help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2008-0655
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Caperuto, L. C.
Right arrow Articles by Bordin, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caperuto, L. C.
Right arrow Articles by Bordin, S.
Endocrinology Vol. 149, No. 12 6326-6335
Copyright © 2008 by The Endocrine Society

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Luciana Chagas Caperuto1, Gabriel Forato Anhê1, Tavane David Cambiaghi, Eliana Hiromi Akamine, Daniella do Carmo Buonfiglio, José Cipolla-Neto, Rui Curi and Silvana Bordin

Department of Biological Sciences (L.C.C.), Federal University of São Paulo, 04023-900 São Paulo, Brazil; and Department of 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 São Paulo, 05508-900 São Paulo, Brazil

Address all correspondence and requests for reprints to: Silvana Bordin, Departamento de Fisiologia e Biofísica, Universidade de São Paulo, Av Lineu Prestes, 1524-ICB 1-Sala 125, 05508-900 São Paulo, Brasil. E-mail: sbordin{at}icb.usp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone morphogenetic protein 9 (BMP-9), a member of the TGF-β superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance (HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting (72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXISTENCE OF a liver-derived humoral factor that regulates glucose homeostasis has emerged from the observation that glucose uptake by skeletal muscle is severely impaired by surgical or pharmacological blockade of hepatic parasympathetic nerves (1, 2, 3). The reversal of the hepatic denervation-induced insulin resistance by intraportal acetylcholine administration reinforced this hypothesis (4).

The synthesis and release of the so-called hepatic insulin-sensitizing substance (HISS) has been deeply investigated during the last years (5, 6, 7, 8). Insulin induces HISS liberation by the liver through a mechanism dependent on acetylcholine-induced nitric oxide generation. In addition, HISS production and regulation by insulin is dependent on the feeding status, fasting duration, and refeeding (6, 7).

Despite the informations above, the nature of HISS remains unknown. Bone morphogenetic protein (BMP)-9 is likely to be a candidate.

BMPs are members of the TGF superfamily (TGF-β), which contains a large number of proteins that have been implicated in a variety of developmental processes such as cartilage and bone formation (9). TGF-β receptors are commonly composed by two Ser/Thr kinase transmembrane chains, types I and II. The combination of types I and II chains can generate a broad range of effects in response to the same ligand. Thus, the tissue-specific effect of any BMP depends on the receptor chains expressed by each cell type (10).

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

A high-throughput pharmacogenomic study identified BMP-9 as a regulator of glucose metabolism (16). In vitro, BMP-9 inhibited the expression of phosphoenolpyruvate carboxykinase and increased the expression of malic enzyme and fatty acid synthase (FAS) in liver rat hepatoma H4IIe cells. In differentiated L6 myoblasts, BMP-9 activates the serine/threonine kinase AKT. In vivo, BMP-9 was shown to reduce glycemia in normal and diabetic mice.

Despite its therapeutic potential, BMP-9 was identified as a pharmacological, rather than a physiological target, and still there is a lack of information regarding the regulation of BMP-9 during the course of physiological and pathological processes (17). Such data would be useful to state the relevance of BMP-9 in the control of normal glucose homeostasis.

To clarify the participation of BMP-9 in physiological and nonphysiological alterations of glucose metabolism, we investigated the effect of two classical models of insulin resistance, i.e. prolonged fasting and dexamethasone treatment, and pinealectomy, on the expression and processing of BMP-9. Pinealectomy was chosen because of the remarkable glucose intolerance, decreased adipocyte responsiveness to insulin, normoinsulinemia and hypercorticosteroidism (18, 19, 20). Exposure of the hepatic tissue to glucose, insulin and glucocorticois further elucidated the control of BMP-9 expression and processing. Finally, serum BMP-9 neutralization revealed its role in the physiological regulation of glucose homeostasis. Together, our results prompt that BMP-9 fulfills the requirements for a putative HISS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The reagents and apparatus for SDS-PAGE and immunoblotting were obtained 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 Biobrás (Minas Gerais, Brazil). Enhanced chemiluminescence detection system and nitrocellulose membrane (0.45 mm) were from Amersham Pharmacia Biotech (Uppsala, Sweden). X-ray-sensitive films and chemicals were from IBF (Rio de Janeiro, 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 (Coralville, IA). GoTaq DNA polymerase and ImProm-II reverse transcriptase were from Promega (Madison, WI). Trizol reagent and Sybr Green were from Invitrogen (Carlsbad, CA). Neutralizing anti-BMP-9 antibody and control IgG Fc chain were developed by Imuny Biotechnology (Campinas, Brazil). The affinity-purified rabbit polyclonal antibody was raised against a peptide mapping at the C terminus of BMP-9 of human origin.

Animals
Wistar rats obtained from the Animal Breeding Center of the Institute of Biomedical Sciences (São Paulo, Brazil) were kept under standard laboratory conditions (12-h light, 12-h dark cycle) with free access to food and water.

Rats weighting about 250 g were subjected to daily ip injection of dexamethasone [Dex; 1 mg/kg of body weight (b.w.)] for 5 consecutive days. 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 injection of 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). After recovery from surgery, rats were provided with food and water ad libitum (three or four animals per cage). Six weeks after surgery, the rats were decapitated at night (Zeitgeber time 18, i.e. 6 h after lights off), and a slice of the liver was removed. The effectiveness of the pinealectomy was then verified.

The rats were fasted for 72 (Ft-72) or 12 (Ft-12) h. Ft-72 rats were submitted 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 then fed for 2 h with standard rodent chow ad libitum. After this period, food was 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 sodium thiopental (50 mg/kg b.w., ip) and killed after approximately 10 min. Abdominal cavity was opened and a slice of the liver was removed and processed for total RNA or protein extraction. The procedures described above were conducted according to the guidelines of the Brazilian 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 a monovascular nonrecirculating system through a cannula inserted into the hepatic portal vein, using Krebs-Henseleit (pH 7.4), at 37 C and saturated with a 95%–5% O2-CO2 mixture. The flow rate was always adjusted according to the weight of the liver (4 ml/min · g of fresh weight), considering that the liver weight 4% of the body weight) (21).

The livers were initially perfused for 10 min with Krebs-Henseleit to remove 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 or presence of insulin (20 U/liter). The experiments were performed with livers that maintained the same shape and color throughout the assay, indicating proper vascular perfusion and viability. At the end of the perfusion period, slices of the liver were removed and processed for total RNA or protein preparation.

RNA extraction and conventional RT-PCR
Total RNA was extracted from approximately 100 mg of liver using Trizol reagent (Invitrogen). Total RNA was reverse transcribed and conventional RT-PCR analysis was performed as previously described (22). The amplification products were run on a 1.2% agarose gel containing ethidium bromide, and the band intensities were determined by digital scanning followed by quantification using the Scion Image analysis software (Scion Corp., Frederick, MD). The results were expressed as a ratio of the target genes to the housekeeping RPL37a. The primer sequences used for RT-PCR analysis with their respective melting point and lengths were as follows: HIF1A, sense, 5'-CCCATCCATGTGACCATGAGG-3' and antisense, 5'-TCAGCACCAAGCACGTCATAGG-3', 56 C, 261 bp; FAS, sense, 5'-AAGCCAGGAAGAGTGGGAGAGC-3' and antisense, 5'-GGTTGGACAGCAGGATACACCG-3', 59.2 C, 311 bp; 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 using ROTOR GENE 3000 equipment (Corbett Research, Mortlake, Australia) and Sybr Green (Invitrogen) as fluorescent dye. Primer sequences and reaction conditions were: BMP-9, sense, 5'-TTCAGGATGAGGGCTGGGAG-3' and antisense, 5'-GGATGTCTTCACAAGCACGGTC-3', 58.5 C. Amplification efficiency of each sample was calculated as described by Ramakers et al. (23). Gene expression was determined by the method of Liu and Saint (24) using RPL37a gene expression as inner control. All samples were compared using the relative cycle threshold. The cycle threshold value is the calculated cycle number by which the fluorescence signal emitted is significantly above background levels.

Protein analysis by Western blotting
Fragments of liver containing approximately 100 mg were homogenized in a boiling extraction buffer [10% sodium dodecyl sulfate, 100 mM Tris (pH 7.4), 10 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM sodium vanadate] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Inc., Westbury, NY) operated at maximum speed for 30 sec.

The extracts were centrifuged at 15,000 x g, 4 C, for 40 min to remove insoluble material. Protein concentrations of the supernatants were determined by the Bradford assay, and an equal amount of total protein from each sample (75 µg) was treated with Laemmli buffer containing dithiothreitol 100 mM. Samples were heated in a boiling water bath for 5 min, after which they were subjected to SDS-PAGE (10% bis-acrylamide).

Electrotransfer of proteins from gel to nitrocellulose membrane was performed for 90 min at 120 V (constant) as described elsewhere (25). Nonspecific protein binding to nitrocellulose was reduced by preincubating the membrane overnight at 4 C in blocking buffer (1% gelatin, 10 mm Tris, 150 mm NaCl, and 0.02% Tween 20). The nitrocellulose blot was incubated with anti-BMP-9 antibody (1:1000) diluted in blocking buffer overnight at 4 C and then washed for 30 min with blocking buffer without gelatin.

Bound antibodies were detected with horseradish peroxidase-conjugated anti-IgG (1:10,000) and visualized by chemiluminescence in x-ray sensitive films. Band intensities were quantified from the developed autoradiographs using Scion Image program.

Intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT)
Male Wistar rats weighting 200 g were fasted for 12 h before the experiments. An iv injection of anti-BMP-9 neutralizing antibody (100 µg/kg b.w.), IgG Fc chain (100 µg/kg b.w.), or NaCl 0.9% (CTL) was administrated in the penin vein. After 15 min the rats were submitted to 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 collected from the tail at 0, 5, 10, 15, 30, 60, 120, 150, and 180 min for measurement of serum glucose. ITT was carried out with an insulin injection (2 IU/kg), and blood samples were collected from the tail at 0, 5, 10, 15, 20, 25, and 30 min for measurement of serum glucose. The constant rate for glucose disappearance (KITT) was calculated using the formula 0.693/half-time. The glucose half-time was calculated from the slope of the least-square analysis of the plasma glucose concentrations during the linear decay phase (26).

Statistical analysis
Results are presented as means ± SEM. Comparisons were performed using one-way ANOVA, with Tukey-Kramer posttest (INStat; Graph Pad Software, Inc., San Diego, CA), and the unpaired Student’s t test when appropriate. The level of significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BMP-9 expression and processing in the liver of insulin-resistant rats
To characterize the putative role of BMP-9 in the regulation of glucose homeostasis, the first approach applied was to analyze BMP-9 mRNA and protein content in the liver of insulin-resistant rats. The models chosen were: 1) 72 h fasting, 2) dexamethasone treatment, and 3) pinealectomy. Two immunoreactive bands, the nonprocessed inactive 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 refer to the secreted mature form and nonprocessed 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. 1AGo) compared with CTL counterparts. This modulation seems to be specific for BMP-9 because no alterations were found in BMP-2 mRNA expression (data not shown).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

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

 
The content of the BMP-9 nonprocessed precursor increased in Ft-72, Dex, and Pnx rats, respectively, 66 ± 14, 140 ± 19, and 58 ± 13% over their control counterparts (P < 0.05; Fig. 1BGo). The content of the BMP-9 mature secreted form was similar in FT-72 and CTL rats but decreased in Dex and Pinx rats to 39 ± 5% and 77 ± 8%, respectively (P < 0.05; Fig. 1CGo). BMP-9 processing was decreased in Ft-72 (Fig. 2AGo), Dex (Fig. 2BGo), and Pnx rats (to 50 ± 6, 19 ± 4, and 52 ± 8%, respectively, compared with their control counterparts; P < 0.05).


Figure 2
View larger version (9K):
[in this window]
[in a new window]

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

 
Glucose and insulin directly affect BMP-9 expression and processing in perfused liver
To investigate the participation of glucose and insulin in the regulation of BMP-9 expression and processing, we performed in situ liver perfusion with these agents. Previously, to assure the efficiency of the perfusion procedure, we assessed insulin-induced AKT phosphorylation, and hypoxia-inducible factor (HIF-1A), and FAS mRNA expression. AKT phosphorylation was increased after a 30-min perfusion with insulin when compared with the noninsulin time-matched perfused livers. AKT phosphorylation peaked after 60 min of perfusion and remained detectable up to 120 min (Fig. 3AGo). Concomitantly, tyrosine phosphorylation of insulin receptor substrate 1/2 increased (data not shown). FAS gene expression is known to be rapidly and directly induced by insulin (27, 28). FAS mRNA expression was not affected by increasing glucose concentration from 5.6 to 11.2 mM in the perfusion medium but was increased by the addition of insulin to the medium (152 ± 7%; P < 0.05) (Fig. 3BGo). Together these results demonstrated that the liver preserved insulin responsiveness during a 2-h perfusion. HIF-1A is a transcription factor up-regulated by hypoxia (29). HIF-1A expression was not altered throughout the perfusion period, ranging from 30 to 120 min, despite variations in insulin concentration (Fig. 3CGo). This result indicated that negligible rates of hypoxia occurred during the perfusions.


Figure 3
View larger version (24K):
[in this window]
[in a new window]

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

 
Figure 4AGo shows that BMP-9 mRNA expression was not modulated by alterations in glucose concentrations. Insulin induced an increase in BMP-9 mRNA expression exclusively when combined to glucose 11.2 mM (39 ± 10% compared with the condition with no glucose; P < 0.05) (Fig. 4BGo). The content of the BMP-9 nonprocessed precursor was similarly increased when perfusion was performed with 11.2 mM glucose, insulin or combination of both (respectively 141 ± 11, 136 ± 18, and 165 ± 21% compared with CTL; P < 0.05) (Fig. 4CGo). As noted for BMP-9 mRNA expression, the content of the BMP-9 mature secreted form was unaltered by the perfusion with 11.2 mM glucose or insulin, whereas the combination of both resulted in its up-regulation (193 ± 33% compared with the condition with no glucose; P < 0.05; Fig. 4DGo). The ratio of mature and immature BMP-9 content also increased with glucose plus insulin (to 132 ± 17%; P < 0.05 vs. CTL) but was decreased by insulin in the absence of glucose (to 51 ± 18%; P < 0.05; Fig. 4EGo).


Figure 4
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 4. BMP-9 expression in livers perfused with glucose and insulin. Livers were perfused for 2 h with Krebs containing glucose 0.0, 2.8, 5.6, or 11.2 mM. Total RNA was extracted and used for real-time PCR analysis of BMP-9 mRNA (A). Livers were perfused for 2 h with Krebs containing glucose, insulin, or the combination of both. Total RNA was extracted and used for real-time PCR analysis of BMP-9 mRNA normalized by the constitutive gene RPL37a (B). Total protein was extracted and used for immunoblotting analysis of BMP-9 nonprocessed precursor (C) and mature secreted form (D). BMP-9 processing was assessed by the value of the ratio mature secreted form/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 without glucose plus insulin (n = 5).

 
Oral glucose administration induced BMP-9 expression in the liver of 12-h fasted rats
The effect of the physiological increase in glucose and insulin concentrations on BMP-9 expression in overnight fasted rats that received a glucose solution (1 g/kg b.w.) by intragastric gavage was investigated. BMP-9 mRNA expression was increased 2 h after glucose oral administration (22 ± 4% compared with CTL; P < 0.05; Fig. 5AGo) Similarly, as shown in Fig. 5Go, B and C, oral glucose induced an increase of both BMP-9 nonprocessed precursor and mature secreted form (by 62 ± 25 and 73 ± 25%, respectively, compared with CTL; P < 0.05). BMP-9 protein processing did not change (Fig. 5DGo) due to the simultaneous increase in 47 kDa precursor content (Fig. 5BGo).


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 5. BMP-9 expression in livers of rats submitted to gavage with glucose. Wistar rats fasted for 12 h were submitted to gavages containing NaCl 0.9% (CTL) or NaCl 0.9% plus glucose (1 g/kg b.w.) After 2 h, fragments of the livers were removed and total RNA was extracted and used for real-time PCR analysis of BMP-9 mRNA normalized by the constitutive gene RPL37a (A). In parallel, another fragment of the liver was removed and total protein was extracted and used for immunoblotting analysis of BMP-9 nonprocessed precursor (B) and mature secreted form (C). BMP-9 processing 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 = 5).

 
Refeeding did not increase BMP-9 expression and processing in the liver of 72-h fasted rats
As an attempt to evaluate whether the prolonged fasting would hamper the effect of glucose and insulin on BMP-9 expression and processing in vivo, 72-h fasted rats were submitted to a refeeding schedule. Reduction of BMP-9 mRNA expression induced by 72 h fasting (65 ± 3% of the CTL values; P < 0.05) was not altered 1 h after the refeeding (70 ± 6% of the CTL values; P < 0.05), and it was only partially restored after 2 h (Fig. 6AGo). Four hours after refeeding, BMP-9 mRNA levels returned to the values before refeeding (± 60 ± 3% of the CTL values; P < 0.05).


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 6. BMP-9 expression in livers of 72-h fasted rats submitted to refeeding. Wistar rats fasted for 72 h (Ft-72) were submitted to refeeding (ad libitum) and killed in different intervals after food became available (1, 2, or 4 h). Fed rats were used as control (CTL). Two fragments of the liver from each animal were removed and separately used for total RNA and protein extraction. Total RNA was used for real time PCR analysis of BMP-9 mRNA normalized by the constitutive gene RPL37a (A). Total protein was used for immunoblotting analysis of BMP-9 nonprocessed precursor (B) and mature secreted form (C). BMP-9 processing 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).

 
BMP-9 nonprocessed precursor increased in 72-h fasted rats, and a further increase was observed 1 and 2 h after refeeding (161 ± 32 and 358 ± 50%, respectively, compared with CTL; P < 0.05) (Fig. 6BGo). Four hours after refeeding, the content of the BMP-9 nonprocessed precursor returned to the values observed in 72-h fasted rats. In addition, 72 h fasting did not alter the levels of the mature secreted form, and no further alterations were observed after refeeding (Fig. 6CGo). The ratio of 13 kDa mature BMP-9 in relation to 47 kDa precursor was decreased in 72-h fasted rats and remained as such after refeeding (Fig. 6DGo).

Dexamethasone down-regulates BMP-9 expression and processing in rat liver
Dexamethasone is well known to induce insulin resistance in peripheral tissues such as skeletal muscle and liver (30, 31). To test whether the impairment of glucose plus insulin-induced BMP-9 expression and processing observed in prolonged fasting rats was due to the high levels of glucocorticoids, we performed the in situ liver perfusion in rats pretreated with dexamethasone. Treatment with dexamethasone 12 h before the liver perfusion reduced the stimulation of BMP-9 mRNA expression induced by the combination of 11.2 mM glucose and insulin (to 31 ± 4%, compared with CTL; P < 0.05) (Fig. 7AGo). Dexamethasone also up-regulated the accumulation of BMP-9 nonprocessed precursor induced by the combination of glucose and insulin (59 ± 22% compared with CTL; P < 0.05) and did not alter the stimulation of the BMP-9 mature secreted form (Fig. 7Go, B and C). As a consequence, the processing of BMP-9 induced by glucose plus insulin was reduced in rats previously treated with dexamethasone (74 ± 9% compared with CTL; P < 0.05; Fig. 7DGo).


Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 7. BMP-9 expression in livers of rats previously treated with dexamethasone and perfused with glucose and insulin. Wistar rats were treated with Dex or vehicle (CTL) 12 h before anesthesia and liver perfusion. Livers were perfused for 2 h with Krebs containing glucose 11.2 mM plus insulin. After perfusion, two fragments of the liver from each animal were removed and separately used for total RNA and protein extraction. Total RNA was used for real-time PCR analysis of BMP-9 mRNA normalized by the constitutive gene RPL37a (A). Total protein was used for immunoblotting analysis of BMP-9 nonprocessed precursor (B) and mature secreted form (C). BMP-9 processing 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 = 6).

 
Serum BMP-9 neutralization with anti-BMP-9 antibody causes glucose intolerance and insulin resistance in 12-h fasted rats
Previous treatment of 12-h fasted rats with anti-BMP-9 antibody resulted in glucose intolerance as observed by a more accentuated ascendant phase of serum glucose values, which was followed by a noncompensatory glucose decay phase (Fig. 8AGo). Therefore, the area under the curve (blood glucose vs. time) was increased in rats treated with anti-BMP-9 antibody (41 ± 13% compared with CTL; P < 0.05; Fig. 8BGo).


Figure 8
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 8. GTT and ITT in 12-h fasted rats submitted to serum BMP-9 neutralization with anti-BMP-9 antibody. Wistar rats were fasted for 12 h and submitted to an iv injection with anti-BMP-9 antibody (100 µg/kg), control IgG Fc chain (100 µg/kg), or NaCl 0.9% (CTL) 15 min before the GTT or the ITT. Rats received an ip glucose injection (2 mg/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 glucose concentration (A). The area under the curve (AUC) obtained from the GTT 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 30 min later for the determination of the blood glucose concentration and calculation of KITT (C). The results are expressed as means ± SEM. *, P < 0.05 vs. CTL (n = 6).

 
To characterize whether this glucose intolerance was due to insulin resistance or an impairment of pancreatic function, we next performed ITT in anti-BMP-9 antibody-treated rats. Figure 8CGo shows that the KITT was decreased in rats treated with anti-BMP-9 antibody (66 ± 5% of the CTL values; P < 0.05). Injection of the same amount of control IgG Fc chain did change glucose tolerance (Fig. 8BGo) and insulin sensitivity (Fig. 8CGo), indicating that the effect of anti-BMP-9 was not due to an unspecific action.

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


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidences presented herein suggest that BMP-9 is a liver-secreted factor that might play an important role in the control of glucose homeostasis. The following observations support this proposition: 1) BMP-9 mRNA and mature secreted form contents were decreased in three distinct models of insulin resistance (72 h fasted rats, Dex-treated rats, and Pinx rats); 2) BMP-9 mRNA and mature secreted form were directly up-regulated by the combination of glucose plus insulin and by glucose oral challenge; 3) prolonged fasting and previous exposure to dexamethasone abrogated the refeeding-induced up-regulation of BMP-9; and 4) serum BMP-9 neutralization with an anti-BMP-9 antibody induced glucose intolerance and insulin resistance in overnight fasted rats.

Chen et al. (16) demonstrated that the treatment with recombinant BMP-9 improves glucose homeostasis in diabetic rodents. This observation suggests that impairment in BMP-9 expression might be associated with generation of glucose intolerance in the above-mentioned models. Therefore, an integrated interpretation of the present data and the results of Chen et al. point to a pathophysiological role of BMP-9 in glucose homeostasis.

One of the most important mechanisms for the control of the biological effects of TGF-β family proteins, and by extension BMPs, is the conversion to an active form (32). BMP-9 is translated as an inactive precursor protein of approximately 48 kDa, which is composed by a hydrophobic secretory leader, an amino-terminal proregion and a carboxyl-terminal mature domain (11, 33, 34, 35). To become active, the proregion of all BMPs suffers a proteolytic cleavage at the multibasic amino acid motif R-X-X-R, releasing the C-terminal mature protein (36). Mature BMP-9 is a homodimer of two 110-amino acid chains with a calculated molecular mass of 13 kDa (www. genenames.org) (12). In most cases, the proregion dissociates and the mature ligand is secreted from the cell. Similarly to growth differentiation factor-8 and TGF-β-1, -2, and -3 (37, 38), the proregion of BMP-9 remains tightly, but not covalently, linked to its C terminus region after secretion from the cell (11). However, different from growth differentiation factor-8 and TGF-βs, in which the proregions remain associated and have been found to be functionally inhibitory (37), both BMP-9 and BMP-9 proregion complexes are equally active, whereas the proregion alone is inactive (11).

Both BMP-9 mRNA expression and processing were reduced in insulin-resistant rats. The content of the mature secreted form of 13 kDa was decreased in insulin-resistant rats, whereas the content of the 47-kDa precursor containing the proregion covalently linked to the C terminus region was augmented. Therefore, the ratio of the secreted mature form by the nonprocessed inactive precursor was decreased in hepatic tissue during insulin resistance. This observation led us to suggest that in insulin resistance states an impairment of BMP-9 processing occurs.

Down-regulation of BMP-9 expression and processing in the liver of insulin-resistant rats guided us to investigate the putative participation of glucose and insulin in these events. Only the combination of glucose and insulin was able to increase BMP-9 mRNA levels and both the inactive precursor and the mature secreted forms of BMP-9, resulting in a higher rate of BMP-9 processing. Unexpectedly, BMP-9 processing rate diminished in the presence of insulin alone. Although we did not investigate the nature of this response, it is possible that insulin per se regulates posttranscriptional modifications of BMP-9, as occurs in the activation of the key hepatic transcription factor sterol-response-element-binding protein-1c (28). We then hypothesized that BMP-9 expression and secretion might be stimulated in the liver when an increase in glucose and insulin portal levels occurs, i.e. in the postprandial state. This observation suggests a common feature in the physiological control of BMP-9 and HISS.

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

Glucose solutions were administrated to overnight fasted rats to simulate refeeding and postprandial events after a physiological period of food deprivation. Two hours after oral glucose administration, there was an increase in BMP-9 mRNA levels together with an increase in both BMP-9 inactive precursor and mature secreted form. The ratio of BMP-9 processing did not change, probably because a simultaneous up-regulation of the inactive precursor and mature secreted form. These results suggest that the in vivo increase of glucose and insulin might activate transcriptional and posttranscriptional mechanisms that control BMP-9 expression to account for its proper secretion.

During a prolonged fasting period, hepatic insulin resistance occurs (39, 40, 41) in parallel with reduced plasma levels of insulin and glucose (39, 40). Prolonged fasting also leads to insulin resistance in skeletal muscles (40, 41). Additionally, it was demonstrated that the progression of fasting is linked to insulin resistance due to impairment in HISS action (5). Refeeding of 72-h fasted rats was not able to induce changes in levels of both BMP-9 mRNA and mature secreted form, although an accumulation of BMP-9 immature precursor was observed. Thus, prolonged fasting periods impair glucose/insulin-induced BMP-9 expression and processing. This is therefore an additional similarity between the regulation of HISS and BMP-9.

What is the determinant event of prolonged fasting periods that abrogates BMP-9 production? The three models of insulin resistance used herein share the high plasma glucocorticoid levels as a common feature. Although melatonin has a direct suppressive effect on insulin secretion (42, 43), Pinx rats display normoinsulinemia and hypercorticosteronemia (20). This is probably due to the fact that melatonin also suppresses adrenal cortex activity (44). These observations lead us to conclude that the absence of melatonin has an overcoming effect on metabolism that results from the lack of a suppressive action on adrenal glands rather than on pancreatic islets.

Hypercorticosteronemia has been associated with increased glucose production by the liver, decreased glucose transport and use by peripheral tissues, and decreased lipogenesis in adipocytes (45, 46, 47). Melatonin suppresses adrenal secretory activity and its ablation results in an increase of glucocorticoid production (48). Prolonged fasting (48–96 h) differentiates from short and physiological periods of food deprivation by marked increased values of plasma glucocorticoid levels (49). Indeed, the accumulation of the BMP-9 inactive precursor in parallel with reduction in the BMP-9 mature secreted form in the liver of insulin-resistant rats was more evident in Dex-treated rats when compared with Pinx and 72-h fasted ones.

To further address this issue, BMP-9 expression and processing were investigated in perfused livers of 12 h fasted rats previously submitted to ip dexamethasone injection. Increase in levels of BMP-9 mRNA and protein induced by glucose plus insulin was abrogated by glucocorticoid administration. This finding supports the proposition that high levels of glucocorticoids during prolonged fasting periods exert a suppressive effect on refeeding-induced BMP-9 expression. These findings are particularly important because a disruption in liver-derived hormonal mechanism is likely to take part in the onset of peripheral 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 overnight (12 h) fasted rats, which resulted in glucose intolerance as observed by GTT analysis. Physiological overnight fasting was chosen to these experiments to reinforce the suggestive HISS nature of BMP-9. According to this theory, HISS is secreted and sensitizes skeletal muscle and liver to insulin after a short period of food deprivation (5). Moreover, we have demonstrated that oral glucose challenge efficiently induces BMP-9 expression and processing.

As demonstrated by Chen et al. (16), two mechanisms must be considered to explain BMP-9 effects on glucose homeostasis. First, in vivo BMP-9 markedly increases insulin serum values after glucose challenge only 24 h after treatment, which was suggested to be a long-term effect. Second, in vitro treatment of skeletal muscle cells with BMP-9 directly increased AKT activity. AKT serine threonine kinase is a key enzyme activated by insulin signaling cascade. Its activation is involved in both suppression of hepatic glucose production and skeletal muscle glucose uptake (51). It is important to note that in the study by Chen et al. (16), no direct effect of BMP-9 on insulin secretion was observed in vitro in pancreatic β-cell lineages. These observations suggest that the in vivo increase of insulin serum levels is a secondary event that might result, for example, from the decrease in blood glucose levels.

Insulin induces several metabolic events aimed at decreasing endogenous glucose production by the liver and increasing glucose uptake by the skeletal muscle. The former effect seems not to be the main BMP-9 response because BMP-9 neutralizing antibody did not alter, at least acutely, fasting glucose levels.

The main mechanism attributed to HISS in the modulation of glucose homeostasis is the increase of skeletal muscle sensitivity to insulin action (5). Therefore, we tested whether insulin resistance accounted for glucose intolerance after BMP-9 neutralization with anti-BMP-9 antibody. ITT revealed that anti-BMP-9 antibody treatment resulted in insulin resistance. It is plausible to affirm, therefore, that BMP-9 controls glucose homeostasis in normal rats at least by improving insulin action in peripheral tissues.

In summary, our results show that BMP-9 plays a role in the control of physiological insulin sensitivity, and its regulation presents similarities to that of HISS. BMP-9 mRNA and protein content were regulated by oral glucose administration, probably as a consequence of the direct and combined actions of insulin and glucose in the liver. Prolonged fasting impaired BMP-9 expression and processing induced by refeeding. Dexamethasone directly down-regulated glucose/insulin-induced BMP-9 expression, suggesting a mechanism that reduces BMP-9 in the liver of insulin-resistant rats. Additionally, serum BMP-9 neutralization in normal rats resulted in glucose intolerance and insulin resistance.


   Acknowledgments
 
The authors thank Luciene Ribeiro, Maristela M. Okamoto, José L. dos Santos, and Julieta S. Falcão for technical assistance, and Dr. Carla R. Oliveira Carvalho for kindly allowing the use of facilities from her laboratory.


   Footnotes
 
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico. L.C.C. was supported during this study by a fellowship of FAPESP.

Disclosure Statement: The authors have nothing to declare.

First Published Online August 14, 2008

1 L.C.C. and G.F.A. contributed equally to this study. Back

Abbreviations: BMP, Bone morphogenetic protein; b.w., body weight; Dex, dexamethasone; FAS, fatty acid synthase; GTT, glucose tolerance test; HIF-1A, hypoxia-inducible factor; HISS, hepatic insulin-sensitizing substance; ITT, insulin tolerance test; KITT, constant rate for glucose disappearance; Pinx, pinealectomy.

Received May 5, 2008.

Accepted for publication August 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Xie H, Lautt WW 1996 Insulin resistance of skeletal muscle produced by hepatic parasympathetic interruption. Am J Physiol 270:E858–E863
  2. Sadri P, Lautt WW 2000 Glucose disposal by insulin, but not IGF-1, is dependent on the hepatic parasympathetic nerves. Can J Physiol Pharmacol 78:807–812[Medline]
  3. Ribeiro RT, Afonso RA, Macedo MP 2007 Hepatic parasympathetic role in insulin resistance on an animal model of hypertension. Metabolism 56:227–233[Medline]
  4. Xie H, Lautt WW 1996 Insulin resistance caused by hepatic cholinergic interruption and reversed by acetylcholine administration. Am J Physiol 271:E587–E592
  5. Lautt WW 1999 The HISS story overview: a novel hepatic neurohumoral regulation of peripheral insulin sensitivity in health and diabetes. Can J Physiol Pharmacol 77:553–562[CrossRef][Medline]
  6. Lautt WW, Macedo MP, Sadri P, Takayama S, Duarte Ramos F, Legare DJ 2001 Hepatic parasympathetic (HISS) control of insulin sensitivity determined by feeding and fasting. Am J Physiol Gastrointest Liver Physiol 281:G29–G36
  7. Latour MG, Lautt WW 2002 Insulin sensitivity regulated by feeding in the conscious unrestrained rat. Can J Physiol Pharmacol 80:8–12[CrossRef][Medline]
  8. Porszasz R, Legvari G, Pataki T, Szilvassy J, Nemeth J, Kovacs P, Paragh G, Szolcsanyi J, Szilvassy Z 2003 Hepatic insulin sensitizing substance: a novel ‘sensocrine’ mechanism to increase insulin sensitivity in anaesthetized rats. Br J Pharmacol 139:1171–1179[CrossRef][Medline]
  9. Wozney JM 1992 The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 32:160–167[CrossRef][Medline]
  10. Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H, Owen TA, Thompson DD 1998 Cloning and characterization of a novel member of the transforming growth factor-β/bone morphogenetic protein family. J Biol Chem 273:13760–13767[Abstract/Free Full Text]
  11. Brown MA, Zhao Q, Baker KA, Naik C, Chen C, Pukac L, Singh M, Tsareva T, Parice Y, Mahoney A, Roschke V, Sanyal I, Choe S 2005 Crystal structure of BMP-9 and functional interactions with pro-region and receptors. J Biol Chem 280:25111–25118[Abstract/Free Full Text]
  12. Miller AF, Harvey SA, Thies RS, Olson MS 2000 Bone morphogenetic protein-9. An autocrine /paracrine cytokine in the liver. J Biol Chem 275:17937–17945[Abstract/Free Full Text]
  13. Lopez-Coviella I, Berse B, Krauss R, Thies RS, Blusztajn JK 2000 Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. Science 289:313–316[Abstract/Free Full Text]
  14. Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, Deyrup AT, Jiang W, Luu HH, Luo J, Szatkowski JP, Vanichakarn P, Park JY, Li Y, Haydon RC, He TC 2004 Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther 11:1312–1320[CrossRef][Medline]
  15. Majumdar MK, Wang E, Morris EA 2001 BMP-2 and BMP-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL-1. J Cell Physiol 189:275–284[CrossRef][Medline]
  16. Chen C, Grzegorzewski KJ, Barash S, Zhao Q, Schneider H, Wang Q, Singh M, Pukac L, Bell AC, Duan R, Coleman T, Duttaroy A, Cheng S, Hirsch J, Zhang L, Lazard Y, Fischer C, Barber MC, Ma ZD, Zhang YQ, Reavey P, Zhong L, Teng B, Sanyal I, Ruben SM, Blondel O, Birse CE 2003 An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis. Nat Biotechnol 21:294–301[CrossRef][Medline]
  17. Groop L 2003 Bringing diabetes therapeutics to the big screen. Nat Biotechnol 21:240–241[CrossRef][Medline]
  18. Lima FB, Machado UF, Bartol I, Seraphim PM, Sumida DH, Moraes SMF, Hell NS, Okamoto MNO, Saad MJ, Carvalho CRO, Cipolla-Neto J 1998 Pinealectomy causes glucose intolerance and decreases adipose cell responsiveness to insulin in rats. Am J Physiol 275:E934–E941
  19. Alonso-Vale MI, Anhê GF, Borges-Silva CN, Andreotti S, Peres SB, Cipolla-Neto J, Lima FB 2004 Pinealectomy alters adipose tissue adaptability to fasting in rats. Metabolism 53:500–506[CrossRef][Medline]
  20. Alonso-Vale MI, Borges-Silva CN, Anhê GF, Andreotti S, Machado MA, Cipolla-Neto J, Lima FB 2004 Light/dark cycle-dependent metabolic changes in adipose tissue of pinealectomized rats. Horm Metab Res 36:474–479[CrossRef][Medline]
  21. Borba-Murad GR, Mario EG, Bassoli BK, Bazotte RB, de Souza HM 2005 Comparative acute effects of leptin and insulin on gluconeogenesis and ketogenesis in perfused rat liver. Cell Biochem Funct 23:405–413[Medline]
  22. Bordin S, Amaral ME, Anhê GF, Delghingaro-Augusto V, Cunha DA, Nicoletti-Carvalho JE, Boschero AC 2004 Prolactin-modulated gene expression profiles in pancreatic islets from adult female rats. Mol Cell Endocrinol 220:41–50[CrossRef][Medline]
  23. Ramakers C, Ruijter JM, Deprez RH, Moorman AF 2003 Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66[CrossRef][Medline]
  24. Liu W, Saint DA 2002 A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302:52–59[CrossRef][Medline]
  25. Anhê GF, Torrão AS, Nogueira TC, Caperuto LC, Amaral ME, Medina MC, Azevedo-Martins AK, Carpinelli AR, Carvalho CR, Curi R, Boschero AC, Bordin S 2006 ERK3 associates with MAP2 and is involved in glucose-induced insulin secretion. Mol Cell Endocrinol 251:33–41[CrossRef][Medline]
  26. Bonora E, Manicardi V, Zavaroni I, Coscelli C, Butturini U 1987 Relationships between insulin secretion, insulin metabolism and insulin resistance in mild glucose intolerance. Diabete Metab 13:116–121[Medline]
  27. Paulauskis JD, Sul HS 1989 Hormonal regulation of mouse fatty acid synthase gene transcription in liver. J Biol Chem 264:574–577[Abstract/Free Full Text]
  28. Foufelle F, Ferré P 2002 New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c. Biochem J 366:377–391[CrossRef][Medline]
  29. Belozerov VE, Van Meir EG 2006 Inhibitors of hypoxia-inducible factor-1 signaling. Curr Opin Investig Drugs 7:1067–1076[Medline]
  30. Venkatesan N, Lim J, Bouch C, Marciano D, Davidson MB 1996 Dexamethasone-induced impairment in skeletal muscle glucose transport is not reversed by inhibition of free fatty acid oxidation. Metabolism 45:92–100[CrossRef][Medline]
  31. Saad MJ, Folli F, Kahn JA, Kahn CR 1993 Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats. J Clin Invest 92:2065–2072[Medline]
  32. Khalil N 1999 TGF-β: from latent to active. Microbes Infect 1:1255–1263[CrossRef][Medline]
  33. Constam DB, Robertson EJ 1999 Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases. J Cell Biol 144:139–149[Abstract/Free Full Text]
  34. Sykaras N, Opperman LA 2003 Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician? J Oral Sci 45:57–73[Medline]
  35. Allendorph GP, Vale WW, Choe S 2006 Structure of the ternary signaling complex of a TGF-β superfamily member. Proc Natl Acad Sci USA 103:7643–7648[Abstract/Free Full Text]
  36. Aono A, Hazama M, Notoya K, Taketomi S, Yamasaki H, Tsukuda R, Sasaki S, Fujisawa Y 1995 Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer. Biochem Biophys Res Commun 210:670–677[CrossRef][Medline]
  37. Thies RS, Chen T, Davies MV, Tomkinson KN, Pearson AA, Shakey QA, Wolfman NM 2001 GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding. Growth Factors 18:251–259[Medline]
  38. Jiang MS, Liang LF, Wang S, Ratovitski T, Holmstrom J, Barker C, Stotish R 2004 Characterization and identification of the inhibitory domain of GDF-8 propeptide. Biochem Biophys Res Commun 315:525–531[CrossRef][Medline]
  39. Olefsky JM 1976 Effects of fasting on insulin binding, glucose transport, and glucose oxidation in isolated rat adipocytes: relationships between insulin receptors and insulin action. J Clin Invest 58:1450–1460[Medline]
  40. Almira EC, Reddy WJ 1979 Effect of fasting on insulin binding to hepatocytes and liver plasma membranes from rats. Endocrinology 104:205–211[Abstract/Free Full Text]
  41. Penicaud L, Kande J, Le Magnen J, Girard JR 1985 Insulin action during fasting and refeeding in rat determined by euglycemic clamp. Am J Physiol 249:E514–E518
  42. Picinato MC, Haber EP, Cipolla-Neto J, Curi R, de Oliveira Carvalho CR, Carpinelli AR 2002 Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets. J Pineal Res 33:156–160[CrossRef][Medline]
  43. Peschke E 2008 Melatonin, endocrine pancreas and diabetes. J Pineal Res 44:26–40[Medline]
  44. Torres-Farfan C, Richter HG, Rojas-García P, Vergara M, Forcelledo ML, Valladares LE, Torrealba F, Valenzuela GJ, Serón-Ferré M 2003 mt1 Melatonin receptor in the primate adrenal gland: inhibition of adrenocorticotropin-stimulated cortisol production by melatonin. J Clin Endocrinol Metab 88:450–458[Abstract/Free Full Text]
  45. Olefsky JM 1975 Effect of dexamethasone on insulin binding, glucose transport, and glucose oxidation of isolated rat adipocytes. J Clin Invest 56:1499–1508[Medline]
  46. Caro JF, Amatruda JM 1982 Glucocorticoid-induced insulin resistance: the importance of postbinding events in the regulation of insulin binding, action, and degradation in freshly isolated and primary cultures of rat hepatocytes. J Clin Invest 69:866–875[Medline]
  47. Amatruda JM, Livingston JN, Lockwood DH 1985 Cellular mechanisms in selected states of insulin resistance: human obesity, glucocorticoid excess, and chronic renal failure. Diabetes Metab Rev 1:293–317[Medline]
  48. Wetterberg L 1983 The relationship between the pineal gland and the pituitary—adrenal axis in health, endocrine and psychiatric conditions. Psychoneuroendocrinology 8:75–80[CrossRef][Medline]
  49. Nazarloo HP, Nishiyama M, Tanaka Y, Asaba K, Hashimoto K 2002 Down-regulation of corticotropin-releasing hormone receptor type 2β mRNA expression in the rat cardiovascular system following food deprivation. Regul Pept 105:121–129[CrossRef][Medline]
  50. Moore MC, Satake S, Baranowski B, Hsieh P, Neal DW, Cherrington AD 2002 Effect of hepatic denervation on peripheral insulin sensitivity in conscious dogs. Am J Physiol 282:E286–E296
  51. Taniguchi CM, Emanuelli B, Kahn CR 2006 Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85–96[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
B. Herrera, M. van Dinther, P. ten Dijke, and G. J. Inman
Autocrine Bone Morphogenetic Protein-9 Signals through Activin Receptor-like Kinase-2/Smad1/Smad4 to Promote Ovarian Cancer Cell Proliferation
Cancer Res., December 15, 2009; 69(24): 9254 - 9262.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Caperuto, L. C.
Right arrow Articles by Bordin, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caperuto, L. C.
Right arrow Articles by Bordin, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals