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An integrated functional genomics screeningprogram reveals a role for BMP-9 in glucose

homeostasisCecil Chen1, Krzysztof J. Grzegorzewski2, Steve Barash3, Qinghai Zhao1, Helmut Schneider1, Qi Wang2,

Mallika Singh1, Laurie Pukac1, Adam C. Bell4, Roxanne Duan4, Tim Coleman4, Alokesh Duttaroy2, Susan Cheng1,Jon Hirsch2, Linyi Zhang2, Yanick Lazard1, Carrie Fischer4, Melisa Carey Barber4, Zhi-Dong Ma5, Ya-Qin Zhang5,

Peter Reavey4, Lilin Zhong1, Baiqin Teng4, Indra Sanyal1, Steve M. Ruben4, Olivier Blondel4, and Charles E. Birse4*

Published online 24 February 2003; doi:10.1038/nbt795

A coordinated functional genomics program was implemented to identify secreted polypeptides with thera-peutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing >1,000 cDNA libraries, using a combination of bioinformaticalgorithms. Subsequently, ∼ 8,000 human proteins were screened in high-throughput cell-based assaysdesigned to monitor key physiological transitions known to be centrally involved in the physiology of type 2 dia-betes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducingphosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differ-entiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activatedexpression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection ofBMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment.BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combinationof bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited forthe treatment of diabetes.

Diabetes is a major health problem, with >5% of the US populationdiagnosed as having impaired insulin function1. There are two majortypes of diabetes. In type 1 diabetes, autoimmune destruction of thepancreatic β-cells results in an absolute requirement for exogenousinsulin in patients. Defects in the secretion of insulin and resistanceto the action of insulin contribute to type 2 diabetes, the more preva-lent form of diabetes that accounts for >90% of cases (reviewed byTaylor2 and Saltiel3).

Substantial progress has been made recently in understanding themolecular mechanisms that underlie the pathophysiology ofdiabetes. Advances include an improved resolution of the insulin sig-naling pathways with respect to hepatic gluconeogenesis4 andperipheral glucose uptake5, a better understanding of the role of fattyacids in the modulation of insulin action6, and improved characteri-zation of the cellular events that control insulin release by the pan-creatic β-cell7. These achievements have resulted in the developmentof new therapeutics that improve insulin action at the level of theliver (metformin) and peripheral tissues (thiazolidinediones) orenhance insulin secretion (sulfonylureas). Despite the arrival ofthese antidiabetic drugs, however, new treatments are still requiredthat will act more specifically, to reduce the occurrence of side effectscommonly associated with current treatments, such as weight gainor overt hypoglycemic episodes.

A near-complete repertoire of proteins expressed from the humangenome has been resolved. These proteins have been identified pri-marily through high-throughput sequencing of ESTs8,9, together withthe complete sequence draft of the human genome10,11. The conver-gence of this sequence information, with improved methods of assayminiaturization and liquid handling, has led to the possibility of effi-ciently screening for hitherto unknown biotherapeutics on a genome-wide scale. We have implemented such a functional genomics discov-ery program to identify secreted proteins with the capacity to modu-late key biochemical steps involved in the pathogenesis of diabetes.

Using these high-throughput approaches, we identify a role forBMP-9 in glucose homeostasis. BMP-9 is a member of the trans-forming growth factor-β (TGF-β) superfamily of cytokines thatcarry out critical functions in differentiation, growth, and apopto-sis12. Here we show that BMP-9 reduces glycemia in both normal anddiabetic murine models. The ability of BMP-9 to lower glucose levelsseems to be associated with its ability to promote insulin release inpancreatic β-cells, to regulate metabolic signaling through glycogensynthase kinase (GSK) in muscle, and to inhibit hepatic glucose pro-duction. The identification of new functions for BMP-9 in modulat-ing key transitions in glucose and lipid metabolism illustrates thefeasibility of efficiently screening thousands of human proteins fortherapeutic applications in a disease-oriented fashion.

1Department of Lead Development and Characterization, 2Department of Preclinical Development, 3Department of Information Technology, 4Department ofPreclinical Discovery, and 5Department of Antibody Development, Human Genome Sciences, Inc., 9800 Medical Center Dr., Rockville, MD 20850, USA.

*Corresponding author (charlie_birse@hgsi.com).

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ResultsSelection of secreted proteins from EST database. To identify secret-ed proteins that would serve as the compound library for subsequentcell-based screening, EST sequences in the Human Genome Sciences(HGS) database were analyzed for the presence of a N-terminalsecretory signal peptide using a combination of methods (Fig. 1).Analyses included hidden Markov model (HMM) classificationusing HMMer software13 running a signal peptide–scoring matrixdeveloped internally14 and the SignalP program15 (using cutoffvalues shown in Table 1). An integrated scoring rule was developedand fine-tuned that correctly classified known secreted proteinsfrom SWISS-PROT version 29 (ref. 16) while falsely recognizing only1.0% of known intracellular nuclear type II membrane proteins assecreted (see Supplementary Table 1 online). The signal peptideHMM was validated experimentally through the generation of artifi-cial signal peptides having various HMM scores, which were eachused to replace the N terminus of secreted alkaline phosphatase(SEAP) to create secretion test constructs. The strengths of the signals were determined by the alkaline phosphatase activitiesreleased from the transfected cells, and correlated closely with theHMM scores14.

Of the 1.4 million novel ESTs in the HGS database, 51% encodedopen reading frames (ORFs), with 5.5% (77,000) encoding N-terminal signal peptides. All cDNA clones that met this initialscreen were fully sequenced to confirm that they contained a completecoding region that still satisfied the classification rule. The averagelength of the ORF in these secreted proteins was 119 amino acids. As aprelude to the diabetes cell-based screening program, 155,000 addi-tional sequences (5% of total) from cDNA libraries prepared fromhepatic, adipose, skeletal muscle, and pancreatic tissues of normal,obese, and diabetic individuals were added to the HGS EST database.

A high-throughput approach was used to clone those cDNAsencoding predicted secreted proteins into a mammalian expressionvector under the control of the cytomegalovirus (CMV) promoter.Constructs were transiently transfected into HEK 293 cells, andsupernatants harvested after 48 hours were tested in a variety ofcell-based assays. Approximately 8,000 conditioned supernatantswere screened.

High-throughput screening. Diabetes is a complex metabolic dis-ease. Central events in disease progression include reduced insulinrelease, elevated hepatic gluconeogenesis, and reduced insulin-mediated glucose uptake by peripheral tissues. A series of assays covering these key biochemical transitions were done to screen forproteins with therapeutic potential for diabetes indications (Fig. 2).Pancreatic cell lines were screened for insulin secretion, calcium flux,and resistance to tumor necrosis factor (TNF)-induced apoptosis.Adipose and muscle cells were tested for activation of signaling path-ways and changes in viability or proliferation. Reporter constructswere developed in hepatocytes and adipocytes to detect modulatorsof glucose production or fatty acid metabolism.

BMP-9 was one of the proteins identified in the high-throughputdiabetes screening program. It scored positively in several cell-basedassays, as summarized in Table 2.

Elevated hepatic glucose production plays a central role in theincreased glycemia observed in type 2 diabetes after overnight fast-ing17. PEPCK catalyzes the reversible decarboxylation of oxaloacetateto yield phosphoenolpyruvate and CO2, and is the rate-limiting stepin hepatic gluconeogenesis. In the PEPCK reporter screen (in theH4IIe rat liver cell background), BMP-9 had a value of 38.2 relativelight units (RLU), almost 3 s.d. below the mean of all the values inthe plate (116.2), indicating substantial inhibition of the PEPCKpromoter activity (Fig. 3A). By comparison, insulin (100 nM), aknown inhibitor of PEPCK activity, had a value of 17.5 RLU.

The serine-threonine kinase Akt mediates some of the effects ofinsulin in skeletal muscle and adipose tissue, such as glucose trans-port and glycogen synthesis. Mice deficient in Akt-2 are impaired inthe ability of insulin to lower blood glucose, indicating the impor-tance of the phosphatidylinositol 3-kinase–Akt pathway in the main-tenance of normal glucose homeostasis18. One of the substrates forAkt is GSK-3, a constitutively active enzyme that regulates glycogensynthesis in response to insulin. Akt phosphorylates and inactivatesGSK-3, thus reducing the rate of phosphorylation of glycogen syn-thase and promoting glycogen synthesis19. We developed an Aktkinase assay using differentiated rat L6 myotubes that measures thephosphorylation of GSK-3 using a phospho-specific antibody. Inthis assay, BMP-9 had a value of 1.4 RLU, >3 s.d. above the mean ofall the values in the plate (0.8 RLU; see Fig. 3B), indicating that BMP-9 activates the Akt–GSK-3 pathway in L6 cells. Insulin (100 nM), aknown activator of Akt kinase in differentiated L6 cells, had a valueof 1.6 RLU. That BMP-9 can activate the Akt–GSK-3 pathway wasconfirmed by western analysis (data not shown).

HMM-SignalP analysis of BMP-9 confirmed the presence of asignal peptide at the N terminus of this 429-amino-acid ORF (Fig. 3C). N-terminal sequencing verified that the secreted form ofthe BMP-9 was formed by cleavage between residues 22 and

Table 1. Prediction of signal peptide cleavage site of BMP-9

Measurea Position Value Cutoff Signal peptide

Max.Y 23 0.747 0.34 YesMax. S 13 0.995 0.88 YesMean S 1–22 0.903 0.48 Yes

aSignal peptide analysis showing max. Y, max. S, and mean S values used toscreen ESTs for putative signal peptides, together with values obtained for BMP-9.The predicted signal peptide cleavage site is between residues 22 and 23.

Figure 1. Selection of secreted proteins. A search of 3.2 million humanESTs (blue filled square) from >1,000 different cDNA libraries in the HGSdatabase was conducted for secretory proteins. ESTs were translated inthree frames. All resulting ORFs (green filled square) starting with ATGwere checked for the presence of an N-terminal secretory signal peptide(red filled square) using two statistical classifiers. The first classifier wasan HMM using a signal-peptide profile. The second classifier was theSignalP program. Those ESTs containing ORFs scoring positive withrespect to both classifiers were assembled together to identify andremove redundant sequences. The cDNAs corresponding to theremaining ESTs were fully sequenced, and those whose finishedsequences contained complete coding regions starting with a putativesignal peptide formed the set of clones that entered high-throughputfunctional screening.

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23 (QG-KP), as predicted by the SignalP algorithm (Table 1). SDS-PAGE analysis of recombinant BMP-9, purified from a Chinesehamster ovary (CHO) line using conventional chromatographicmethods, showed N-terminal (40 kDa) and C-terminal (14 kDa)fragments (Fig. 4A). These polypeptides, generated through furin-mediated cleavage, formed a tight, noncovalent complex thatremained associated through purification. This protein preparationwas used for all subsequent studies.

Hepatic glucose production. Purified recombinant BMP-9markedly reduced the transcriptional activity of PEPCK-SEAPreporter in H4IIe rat hepatoma cells in a dose-dependent fashion(Fig. 4B), with a half-maximal inhibitory concentration (IC50) of455 pM. The result confirmed that inhibition of PEPCK transcrip-tion observed in the high-throughput screening program was adirect result of BMP-9 treatment.

To support this hypothesis further, we investigated the effect of BMP-9 on glucose production in H4IIe cells. Both insulin and BMP-9 potent-ly inhibited hepatic glucose production, with IC50 values of 34 pM and81 pM, respectively (Fig. 4C). The result confirmed that BMP-9, likeinsulin, regulates directional glucose metabolism in hepatocytes.Although insulin seems to be a more potent inhibitor of PEPCK activi-ty than BMP-9, their effect on total glucose production is equivalent,indicating the involvement of other glucose homeostatic pathways.Indeed, preliminary cDNA arraying data indicated the early activationof the activin receptor-like kinase/SMAD pathway by BMP-9 and notinsulin, suggesting that other genes responsive to this signaling cascademay be capable of modulating glucose concentration (data not shown).

Lipid metabolism. The liver is the primary organ responsible forconverting excess carbohydrates to fatty acids that can be stored astriglycerides or consumed in muscle. Stringent regulation of lipidmetabolism and balanced levels of free fatty acids in the serum areimportant variables in maintaining glucose homeostasis. Improper reg-ulation of fatty acid metabolism is believed to have a central function inmediating insulin resistance in the peripheral tissues20,21. Reporterassays were therefore constructed to investigate the effect of BMP-9 onthe transcription of two key enzymes involved in hepatic fatty acidmetabolism: malic enzyme (ME) and fatty acid synthase (FAS). ME cat-alyzes the oxidative decarboxylation of malate to pyruvate and providesNADPH for fatty acid synthesis. Functional response elements for per-oxisome proliferator-activated receptors22, thyroid hormone recep-tors23, and insulin24 have been identified in the ME promoter.

FAS catalyzes all reactions for the synthesis of palmitate from acetyl-CoA and malonyl-CoA. Transcription of FAS gene is regulated by thetranscription factor sterol regulatory element–binding protein-1c(SREBP-1c), a key regulator of genes required for fatty acid synthesisthat is highly expressed in the liver25. Overexpression of SREBP-1cmimics insulin’s effects on hepatic gene expression in diabetic mice26,indicating its pivotal requirement in insulin action. Treatment of FASand ME reporters with BMP-9 strongly stimulated the secretion ofSEAP in a dose-dependent manner, with half-maximal effective con-

centration (EC50) values of79 pM and 104 pM, respectively (Figs. 4D,E). These data indicatedthat BMP-9 activates the tran-scription of FAS and ME andthus seems to have an importantfunction in regulating fatty acidsynthesis in the liver.

In addition to the activitiesobserved for BMP-9 in the dia-betic cell-based screens (Table2), additional assays were doneto investigate potential inflam-matory and proliferative properties of the factor. Noinflammatory effects of BMP-9were seen in >100 high-throughput cell-based assaysusing T and B cells, naturalkiller cells, basophils, eosin-ophils, and mast cells (usingboth primary immune cells andcell lines) (data not shown).

Table 2. Effects of BMP-9 on various cell-based assays

Assay Cell type Result Positive control

PEPCK-SEAP reporter H4IIe hepatoma cell line Decreased InsulinME-SEAP reporter H4IIe hepatoma cell line Increased InsulinFAS-SEAP reporter H4IIe hepatoma cell line Increased InsulinSREBP-SEAP reporter H4IIe hepatoma cell line Increased InsulinMEF-SEAP reporter Differentiated 3T3-L1 adipocyte cell line No effect InsulinAkt Differentiated L6 myoblast cell line Increased InsulinERK Differentiated 3T3-L1 adipocyte cell line No effect InsulinERK H4IIe hepatoma cell line No effect InsulinGlucose production H4IIe hepatoma cell line Decreased InsulinGlucose uptake 3T3-L1 pre-adipocyte cell line No effect InsulinInsulin secretion HITT15 SV40-transformed pancreatic β-cell line No effect GLP-1, glucoseInsulin secretion INS-1 pancreatic β-cell line No effect GLP-1, glucoseCalcium flux HITT15 SV40-transformed pancreatic β-cell line No effect A23187cAMP levels 3T3-L1 pre-adipocyte cell line No effect ForskolinProliferation L6 myoblast cell line Increased Serum, PDGFBBProliferation 3T3-L1 pre-adipocyte cell line Increased Serum, bFGFProliferation H4IIe hepatoma cell line No effect SerumProliferation HepG2 hepatocellular carcinoma cell line No effect SerumInhibition of caspase-3, ARIP pancreatic ductal cell line No effect Serum

-7 activation

Figure 2. Overview of diabetic screening. Schematic representation ofhigh- and low-throughput biological screen (HTBS, LTBS) assays used inscreening secreted proteins for ability to modulate fat metabolism,glucose transport, glucose synthesis, and insulin release. Cell lines usedare also shown. ERK, extracellularly regulated kinase; PPAR, peroxisomeproliferator-activated receptors; FAS, fatty acid synthase; SREBP, sterolregulatory element–binding protein; MEF, myocyte enhancer factor-2abinding site from Glut4 gene promoter; PEPCK, phosphoenolpyruvatecarboxykinase; FMAT, fluorometric microvolume assay technology;FLIPR, fluorometric imaging plate reader.

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BMP-9 stimulates proliferation in L6 myoblasts and 3T3-L1 pre-adipocytes; no proliferative effects were seen on other primary cells(e.g., dermal fibroblasts, aortic smooth muscle cells, skeletal musclecells, and endothelial cells) or in a panel of cell lines (includinghepatocellular bone marrow stromal cells, and human pancreaticcarcinoma cells). Notably, the ability of BMP-9 to regulate prolifer-ation of 3T3-L1 pre-adipocytes is similar to that observed withinsulin27, leptin28, and resistin29. For example, leptin, which has beenshown to contribute to improved glucose and lipid homeostasis,promotes pre-adipocyte proliferation, whereas resistin and resistin-like proteins, which are thought to contribute to the development ofperipheral insulin resistance, inhibit the ability of adipocyte precur-sors to divide and differentiate.

BMP-9 lowers glycemia levels in normal mice and diabetic model.The insulin-like properties of BMP-9 prompted the evaluation of itsability to modulate glucose levels in vivo.

Regularly fed C57BL/6 mice were treated with a single dose ofBMP-9 at either 1 or 5 mg/kg. A positive control group received a

single subcutaneous (s.c.) dose of Humulin-R (3 U/kg), whereas thenegative control received the vehicle. Treatment with Humulin-Rresulted in a rapid and transient decline in glycemia, with maximaleffect observed after 30 minutes (Fig. 5A). Mice receiving BMP-9 at 5 mg/kg showed little change in blood glucose concentration overthe first 4 hours, but showed a delayed dose-dependent response,with a substantial hypoglycemic effect 24–48 hours after administra-tion. Glucose concentrations fell to 63.9 ± 2.1% of control (P < 0.0001) at 24 hours and to 79.9 ± 2.0% of the control level (P < 0.001) at 48 hours, and returned to normal by 72 hours.

To determine if BMP-9 was capable of altering the glucose con-centration in diabetic mice, we implemented a similar regimen withdb/db mice, with animals receiving a single s.c. dose of either BMP-9(5 mg/kg) or Humulin-R (3 U/kg). Insulin induced a rapid and tran-sient reduction of glucose concentration, which fell to 45.0 ± 11.7%(P < 0.005) of the control at 30 minutes. Glucose concentrations hadrebounded to the point of being similar to control 2 hours afterinjection (Fig. 5B). Treatment with BMP-9 caused a significantdecline in glucose concentration (45.8 ± 12.2%, P < 0.01) within thefirst 30 hours, compared with the amount of glycemia in buffer-treated mice. The effect of BMP-9 lasted at least 52 hours.

Normal mice treated with BMP-9 (5 mg/kg) showed lower foodintake than did mice receiving placebo (Fig. 5C). To investigate if theobserved glucose modulation in the plasma of BMP-9-treated mice isdue to altered food consumption, we carried out a pair-fed analysis.One group received an unrestricted amount of food (ad libitumcontrol), whereas the other group received a restricted amount offood (pair-fed control) determined by the amount of food consumedby BMP-9-treated mice shown in Figure 5C. The experimental group(pair-fed BMP-9) received a single administration of BMP-9 and arestricted amount of food. The comparison between control groupsshowed that restriction of food intake had no effect on the plasmaglucose concentration. However, treatment of food-restricted micewith BMP-9 again resulted in significantly lowered glycemia (P < 0.005) when compared with control mice on restricted foodintake (Fig. 5D).

BMP-9 promotes improved plasma glucose elimination. The pos-sibility that BMP-9 affects glycemia by modulating insulin releasewas investigated. Normal rats were treated with BMP-9 and thenchallenged with glucose after 2 hours and 26 hours, resulting in thepredicted spike of glucose concentration at time 0 (Fig. 6A). On

Figure 3. High-throughput screening reveals previously unknown activitiesfor BMP-9 in gluconeogenesis and Akt signaling. (A) Identification of BMP-9as an inhibitor of PEPCK-SEAP reporter activity in H4IIe cells in a high-throughput screen. Cells were treated with transient transfectionsupernatants of secreted proteins, and SEAP activities induced were plottedagainst row number in a 96-well plate. The red dashed line denotes thethreshold, which is defined as the mean of all the values in the plate minus 2s.d. BMP-9 (blue square) strongly inhibited the reporter activity relative tothe vector control (black square). Insulin (red square) was used as thepositive control. (B) Identification of BMP-9 as an activator of Akt kinaseactivity in differentiated L6 myotubes in a high-throughput screen. Transienttransfection supernatants of secreted proteins were tested for their ability toactivate Akt kinase activity. Red dashed line denotes the threshold, which isdefined as the mean of all the values in the plate plus 2 s.d. BMP-9 (bluesquare) induced Akt kinase activity relative to the vector control (blacksquare). Insulin (red square) was used as the positive control. (C) Signalpeptide analysis of BMP-9 ORF. SignalP calculated various scores usingneural networks operating on sliding windows around each of the residuesin the input peptide sequence. The C score at a given residue was anestimate of the probability that the residue is +1 of a signal peptide cleavagesite, the S score was an estimate of the probability that the residue is withina signal peptide, and the Y score combines both these measures into asingle quantity based on the C score at the residue and the decrease in Sscore in a window surrounding the residue. The mean S score was the meanof the S score from position 1 to the predicted cleavage site, which is theposition at which the Y score has maximal value.

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day 0, 2 hours after BMP-9 treatment, no significant differencebetween control and experimental groups was observed in eitherglucose tolerance (Fig. 6A) or insulin release (Fig. 6B). However, onday 1, 26 hours after treatment, glucose tolerance tests showed signif-icantly improved plasma glucose elimination in rats treated with 5 mg/kg of BMP-9 (Fig. 6A). The enhanced glucose uptake (29.2 ±9.7%, P < 0.05), associated with the highest dose of BMP-9, is givenin the inset plot showing area under the curve.

Notably, in blood sera drawn from rats 26 hours after treatmentwith 5 mg/kg of BMP-9, insulin was markedly increased at 2 minutes(340 ± 27%, P < 0.001) and 7 minutes (388 ± 71%, P < 0.01) afterglucose challenge, before returning to the normal concentrationwithin 30 minutes (Fig. 6B). It therefore seems that the long-termaction of BMP-9 in modulating glucose concentrations in mice andrats may be mediated through elevated amounts of insulin release.Similar increases in serum insulin were observed after treatment ofZuker diabetic rats, a rodent model of type 2 diabetes (data notshown). Although in vitro analyses performed with pancreatic β-celllines (INS-1 and HITT15) suggest that BMP-9 may be acting indi-rectly (Table 2), additional studies are required to clarify the mecha-nism of action of BMP-9-mediated insulin release.

DiscussionDiabetes is a complex multigenic metabolic disorder of impaired glu-cose homeostasis. A number of secreted factors have central roles inbalancing glucose concentration, including insulin, leptin,adiponectin, and glucagon-like peptide-1 (GLP-1). These proteinshave either been developed or are being evaluated as potential diabet-ic therapeutics. To identify additional natural biomolecules withapplications in the treatment of diabetes, we have adopted anapproach integrating functional genomics and high-throughputscreening methods. This coordinated program combines the accurate

identification of secreted proteins, the expression of these polypep-tides in a mammalian system, and the screening of this library foractivity. As well as elucidating function to hitherto uncharacterizedproteins, these methods also permit the functional reclassification ofproteins already described, such as BMP-9.

The cell-based assays used in the screening processes weredesigned to identify proteins that mimic the actions of insulin (suchas modulators of metabolic enzymes), to activate signaling pathwaysknown to be important in glucose homeostasis (such as Akt–GSK-3kinase and ras-MEK-ERK pathways), or to affect the functions orviability of insulin-secreting pancreatic cells (calcium flux, apoptosisassays). The progress of these assays was monitored through theinclusion of many proteins of known function. For example, in thekinase assays, growth hormone and platelet-derived growth factorwere shown to activate Akt kinase in differentiated L6 myotubes andto activate ERK in differentiated 3T3-L1 cells, respectively.

Several criteria were evaluated in the selection of clones for fur-ther characterization: (i) whether the protein expressed by transienttransfection gave consistent activity in the in vitro assays;(ii) whether the EST contained an ORF that is predicted to encode asecreted protein; (iii) whether the ORF encodes an unfamiliar oralready characterized protein, or a known protein where the detectedactivity has not before been associated with the protein (as with theglucose-lowering capacity of BMP-9). The final steps in the evalua-tion process reviewed protein homology and transcript expressionprofiles. In the screening assays listed in Figure 2, BMP-9 was identi-fied as the most potent and wide-acting protein (Table 2), and thuswas the initial clone developed. However, 11 additional independentclones were also found to have consistent activity and are being fur-ther evaluated: 3 hits in the kinase assays, 1 hit from the glucose pro-duction assay and 8 hits from the proliferation assays (one of theclones scored positively in both kinase and proliferation assays).

Figure 4. BMP-9 regulates hepaticgluconeogenesis and lipid metabolism invitro. All results are presented relative tothe untreated control. (A) SDS-PAGEshowing purified N-terminal (40 kDa) andC-terminal (14 kDa) forms of the BMP-9protein. Full-length recombinant BMP-9protein was cleaved between residues 319and 320 to produce N-terminal (23–319)and C-terminal (320–429) polypeptides.The two fragments formed a tightly associated, noncovalent complex that remained associated through purification. The identity of both fragments wasconfirmed by N-terminal sequencing. (B) BMP-9 inhibits transcriptional activity of PEPCK-SEAP reporter in H4IIe rat hepatoma cells. Serum-deprivedreporter cells were treated with purified, recombinant BMP-9 at various concentrations. SEAP activity in the conditioned media was measured after a 48 hincubation period. Activity of BMP-9 (blue open triangle) is compared with insulin (red filled circle). (C) BMP-9 inhibits glucose production in H4IIehepatocytes. Cells were incubated overnight with various concentrations of BMP-9 (blue open triangle) or insulin (red open circle) in the presence of 25 mM glucose. Glucose-containing medium was then replaced with glucose-free production medium, and glucose concentration in the supernatants was measured after 5 h. (D, E) BMP-9 induces transcriptional activity of ME-SEAP (D) and FAS-SEAP (E) reporters in H4IIe hepatocytes. Serum-deprived reporter cells were treated with purified, recombinant BMP-9 at various concentrations. SEAP activity in the conditioned medium was measured after a 48 h incubation period. Activity of BMP-9 (blue open triangle) is compared with insulin (red filled circle).

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Members of the TGF-β–BMP superfamily represent a highly con-served set of cytokines that play important roles in differentiation,growth, and apoptosis12. Despite the pleiotropic effects of these pro-teins, certain members show a highly restricted expression profile,suggesting a more confined mode of action. For example, BMP-7and GDF-9 are primarily expressed in the kidney and ovary, respec-tively. Indeed, deletion of the gene encoding BMP-7 results in deathsoon after birth with underdeveloped kidneys that lack glomeruli30,whereas removal of growth differentiation factor-9 (GDF-9)revealed its role during early ovarian folliculogenesis31.

BMP-9 shows a highly restricted hepatic expression profile32, withthe mature protein expressed in liver endothelial, stellate, andKupffer cells33. BMP-9 binds to hepatic endothelial cells and Kupffercells33 as well as to primary hepatocytes and various liver cell linesincluding HepG2 (ref. 32).

Our studies extend these findings, providing evidence for hepaticfunction of BMP-9, inhibiting gluconeogenesis and modulatingexpression of pivotal regulators of lipid metabolism. It is likely thatthe key functions of BMP-9 are mediated in an autocrine-paracrinefashion, as earlier predicted33. It is possible that the additional activi-ties of this cytokine are observed when BMP-9 is released in anendocrine fashion, facilitating its action on muscle and pancreatictissues. Release from its hepatocellular environment may also pro-vide an opportunity for BMP-9 to mediate already reported effectson bone morphogenesis34–36, hematopoietic proliferation and inhibi-tion37, and neuronal differentiation38.

In contrast to the rapid hypoglycemic effect of insulin, whereinsubstantial reductions in glucose concentration are observed in min-utes, BMP-9 affects glucose homeostasis over a considerably extend-ed period, with maximal reduction in glucose concentrations seenafter 24–30 hours in normal and diabetic rodent models. Thisresponse does not seem to be the result of retention of BMP-9 in theserum for an extended period. Whereas serum concentrations ofBMP-9 were 1.7–3.6 µg/ml at 6 hours after treatment (5 mg/kg),concentrations in all control mice and in mice assayed 24 hours aftertreatment were below the sensitivity threshold of the assay (25 ng/ml) (data not shown). This delayed response to treatmentwith BMP-9 is interesting not only from a clinical standpoint, withthe possible emergence of a new class of long-acting hyperglycemicagents, but also from a mechanistic viewpoint. Although it is notclear how BMP-9 mediates its effect on glucose balance, at least partof the mechanism seems to involve enhanced insulin release. It ispossible that BMP-9 triggers a signaling response that leads to elevat-ed levels of insulin synthesis or improved β-cell glucorecognition.

The liver is a critical organ in the regulation of glucose homeosta-sis, combining glucose-sensing and glucose-producing capabilities.BMP-9 is the first liver-specific factor shown to regulate blood glu-cose concentration. The existence of a hepatic factor regulatingperipheral glucose disposal has been proposed by numerous experi-mental paradigms39,40. It was postulated that postprandial elevationof serum insulin results in the parasympathetically controlled releaseof a hepatic insulin-sensitizing substance (HISS) that activates glu-cose uptake in skeletal muscle41. However, the molecular identity ofthis factor is not known.

We describe high-throughput methods for identifying and func-tionally screening thousands of human secreted proteins for thera-peutic applications in the treatment of diabetes. Although theapproaches outlined here have focused on identifying factors thatmodulate key transitions in glucose and lipid metabolism, clearlythese functional screening methods could be applied to other diseaseindications through the construction of alternative cell-basedassays42. Because the bioinformatic algorithms used here to recog-nize secreted proteins can be scaled to other large EST databases43,and the expression of protein using transient transfection overcomes

Figure 5. BMP-9 is a hypoglycemic agent in diabetic mice. (A) Single doseof BMP-9 lowers glucose levels after 24 h in normal mice. C57BL/6 micewere injected with a single s.c. dose of BMP-9 at 1 mg/kg (blue opentriangle) or 5 mg/kg (green open triangle), or with 3 U/kg of Humulin-R (red filled circle). Glucose concentration in the blood sample obtained from the tail vein was measured at the time indicated (x-axis). (B) Singleadministration of BMP-9 lowers glycemia in db/db model. db/db mice wereinjected with a single s.c. dose of BMP-9 at 5 mg/kg (green open triangle) orwith 3 U/Kg of Humulin-R (red filled circle). Blood glucose concentration wasdetermined at the time indicated (x-axis). (C) Food intake is reduced inBMP-9-treated normal mice. Normal mice with an unlimited food supplywere injected with a single s.c. dose of BMP-9 (5 mg/kg). Food intake wasmonitored after 24 h. (D) Restricted food intake does not affect glucoseconcentration. Glucose concentrations of normal animals with unrestrictedor restricted food supply were monitored. Readings were also taken for micewith a restricted diet after treatment with BMP-9 (5 mg/kg).

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the need for labor-intensive evaluation of polypeptide expressionand purification, it is foreseen that the application of these methodsmay become widely used in accelerating efforts to characterize thehuman proteome44,45.

Experimental protocolBioinformatics analysis. Each EST sequence in the HGS database was ana-lyzed for the presence of an ORF encoding a putative N-terminal signal pep-tide as scored by the HMM13,14 and the SignalP program15 using the cutoffvalues shown in Table 1. cDNA clones with ESTs meeting both of these crite-ria were fully sequenced, and those clones with completed sequences stillmeeting these criteria were selected for high-throughput screening.

Transient transfection. 293T cells grown in DMEM supplemented with 10% FBS were plated on poly-D-lysine-coated plates before transfection.Clones were transfected using Lipofectamine Plus (Invitrogen, Carlsbad, CA)following the manufacturer’s instructions. Supernatants were collected andassayed after 48 h.

PEPCK-SEAP reporter assay. The PEPCK-SEAP reporter construct is basedon the promoterless pSEAP2-neo vector, which contains the wild-typePEPCK promoter sequence from –600 to +69 fused to the SEAP-encodinggene. Stable H4IIe cells containing the PEPCK-SEAP construct were treatedwith 0.5 µM dexamethasone for 18 h to activate the PEPCK promoter beforeaddition of transient transfection supernatants. After a 48 h incubation

period, conditioned media were removed and SEAP activity was deter-mined using the manufacturer’s recommended protocol (TropixPhospha-Light System; Applied Biosystems, Bedford, MA).

ME- and FAS-SEAP reporter assays. Human ME (–1183 to –77) and FAS(–444 to +8) promoters were cloned into pSEAP2-neo. The constructswere transfected into H4IIe cells, and stable clones were selected. Cellswere deprived of serum for 18–24 h before being treated with BMP-9 orcontrols. After a 48 h incubation, conditioned media were harvested andSEAP activity was measured.

Akt kinase assay. Akt kinase assay was done using a modified protocol46,using 5 µg/ml paramyosin–GSK-3 fusion protein (Cell SignalingTechnologies, Beverly, MA) as the kinase substrate, and a phospho-specificGSK-3b (Ser9) antibody (Cell Signaling Technologies).

Glucose production assay. An earlier described protocol47 was adapted fora 96-well format. Glucose concentrations were measured using theAmplex Red kit from Molecular Probes (Eugene, OR) and corrected forthe protein concentration in the cell lysate.

In vivo studies. All animal experimentation was done in accordance withthe Animal Welfare Act and the Guide for the Care and Use of LaboratoryAnimals48, and under the supervision and approval of the InstitutionalAnimal Care and Use Committees.

Specific pathogen–free, 12- to 16-week-old male db/db (C57BL6/J+/+Leprdb) and normal C57BL6 mice at 9 weeks of age (JacksonLaboratories, Bar Harbor, ME) were housed in static microisolators andallowed ad libitum access to pelleted chow and water. The animal room wasmaintained on a 12 h light–12 h dark cycle. C57BL/6 mice (n = 3) receiveda single subcutaneous (s.c.) dose of BMP-9 (1 or 5 mg/kg) or 3 U/kg ofHumulin-R (Eli Lilly, Indianapolis, IN). Glucose concentration in theblood sample obtained from the tail vein was tested using Glucometer EliteXL (Bayer, Tarrytown, NY). db/db mice received a single s.c. dose of BMP-9(5 mg/kg) or 3 U/kg of Humulin-R Blood glucose concentration was deter-mined twice daily for 3 consecutive days as described earlier.

In the pair-fed experiment, male C57BL/6 mice (n = 5) were housedindividually and monitored for weight, food consumption, and glucoseconcentration. Before the pair-fed experiment, the level of food consump-tion by BMP-9-treated mice was determined. Mice received a single s.c.dose of BMP-9 at 5 mg/kg and received unrestricted access to food intake.The amount of food consumed by this group was measured 24 h later.

This value was used to calculate the restricted amount of food in the pair-fedexperiment, in which one control group received food ad libitum whereas thesecond control group and the BMP-9-treated group (5 mg/kg) received arestricted amount of food determined as just described. The plasma glucoseconcentration was determined 24 h later.

Wistar rats (330–370 g), catheterized in their carotid artery and jugularvein, were fasted 18 h before the BMP-9 treatment and throughout the exper-imental period (42 h). Vehicle or BMP-9 (0.5, 1.5, and 5 mg/kg) was adminis-tered intravenously (i.v.) (n = 4). D-Glucose (70% wt/vol dextrose, 2 g/kg i.v.)was injected 2 h later. Blood was drawn at 2, 7, 15, 30, 60, and 120 min fromthe jugular vein. Twenty-four hours after BMP-9 treatment, an equivalentglucose tolerance test was taken.

Note: Supplementary information is available on the Nature Biotechnologywebsite.

AcknowledgmentsWe thank Viktor Roschke, Partha Chowdhury, Michael Bloom, and KathyMcCormick for technical assistance, and Paul Moore for a critical review of themanuscript.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 1 July 2002; accepted 12 December 2002

Figure 6. BMP-9 mediates insulin release and improves glucose tolerance.Glucose tolerance test in Wistar rats was done as described in ExperimentalProtocol. (A, B) Plasma concentrations of (A) glucose and (B) insulin weremeasured before and after an i.v. glucose challenge (2 mg/kg) at day 0 and +1after treatment with vehicle (black filled square), BMP-9 at 0.5 mg/kg (red opencircle), 1.5 mg/kg (blue open triangle), or 5 mg/kg (green open triangle).Asterisks indicate statistically significant differences from the vehicle control.The area under the curve for the different treatment groups is also plotted(inset), using the same color code as in the main panel.

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