A hormone complex of FABP4 and nucleoside kinases regulates islet function

Abstract

The liberation of energy stores from adipocytes is critical to support survival in times of energy deficit; however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency disrupts metabolic homeostasis^1,2. Coupled to lipolysis is the release of a recently identified hormone, fatty-acid-binding protein 4 (FABP4)³. Although circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans^4-7, no mechanism of action has yet been described^8-10. Here we show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose-beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4-ADK-NDPK complex, Fabkin, represents a previously unknown hormone and mechanism of action that integrates energy status with the function of metabolic organs, and represents a promising target against metabolic disease.

Figure 1.. Depletion of FABP4 increases beta-cell mass and function

(a) Gross pancreas images in lean wild type (WT) and FABP4^−/− mice. (b) Insulin immunohistochemistry (IHC) in pancreatic sections from 7-wk-old WT or FABP4^−/− mice (N=4/group). (c) Quantification of percentage insulin positive area per total pancreatic area based on IHC (N=4/group; P=0.0318). (d) Total pancreatic insulin content from 7-wk-old WT and FABP4^−/− mice (N=3/group; P=0.0198). (e) Glucose-stimulated insulin secretion (GSIS) from islets ex vivo under low glucose (2.8mM; LG) and high glucose (16.7mM; HG) conditions (N=8/group; P=0.006008). (f) 6hr fasting blood glucose from diet-induced obese (DIO) mice before treatment (wk 0) and following a-Ab for 3 wks (N=10/group; P=0.000064). (g) Glucose tolerance test (GTT) in DIO mice treated for 2 wks with PBS or a-Ab (N=10/group). (h) Insulin IHC in pancreatic sections from DIO mice treated with PBS or a-Ab for 3 wks (N=6/group) with (i) quantification of total islet number per pancreatic section (N=8/group; P=0.0157), and (j) percentage of insulin positive area per total pancreatic area (N=4/group). (k) Plasma FABP4 levels in autoantibody positive and negative normal glucose tolerant (NGT) individuals compared to new-onset T1D patients (<1-year duration; BABYDIAB and DiMELLI cohorts) (N=30/group; Ab+ vs. T1D P=0.0049; Ab- vs. T1D P=0.0047). (l) Correlation of plasma FABP4 with HbA1c percentage in established T1D patients (BRI cohort; N=50/group). (m) Plasma FABP4 levels in NOD mice while NGT, one week prior to hyperglycemia (Prior), or at time of T1D onset (N=35 (NGT), 16 (Prior), 10 (T1D); NGT vs. Prior P<0.00001; NGT vs. T1D P=0.0193). (n) Incidence curve for NOD model of T1D following treatment with PBS, a-Ab, or c-Ab beginning at 10 wks of age (N=36/group; P=0.0079). (o) Average blood glucose of NOD mice at the time of T1D diagnosis (N=23 (PBS), 11 (a-Ab), 19 (c-Ab); PBS vs. a-Ab P=0.0491; a-Ab vs. c-Ab P=0.0072). (p) Plasma insulin levels prior to T1D diagnosis in NOD mice (N=22 (PBS), 10 (a-Ab), 18 (c-Ab); PBS vs. a-Ab P=0.0350; a-Ab vs. c-Ab P=0.0055). (q) GTT and (r) corresponding plasma insulin values in non-diabetic NOD mice treated with PBS or a-Ab (N=6/group). (s) GSIS from islets isolated from NOD mice treated with PBS or a-Ab for 15 wks (N=4/group; P=0.0452). (t) Insulin IHC and quantification of (u) percentage of insulin positive area per total pancreatic area (P=0.0125), and (v) islet number per pancreatic section from mice treated with a-Ab or c-Ab for 5 wks (N=5/group; P=0.0085). (w) Quantification of insulin and glucagon immunofluorescence (IF) in pancreatic sections of NOD mice treated with a-Ab or c-Ab for 5 wks (N=4/group; P=0.0409). (x) IF and (y) quantification of insulin (green) and glucagon (red) staining in pancreatic sections of DIO mice treated with a-Ab or PBS for 3 wks (N=26 (PBS) and 30 (a-Ab) islets from 5 mice/group; P=0.0044). Scale bars for panels 500um (b,h,t) and 200um (x). *P<0.05, **P<0.01, ***P<0.001. Data are mean +/− SEM. Two-tailed unpaired t-test (c-f,I,j,u,v,w,y); One-way ANOVA (k,m,o,p); Two-way ANOVA (g,q,r,s); Simple linear regression correlation (l); Log-rank Mantel-Cox test (n).

Figure 2.. Circulating FABP4 forms a hormonal complex with NDPK and ADK to regulate extracellular nucleosides.

(a) Results of three independent immune-precipitation (IP) and mass spectrometry experiments from WT or FABP4^−/− DIO serum with a-Ab or c-Ab with top hits based on enrichment and spectral counts. Spectral counts of (b) ADK and (c) NDPK from Screen 1 (N=3/group). (d) Summary of MST among proposed complex components (N=6/interaction). (e) IP of GST-tagged NDPK with each of the proposed complex components and a-Ab (LC, light chain; HC, heavy chain; N=4). (f) On-bead ADK kinase assay for ATP production from WT or FABP4^−/− mouse serum (N=5/group). (g) ATP production from recombinant ADK in the presence of complex components (N=3/group; ADK vs. ADK FABP4 P=0.0113; ADK vs FABP4-ADK-NDPK P=0.0154; ADK-NDPK vs. ADK FABP4 P=0.0360). (h) On-bead NDPK kinase assay for ADP production from WT or FABP4^−/− mouse serum (N=5/group). (i) ADP production from recombinant NDPK in the presence of complex components (N=3/group; NDPK vs. NDPK ADK P=0.0001; NDPK vs. NDPK FABP4 P=0.0221; NDPK vs. FABP4-ADK-NDPK P=0.0001). (j) ADK activity to produce ATP in the presence of complex components with a-Ab or c-Ab (N=3/group; ADK vs. FABP4-ADK-NDPK P=0.0076; FABP4-ADK-NDPK vs. a-Ab P=0.0340; ADK vs. c-Ab P<0.0001; FABP4-ADK-NDPK vs. c-Ab P<0.0001; a-Ab vs. c-Ab P<0.0001). (k) NDPK activity to produce ADP in the presence of complex components with a-Ab or c-Ab (N=3/group; NDPK vs. FABP4-ADK-NDPK P<0.0001; NDPK vs. c-Ab P=0.0002; FABP4-ADK-NDPK vs. a-Ab P=0.0002; a-Ab vs. c-Ab P=0.0003). (l) Proposed model of extracellular nucleoside regulation from the complex. *P<0.05, **P<0.01, ***P<0.001. Data are mean +/− SEM. Two-way ANOVA (f,h); One-way ANOVA (g,i-k).

Figure 3.. The FABP4-ADK-NDPK complex inhibits GSIS and is neutralized by a-Ab.

GSIS from (a) WT primary mouse islets (N=3/group; P<0.0001), (b) human islets (N=3/group; Control vs. NDPK-ADK P=0.0038; NDPK-ADK vs. FABP4-ADK-NDPK P=0.0049; Control c-Ab vs. NDPK-ADK c-Ab P=0.0043; NDPK-ADK c-Ab vs. FABP4-ADK-NDPK c-Ab P=0.0230; Control a-Ab vs. NDPK-ADK a-Ab 0.0031; NDPK-ADK a-Ab vs. FABP4-ADK-NDPK a-Ab P=0.0067) and (c) INS1 cells with complex components (N=3/group; Control vs. NDPK-ADK P=0.0012; NDPK-ADK vs. FABP4-ADK-NDPK P=0.0002; FABP4-ADK-NDPK vs. a-Ab P=0.0009; c-Ab vs. a-Ab P=0.0066). (d) Proposed model for the activity of FABP4-ADK-NDPK on P2Y1. (e) GSIS from WT mouse islets treated with high ratio (ATP degrading) or low ratio (ATP and ADP degrading) apyrase (N=3/group). GSIS from INS1 cells treated with control, NDPK-ADK, or FABP4-ADK-NDPK in the presence of (f) 5uM ATP or non-metabolizable ATPγS (N=7/group) and (g) 5uM of ADP (N=3/group; Control vs. NDPK-ADK P=0.0132; NDPK-ADK vs. FABP4-ADK-NDPK P=0.0307; FABP4-ADK-NDPK vs. FABP4-ADK-NDPK ADP P=0.0273). (h) GSIS from INS1 cells with ADP in the presence or absence of P2Y1 antagonist MRS2179 (N=4/group). (i) GSIS from INS1 cells treated with NDPK-ADK or FABP4-ADK-NDPK in the presence of MRS2179 (N=3/group; Control vs. NDPK-ADK P=0.0232; NDPK-ADK vs. FABP4-ADK-NDPK P=0.0038; NDPK-ADK vs. NDPK-ADK MRS2179 P=0.0026). (j) Kinase activity of WT NDPK or kinase dead H118N NDPK to produce ADP (N=4/group). (k) GSIS from INS1 cells treated with WT NDPK or H118N NDPK in combination with ADK and FABP4 (N=4/group). (l) Kinase activity of ADK to generate ATP in the presence of ADK inhibitor (N=4/group). (m) GSIS from INS1 cells treated with NDPK-ADK or FABP4-ADK-NDPK in the presence of ADK inhibitor (N=3). In all cases, low glucose (LG; 2.8mM), high glucose (HG; 16.7mM). *P<0.05, **P<0.01, ***P<0.001. Data are mean +/− SEM. Two-way ANOVA (a,b,e-m); One-way ANOVA (c).

Figure 4.. The FABP4-ADK-NDPK complex alters beta-cell calcium dynamics and promotes cell death.

(a) Western blot (WB) and quantification of pPKA substrate phosphorylation in human islets treated with NDPK-ADK or FABP4-ADK-NDPK (N=6; Control vs. NDPK-ADK P<0.0001; NDPK-ADK vs. FABP4-ADK-NDPK P=0.0007; Control vs. FABP4-ADK-NDPK P=0.0095). (b) WB and quantification of pIP3R in INS1 cells treated with FABP4-ADK-NDPK (N=4; P=0.0034). Cytosolic calcium flux in INS1 cells from (c,d) the extracellular space in response to glucose (N=3 coverslips, 150 cells/condition except NDPK-ADK 144 cells) and (e,f) the ER in response to 1uM thapsigargin by Fura-2 AM staining (N=3 coverslips, 75 cells/condition except NDPK-ADK 100 cells). (g) WB for cleaved caspase 3 (CC3), pJNK, Chop, and B-Tubulin from INS1 cells treated with FABP4-ADK-NDPK for 24hrs (N=4). (h) Gene expression of ER stress marker Chop following 2hr treatment with complex components in the presence or absence of Tg (N=3; Control vs. FABP4-ADK-NDPK P<0.0001; NDPK-ADK vs. FABP4-ADK-NDPK P=0.0012; FABP4-ADK-NDPK vs. a-Ab P=0.0003). (i) Cleaved caspase 3/7 activity in INS1 cells treated with cytokine cocktail (TNFa, IFNg and IL-1b) with or without FABP4-ADK-NDPK (N=4/group). (j) IF for insulin (green), BrdU (red), and nuclei (DAPI; blue) and (k) quantification of pancreatic sections from NOD mice treated with BrdU and a-Ab or c-Ab for 5wks (N=5 mice/group; 48 (c-Ab), 46 (a-Ab) islets). (l) IF for insulin (green), Ki67 (red), and nuclei (DAPI; blue) and (m) quantification of pancreatic sections from DIO mice treated with PBS or a-Ab for 3 weeks (N=5 mice/group; 39 (PBS), 45 (a-Ab) islets; P=0.0437). (n) IF staining of pancreatic sections from NOD mice treated with a-Ab or c-Ab for insulin (red), CC3 (green) and nuclei (DAPI; blue) with (o) quantification of CC3 and insulin co-stained cells (N=5 mice/group; 33 (c-Ab), 45 (a-Ab) islets; P=0.0010). (p) IF of pancreatic sections from DIO mice treated with PBS or a-Ab for insulin (green), CC3 (red) and nuclei (DAPI; blue) with (q) quantification of CC3 and insulin co-stained cells (N= 5 mice/group; 25 (PBS), 34 (a-Ab) islets; P<0.0001). Scale bars are 150um (j,l,p) and 100um (n). *P<0.05, **P<0.01, ***P<0.001. Data are mean +/− SEM. One-way ANOVA (d,f); Two-tailed unpaired t-test (a,b,k,m,o,q); Two-way ANOVA (h,i).