A liver HIF-2α/IRS2 pathway sensitizes hepatic insulin signaling and is modulated by VEGF inhibition

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1 A liver HIF-2α/IRS2 pthwy sensitizes hepti insulin signling n is moulte y VEGF inhiition Kevin Wei1,1, Stephnie M. Pieewiz1,1, Lis M. MGinnis1,1, Cullen M. Tniguhi2, Stnley J. Wiegn3, Keith Anerson3, Crol W-M. Chn1, Kimerly X. Mullign4, Dvi Kuo1, Jenny Yun1, Mrio Vllon1, Lori Morton3, Etienne Lefi5, M. Celeste Simon6, Jquelyn J. Mher7, Gilles Mithieux8, Fienne Rjs8, Justin Annes9, Owen P. MGuinness4, Gvin Thurston3, Amto J. Gii2, n Clvin J. Kuo1 1 Division of Hemtology, Stnfor University Shool of Meiine, Stnfor, Cliforni, USA. 2 Division of Rition Onology, Stnfor University Shool of Meiine, Stnfor, Cliforni, USA. 3 Regeneron Phrmeutils, Trrytown, New York, USA. 4 Deprtment of Moleulr Physiology n Biophysis, Vnerilt University Shool of Meiine, Nshville, Tennessee, USA. 5 INSERM U16, Institut ntionl e l Reherhe Agronomique 1235, Université e Lyon, Lyon, Frne. 6 Armson Fmily Cner Reserh Institute, Howr Hughes Meil Institute, University of Pennsylvni Shool of Meiine, Philelphi, Pennsylvni, USA. 7 University of Cliforni Sn Frniso Liver Center, Sn Frniso Generl Hospitl, Sn Frniso, Cliforni, USA. 8 INSERM U855, Université e Lyon, Lyon, Frne. 9 Division of Enorinology n Metolism, Stnfor University Shool of Meiine, Stnfor, Cliforni, USA. 1 These uthors ontriute eqully to this work. Corresponene shoul e resse to C.J.K. (jkuo@stnfor.eu). Supplementry Informtion Inluing Supplementry Figures 1-8 Nture Meiine: oi:1.138/nm.3295

2 Supplementry Figure 1 C57Bl/6 GTT C57Bl/6 ITT / GTT ITT AUC A-F A-sFlt1 A-F A-sFlt1 A-F A-sFlt1 A-sFlk1 e f g ITT AUC A-F / ITT C57Bl/6 GTT / GTT A-sFlt1 A-sFlk1 GTT / Rgwee B h GTT C57Bl/6 IgG DC Supplementry Figure 1. -f. The AUC vlues from the GTT n ITT stuies from A-sFlt1, A-sFlk1 n reominnt -trete mie in Figure 1 re presente. g. 8-1 week ol / mie reeive i.p. injetion of the nti-vegf ma B for 14 ys t 5mg/kg 3x/week followe y GTT. h. C57Bl/6 mie (n=5) reeive nti VEGFR2 ma DC11 s.. 4 mg/kg 2x/week for 14 ys followe y GTT. = p<.5 versus ontrols. Vlues re men ± s.e.m. Nture Meiine: oi:1.138/nm.3295

Supplementry Figure 2 A sflt1 C57Bl/6 Aflierept SCID Gluose (mg/l) 14 12 1 8 6 4 2 A-F A-sFlt1 fst Gluose (mg/l) 16 14 12 1 8 6 4 2 Humn F Aflierept 1mg/kg Aflierept 25mg/kg Aflierept 4mg/kg fst f

A li n fsting loo gluose levels in () 1 12 week-ol C57Bl/6 (n = 7) mie injete with 5 x 1 8 p.f.u. A-F or A-sFlt1 n () 8 1 week-ol SCID (n = 8) mie 6 weeks fter twie-weekly s.

3 Supplementry Figure 2 A sflt1 C57Bl/6 Aflierept SCID Gluose (mg/l) A-F A-sFlt1 fst Gluose (mg/l) Humn F Aflierept 1mg/kg Aflierept 25mg/kg Aflierept 4mg/kg fst f Aflierept / fste / / /+ /+ Anti-VEGFR2 ma C57Bl/6 Gluose (mg/l) g Rt IgG DC11 fst e Aflierept / ys tretment / / /+ /+ fli Supplementry Figure 2.,. A li n fsting loo gluose levels in () 1 12 week-ol C57Bl/6 (n = 7) mie injete with 5 x 1 8 p.f.u. A-F or A-sFlt1 n () 8 1 week-ol SCID (n = 8) mie 6 weeks fter twie-weekly s.. injetion with or ontrol t the inite oses week ol / (n=5) or /+ mie trete with or ontrol protein twie weekly with 25 mg kg -1 suutneously for 15 ys.. A liitum n fsting gluose levels of C57Bl/6 mie trete with the nti-vegfr2 DC11 s.. 4 mg kg -1 twie weekly for 2 weeks. e. VEGF inhiitor tretment inues sustine orretion of hyperglyemi in / mie. A liitum loo gluose levels from -6 weeks re epite. f. A liitum n fste (16 hour) plsm insulin level in SCID mie. There is tren towr erese insulin levels in the fe stte (P=.21) with no eviene for ny hyperinsulinemi s use for erese loo gluose. g. Fsting plsm glugon levels in SCID mie. Vlues re men ± s.e.m. = P <.5. Nture Meiine: oi:1.138/nm.3295

4 Supplementry Figure 3 Gluose infusion rte Bloo gluose lmp gluose infusion (mg kg -1 min -1 ) insulin infusion strt Time (minutes) Time (minutes) Gluose turnover rte Gluose uptke gluose turnover (mg kg -1 min -1 ) gluose turnover rte Rg (ug/min/mg) fli Supplementry Figure 3. Mesurements of tissue-speifi gluose uptke following meite Vegf inhiition from the mie in Fig. 1e in whih insulin-suppresse hepti gluose proution (HGP) ws etermine y euglyemi hyperinsulinemi lmp nlyses. HGP (Figure 1e) ws etermine y sutrting the gluose infusion rte. neessry to mintin onstnt loo gluose level (lmp). from the whole oy gluose uptke. The gluose turnover rte is epite in. Whole oy gluose uptke n insulin-stimulte gluose uptke ws etermine s the rtio of the [3-3 H] gluose infusion rte () to the speifi tivity of plsm gluose uring the sl n lmpe perio, respetively. Insulin-stimulte gluose uptke in iniviul orgns ws unltere n ws etermine y estimting the orgn-speifi ontent of non-metolizle gluose nlog. Insulin onentrtion uring lmp ws 1.4±.2 vs 1.8±.4 ng/ml in n, respetively. Vlues re men ± s.e.m. Nture Meiine: oi:1.138/nm.3295

Supplementry Figure 4 A-F p-ampk Ampk p-ampk Ampk nuler Cre WB p-a1 A1 A-sFlt1

sflt1 sflk1 PAS (glyogen) G6p F Supplementry Figure 4.

. Ault C57Bl/6 mie (n=3) reeive single i.v. injetion of 5x18 p.f.u. A-sFlt1 or

with 25 mg kg-1 suutneously for 15 ys (right pnel) followe y Western lotting

er extrt for Cre.. / mie reeive single i.v. injetion of 19 p.f.u.

injetion of 19 pfu of the inite enoviruses followe y liver hrvest in the fste

In the top row, note sl (F) G6p immunoretivity (rown) tht is signifintly erese

5 Supplementry Figure 4 A-F p-ampk Ampk p-ampk Ampk nuler Cre WB p-a1 A1 A-sFlt1 nuler Cre WB F sflt1 sflk1 p-gsk3 Gsk3 p-akt F sflt1 sflk1 Irs2 -tin Akt e sflt1 sflk1 PAS (glyogen) G6p F Supplementry Figure 4.. Ault SCID mie (n=5) were trete with or ontrol protein twie weekly with 25 mg kg-1 suutneously for 15 ys. This ws followe y Western lotting of liver extrts for the inite ntioies.. Ault C57Bl/6 mie (n=3) reeive single i.v. injetion of 5x18 p.f.u. A-sFlt1 or A-F (left pnel), or ult SCID mie (n=3) reeive or ontrol protein twie weekly with 25 mg kg-1 suutneously for 15 ys (right pnel) followe y Western lotting of liver nuler extrt for Cre.. / mie reeive single i.v. injetion of 19 p.f.u. of the inite enoviruses followe y liver Western lot fter 15 ys, ium.. C57Bl/6 mie reeive 19 p.f.u. of the inite enoviruses followe y liver Western lot fter 15 ys, ium. e. Vegf inhiition represses G6p n inreses glyogen synthesis in liver. C57Bl/6 mie reeive single i.v. injetion of 19 pfu of the inite enoviruses followe y liver hrvest in the fste stte fter 14 ys n IHC using nti-g6p (top) or PAS stining for glyogen (ottom). In the top row, note sl (F) G6p immunoretivity (rown) tht is signifintly erese y VEGF inhiition (sflt1, sflk1). In the ottom row, note low-level PAS/glyogen positivity (F) whih is preferentilly inrese y VEGF inhiition (sflt1, sflk1) (rk purple stin). These results re onsistent with VEGF inhiition sensitizing hepti insulin signling with repression of the gluoneogeni enzyme G6p n inrese of liver glyogen stores.. Nture Meiine: oi:1.138/nm.3295

Supplementry Figure 5 Aflierept ose-epenene A-F 5 A- 14 A-

Aflierept reversiility 25 Vsulr re ensity % 14 12 1 8 6 4

2x/week for 14 ys, followe y CD31 stining.

Aitionlly, quntittion of CD31+ sinusois in 1 iniviul fiels

injetion of enovirus enoing to hieve trnsient plsm

, 26); or ontrol enovirus enoing mouse IgG2 F followe y

p, 3x/week) for 14 ys, followe y CD31 stining of the hepti

6 Supplementry Figure 5 Aflierept ose-epenene A-F 5 A- 14 A- 5 A- 21 A- 1 A- 111 CD31 IgG Anti-rgwee Vsulr re ensity % Aflierept reversiility 25 Vsulr re ensity % DC11 B Supplementry Figure 5.. C57Bl/6 mie reeive the inite s.. oses of ( Trp ), humn ntioy IgG1 F frgment () or vehile 2x/week for 14 ys, followe y CD31 stining. Hepti vsulture surrouning the entrl vein is epite. Aitionlly, quntittion of CD31+ sinusois in 1 iniviul fiels is presente in intervessel n entrl vein regions.. C57Bl/6 mie reeive single i.v. injetion of enovirus enoing to hieve trnsient plsm expression of tht lsts ~3 ys (Tm et l, Nt. Me., 26); or ontrol enovirus enoing mouse IgG2 F followe y hrvest n CD31 immunofluoresene nlysis of liver sinusois t the inite times. The reversiility of the hepti sinusoil regression is epite over ys C57Bl/6 mie reeive the nti-vegfr2 ma DC11 or ontrol rt IgG (4 mg/kg, i. p, 3x/week) for 14 ys, followe y CD31 stining of the hepti vsulture. 1x mgnifition.. C57Bl/6 mie reeive reeive i. p. injetion of the nti-vegf ma B or ontrol nti-rgwee ma for 14 ys t 5 mg/kg 3x/week followe y CD31 stining of the hepti vsulture. 2x mgnifition. Quntittion of vsulr re ensity ws performe s in Fig. 2. Vlues re men ± s.e.m. Nture Meiine: oi:1.138/nm.3295

Supplementry Figure 6 HIF trget genes fli 1 2 3 4 5 1 2 3 4 Col61 Trfr2 Hk1 Loxl2 Col12 Nr1 Cite2 Epo Igfgp1 Reltive gene expression 2 1.5 1.5 -.5-1 -1.

. injetions of (n=4) or ontrol protein (n=5) twie weekly t 25 mg/kg for 15 ys. Liver RNA ws extrte for mirorry nlysis s esrie in the Supplementry Mterils.

7 Supplementry Figure 6 HIF trget genes fli Col61 Trfr2 Hk1 Loxl2 Col12 Nr1 Cite2 Epo Igfgp1 Reltive gene expression Llr As Aly Dhr7 Hmgs1 Li1 Nshl Sqle Insig1 Str4 Fsn Supplementry Figure 6.. Ault SCID mie reeive s.. injetions of (n=4) or ontrol protein (n=5) twie weekly t 25 mg/kg for 15 ys. Liver RNA ws extrte for mirorry nlysis s esrie in the Supplementry Mterils. The upregultion of HIF trget genes is epite in the right pnel.. Ault SCID mie reeive s.. injetions of (n=4) or ontrol protein (n=5) twie weekly t 25 mg/kg for 15 ys. Liver RNA ws extrte for mirorry nlysis s esrie in the Supplementry Mterils. Expression of Srep-1 trget genes re shown here. Nture Meiine: oi:1.138/nm.3295

Supplementry Figure 7 16 Hif-2 flox/flox (wt) 16 Hif-2 flox/flox ; l-cre (LKO) NS A shrna: Lu Hif-2 Hif-1 Hif-2 RBP5 5 liver WB / GTT AUC 12 8 4 A-sFlt1: - + AUC 12 8 4 A-sFlt1: - + 4 3 2 1 3 6 9 12

. AUC nlysis of Fig 2f emonstrting reution of A-sFlt1 effets on GTT y Hif-2 liver-speifi knokout in Hif-2 flox/flox ; lumin-cre mie. -e.

8 Supplementry Figure 7 16 Hif-2 flox/flox (wt) 16 Hif-2 flox/flox ; l-cre (LKO) NS A shrna: Lu Hif-2 Hif-1 Hif-2 RBP5 5 liver WB / GTT AUC A-sFlt1: - + AUC A-sFlt1: F + Lu shrna F + Hif2 shrna sflt1 + Lu shrna sflt1 + Hif2 shrna 5 e 4 2. AUC Fol-inrese (WB ensitometry) Supplementry Figure 7.. AUC nlysis of Fig 2f emonstrting reution of A-sFlt1 effets on GTT y Hif-2 liver-speifi knokout in Hif-2 flox/flox ; lumin-cre mie. -e. Inhiition of A-sFlt1 effets on GTT y enovirl liver Hif-2 shrna knokown in / mie. D/ mie reeive 5x1 8 pfu i.v. of A-F or AsFlt1, n A-shRNA Lu or A-shRNA Irs2 (n=6/onition) followe y GTT fter 7.. Effiy of shrna knokown of enogenous mouse liver Hif-2 protein. Western immunolot of liver protein normlize to RBP5, n=3 nimls/group. The / mie in -e. were nlyze y GTT fter 5.. AUC lultion. e. Densitometry quntifition of Western immunolot of / liver extrts (n=3) trete s ove n stimulte with 1 U kg -1 insulin for 5 min fter 5 hour fst; vlues were normlize to loing ontrol. Insulin-stimulte p- Akt n p-gsk3 shown reltive to unstimulte. The hnges re expresse in ritrry units. Nture Meiine: oi:1.138/nm.3295

9 Supplementry Figure 8 GTT AUC % sl gluose A-F A-HIF-2 PN A-HIF-1 ODD ITT AUC fst F HIF-2 PN HIF-1 ODD e e A-GFP A-HIF-1 ODD A-HIF-2 ODD f GTT AUC g Aeno: HIF-1 HIF-2 Irs2 p-akt Akt p-foxo1 Foxo1 GFP HIF-1 ODD HIF-2 ODD mouse liver WB - fste h mrna (fol inrese) GFP HIF2- ODD HIF-1 ODD G6p Pk1 Pprg1 i 6 j 6 GTT AUC GTT AUC Supplementry Figure 8.. AUC from GTT in Fig. 3 is epite.,. C57Bl/6 mie (n=8) reeive single i.v. injetion of the inite enoviruses.. ITT t y 15. The initil loo gluose levels in. were F=115, HIF-1 =124, HIF-2 =98.6 mg/l.. AUC from the ITT in.. Hepti tivtion of HIF-2 ut not HIF-1 ereses n fsting gluose, n=5 fter enovirus injetion. e-h. C57Bl/6 mie reeive single i.v. injetion of 3 x 1 8 pfu of A GFP, A HIF-1 ODD or A HIF-2 ODD followe y hrvest in the fste stte. e, f. GTT n AUC nlysis fter 3. g. Liver Western lot with the inite ntioies. h. qrt-pcr for gluogeneogeni genes G6p n Pk1. Vlues expresse s men ± s.e.m. = P<.5. i. AUC nlysis of the / mie in Fig. 3g. inites P<.5 versus ll other groups, n=8/group. HIF-2 PN + shrna Irs2 ws not signifintly ifferent from F + shrna Lu or F + shrna Irs2 y ANOVA. j. AUC nlysis of / mie in Fig. 3i, n=6/group. All vlues expresse s men ± s.e.m. = P<.5. Nture Meiine: oi:1.138/nm.3295

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION { OI: 1.138/n31 Srifie n nlyze APs on week 1 s of iet 1 4 6 High-ft iet BrU High-ft iet BrU 4 High-ft iet BrU 6 High-ft iet BrU Lin - Lin - : C34 + : C9 + 1 1 3 1 4 1 5 C45 1 C34 1 1 1 1 3 1 4 1 5 S-1