Cell Metabolism, Volume 27 Supplemental Information LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor Xuecai Ge, Hong Yang, Maria A. Bednarek, Hadas Galon-Tilleman, Peirong Chen, Michael Chen, Joshua S. Lichtman, Yan Wang, Olivier Dalmas, Yiyuan Yin, Hui Tian, Lutz Jermutus, Joseph Grimsby, Cristina M. Rondinone, Anish Konkar, and Daniel D. Kaplan
A Sham VSG Ghrl expression in stomach detected by RNAseq 12 1 8 6 4 2 Gene Expression (RPKM) Ghrl expression in stomach detected by qpcr 1.8.6.4.2 Relative gene expression level (normalized to GAPDH) Relative gene expression level (normalized to GAPDH) Leap2 expression level detected by qpcr.4.3.2.1 Stomach ** Duodenum *.4.3.2.1 Relative gene expression level (normalized to GAPDH) B Sequence and disulfide bond structure of murine LEAP2 Disulfide Bonds MLQLKLFAVLLTCLLLLGQVNSSPVPEVSSAKRSRRMTPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE-COOH Signal peptide Propeptide Mature peptide C Multiple species alignment of LEAP2 Homo sapiens Pan troglodytes Macaca mulatta Cavia porcellus Bos taurus Mus musculus Rattus norvegicus Oryctolagus cuniculus Gallus gallus Ctenopharyngodon idella Mature Peptide D Identification of LEAP2 from tissue extracts MS of Synthetic LEAP2 IP-MS of 1 nm Synthetic LEAP2 in PBS IP-MS of 1 nm Synthetic LEAP2 in Stomach Extract IP-MS of Endogenous LEAP2 from Stomach Extract IP-MS of Endogenous LEAP2 from Liver Extract IP-MS of Endogenous LEAP2 from Small Intestine Extract Wash Ge et al. Supplementary Figure 1. LEAP2 is a secreted peptide with two conserved disulfide bonds 1
Ge et al. Supplementary Figure 1, related to Figure 1. LEAP2 is a secreted peptide with two conserved disulfide bonds (A) Expression level of Ghrl and Leap2 detected by RNA-seq or qpcr before and after vertical sleeve gastrectomy (VSG) surgery. **, p<.1., p<.1 vs. sham. Data are from one experiment that is representative of three separate experiments. Data are represented as mean ± SEM. (B) Amino acid sequence of LEAP2 - disulfide bonds are indicated. (C) Multiple species alignment of LEAP2 showing conserved cysteine residues (highlighted). (D) To examine endogenous forms of LEAP2, LEAP2 was immunoprecipitated from stomach, intestine, and liver extracts using an antibody recognizing the C-terminus of the peptide, followed by a search for LEAP2 isoforms using LC-MS. The search included all likely charge states of the 76 aa prepro-peptide precursor, the 54 aa pro-peptide, the mature 4 aa peptide, and smaller potential LEAP2 forms that could be generated by cleavage following basic residues and that would leave the disulfide-bonded core of the peptide intact. The peptide forms that were examined are as follows: MLQLKLFAVL LTCLLLLGQV NSSPVPEVSS AKRSRRMTPF WRGVSLRPIG ASCRDDSECI TRLCRKRRCS LSVAQE SPVPEVSS AKRSRRMTPF WRGVSLRPIG ASCRDDSECI TRLCRKRRCS LSVAQE MTPF WRGVSLRPIG ASCRDDSECI TRLCRKRRCS LSVAQE GVSLRPIG ASCRDDSECI TRLCRKRRCS LSVAQE PIG ASCRDDSECI TRLCRKRRCS LSVAQE From all three tissues, the mature, 4 aa form of LEAP2 was detected, but none of the other forms were found. The extracted ion chromatogram for the z=6 charge state representing mature LEAP2 peptide from the indicated samples is shown in the top panel. A representative isotopic distribution of the predominant (+6 charge state) peak representing the mature form of LEAP2 (4581.29 Da) is shown in the bottom panel. 2
A GHSR tagged with enzyme donor GHSR Activating ligand binds to GHSR B Activating ligand binds to GHSR GHSR GHSR PLC Gq Enzyme donor IP3 Gq β-arrestin recruited to GHSR β-arrestin & Enzyme Acceptor Calcium dye Substrate C Antagonist mode agonist LEAP2 E 75 5 25-25 -1-9 -8-7 -6 LEAP2 inhibits ghrelin-induced GHSR activity in calcium mobilization assay 1 GHSR Activation (%) GHSR Activation (%) Percentage activation Ghreilin-induced GHSR activation in calcium mobilization assay 75 5 25-25 -1-8 -7U 2 O -6-4 S G H S-5 R Log [LEAP2], C a lc iu m M M o b iliz a tio n [With (13 [D -A r gghrelin 1,D -P h e at 5,DEC -T r 8 p 7,9,L enm)] u 1 1 ]-S u b s ta n c e -5 Log [Ghrelin], M LEAP2 compete-calcium-3parameter Ghrelin (um) GHSR Activation (RLU) 4 LEAP2 (nm) G 1.23 3.7 11.1 33.3 1 3 2 1-11 35 35 LEAP2 P o m p o u n d ] (M ) [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-Substance P[C(nM) 1.2 3 E - 8 3.7 E - 8 25 25. 1 2 2 15 15 1 1 5 5-1 -9-8 -7-6 -5-4 -3-1 1 1-11 GHSR -1 1-1 1-9 -9 1-8 -8 1-7 -7 1-6 -6 1-5 -5 1-4 -4 1-3 -3 [G h r e lin ] (M ) (GHrelin),M Loglog[Ghrelin], M H -9 3 3 RLU GHSR Activation (RLU) F Read GPCR activity 9min 3min 1-11 agonist Read GPCR activity 9min 125 Ca2+ Luminescence Agonist mode D ER Dye chelated to calcium Bottom Top LogEC5 HillSlope EC5 15.2 = 29. -8.367 ~ 1. 4.298e-9 Log [Ghrelin], M 1.23E-8 17.8 = 29. -8.253.9574 5.578e-9 3.7E-8 17.9 = 29. -8.22.979 6.286e-9 LEAP2/GHSR/Hoechst 1.11E-7 16.1 = 29. -8.53.8175 8.857e-9 3.33E-7 18.2 = 29. -7.656.7852 2.27e-8.1 17.7 = 29. -7.284.7256 5.198e-8 1μm Ge et al. Supplementary Figure 2. LEAP2 Antagonizes Ghrelin-induced Activation of Growth Hormone Secretagogue Receptor (GHSR) 3
Ge et al. Supplementary Figure 2, related to Figure 2. LEAP2 Antagonizes Ghrelin-induced Activation of Growth Hormone Secretagogue Receptor (GHSR) (A) Diagram showing the detection of β-arrestin recruitment following GHSR activation in PathHunter cells. GHSR is tagged with a small enzyme donor, and is stably co-expressed with a fusion protein of β-arrestin and truncated β- galactosidase. Activation of GHSR leads to β-arrestin recruitment, which then allows complementation of the two enzyme fragments, generating active β-galactosidase. The active enzyme converts non-luminescent substrate to a chemiluminescent product. (B) Diagram showing the detection of calcium mobilization following GHSR activation. Cells stably-express wild-type GHSR. Activation of GHSR triggers G q activation, resulting in phospholipase C (PLC) activation, and ultimately, mobilization of calcium from intracellular stores. The calcium concentration is detected using the calcium-sensitive dye FLUO-3. (C) Experimental procedure for evaluating GHSR activation in agonist and antagonist modes. (D) Concentration-response curve showing ghrelin-induced GHSR activation, detected using calcium mobilization assay (EC 5 = 4.25 ±.67 nm). (E) Concentration-response curve of LEAP2 on GHSR activation in the presence of ghrelin (at EC 8 ) detected by calcium mobilization (LEAP2 IC 5 = 11.6 ± 1.3 nm). (F) Concentration-response curve of ghrelin-induced GHSR activation (using calcium mobilization assay) in the presence of LEAP2, demonstrating that LEAP2 is a non-competitive antagonist of GHSR. (G) Concentration-response curve of ghrelin-induced GHSR activation (using calcium mobilization assay) in the presence of a known competitive antagonist, [D-Arg 1, D-Phe 5, D-Trp 7,9, Leu 11 ]- Substance P. (H) Binding of LEAP2 to GHSR in COS7 cells transiently transfected with GHSR. LEAP2 binds to transfected cells (arrows), but not non-transfected cells (arrow heads). Data are from one experiment that is representative of three separate experiments. Data are represented as mean ± SEM. 4
B 1.6 1.4 1.2 1..8.6.4.2. Leap2 mrna expression is greater in jejunum than liver Jejunum Liver brain stem hippocampus hypothalamus esophagus stomach duodenum jejunum ileum colon liver ventricle atrium lung spleen pancreas adrenal testis kidney bone muscle skin fat cortex cerebellum Relative Leap2 mrna level (normalized to GAPDH) A C In situ hybridization of Leap2 in liver D In situ hybridization of Leap2 in jejunum villus hepatocytes 2 μm 2 μm 2 μm crypt 2 μm F Development of LEAP2 Sandwich ELISA (2) Capture: rabbit-anti-leap2 Detection: HRP-goatanti-rabbit (3) (4) Narrow range of LEAP2 concentrations R2=.9951 (5) Log [LEAP2] ng/ml H Fasting reduces serum LEAP2 levels, increases Ghrelin levels 3 ** 12 2.5 1 2 8 6 1.5 1. 4.5 2 Before fast After fast After refeed Serum Ghrelin (ng/ml) 14 Minutes after LEAP2 injection Synthetic LEAP2 (ng/ml) Serum LEAP2 (nm) LEAP2 levels in serum after single i.v. injection (LEAP2: 4 µg/kg BW) Serum LEAP2 (ng/ml) Serum LEAP2 (ng/ml) G R2=.9995 3.8 **.7 2.5.6 2.5.4 1.5.3 1.2.5.1 Serum Ghrelin (nm) (1) Wide range of LEAP2 concentrations OD45 nm LEAP2 in serum Coating: goat-anti-leap2 LEAP2 ELISA Standard Curve OD45 nm E Before fast After fast After refeed Ge et al. Supplementary Figure 3. LEAP2 is produced in enterocytes and hepatocytes; detection of endogenous LEAP2 5
Supplementary Figure 3, related to Figure 3. LEAP2 is produced in enterocytes and hepatocytes; detection of endogenous LEAP2 (A) Quantitative PCR showing that Leap2 is expressed in the small intestine and the liver, but not other tissues. Leap2 expression level is the highest in jejunum. Not shown: Separately, Leap2 expression was measured in RNA isolated from cerebral cortex, hypothalamus, and pituitary using RNA-seq. Leap2 was undetectable in cerebral cortex and hypothalamus, and was detected at a very low level in the pituitary (.1 FPKM). (B) In situ hybridization showing expression levels of Leap2 in jejunum and liver. (C-D) Expression pattern of Leap2 in liver and jejunum. In liver, Leap2 is ubiquitously expressed in all hepatocytes. Note that in jejunum, Leap2 is highly expressed in the outer layer of villi (arrows in D), but not in crypts. Leap2 is absent from the lamina propria layer of the villus. (E) Diagram showing the development of LEAP2 Sandwich ELISA. (F) Detection of synthetic LEAP2. The wide range LEAP2 concentration curve is fitted with sigmoidal four parameter logistic (4PL) regression. The narrow range LEAP2 concentration curve is fitted with a second order polynomial. (G) LEAP2 ELISA used to detect the levels of LEAP2 in serum after a single dose of peptide was administered to mice intravenously. (H) Effects of fasting and refeeding on serum levels of LEAP2 and ghrelin. LEAP2 and ghrelin levels are shown in ng/ml (left axes) and nm (right axes) **, p<.1,, p<.1. Student s t-test. N=5 mice/group. Data in (A) and (F-H) are from one experiment that is representative of three separate experiments. Data are represented as mean ± SEM. 6
A Fat mass / Body weight.16.14.12.1.8.6.4.2. CR AL CR AL AAV: GFP LEAP2 Day Day 12 (Day 8 for LEAP2-CR).9.8.7.6.5.4.3.2.1. CR AL CR AL GFP LEAP2 Lean mass / Body weight B Free Fatty Acids (μm) 7 6 5 4 3 2 1 AL CR AAV: GFP LEAP2 GFP LEAP2 12 1 8 6 4 2 Total Ketone Bodies (μm) C Serum LEAP2 (ng/ml) 35 3 25 2 15 1 5 AAV: GFP LEAP2 AL CR D Serum GH (ng/ml) 8 6 4 2 GH pump Veh pump 2 4 6 1 14 Days after implant E Serum LEAP2 (ng/ml) 6 5 4 3 2 1 * pump GH Veh F Serum GH (ng/ml) 6 5 4 3 2 1 GH pump Veh pump G Serum Ghrelin (ng/ml) 3 25 2 15 1 5 * GH pump Veh pump AAV: GFP LEAP2 AAV: GFP LEAP2 AAV: GFP LEAP2 H Fat mass / Body weight.25.2.15.1.5 Day Day 12 1.2 1.8.6.4.2 Lean mass / Body weight Pump: GH Veh GH Veh GH Veh GH Veh AAV: GFP LEAP2 GFP LEAP2 AAV: GFP LEAP2 GFP LEAP2 I Free Fatty Acids (μm) 7 6 5 4 3 2 1 GH pump Veh pump 12 1 8 6 4 2 Total Ketone Bodies (μm) Supplementary Figure 4. Hormone levels and body composition after chronic caloric restriction 7
Ge et al. Supplementary Figure 4, related to Figure 4. Hormone levels and body composition after chronic caloric restriction (A) Ratio of fat or lean mass to body weight at the end of the chronic caloric restriction experiment, as measured by MRI. Caloric restriction caused reduction of fat mass, but had minimal effect on lean mass. (B) Serum levels of free fatty acids and total ketone bodies at the end of the chronic calorie restriction experiment. Both free fatty acid and total ketone body levels are dramatically decreased by chronic caloric restriction, indicating depletion of body fat and reduced ketogenesis. *, p<.5; **, p<.1;, p<.1. Student s t-test. N=5 mice/group. (C) Serum levels of LEAP2 following chronic caloric restriction. (D) Serum levels of GH after osmotic pump implantation in naïve mice. The osmotic pumps stably delivered GH over the entire experimental period. (E-G) Serum levels of LEAP2, GH, and Ghrelin at the end of the experiment. (H) Ratio of fat or lean mass to body weight at the end of the experiment, as measured by MRI. Caloric restriction caused reduction of fat mass. (I) Levels of free fatty acids and total ketone bodies at the end of the assay, both of which are dramatically decreased by chronic caloric restriction. The normal levels are shown in panel (B). *, p<.5; **, p<.1;, p<.1. Student s t-test. N=7 mice/group. *, p<.5; **, p<.1;, p<.1. Student t-test. N=5-7. Figure S4: Data are from one experiment that is representative of three separate experiments. Data are represented as mean ± SEM. 8