Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and...

Modulation of Bone Morphogenetic Protein-9 Expressionand Processing by Insulin, Glucose, and Glucocorticoids:Possible Candidate for Hepatic Insulin-SensitizingSubstance

Luciana Chagas Caperuto,* Gabriel Forato Anhe,* Tavane David Cambiaghi, Eliana Hiromi Akamine,Daniella do Carmo Buonfiglio, Jose Cipolla-Neto, Rui Curi, and Silvana Bordin

Department of Biological Sciences (L.C.C.), Federal University of Sao Paulo, 04023-900 Sao Paulo, Brazil; and Departmentof Physiology and Biophysics (G.F.A., T.D.C., E.H.A., D.C.B., J.C.-B., R.C., S.B.), Institute of Biomedical Sciences,University of Sao Paulo, 05508-900 Sao Paulo, Brazil

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-�superfamily predominantly expressed in nonparenchymal livercells, has been demonstrated to improve glucose homeostasis indiabetic mice. Along with this therapeutic effect, BMP-9 wasproposed as a candidate for the hepatic insulin-sensitizing sub-stance (HISS). Whether BMP-9 plays a physiological role in glu-cose homeostasis is still unknown. In the present study, we showthat BMP-9 expression and processing is severely reduced in theliver of insulin-resistant rats. BMP-9 expression and processingwas directly stimulated by in situ exposition of the liver to thecombinationofglucoseandinsulinandoralglucose inovernightfasted rats. Additionally, prolonged fasting (72 h) abrogatedrefeeding-induced BMP-9 expression and processing. Previous

exposition to dexamethasone, a known inductor of insulin re-sistance, reduced BMP-9 processing stimulated by the combi-nation of insulin and glucose. Finally, we show that neutraliza-tion of BMP-9 with an anti-BMP-9 antibody induces glucoseintolerance and insulin resistance in 12-h fasted rats. Collec-tively, the present results demonstrate that BMP-9 plays an im-portant role in the control of glucose homeostasis of the normalrat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resis-tance. BMP-9 regulation according to the feeding status and thepresence of diabetogenic factors reinforces the hypothesis thatBMP-9 might exert the role of HISS in glucose homeostasisphysiology. (Endocrinology 149: 6326–6335, 2008)

THE EXISTENCE OF a liver-derived humoral factorthat regulates glucose homeostasis has emerged

from the observation that glucose uptake by skeletal mus-cle is severely impaired by surgical or pharmacologicalblockade of hepatic parasympathetic nerves (1–3). Thereversal of the hepatic denervation-induced insulin resis-tance by intraportal acetylcholine administration rein-forced this hypothesis (4).

The synthesis and release of the so-called hepatic insulin-sensitizing substance (HISS) has been deeply investigatedduring the last years (5–8). Insulin induces HISS liberationby the liver through a mechanism dependent on acetylcho-line-induced nitric oxide generation. In addition, HISS pro-duction and regulation by insulin is dependent on the feed-ing status, fasting duration, and refeeding (6, 7).

Despite the informations above, the nature of HISS re-mains unknown. Bone morphogenetic protein (BMP)-9 islikely to be a candidate.

BMPs are members of the TGF superfamily (TGF-�), whichcontains a large number of proteins that have been impli-cated in a variety of developmental processes such as carti-lage and bone formation (9). TGF-� receptors are commonlycomposed by two Ser/Thr kinase transmembrane chains,types I and II. The combination of types I and II chains cangenerate a broad range of effects in response to the sameligand. Thus, the tissue-specific effect of any BMP dependson the receptor chains expressed by each cell type (10).

BMPs regulate a diverse array of cellular functions duringdevelopment and in the adult life (11). Among these proteins,BMP-9 has been shown to induce proliferation of culturedliver cells (12) and promote cholinergic differentiation andthe synthesis of acetylcholine in cholinergic neurons (13).BMP-9 also produces ectopic bone growth and directs thedifferentiation of mesenchymal cells into cartilage (14, 15).BMP-9 is predominantly expressed in nonparenchymal livercells, i.e. endothelial, Kupffer, and stellate cells (12).

A high-throughput pharmacogenomic study identifiedBMP-9 as a regulator of glucose metabolism (16). In vitro,BMP-9 inhibited the expression of phosphoenolpyruvate car-boxykinase and increased the expression of malic enzymeand fatty acid synthase (FAS) in liver rat hepatoma H4IIecells. In differentiated L6 myoblasts, BMP-9 activates theserine/threonine kinase AKT. In vivo, BMP-9 was shown toreduce glycemia in normal and diabetic mice.

Despite its therapeutic potential, BMP-9 was identified asa pharmacological, rather than a physiological target, and

First Published Online August 14, 2008* L.C.C. and G.F.A. contributed equally to this study.Abbreviations: BMP, Bone morphogenetic protein; b.w., body weight;

Dex, dexamethasone; FAS, fatty acid synthase; GTT, glucose tolerancetest; HIF-1A, hypoxia-inducible factor; HISS, hepatic insulin-sensitizingsubstance; ITT, insulin tolerance test; KITT, constant rate for glucosedisappearance; Pinx, pinealectomy.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/08/$15.00/0 Endocrinology 149(12):6326–6335Printed in U.S.A. Copyright © 2008 by The Endocrine Society

doi: 10.1210/en.2008-0655

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still there is a lack of information regarding the regulation ofBMP-9 during the course of physiological and pathologicalprocesses (17). Such data would be useful to state the rele-vance of BMP-9 in the control of normal glucose homeostasis.

To clarify the participation of BMP-9 in physiological andnonphysiological alterations of glucose metabolism, we inves-tigated the effect of two classical models of insulin resistance,i.e. prolonged fasting and dexamethasone treatment, and pi-nealectomy, on the expression and processing of BMP-9. Pi-nealectomy was chosen because of the remarkable glucoseintolerance, decreased adipocyte responsiveness to insulin,normoinsulinemia and hypercorticosteroidism (18–20). Ex-posure of the hepatic tissue to glucose, insulin and glucocor-ticois further elucidated the control of BMP-9 expression andprocessing. Finally, serum BMP-9 neutralization revealed itsrole in the physiological regulation of glucose homeostasis.Together, our results prompt that BMP-9 fulfills the require-ments for a putative HISS.

Materials and MethodsMaterials

The reagents and apparatus for SDS-PAGE and immunoblotting wereobtained from Bio-Rad (Richmond, CA). Tris, dithiothreitol, Tween 20,glycerol, and dexamethasone were obtained from Sigma Chemical Co.(St. Louis, MO). Human insulin was from Biobras (Minas Gerais, Brazil).Enhanced chemiluminescence detection system and nitrocellulose mem-brane (0.45 mm) were from Amersham Pharmacia Biotech (Uppsala,Sweden). X-ray-sensitive films and chemicals were from IBF (Rio deJaneiro, Brazil). Antibodies against BMP-9 (sc-27821) and pAKT1/2/3-Ser were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).PCR primers were manufactured by Integrated DNA Technologies (Cor-alville, IA). GoTaq DNA polymerase and ImProm-II reverse transcrip-tase were from Promega (Madison, WI). Trizol reagent and Sybr Greenwere from Invitrogen (Carlsbad, CA). Neutralizing anti-BMP-9 antibodyand control IgG Fc chain were developed by Imuny Biotechnology(Campinas, Brazil). The affinity-purified rabbit polyclonal antibody wasraised against a peptide mapping at the C terminus of BMP-9 of humanorigin.

Animals

Wistar rats obtained from the Animal Breeding Center of the Instituteof Biomedical Sciences (Sao Paulo, Brazil) were kept under standardlaboratory conditions (12-h light, 12-h dark cycle) with free access to foodand water.

Rats weighting about 250 g were subjected to daily ip injection ofdexamethasone [Dex; 1 mg/kg of body weight (b.w.)] for 5 consecutivedays. The experiments were performed on the morning of the sixth day.Control counterparts received vehicle for the same period of time.

Seven-week-old male Wistar rats were anesthetized with ip injectionof sodium thiopental (50 mg/kg b.w.) and subjected to pinealectomy(Pinx) or to a sham operation (CTL) as described by Lima et al. (18). Afterrecovery from surgery, rats were provided with food and water adlibitum (three or four animals per cage). Six weeks after surgery, the ratswere decapitated at night (Zeitgeber time 18, i.e. 6 h after lights off), anda slice of the liver was removed. The effectiveness of the pinealectomywas then verified.

The rats were fasted for 72 (Ft-72) or 12 (Ft-12) h. Ft-72 rats weresubmitted to refeeding with standard rodent chow ad libitum (1, 2, or 4 h)before the experiments. Ft-12 rats were food deprived for 12 h and thenfed for 2 h with standard rodent chow ad libitum. After this period, foodwas withdrawn for 12–14 h before administration of glucose by gavage(1 g/kg b.w., 2 h before the experiment) or liver perfusion.

Rats not used for in situ liver perfusion were anesthetized with so-dium thiopental (50 mg/kg b.w., ip) and killed after approximately 10min. Abdominal cavity was opened and a slice of the liver was removedand processed for total RNA or protein extraction. The procedures

described above were conducted according to the guidelines of theBrazilian College for Animal Experimentation.

In situ liver perfusion

Rats were anesthetized with sodium thiopental (50 mg/kg b.w., ip)and then subjected to laparotomy. Livers were perfused in situ in amonovascular nonrecirculating system through a cannula inserted intothe hepatic portal vein, using Krebs-Henseleit (pH 7.4), at 37 C andsaturated with a 95%–5% O2-CO2 mixture. The flow rate was alwaysadjusted according to the weight of the liver (4 ml/min � g of freshweight), considering that the liver weight 4% of the body weight) (21).

The livers were initially perfused for 10 min with Krebs-Henseleit toremove blood. Next, the livers were perfused for 120 min with Krebs-Henseleit containing glucose (2.8, 5.6, or 11.2 mm) in the absence orpresence of insulin (20 U/liter). The experiments were performed withlivers that maintained the same shape and color throughout the assay,indicating proper vascular perfusion and viability. At the end of theperfusion period, slices of the liver were removed and processed for totalRNA or protein preparation.

RNA extraction and conventional RT-PCR

Total RNA was extracted from approximately 100 mg of liver usingTrizol reagent (Invitrogen). Total RNA was reverse transcribed and con-ventional RT-PCR analysis was performed as previously described (22).The amplification products were run on a 1.2% agarose gel containingethidium bromide, and the band intensities were determined by digitalscanning followed by quantification using the Scion Image analysis soft-ware (Scion Corp., Frederick, MD). The results were expressed as aratio of the target genes to the housekeeping RPL37a. The primersequences used for RT-PCR analysis with their respective melting pointand lengths were as follows: HIF1A, sense, 5�-CCCATCCATGTGAC-CATGAGG-3� and antisense, 5�-TCAGCACCAAGCACGTCATAGG-3�, 56 C, 261 bp; FAS, sense, 5�-AAGCCAGGAAGAGTGGGAGAGC-3�and antisense, 5�-GGTTGGACAGCAGGATACACCG-3�, 59.2 C, 311bp; RPL37a, sense, 5�-CAAGAAGGTCGGGATCGTCG-3� and antisense,5�-ACCAGGCAAGTCTCAGGAGGTG-3�, 57 C, 290 bp.

Real-time PCR

Real-time RT-PCR was used to detect BMP-9 RNA expression usingROTOR GENE 3000 equipment (Corbett Research, Mortlake, Australia)and Sybr Green (Invitrogen) as fluorescent dye. Primer sequences andreaction conditions were: BMP-9, sense, 5�-TTCAGGATGAGGGCTGG-GAG-3� and antisense,5�-GGATGTCTTCACAAGCACGGTC-3�, 58.5C.Am-plification efficiency of each sample was calculated as described by Ra-makers et al. (23). Gene expression was determined by the method of Liuand Saint (24) using RPL37a gene expression as inner control. All sampleswere compared using the relative cycle threshold. The cycle threshold valueis the calculated cycle number by which the fluorescence signal emitted issignificantly above background levels.

Protein analysis by Western blotting

Fragments of liver containing approximately 100 mg were homog-enized in a boiling extraction buffer [10% sodium dodecyl sulfate, 100mm Tris (pH 7.4), 10 mm EDTA, 10 mm sodium pyrophosphate, 100 mmsodium fluoride, 10 mm sodium vanadate] with a Polytron PTA 20Sgenerator (model PT 10/35; Brinkmann Instruments, Inc., Westbury,NY) operated at maximum speed for 30 sec.

The extracts were centrifuged at 15,000 � g, 4 C, for 40 min toremove insoluble material. Protein concentrations of the supernatantswere determined by the Bradford assay, and an equal amount of totalprotein from each sample (75 �g) was treated with Laemmli buffercontaining dithiothreitol 100 mm. Samples were heated in a boilingwater bath for 5 min, after which they were subjected to SDS-PAGE(10% bis-acrylamide).

Electrotransfer of proteins from gel to nitrocellulose membrane wasperformed for 90 min at 120 V (constant) as described elsewhere (25).Nonspecific protein binding to nitrocellulose was reduced by prein-cubating the membrane overnight at 4 C in blocking buffer (1%gelatin, 10 mm Tris, 150 mm NaCl, and 0.02% Tween 20). The nitro-

Caperuto et al. • Regulation of Glucose Homeostasis by BMP-9 Endocrinology, December 2008, 149(12):6326–6335 6327


cellulose blot was incubated with anti-BMP-9 antibody (1:1000) di-luted in blocking buffer overnight at 4 C and then washed for 30 minwith blocking buffer without gelatin.

Bound antibodies were detected with horseradish peroxidase-conju-gated anti-IgG (1:10,000) and visualized by chemiluminescence in x-raysensitive films. Band intensities were quantified from the developed au-toradiographs using Scion Image program.

Intraperitoneal glucose tolerance test (GTT) and insulintolerance test (ITT)

Male Wistar rats weighting 200 g were fasted for 12 h before theexperiments. An iv injection of anti-BMP-9 neutralizing antibody (100�g/kg b.w.), IgG Fc chain (100 �g/kg b.w.), or NaCl 0.9% (CTL) wasadministrated in the penin vein. After 15 min the rats were submittedto an ip GTT or an ip ITT. GTT was carried out with a glucose injection(2 g/kg of a 20% solution of d-glucose). The blood samples were col-lected from the tail at 0, 5, 10, 15, 30, 60, 120, 150, and 180 min formeasurement of serum glucose. ITT was carried out with an insulininjection (2 IU/kg), and blood samples were collected from the tailat 0, 5, 10, 15, 20, 25, and 30 min for measurement of serum glucose.The constant rate for glucose disappearance (KITT) was calculatedusing the formula 0.693/half-time. The glucose half-time was calcu-

lated from the slope of the least-square anal-ysis of the plasma glucose concentrations dur-ing the linear decay phase (26).

Statistical analysis

Results are presented as means � sem. Com-parisons were performed using one-wayANOVA, with Tukey-Kramer posttest (INStat;Graph Pad Software, Inc., San Diego, CA), andthe unpaired Student’s t test when appropriate.The level of significance was set at P � 0.05.

ResultsBMP-9 expression and processing in theliver of insulin-resistant rats

To characterize the putative role ofBMP-9 in the regulation of glucose ho-meostasis, the first approach appliedwas to analyze BMP-9 mRNA and pro-tein content in the liver of insulin-resistant rats. The models chosen were:1) 72 h fasting, 2) dexamethasone treat-ment, and 3) pinealectomy. Two immu-noreactive bands, the nonprocessedinactive precursor of BMP-9 (47 kDa)and the mature secreted form (13 kDa),were identified by Western blot (12).Thus, the BMP-9 processing results referto the secreted mature form and non-processed inactive precursor ratio.

BMP-9 mRNA decreased in Ft-72 rats(35 � 3%; P � 0.05), Dex rats (35 � 7%;P � 0.05), and Pnx rats (21 � 1%; P �0.05; Fig. 1A) compared with CTL coun-terparts. This modulation seems to bespecific for BMP-9 because no alterationswere found in BMP-2 mRNA expression(data not shown).

The content of the BMP-9 nonprocessedprecursor increased in Ft-72, Dex, and Pnxrats, respectively, 66 � 14, 140 � 19, and58 � 13% over their control counterparts

(P � 0.05; Fig. 1B). The content of the BMP-9 mature secretedform was similar in FT-72 and CTL rats but decreased in Dexand Pinx rats to 39 � 5% and 77 � 8%, respectively (P � 0.05;Fig. 1C). BMP-9 processing was decreased in Ft-72 (Fig. 2A),Dex (Fig. 2B), and Pnx rats (to 50 � 6, 19 � 4, and 52 � 8%,respectively, compared with their control counterparts; P �0.05).

Glucose and insulin directly affect BMP-9 expression andprocessing in perfused liver

To investigate the participation of glucose and insulin inthe regulation of BMP-9 expression and processing, we per-formed in situ liver perfusion with these agents. Previously,to assure the efficiency of the perfusion procedure, we as-sessed insulin-induced AKT phosphorylation, and hypoxia-inducible factor (HIF-1A), and FAS mRNA expression. AKTphosphorylation was increased after a 30-min perfusion withinsulin when compared with the noninsulin time-matched

FIG. 1. BMP-9 expression in liver of insulin-resistant rats. Total RNA extracted from liverof FT-72, Dex, and Pinx rats and CTL counterparts were used for real-time PCR analysis ofBMP-9 mRNA expression normalized by the constitutive gene RPL37a (A). Protein extractsfrom liver of FT-72, Dex, and Pinx rats and CTL counterparts were used for immunoblottinganalysis of BMP-9 nonprocessed precursor (B) and mature secreted form (C). The results areexpressed as means � SEM. *, P � 0.05 vs. CTL counterparts (n � 4).

6328 Endocrinology, December 2008, 149(12):6326–6335 Caperuto et al. • Regulation of Glucose Homeostasis by BMP-9


perfused livers. AKT phosphorylation peaked after 60 min ofperfusion and remained detectable up to 120 min (Fig. 3A).Concomitantly, tyrosine phosphorylation of insulin receptorsubstrate 1/2 increased (data not shown). FAS gene expres-sion is known to be rapidly and directly induced by insulin(27, 28). FAS mRNA expression was not affected by increas-ing glucose concentration from 5.6 to 11.2 mm in the perfu-sion medium but was increased by the addition of insulin tothe medium (152 � 7%; P � 0.05) (Fig. 3B). Together theseresults demonstrated that the liver preserved insulin respon-siveness during a 2-h perfusion. HIF-1A is a transcription factor

up-regulated by hypoxia (29). HIF-1A ex-pression was not altered throughout theperfusion period, ranging from 30 to 120min, despite variations in insulin concen-tration (Fig. 3C). This result indicated thatnegligible rates of hypoxia occurred duringthe perfusions.

Figure 4A shows that BMP-9 mRNAexpression was not modulated by alter-ations in glucose concentrations. Insulininduced an increase in BMP-9 mRNAexpression exclusively when combinedto glucose 11.2 mm (39 � 10% comparedwith the condition with no glucose; P �0.05) (Fig. 4B). The content of the BMP-9nonprocessed precursor was similarlyincreased when perfusion was per-

formed with 11.2 mm glucose, insulin or combination of both(respectively 141 � 11, 136 � 18, and 165 � 21% comparedwith CTL; P � 0.05) (Fig. 4C). As noted for BMP-9 mRNAexpression, the content of the BMP-9 mature secreted formwas unaltered by the perfusion with 11.2 mm glucose orinsulin, whereas the combination of both resulted in its up-regulation (193 � 33% compared with the condition with noglucose; P � 0.05; Fig. 4D). The ratio of mature and immatureBMP-9 content also increased with glucose plus insulin (to132 � 17%; P � 0.05 vs. CTL) but was decreased by insulin

in the absence of glucose (to 51 � 18%;P � 0.05; Fig. 4E).

Oral glucose administration inducedBMP-9 expression in the liver of 12-hfasted rats

The effect of the physiological in-crease in glucose and insulin concentra-tions on BMP-9 expression in overnightfasted rats that received a glucose solu-tion (1 g/kg b.w.) by intragastric gavagewas investigated. BMP-9 mRNA expres-sion was increased 2 h after glucose oraladministration (22 � 4% compared withCTL; P � 0.05; Fig. 5A) Similarly, asshown in Fig. 5, B and C, oral glucoseinduced an increase of both BMP-9 non-processed precursor and mature se-creted form (by 62 � 25 and 73 � 25%,respectively, compared with CTL; P �0.05). BMP-9 protein processing did notchange (Fig. 5D) due to the simultaneousincrease in 47 kDa precursor content(Fig. 5B).

Refeeding did not increase BMP-9expression and processing in the liver of72-h fasted rats

As an attempt to evaluate whether theprolonged fasting would hamper the ef-fect of glucose and insulin on BMP-9 ex-pression and processing in vivo, 72-h

FIG. 2. BMP-9 processing in liver of insulin-resistant rats. Protein extracts from liver ofFt-72, Dex, and Pinx rats and CTL counterparts were used for immunoblotting analysis ofBMP-9 processing (A, B, and C, respectively). The results of densitometry analysis are shownas the value of the ratio mature secreted form/nonprocessed precursor and express themeans � SEM. *, P � 0.05 vs. CTL counterparts (n � 4).

FIG. 3. FAS and HIF-1A mRNA expression and serine phosphorylation of AKT in in situperfused livers. Livers were perfused with Krebs in the presence or absence of insulin duringdifferent intervals. Protein extracts were used for immunoblotting analysis of AKT serinephosphorylation (A). Livers were perfused for 2 h with Krebs containing glucose 5.6 or 11.2mM or glucose 11.2 mM plus insulin. Total RNA was extracted and used for conventional PCRanalysis of FAS mRNA normalized by the constitutive gene RPL37a (B). Livers were perfusedwith Krebs containing glucose 5.6 mM in the presence (black bars) or absence (white bars)of insulin during different intervals. Total RNA was extracted and used for conventional PCRanalysis of HIF-1A mRNA normalized by the constitutive gene RPL37a (C). The results areexpressed as means � SEM. *, P � 0.05 vs. G5.6 and G11.2 (n � 3).



fasted rats were submitted to a refeeding schedule. Re-duction of BMP-9 mRNA expression induced by 72 h fast-ing (65 � 3% of the CTL values; P � 0.05) was not altered1 h after the refeeding (70 � 6% of the CTL values; P �0.05), and it was only partially restored after 2 h (Fig. 6A).Four hours after refeeding, BMP-9 mRNA levels returnedto the values before refeeding (� 60 � 3% of the CTLvalues; P � 0.05).

BMP-9 nonprocessed precursor increased in 72-h fastedrats, and a further increase was observed 1 and 2 h afterrefeeding (161 � 32 and 358 � 50%, respectively, comparedwith CTL; P � 0.05) (Fig. 6B). Four hours after refeeding, thecontent of the BMP-9 nonprocessed precursor returned to thevalues observed in 72-h fasted rats. In addition, 72 h fastingdid not alter the levels of the mature secreted form, and nofurther alterations were observed after refeeding (Fig. 6C). Theratio of 13 kDa mature BMP-9 in relation to 47 kDa precursorwas decreased in 72-h fasted rats and remained as such afterrefeeding (Fig. 6D).

Dexamethasone down-regulatesBMP-9 expression and processingin rat liver

Dexamethasone is well knownto induce insulin resistance in pe-ripheral tissues such as skeletalmuscle and liver (30, 31). To testwhether the impairment of glu-cose plus insulin-induced BMP-9expression and processing ob-served in prolonged fasting ratswas due to the high levels of glu-cocorticoids, we performed the insitu liver perfusion in rats pre-treated with dexamethasone.Treatment with dexamethasone12 h before the liver perfusionreduced the stimulation ofBMP-9 mRNA expression in-duced by the combination of 11.2mm glucose and insulin (to 31 �4%, compared with CTL; P �0.05) (Fig. 7A). Dexamethasonealso up-regulated the accumulationof BMP-9 nonprocessed precursorinduced by the combination of glu-cose and insulin (59 � 22% com-pared with CTL; P � 0.05) and didnot alter the stimulation of theBMP-9 mature secreted form (Fig. 7,B and C). As a consequence, the pro-cessing of BMP-9 induced by glu-cose plus insulin was reduced in ratspreviously treated with dexameth-asone (74 � 9% compared with CTL;P � 0.05; Fig. 7D).

Serum BMP-9 neutralization withanti-BMP-9 antibody causesglucose intolerance and insulin

resistance in 12-h fasted rats

Previous treatment of 12-h fasted rats with anti-BMP-9antibody resulted in glucose intolerance as observed by amore accentuated ascendant phase of serum glucose values,which was followed by a noncompensatory glucose decayphase (Fig. 8A). Therefore, the area under the curve (bloodglucose vs. time) was increased in rats treated with anti-BMP-9 antibody (41 � 13% compared with CTL; P � 0.05;Fig. 8B).

To characterize whether this glucose intolerance was due toinsulin resistance or an impairment of pancreatic function, wenext performed ITT in anti-BMP-9 antibody-treated rats. Figure8C shows that the KITT was decreased in rats treated withanti-BMP-9 antibody (66 � 5% of the CTL values; P � 0.05).Injection of the same amount of control IgG Fc chain did changeglucose tolerance (Fig. 8B) and insulin sensitivity (Fig. 8C),indicating that the effect of anti-BMP-9 was not due to an un-specific action.

FIG. 4. BMP-9 expression in livers perfused with glucose and insulin. Livers were perfused for 2 hwith Krebs containing glucose 0.0, 2.8, 5.6, or 11.2 mM. Total RNA was extracted and used forreal-time PCR analysis of BMP-9 mRNA (A). Livers were perfused for 2 h with Krebs containingglucose, insulin, or the combination of both. Total RNA was extracted and used for real-time PCRanalysis of BMP-9 mRNA normalized by the constitutive gene RPL37a (B). Total protein wasextracted and used for immunoblotting analysis of BMP-9 nonprocessed precursor (C) and maturesecreted form (D). BMP-9 processing was assessed by the value of the ratio mature secretedform/nonprocessed precursor (E). The results are expressed as means � SEM. *, P � 0.05 vs.perfusion without glucose and insulin or insulin alone; #, P � 0.05 vs. perfusion with or withoutglucose plus insulin (n � 5).



Fasting blood glucose levels measured before GTT and ITTwere identical in rats injected with anti-BMP-9 neutralizingantibody, control IgG, or saline (104.7 � 4.8 and 95.8 � 5.0,and 100.1 � 3,7 mg/dl, respectively).

Discussion

Evidences presented herein suggest that BMP-9 is a liver-secreted factor that might play an important role in the con-trol of glucose homeostasis. The following observations sup-port this proposition: 1) BMP-9 mRNA and mature secretedform contents were decreased in three distinct models ofinsulin resistance (72 h fasted rats, Dex-treated rats, and Pinxrats); 2) BMP-9 mRNA and mature secreted form were di-rectly up-regulated by the combination of glucose plus in-sulin and by glucose oral challenge; 3) prolonged fasting andprevious exposure to dexamethasone abrogated the refeeding-induced up-regulation of BMP-9; and 4) serum BMP-9 neutral-ization with an anti-BMP-9 antibody induced glucose intoler-ance and insulin resistance in overnight fasted rats.

Chen et al. (16) demonstrated that the treatment withrecombinant BMP-9 improves glucose homeostasis in di-abetic rodents. This observation suggests that impairmentin BMP-9 expression might be associated with generation

of glucose intolerance in the above-mentioned models.Therefore, an integrated interpretation of the present dataand the results of Chen et al. point to a pathophysiologicalrole of BMP-9 in glucose homeostasis.

One of the most important mechanisms for the control ofthe biological effects of TGF-� family proteins, and by ex-tension BMPs, is the conversion to an active form (32). BMP-9is translated as an inactive precursor protein of approxi-mately 48 kDa, which is composed by a hydrophobicsecretory leader, an amino-terminal proregion and a car-boxyl-terminal mature domain (11, 33–35). To becomeactive, the proregion of all BMPs suffers a proteolyticcleavage at the multibasic amino acid motif R-X-X-R, re-leasing the C-terminal mature protein (36). Mature BMP-9is a homodimer of two 110-amino acid chains with a cal-culated molecular mass of 13 kDa (www. genenames.org) (12).In most cases, the proregion dissociates and the mature ligandis secreted from the cell. Similarly to growth differentiationfactor-8 and TGF-�-1, -2, and -3 (37, 38), the proregion of BMP-9remains tightly, but not covalently, linked to its C terminusregion after secretion from the cell (11). However, different fromgrowth differentiation factor-8 and TGF-�s, in which the pro-regions remain associated and have been found to be function-ally inhibitory (37), both BMP-9 and BMP-9 proregion com-plexes are equally active, whereas the proregion alone isinactive (11).

Both BMP-9 mRNA expression and processing were re-duced in insulin-resistant rats. The content of the maturesecreted form of 13 kDa was decreased in insulin-resistantrats, whereas the content of the 47-kDa precursor containingthe proregion covalently linked to the C terminus region wasaugmented. Therefore, the ratio of the secreted mature formby the nonprocessed inactive precursor was decreased inhepatic tissue during insulin resistance. This observation ledus to suggest that in insulin resistance states an impairmentof BMP-9 processing occurs.

Down-regulation of BMP-9 expression and processing inthe liver of insulin-resistant rats guided us to investigate theputative participation of glucose and insulin in these events.Only the combination of glucose and insulin was able toincrease BMP-9 mRNA levels and both the inactive precursorand the mature secreted forms of BMP-9, resulting in a higherrate of BMP-9 processing. Unexpectedly, BMP-9 processingrate diminished in the presence of insulin alone. Althoughwe did not investigate the nature of this response, it is pos-sible that insulin per se regulates posttranscriptional modi-fications of BMP-9, as occurs in the activation of the keyhepatic transcription factor sterol-response-element-bindingprotein-1c (28). We then hypothesized that BMP-9 expressionand secretion might be stimulated in the liver when an in-crease in glucose and insulin portal levels occurs, i.e. in thepostprandial state. This observation suggests a common fea-ture in the physiological control of BMP-9 and HISS.

HISS secretion is immediately increased in postprandialstate triggered by increased plasma insulin levels. In thissituation, HISS liberation enhances skeletal muscle glucoseuptake, prompting it to approximately 50–60% of the whole-body insulin-stimulated glucose uptake (5). Treatment ofskeletal muscle cells with BMP-9 in vitro increased insulinsignaling that mediates glucose uptake (16).

FIG. 5. BMP-9 expression in livers of rats submitted to gavage withglucose. Wistar rats fasted for 12 h were submitted to gavages con-taining NaCl 0.9% (CTL) or NaCl 0.9% plus glucose (1 g/kg b.w.) After2 h, fragments of the livers were removed and total RNA was ex-tracted and used for real-time PCR analysis of BMP-9 mRNA nor-malized by the constitutive gene RPL37a (A). In parallel, anotherfragment of the liver was removed and total protein was extracted andused for immunoblotting analysis of BMP-9 nonprocessed precursor(B) and mature secreted form (C). BMP-9 processing was assessed bythe value of the ratio mature secreted form/nonprocessed precursor(D). The results are expressed as means � SEM. *, P � 0.05 vs. CTL(n � 5).



Glucose solutions were administrated to overnight fastedrats to simulate refeeding and postprandial events after a phys-iological period of food deprivation. Two hours after oral glu-cose administration, there was an increase in BMP-9 mRNAlevels together with an increase in both BMP-9 inactive pre-cursor and mature secreted form. The ratio of BMP-9 processingdid not change, probably because a simultaneous up-regulation ofthe inactive precursor and mature secreted form. These resultssuggest that the in vivo increase of glucose and insulin mightactivate transcriptional and posttranscriptional mechanisms thatcontrol BMP-9 expression to account for its proper secretion.

During a prolonged fasting period, hepatic insulin resis-tance occurs (39–41) in parallel with reduced plasma levelsof insulin and glucose (39, 40). Prolonged fasting also leadsto insulin resistance in skeletal muscles (40, 41). Additionally,it was demonstrated that the progression of fasting is linked

to insulin resistance due to impairmentin HISS action (5). Refeeding of 72-hfasted rats was not able to induce changesin levels of both BMP-9 mRNA and ma-ture secreted form, although an accumu-lation of BMP-9 immature precursor wasobserved. Thus, prolonged fasting periodsimpair glucose/insulin-induced BMP-9expression and processing. This is there-fore an additional similarity between theregulation of HISS and BMP-9.

What is the determinant event of pro-longed fasting periods that abrogatesBMP-9 production? The three models ofinsulin resistance used herein share thehigh plasma glucocorticoid levels as acommon feature. Although melatoninhas a direct suppressive effect on insulinsecretion (42, 43), Pinx rats display nor-moinsulinemia and hypercorticosteron-emia (20). This is probably due to the factthat melatonin also suppresses adrenalcortex activity (44). These observationslead us to conclude that the absence ofmelatonin has an overcoming effect onmetabolism that results from the lack ofa suppressive action on adrenal glandsrather than on pancreatic islets.

Hypercorticosteronemia has been as-sociated with increased glucose produc-tion by the liver, decreased glucosetransport and use by peripheral tissues,and decreased lipogenesis in adipocytes(45–47). Melatonin suppresses adrenalsecretory activity and its ablation resultsin an increase of glucocorticoid produc-tion (48). Prolonged fasting (48–96 h)differentiates from short and physiolog-ical periods of food deprivation bymarked increased values of plasma glu-cocorticoid levels (49). Indeed, the accu-mulation of the BMP-9 inactive precur-sor in parallel with reduction in theBMP-9 mature secreted form in the liverof insulin-resistant rats was more evi-

dent in Dex-treated rats when compared with Pinx and 72-hfasted ones.

To further address this issue, BMP-9 expression and pro-cessing were investigated in perfused livers of 12 h fasted ratspreviously submitted to ip dexamethasone injection. Increase inlevels of BMP-9 mRNA and protein induced by glucose plusinsulin was abrogated by glucocorticoid administration. Thisfinding supports the proposition that high levels of glucocor-ticoids during prolonged fasting periods exert a suppressiveeffect on refeeding-induced BMP-9 expression. These findingsare particularly important because a disruption in liver-derivedhormonal mechanism is likely to take part in the onset of pe-ripheral insulin resistance (4, 50).

To ensure the relevance of BMP-9 in glucose homeostasis,we neutralized serum BMP-9 with anti-BMP-9 antibody in

FIG. 6. BMP-9 expression in livers of 72-h fasted rats submitted to refeeding. Wistar ratsfasted for 72 h (Ft-72) were submitted to refeeding (ad libitum) and killed in differentintervals after food became available (1, 2, or 4 h). Fed rats were used as control (CTL). Twofragments of the liver from each animal were removed and separately used for total RNA andprotein extraction. Total RNA was used for real time PCR analysis of BMP-9 mRNA nor-malized by the constitutive gene RPL37a (A). Total protein was used for immunoblottinganalysis of BMP-9 nonprocessed precursor (B) and mature secreted form (C). BMP-9 pro-cessing was assessed by the value of the ratio mature secreted form/nonprocessed precursor(D). The results are expressed as means � SEM. *, P � 0.05 vs. CTL (n � 4).



overnight (12 h) fasted rats, which resulted in glucose intol-erance as observed by GTT analysis. Physiological overnightfasting was chosen to these experiments to reinforce thesuggestive HISS nature of BMP-9. According to this theory,HISS is secreted and sensitizes skeletal muscle and liver toinsulin after a short period of food deprivation (5). Moreover,we have demonstrated that oral glucose challenge efficientlyinduces BMP-9 expression and processing.

As demonstrated by Chen et al. (16), two mechanisms mustbe considered to explain BMP-9 effects on glucose homeosta-sis. First, in vivo BMP-9 markedly increases insulin serumvalues after glucose challenge only 24 h after treatment,which was suggested to be a long-term effect. Second, in vitrotreatment of skeletal muscle cells with BMP-9 directly in-creased AKT activity. AKT serine threonine kinase is a keyenzyme activated by insulin signaling cascade. Its activationis involved in both suppression of hepatic glucose produc-tion and skeletal muscle glucose uptake (51). It is importantto note that in the study by Chen et al. (16), no direct effectof BMP-9 on insulin secretion was observed in vitro in pan-creatic �-cell lineages. These observations suggest that the in

vivo increase of insulin serum levels is a secondary event thatmight result, for example, from the decrease in blood glucoselevels.

Insulin induces several metabolic events aimed at decreas-ing endogenous glucose production by the liver and increas-ing glucose uptake by the skeletal muscle. The former effectseems not to be the main BMP-9 response because BMP-9neutralizing antibody did not alter, at least acutely, fastingglucose levels.

The main mechanism attributed to HISS in the modulationof glucose homeostasis is the increase of skeletal musclesensitivity to insulin action (5). Therefore, we tested whetherinsulin resistance accounted for glucose intolerance afterBMP-9 neutralization with anti-BMP-9 antibody. ITT re-vealed that anti-BMP-9 antibody treatment resulted in insu-lin resistance. It is plausible to affirm, therefore, that BMP-9controls glucose homeostasis in normal rats at least by im-proving insulin action in peripheral tissues.

In summary, our results show that BMP-9 plays a role inthe control of physiological insulin sensitivity, and its reg-

FIG. 7. BMP-9 expression in livers of rats previously treated withdexamethasone and perfused with glucose and insulin. Wistar ratswere treated with Dex or vehicle (CTL) 12 h before anesthesia andliver perfusion. Livers were perfused for 2 h with Krebs containingglucose 11.2 mM plus insulin. After perfusion, two fragments of theliver from each animal were removed and separately used for totalRNA and protein extraction. Total RNA was used for real-time PCRanalysis of BMP-9 mRNA normalized by the constitutive geneRPL37a (A). Total protein was used for immunoblotting analysis ofBMP-9 nonprocessed precursor (B) and mature secreted form (C).BMP-9 processing was assessed by the value of the ratio maturesecreted form / nonprocessed precursor (D). The results are expressedas means � SEM. *, P � 0.05 vs. CTL (n � 6).

FIG. 8. GTT and ITT in 12-h fasted rats submitted to serum BMP-9neutralization with anti-BMP-9 antibody. Wistar rats were fasted for12 h and submitted to an iv injection with anti-BMP-9 antibody (100�g/kg), control IgG Fc chain (100 �g/kg), or NaCl 0.9% (CTL) 15 minbefore the GTT or the ITT. Rats received an ip glucose injection (2mg/kg), and samples were collected from the tail 0, 15, 30, 60, 90, 120,150, and 180 min later for the determination of the blood glucoseconcentration (A). The area under the curve (AUC) obtained from theGTT was calculated (B). Rats received an ip insulin injection (2 IU/kg)and samples were collected from the tail 0, 5, 10, 15, 20, 25, and 30min later for the determination of the blood glucose concentration andcalculation of KITT (C). The results are expressed as means � SEM. *,P � 0.05 vs. CTL (n � 6).



ulation presents similarities to that of HISS. BMP-9 mRNAand protein content were regulated by oral glucose admin-istration, probably as a consequence of the direct and com-bined actions of insulin and glucose in the liver. Prolongedfasting impaired BMP-9 expression and processing inducedby refeeding. Dexamethasone directly down-regulated glu-cose/insulin-induced BMP-9 expression, suggesting a mech-anism that reduces BMP-9 in the liver of insulin-resistantrats. Additionally, serum BMP-9 neutralization in normalrats resulted in glucose intolerance and insulin resistance.

Acknowledgments

The authors thank Luciene Ribeiro, Maristela M. Okamoto, Jose L. dosSantos, and Julieta S. Falcao for technical assistance, and Dr. Carla R.Oliveira Carvalho for kindly allowing the use of facilities from herlaboratory.

Received May 5, 2008. Accepted August 7, 2008.Address all correspondence and requests for reprints to: Silvana

Bordin, Departamento de Fisiologia e Biofísica, Universidade de SaoPaulo, Av Lineu Prestes, 1524-ICB 1-Sala 125, 05508-900 Sao Paulo,Brasil. E-mail: [email protected].

This work was supported by grants from the Fundacao de Amparoa Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional deDesenvolvimento Científico e Tecnologico. L.C.C. was supported duringthis study by a fellowship of FAPESP.

Disclosure Statement: The authors have nothing to declare.

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