liver Reveals AMPK-Dependent and -Independent Signaling Networks

Abstract

Metformin is the front-line treatment for type 2 diabetes worldwide. It acts via effects on glucose and lipid metabolism in metabolic tissues, leading to enhanced insulin sensitivity. Despite significant effort, the molecular basis for metformin response remains poorly understood, with a limited number of specific biochemical pathways studied to date. To broaden our understanding of hepatic metformin response, we combine phospho-protein enrichment in tissue from genetically engineered mice with a quantitative proteomics platform to enable the discovery and quantification of basophilic kinase substrates in vivo. We define proteins whose binding to 14-3-3 are acutely regulated by metformin treatment and/or loss of the serine/threonine kinase, LKB1. Inducible binding of 250 proteins following metformin treatment is observed, 44% of which proteins bind in a manner requiring LKB1. Beyond AMPK, metformin activates protein kinase D and MAPKAPK2 in an LKB1-independent manner, revealing additional kinases that may mediate aspects of metformin response. Deeper analysis uncovered substrates of AMPK in endocytosis and calcium homeostasis.

Keywords: AMPK3; LKB1; PKD1; STIM1; aging; calcium; diabetes; kinases; liver; metformin.

Figure 1.. Quantitative In Vivo Proteomics of Differential 14-3-3 Interactors in Murine Liver

Experimental design and workflow of the proteomic analysis of genetic and pharmacological sensitivity to LKB1 and Metformin in murine liver. Three biological replicates for each condition are tested.

Figure 2.. Efficacy of Proteomic Screen Compared to Traditional Phospho-Proteomic Approach

(A) Western blot analysis in biological triplicate of extracted mouse livers for proteomic screen. Hepatic deletion and metformin induction of signaling validated by downstream substrates of AMPK and mTOR. (B) Volcano plot for comparison of genetic sensitivity in vehicle treated 14-3-3 pull-downs from 3 biological replicates. Proteins that pass statistical and fold change criteria are highlighted in red and blue dots and found in the regions of the plot highlighted in yellow. Yellow lines represent the p value (<0.05) and fold change thresholds (>1.5). (C) Venn diagram of total proteins identified in lysate analysis of liver homogenate across 4 biological conditions versus high confidence 14-3-3 interactors following background subtraction and statistical filtering. (D) Venn diagram of proteins that contain the derived AMPK consensus motif to previous SCX-IMAC study. (E) Hierarchical clustering of 1,022 high confidence 14-3-3 interactors by trend within enriched samples

Figure 3.. Comprehensive Global Analysis of Differential 14-3-3 Interactors in Murine Liver

(A) Schematic of pathways contributing to potential 14-3-3 interactors within the screen. (B) Corresponding expected patterns for contributing pathways within normalized heatmaps and hierarchical clustering analysis. 14-3-3 enrichment patterns are depicted in red, lysate analysis for expression profiles are depicted in blue. Trend bar depicts correlation between 14-3-3 enrichment and lysate patterns across conditions. (C) Western blot analysis in biological triplicate of extracted mouse liver homogenate of activation status of additional basophilic kinases. (D) Representative cluster of potential MK2/PKD substrates. (E) Western blot analysis of extracted mouse liver homogenate extending MK2/PKD activation to phenformin. (F) Representative cluster of potential AMPK substrates. (G) Representative cluster of potential AMPKR substrates. (H) Representative cluster of potential AKT substrates.

Figure 4.. Differential 14-3-3 Interactors in Response to Metformin Action and Hepatic LKB1 Loss in Murine Liver, Potential AMPK Substrates

(A) Venn diagram depicting proteins of both metformin and LKB1 dependence. Of 1,022 high confidence 14-3-3 interactors, 250 increase in binding to 14-3-3 1.25-fold or greater in response to metformin administration (dark green). Of those 250 proteins, 110 proteins are reduced in binding by 1.25-fold or more (sum of blue and yellow) and of theses 110 proteins, 56 are reduced in 14-3-3 binding by 1.5-fold or greater in response to LKB1 loss (yellow). (B) Heatmap depicting 56 high confidence 14-3-3 interactors with both genetic (LKB1) and pharmacological sensitivity (metformin) as defined by previous thresholds (1.25-fold metformin induction, 1.5-fold loss upon LKB1 deletion). Purple arrows indicate known AMPK substrates from previous studies that satisfy the derived criteria.

Figure 5.. AMPK Regulates Endocytosis through Phosphorylation of Rabep1

(A) Heatmap of Gene Ontology process: endocytosis. (B) Dose-response curve of metformin at 2 h and 5 h time points in isolated primary hepatocytes from hepatic AMPK WT and conditional AMPK DKO mouse livers. (C) Schematic of domain architecture of Rabep1 highlighting location of S407 and clustal alignment of Rabep1 S407 across species showing conservation of AMPK consensus motif. (D) HEK293T cells transfected with Myc-Rabep1 WT or Ser407Ala as indicated and treated for 60 min with vehicle or 50 μM 991. Myc immunoprecipitations conducted in lysates and probed with anti-AMPK pMOTIF antibody. (E) Phosphorylation sites on Rabep1 identified by immunoprecipitation of transiently transfected Myc-Rabep1 WT in HEK293T cells after treatment with vehicle or 2 mM phenformin for 1 h and analyzed by MS/MS. Data reported in spectral counts indicated as stacked bars and graphed as a ratio of the observed modified versus unmodified spectra per site. Numbers in black overlay onto bars indicate number of observed spectral counts for each version of a given site. Blue portions of bar indicated modified, red portions indicate unmodified. (F) HEK293T cells transfected with Myc-Rabep1 WT or Myc-Rabep1 Ser407Ala, as indicated, pre-treated for 30 min with DMSO or 1.3 mM Gö6976 for 30 min prior to co-treatment with 200 nM TPA and/or 2 mM phenformin or vehicle for an additional 60 min. Myc immunoprecipitates for Rabep1 immunoblotted with anti-PKD pMOTIF and anti-AMPK pMOTIF antibodies and re-probed with anti Myc for loading controls. Lysates probed for pPKD Ser744/748 and pPKD Ser916 as read outs of PKD activation and pJNK as readout of downstream PKD activity.

Figure 6.. AMPK Phosphorylates Stim1 and Stim2 in Response to Metformin in Mouse Liver

(A) Heatmap of Gene Ontology process: calcium ion binding, purple arrows indicate STIM1 and STIM2. (B) Recombinant 14-3-3-GST or GST pull-downs in AMPK WT or AMPK DKO liver homogenates in biological duplicate for each biological condition. (C) Co-immunoprecipitation in mouse embryonic fibroblasts either WT or AMPK DKO for STIM2 and AMPKα using two different polyclonal antibodies against Stim2 full-length sequence. (D) Primary mouse hepatocytes from wild-type or AMPK DKO mice extracted and treated with a dose-response curve of 991 at 0, 10, 25, and 50 μM and compared to 2 mM phenformin for 60 min. (E) AMPK WT and DKO mice treated with 30 mg/kg MK8722 for 4 h, and liver homogenates analyzed by western blot analysis.

Figure 7.. AMPK Regulates Calcium Homeostasis through Phosphorylation of Stim1 and Stim2 to Terminate Store Operated Calcium Entry

(A) Clustal alignments of STIM1 S257, S512, S521, and STIM2 S261, S346, S680 across species showing conservation of AMPK consensus motif at identified potential sites of regulation. (B) Schematic depicting location of potential AMPK sites of regulation on STIM1 and STIM2 on structure of each protein relative to orientation to cytosol and ER lumen. (C) Representative MS/MS trace for identified STIM1 Ser521 site in STIM1 IP-MS in AMPK WT MEFs. (D) Regulated phosphorylation sites on STIM1 identified in STIM2 endogenous IP-MS in primary hepatocytes reported by spectral count. (E) Mouse embryonic fibroblasts stably expressing empty vector, flag-STIM1-WT, flag-STIM1-Ser521Ala in a WT or AMPK DKO background treated with vehicle, 50 μM MK-991 (9) or 2 mM phenformin (P) for 1 h. Flag immunoprecipitates blotted with antibody raised against the STIM1 Ser521 site and re-probed with antiflag antibody for loading. (F) Reduction of SOCE in AMPK WT MEFs but not AMPK DKO MEFs in response to AMPK activation quantitated by single cell microscopy using fura2-AM. Cells treated with vehicle or 2 mM phenformin in DMEM + 10% FBS for 30 min prior to analysis. Following pre-treatment, media was removed and replaced with Ca²⁺-free HBSS containing 2 mM phenformin prior to analysis. ER Ca²⁺ stores were emptied with 2 μM thapsigargin in Ca²⁺-free conditions before addition of buffer containing 2 mM Ca²⁺. (G) Same Fura2-AM measurements without Thapsigargin treatment to empty ER calcium stores. Induction of SOCE in response to calcium addition only seen in AMPK DKO cells in the absence of Tg treatment in phenformin-treated cells. (H) Controls traces for (F), switching the media from calcium-free to calcium-containing media in the absence of phenformin treatment. (I) Control traces for (F) with treatment of phenformin in the presence of calcium containing media at all times. All Fura2-AM graphs (F–I) show mean fura2 ratio of 60 (WT) and 60 (KO) cells across 2 biological replicates containing 30 single-cell readings each.