Abstract
Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β (TGF-β) family, plays pivotal roles in energy expenditure. However, whether and how BMP7 regulates hepatic insulin sensitivity is still poorly understood. Here, we show that hepatic BMP7 expression is reduced in high-fat diet (HFD)-induced diabetic mice and palmitate (PA)-induced insulin-resistant HepG2 and AML12 cells. BMP7 improves insulin signaling pathway in insulin resistant hepatocytes. On the contrary, knockdown of BMP7 further impairs insulin signal transduction in PA-treated cells. Increased expression of BMP7 by adenovirus expressing BMP7 improves hyperglycemia, insulin sensitivity and insulin signal transduction. Furthermore, BMP7 inhibits mitogen-activated protein kinases (MAPKs) in both the liver of obese mice and PA-treated cells. In addition, inhibition of MAPKs recapitulates the effects of BMP7 on insulin signal transduction in cultured hepatocytes treated with PA. Activation of p38 MAPK abolishes the BMP7-mediated upregulation of insulin signal transduction both in vitro and in vivo. Together, our results show that hepatic BMP7 has a novel function in regulating insulin sensitivity through inhibition of MAPKs, thus providing new insights into treating insulin resistance-related disorders such as type 2 diabetes.
Introduction
Obesity, a global health-threatening pandemic, is often accompanied by high rates of metabolic syndromes and disorders, including endocrine abnormalities, type 2 diabetes (T2D), high blood pressure, and fatty blood and liver (Mokdad et al. 2003). The pathogenesis of obesity is that the subcutaneous fat reservoir overload leads to fat spillage and deposits in the muscle, omentum, heart, and liver, which leads to increased inflammation, lipid production and insulin resistance (IR) (Dulloo & Montani 2012). IR is a hallmark of T2D, in which insulin at normal physiological concentrations fails to effectively activate its downstream signal transduction in the skeletal muscle, liver and adipose tissue (Hartstra et al. 2015). Subsequently, most blood glucose in the circulation cannot be translocated into cells and eventually results in hyperglycemia, which further worsens insulin sensitivity in peripheral tissues (Samuel & Shulman 2012, Guo 2014). Thus, strategies to increase insulin sensitivity are promising useful ways to alleviate insulin resistance and hyperglycemia (Samuel & Shulman 2012, Guo 2014). Indeed, several chemicals that target insulin sensitivity, such as metformin and rosiglitazone, were developed as clinical insulin sensitizers (Staels 2006). Several recent studies indicate that these drugs possess side effects including hypoglycemia, lactic acidosis and cardiovascular risks (Phung et al. 2013, Kaiser & Oetjen 2014). Novel insulin sensitizers free of such side effects are urgently needed to cure the increasing number of T2D patients.
Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β (TGF-β) family, has been reported to induce the conversion of white adipose tissue (WAT) to brown adipose tissue (BAT) (Cannon & Nedergaard 2004, Tseng et al. 2008, Schulz et al. 2011, Boon et al. 2013, Schulz & Tseng 2013). Because BAT burns fatty acids and glucose to generate heat, it is a promising therapeutic target against obesity and T2D (Cypess & Kahn 2010, Townsend & Tseng 2012). As a result, BMP7-induced conversion of WAT into BAT has been intensively studied (Tseng et al. 2008). Several recent studies have shown that BMP7 can increase energy consumption and reduce appetite and food intake (Townsend et al. 2012, 2013, Boon et al. 2013), suggesting that BMP7 is a potent candidate for combating obesity and obesity-related disorders such as T2D. However, a recent study also showed that deletion of BMP7 receptor type 1A in adipose tissue attenuates age-related onset of insulin resistance (Schulz et al. 2016). Thus, it is still unclear whether and how BMP7 regulates insulin signal transduction in the liver.
In this study, we increased the expression of BMP7 in the liver and cultured hepatocytes and examined the effects of BMP7 on glucose homeostasis and insulin signaling pathway. Our results showed that BMP7 plays euglycemic roles in obese mice and improves insulin signal transduction in the liver and cultured hepatocytes. We further investigated the underlying molecular mechanism and revealed that BMP7 inhibits mitogen-activated protein kinases (MAPKs) and thus improves insulin signal transduction in hepatocytes.
Materials and methods
Bioreagents
Rabbit anti-phospho-AKT (Ser473) (Cat#4060), rabbit anti-AKT (Cat#4685), rabbit anti-phospho-GSK3β (Ser9) (Cat#5558), rabbit anti-GSK3β (Cat#12456), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (Cat#9215), rabbit anti-p38MAPK (Cat#8690), rabbit anti-phospho-ERK (Thr202/Tyr204) (Cat#4370), rabbit anti-ERK (Cat#4695), rabbit anti-phospho-JNK (Thr183/Tyr185) (Cat#4668), rabbit anti-JNK (Cat#9252), rabbit anti-phospho-ATF2 (Thr71) (Cat#24329), rabbit anti-ATF2 (Cat#35031), rabbit anti-Na/K-ATPase (Cat#3010), rabbit anit-PTEN (Cat#9188), rabbit anti-P110 (Cat#4249) and rabbit anti-c-Jun (Cat#9165) were all purchased from Cell Signaling Technology. Rabbit anti-BMP7 (Cat#ab56023), rabbit anti-phospho-P85 (Tyr607) (Cat#ab182651) and rabbit anti-P85 (Cat#ab191606) were purchased from Abcam. PVDF membrane (Cat#IPFL00010), SB203580 (Cat#559389), U0126 (Cat#662005), and SP600125 (Cat#420119) were from Millipore. Mouse anti-GAPDH (Cat#G8795), mouse anti-GLUT4 (Cat#G4048) and palmitate (Cat#P9767) were purchased from Sigma-Aldrich.
Animal experiments
Six-week-old male C57BL/6J mice (weight, 19 ± 1 g) were provided by the Experimental Animal Center of Nantong University and fed with normal chow diet (NCD) or high-fat diet (HFD, 45% of calories from fat, Research Diets) for 16 weeks in a temperature-controlled (22–24°C) and humidity-controlled (45–55%) environment (12:12 h light-dark cycle). Adenoviruses expressing BMP7 (Ad-BMP7) or GFP (Ad-GFP) was diluted in PBS and injected into mice under HFD for 14 weeks via tail vein injection using 5 × 108 plaque-forming unit (PFU) per mouse, and then fed these mice with an HFD or chow diet continuously until killing. Body weight and fasting blood glucose were monitored weekly throughout the whole experiments. For testing the potential roles of p38 MAPK in BMP7-mediated euglycemic effects, obese mice induced by HFD were injected with Ad-GFP or Ad-BMP7 or Ad-BMP7 together with Ad-MKK6Glu. All animal experiments were performed two times independently and 7–8 mice were included in each group. All experiments involving animals conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Nantong University (Approval ID: SYXK (SU) 2017-0046).
Cell culture and treatments
Human hepatoma cell line HepG2 cells were obtained from the Chinese Academy of Science (Shanghai, China). Murine AML12 cells were from ATCC (Cat#CRL-2254). Cells were cultured in DMEM containing 10% fetal bovine serum (FBS) with 1% penicillin-streptomycin. Cells were maintained at 37°C with humidified air and 5% CO2, with medium changes three times a week. In order to establish a hepatocellular model of insulin resistance, cells were stimulated by 0.4 mM palmitate (PA) for 24 h. For virus-mediated transduction, cells were plated in six-well plates and transduced with Ad-BMP7 or Ad-GFP (Vigene Bioscience, Shanghai, China). 24 h post viral transduction, cells were treated with or without 0.4 mM PA for 24 h. To stimulate insulin signal transduction, cells were treated with insulin (10 nM) for 5 min before harvest. To inhibit p38 MAPK, ERK or JNK, cells were treated with 10 μM sb203580, 10 μM U0126 or 10 μM SP600125, respectively. To stimulate p38 MAPK, cells were transfected with MKK6Glu plasmid (Addgene, plasmid#13518) by using Lipofectamine 2000 (Cat#11668019) (Invitrogen) according to the manual instructions.
Knockdown of BMP7
For gene silence, three pairs of siRNAs against Bmp7 were synthesized (Ribobio Co., Ltd, Guangzhou). The sequences of siRNAs were as follows: CTCTGAACTCCTACATGAA (siRNA-1); AGAACAAGCAACCCTTCAT (siRNA-2); TCATGTTGGACCTGTACAA (siRNA-3). The siRNAs were transfected into AML12 cells by RNAi MAX (Cat#13778150) (Life Technologies) according to the manual instructions. Cells transfected with negative control (NC) siRNA were used as control.
Blood glucose, serum insulin, glucose tolerance test, insulin tolerance test, and HOMA of insulin resistance index
Levels of blood glucose and serum insulin were measured using a Glucometer Elite monitor and a Rat/Mouse Insulin ELISA kit (Cat#EZRMI-13K) (Millipore), respectively. Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed by administering an intraperitoneal injection of 1 g/kg glucose following overnight fasting and 0.75 U/kg insulin after 6 h of fasting, respectively. The concentration of blood glucose was measured at various time points. The HOMA of insulin resistance (HOMA-IR) index was calculated using the following formula: (fasting glucose levels (mmol/L)) × (fasting serum insulin (ng/mL))/22.5.
Biochemical analysis
Triglyceride and cholesterol levels in the liver were assayed by commercial kits (Cat#TR0100; Cat#MAK043) (Sigma-Aldrich).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells or animal tissues using the TRIzol reagent (Invitrogen) and transcribed into cDNA using a synthesis kit (Roche). The gene expression analysis was performed with iQ5 Multicolor Real-Time PCR Detection System (Roche) with SYBR Green Supermix (Roche). The mRNA level was normalized to 18S as a house keeping gene for each sample. Primers for qRT-PCR used in this study were as follows: 18S, 5′-AGTCCCTGCCCTTTGTACACA-3′ (forward) and 5′-CGTTCCGAGGGCCTCACT-3′ (reverse); G6pc (human), 5′-TGGTTGGGATTCTGGGCTCT-3′ (forward) and 5′-TCTACACCCAGTCCCTTGAG-3′ (reverse); Pck1 (human), 5′-GTTCAATGCCAGGTTCCCAG-3′ (forward) and 5′-TTGCAGGCCAGTTGTTGAC-3′ (reverse); Bmp7 (human), 5′-CTTCGTCAACCTCGTGGAAC-3′ (forward) and 5′-AACGTCTCATTGTCGAAGCG-3′ (reverse); Bmp7 (mouse), 5′-ACGGACAGGGCTTCTCCTAC-3′ (forward) and 5′-ATGGTGGTATCGAGGGTGGAA-3′ (reverse).
Protein extraction from tissues and cells
Liver tissues or cells were homogenized with a bench-top homogenizer (Polytron, PT2100) in an ice-cold tissue lysis buffer (25 mM Tris–HCl, pH 7.4; 100 mM NaF; 50 mM Na4P2O7; 10 mM Na3VO4; 10 mM EGTA; 10 mM EDTA; 1% NP-40; 10 μg/mL Leupeptin; 10 μg/mL Aprotinin; 2 mM PMSF and 20 nM Okadaic acid). After homogenization, lysates were centrifuged at 13,800 g for 20 min at 4°C. The supernatant was transferred into Eppendorf tubes. Protein fraction in plasma membrane was prepared using a commercial kit (Cat#P0033) (Beyotime). Protein concentration was quantified by using a Protein Assay Kit (Bio-Rad). Equivalent protein concentration in each sample was prepared and boiled at 100°C for 5 min in 1× Laemmli buffer. Lysates were cooled to room temperature before western blot analysis.
Western blot analysis
Western blot analysis was performed using a procedure as detailed elsewhere (Sun et al. 2014). Briefly, samples from cell lysates or tissue lysates were first resolved by SDS-PAGE and then transferred to a PVDF membrane. After 1 h blocking at room temperature using 5% milk in a buffer containing 10 mM Tris–HCl pH 7.5, 150 mM NaCl and 0.2% Tween 20 (TBST), membrane was incubated overnight with the primary antibody in TBST at 4°C. After the incubation, membrane was washed three times in TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000, Southern Biotech) for 1 h at room temperature. After three-time washing in TBST, membrane was visualized using an enhanced chemiluminescence system (Cat#TL275748) (Pierce). Relative protein levels were quantified using ImageJ (NIH).
Histopathological analysis
Liver tissues were isolated and fixed with 4% paraformaldehyde for 24–30 h at room temperature and embedded in paraffin. 5-μm thick paraffin-embedded tissue sections were then deparaffinized and rehydrated in graduated alcohol in distilled water. Digital images of hepatic tissue sections were captured using a light microscope. Liver sections were stained with hematoxylin and eosin (H&E) using standard protocols and analyzed by microscopy (Olympus) at 20× magnification. Immunohistochemistry was performed to detect the expression of BMP7 in each group. 5-μm paraffin sections were deparaffinized and rehydrated through a descending ethanol gradient. Enzymatic digestion with protease K was used for antigen retrieval. Sections were first incubated with BMP7 antibody (1:100 dilution) at 4°C overnight and then HRP-conjugated secondary antibodies. Positive staining was visualized as brown color using DAB substrate (Cat#SP-9000) (ZSGB-BIO, Beijing, China). Digital images were captured using a light microscope (Olympus) at 20× magnification.
Glucose uptake assay
Insulin-induced glucose uptake in HepG2 cells was measured by Glucose Uptake Cell-Based Assay kit (Cat#600470) (Cayman). HepG2 cells were first seeded with 5 × 104 cells/well in 100 μL culture medium overnight and then treated next day using a procedure as described previously (Jiang et al. 2016). Before completion of the treatment, cells were starved for 2 h in a glucose-free medium and then mixed with 100 μg/mL 2-NBDG (in a glucose-free medium) and 10 nM insulin. The reaction was incubated at 37°C in the darkness for 30 min. Upon completion of the reaction, supernatant was removed via aspiration, and cells were washed with 200 μL of Cell-Based Assay Buffer for two times. Insulin-induced glucose uptake in HepG2 cells was quantified by measuring fluorescence (Ex/Em = 485/535 nm) using a microplate reader (BioTek, Synergy H1).
Oil Red O staining
HepG2 cells were transfected with Ad-BMP7 or Ad-GFP for 24 h and then treated with 0.4 mM PA for additional 24 h. Confluent cells were fixed in phosphate-buffered formalin (10%) for 1 h at room temperature. After removal of formalin, cells were first rinsed with cold PBS and then stained with an Oil Red O solution (three parts of saturated Oil Red O dye in isopropyl alcohol and two parts of water) for 1 h at room temperature, and excess stain was removed by extensive wash with 70% ethanol. Cells were next washed with PBS and then counterstained with hematoxylin and mounted with glycerin-gelatin before imaging (Olympus). Liver tissue was isolated and dehydrated in graduated sucrose, and then embedded in optimum cutting temperature (OCT) compound; and cryo-sections (12 μm thick) were obtained at −20°C. Liver cryo-sections were stained with Oil Red O dye using standard protocols and analyzed by microscopy (Olympus) at 20× magnification.
Statistical analysis
Data are presented as means ± standard error of the mean (s.e.m.). Comparisons between two groups were performed using unpaired two-tailed Student’s t-test. For multiple-group comparisons, one-way ANOVA with Bonferroni’s post hoc test was used. Significance was accepted at the level of P < 0.05.
Results
The correlation between BMP7 expression and insulin signal transduction in the insulin-resistant liver and HepG2 cells
To investigate the potential role(s) that BMP7 plays in the insulin signaling pathway, we analyzed the expression of BMP7 and insulin signal transduction in the liver of HFD-induced obese mice. The body weight gain and fasting blood glucose levels were significantly higher in the mice fed with HFD (Fig. 1A and B). Glucose tolerance was impaired in obese mice (Fig. 1C). Triglyceride in the liver was dramatically increased (Fig. 1D). Next, we analyzed the expression of BMP7 in the liver, and the results showed that BMP7 was dramatically decreased in obese mice both in terms of the protein and mRNA levels (Fig. 1E, F and G). Similar to BMP7, insulin signal transduction was also decreased in the obese mice liver, i.e., the phosphorylated protein levels of Akt (p-Akt) and GSK3β (p-GSK3β) were markedly reduced in the liver of obese mice (Fig. 1E and F). To further confirm these results, we repeated these experiments in insulin-resistant hepatocytes. To that end, we treated HepG2 cells with palmitate to induce insulin resistance. Western blot analysis showed that insulin signal transduction was decreased in the palmitate-treated cells as evidenced by the decreases in p-Akt and p-GSK3β (Fig. 1H and I). We also found that protein levels of BMP7 were concomitantly reduced in the insulin-resistant cells (Fig. 1H and I). mRNA level of BMP7 was observed to exhibit a similar change pattern (Fig. 1J). Collectively, these results revealed similar changes between BMP7 expression and insulin signal transduction in both insulin-resistant livers and hepatocytes, suggesting BMP7 might be involved in regulating the insulin signal pathway.
BMP7 expression is reduced in insulin-resistant liver and hepatocyte. (A) Body weight changes over 14 weeks. Male C57BL/6J mice were fed with NCD or HFD diet for 14 weeks. Body weight and blood glucose were monitored throughout the whole treatment. At the end of treatments, mice were killed and livers were sampled for further analyses. (B) Fasting blood glucose levels. For analyzing blood glucose, mice were fasted for 6 h (8:00–14:00) and blood samples were collected from the tail vein. Blood glucose was measured by a glucometer. (C) Glucose tolerance tests. (D) Triglyceride levels in the liver. (E) The protein levels of BMP7 and insulin signal transduction in the liver were analyzed by Western blot. GAPDH was used as a loading control. (F) Quantitative analysis of the blots as shown in (E). (G) BMP7 expression was reduced in the liver of obese mice. Total RNA was extracted for measuring mRNA levels of BMP7 by qRT-PCR. 18S was used as a house keeping gene. (H) Effects of palmitate on BMP7 expression and insulin signal pathway. HepG2 cells were incubated with or without 0.4 mM palmitate (PA) for 24 h. Insulin signal transduction was stimulated by 10 nM insulin for 5 min. Total cell lysates were prepared and subjected to Western blot. GAPDH was used as a loading control. (I) Quantitative analysis of the blots as shown in (H). (J) Palmitate reduces BMP7 expression induced by insulin in HepG2 cells. Cell treatments were described in (I). Total RNA was extracted for measuring mRNA levels of BMP7 by qRT-PCR. 18S was used as a house keeping gene. NCD, normal chow diet; HFD, high-fat diet; CON, control; INS, insulin; PA, palmitate. Data are expressed as mean ± s.e.m. from n = 3 independent experiments. *P < 0.05, ***P < 0.001 vs NCD in (A, B, C, D, F and G). *P < 0.05 vs CON, # P < 0.05 vs INS in (I and J).
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
Increased BMP7 expression stimulates insulin signal transduction in palmitate-treated hepatocytes
To examine whether BMP7 regulates the insulin signaling pathway, we increased BMP7 expression in HepG2 cells by transducing adenovirus expressing BMP7 (Ad-BMP7). We found that while treatment of palmitate decreased the protein levels of BMP7 in HepG2 cells, application of Ad-BMP7 greatly enhanced the expression of BMP7 (Fig. 2A). For insulin signal transduction, increased expression of BMP7 was observed to prevent the reductions in p-P85, p-Akt and p-GSK3β induced by palmitate (Fig. 2A and B). PTEN and P110 were not altered by BMP7 (Fig. 2A and B). We also analyzed the glucose uptake in these cells and found that treatment of palmitate reduced glucose translocation into the HepG2 cells and that BMP7 largely counteracted the effects of palmitate (Fig. 2C). GLUT4 is an insulin-dependent glucose transporter and our results showed that it was decreased in the presence of palmitate, and BMP7 application prevented this decrease (Fig. 2D and E). Because ectopic upregulation of hepatic gluconeogenesis is a causative factor for hyperglycemia and insulin resistance, we further analyzed hepatic gluconeogenesis. We chose to measure mRNA levels of G6Pase and PEPCK, as both are rate-limiting enzymes in hepatic gluconeogenesis. The results showed that treatment of palmitate stimulated the expression of G6Pase and PEPCK. Increased expression of BMP7 abolished such stimulations (Fig. 2F). In addition, lipid accumulation was enhanced in palmitate-treated HepG2 cells, which could be largely blocked by BMP7 (Fig. 2G).
Increased expression of BMP7 ameliorates palmitate-induced insulin resistance in HepG2 cells. HepG2 cells were transduced with adenovirus expressing GFP (Ad-GFP) or BMP7 (Ad-BMP7) for 24 h and then cells were incubated with or without 0.4 mM palmitate (PA) for 24 h. Insulin signal transduction was stimulated by 10 nM insulin for 5 min. (A) Effects of BMP7 on insulin signal transduction. The protein levels were detected by Western blot. GAPDH was used as a loading control. (B) Quantitative analysis of the blots as shown in (A). (C) BMP7 promotes glucose uptake in PA-treated HepG2 cells. (D) BMP7 stimulates GLUT4 translocation in plasma membrane of HepG2 cells. At the end of treatments, protein fraction in plasma membrane was isolated and subjected to Western blot analysis. Na/K-ATPase was used as a loading control. (E) Quantitative analysis of the blots as shown in (D). (F) BMP7 reduces G6Pase and PEPCK expression. Gene expression was analyzed by qRT-PCR. 18S was used as a house keeping gene. (G) Lipid accumulation was determined by Oil red O staining. CON, control; PA, palmitate; INS, insulin. Data are expressed as mean ± s.e.m. from n = 3 independent experiments. *P < 0.05 vs INS, # P < 0.05 vs INS + PA. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0693.
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
To further confirm the above results in HepG2 cells, AML12 cells, a murine hepatocyte cell line, were employed to examine the potential roles of BMP7 on insulin actions. Similar to the findings in HepG2 cells, PA treatment induced a reduction in BMP7 expression in AML12 cells and the transduction of Ad-BMP7 markedly stimulated expression of BMP7 (Fig. 3A and B). The decreased insulin signal transduction induced by PA was improved by BMP7 (Fig. 3A and B). Next, we decreased BMP7 expression by siRNAs and examined whether knockdown of BMP7 affects insulin signaling pathway. BMP7 mRNA levels were greatly reduced by siRNA-1 and siRNA-2 (Fig. 3C). Western blot analysis showed that BMP7 protein levels were deceased by siRNA-2 (Fig. 3D). Therefore, BMP7 siRNA-2 was chosen for the following experiments. As shown in Fig. 3E, PA treatment impeded insulin signal transduction in AML12 cells. BMP7 knockdown further strengthened this trend (Fig. 3E and F). Taken together, these results clearly indicate that BMP7 has an ability for stimulating insulin signal transduction in palmitate-induced insulin resistant hepatocytes.
BMP7 improves insulin signal transduction in AML12 cells treated with palmitate. (A) BMP7 potentiates insulin signal transduction in insulin-resistant AML12 cells induced by palmitate (PA). AML12 cells were transduced with adenovirus expressing GFP (Ad-GFP) or BMP7 (Ad-BMP7). 24 h post-viral transduction, cells were treated with 0.4 mM PA for additional 24 h. At the end of treatments, cells were incubated with insulin (10 nM) for 5 min to stimulate insulin signal pathway. Total cell lysates were prepared and subjected to Western blot analysis. GAPDH was used as a loading control. (B) Quantitative analysis of the blots as shown in (A). *P < 0.05 vs INS; # P < 0.05 vs INS + PA + Ad-GFP. (C) Effects of BMP7 siRNAs on mRNA levels of BMP7. AML12 cells were transfected with three pairs of siRNAs targeting Bmp7. 72 h post-transfection, total RNA was extracted for measuring mRNA levels of BMP7 by qRT-PCR. 18S was used as a house-keeping gene. ***P < 0.001 vs NS. (D) Effects of BMP7 siRNAs on protein levels of BMP7. AML12 cells were treated as mentioned in (C). At the end of treatment, total cell lysates were prepared and subjected to Western blot analysis. GAPDH was used as a loading control. *P < 0.05 and ***P < 0.001 vs NC. (E) Knockdown of BMP7 further impedes insulin signal transduction in PA-treated AML12 cells. Cells were transfected with BMP7 siRNA-2 for 48 h and then cells were treated with 0.4 mM PA for additional 24 h. To stimulate insulin signal pathway, cells were incubated with insulin (10 nM) for 5 min. Total cell lysates were prepared and subjected to Western blot analysis. GAPDH was used as a loading control. (F) Quantitative analysis of the blots as shown in (E). *P < 0.05 vs INS; # P < 0.05 vs INS + PA + NC. INS, insulin; PA, palmitate; NC, negative control. Data are represented as mean ± s.e.m. from n = 3 independent experiments.
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
Increased BMP7 expression in the liver alleviates insulin resistance in obese mice
Next, we investigated whether BMP7 plays similar roles in vivo. Expression of BMP7 in the liver was increased by Ad-BMP7 via tail vein injection (Fig. 4A). As expected, reduction of p-Akt and p-GSK3β induced by HFD was nearly completely restored by the increased expression of BMP7 (Fig. 4A and B). Blood glucose levels at fasting and feeding states were both increased in HFD-treated mice, which were prevented partially by BMP7 (Fig. 4C). Serum insulin concentrations were increased by HFD, whereas the increased expression of BMP7 prevented this trend, although the difference lacked statistical significance (Fig. 4D). As a consequence, the increase in HOMA-IR in HFD-treated mice was greatly attenuated by BMP7 (Fig. 4E). BMP7 increased insulin-stimulated GLUT4 translocation in plasma membrane in hepatic cells in obese mice (Fig. 4F). Moreover, the glucose tolerance was improved by the increased expression of BMP7 in the liver of these obese mice, while insulin tolerance was not altered (Fig. 4G and H). BMP7 expression in skeletal muscle and white adipose tissues was not affected by the tail injection of Ad-BMP7 (Fig. 4I and J). These data strongly indicate that the increasing BMP7 expression in the liver plays beneficial roles against insulin resistance in obese mice.
Increased expression of BMP7 ameliorates insulin resistance in obese mice. Male C57BL/6J mice were fed with NCD or HFD diet for 14 weeks and then injected with Ad-BMP7 or Ad-GFP via the tail vein. Blood glucose, insulin, GTT and ITT were assayed at different time points as indicated. (A) Hepatic BMP7 expression and insulin signaling pathway at 14 days post-adenovirus transduction. (B) Quantitative analysis of the blots as shown in (A). (C) BMP7 decreases fasting and fed blood glucose levels in obese mice. For fasting, mice deprived of food for 6 h (8:00 h–14:00 h). Blood samples were collected from the tail vein at 7 days post-adenovirus injection. (D) BMP7 reduces fasting and fed serum insulin levels in obese mice. For fasting, mice deprived of food for 6 h (8:00 h–14:00 h). Blood samples were collected from the tail vein at 7 days post-adenovirus injection. (E) Homeostasis model assessment-insulin resistance (HOMA-IR) index. (F) BMP7 increases GLUT4 expression in plasma membrane of hepatic cells. Protein fraction in plasma membrane was isolated from the liver and subjected to Western blot analysis. Na/K-ATPase was used as a loading control. (G) BMP7 improves glucose tolerance in obese mice. GTT was performed at 9 days post-adenovirus injection. (H) BMP7 improves insulin tolerance in obese mice. ITT was performed at 11 days post-adenovirus injection. (I–J) Expression of BMP7 in skeletal muscle (I) and white adipose tissues (J) in obese mice transduced with adenovirus expressing BMP7. Total cell lysates were prepared from gastrocnemius muscle and epididymal fat tissues. Protein levels were analyzed by Western blot and GAPDH was used as a loading control. NCD, normal chow diet; HFD, high-fat diet; Ad-GFP, adenovirus expressing GFP; Ad-BMP7, adenovirus expressing BMP7. Data are expressed as mean ± s.e.m. (n = 7). The experiments were performed two times independently. *P < 0.05, ***P < 0.001 vs NCD; # P < 0.05, ## P < 0.01 vs HFD + Ad-GFP.
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
Increased BMP7 expression attenuates lipid accumulation in the liver of obese mice
In cultured hepatocytes, we observed that BMP7 appeared to prevent lipid accumulation (Fig. 2G). To confirm this finding, we analyzed lipid deposition in the liver in the following experiments. First, we quantified the expression level of BMP7 in the liver by immunohistochemical analysis. As shown in Fig. 5A, the signal corresponding to BMP7 was dramatically reduced in the liver of obese mice; tail vein injection with Ad-BMP7 successfully increased the expression of BMP7 in the liver. H&E staining, together with oil red analysis, showed that lipid deposition in the liver was greatly upregulated in obese mice. However, the increased expression of BMP7 in the liver attenuated lipid accumulation (Fig. 5B). In agreement with these morphological changes, the triglyceride and free cholesterol levels were decreased by the increased expression of BMP7 in the liver (Fig. 5C and D).
Increased expression of BMP7 ameliorates lipid deposition in the liver of obese mice. Male C57BL/6J mice were fed with NCD or HFD diet for 14 weeks and then injected with Ad-BMP7 or Ad-GFP via the tail vein. At 14 days post-adenovirus injection, mice were killed and liver samples were collected for histological analyses and biochemical assays. (A) Immunochemical analysis of BMP7 expression in the liver. (B) H&E staining (upper panel) and Oil Red O staining of the liver (lower panel). (C) Liver triglyceride concentrations. (D) Liver free cholesterol levels. NCD, normal chow diet; HFD, high-fat diet; Ad-GFP, adenovirus expressing GFP; Ad-BMP7, adenovirus expressing BMP7. Data are presented as means ± s.e.m. (n = 7). The experiments were performed two times independently. *P < 0.05, ***P < 0.001 vs NCD; # P < 0.05, ### P < 0.001 vs HFD + Ad-GFP. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0693.
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
BMP7 inhibits MAPK activation in the liver of obese mice
A number of studies showed that MAPK activation plays important roles in insulin signal transduction (Hirosumi et al. 2002, Gao et al. 2010, Jager et al. 2011, Ozaki et al. 2016, Pereira et al. 2016). We thus analyzed whether MAPK is involved in BMP7-mediated regulation in the insulin signaling pathway. We chose to analyze the protein levels of JNK, ERK and p38 MAPK, as they are the main components of the MAPK pathway. Our results showed that while the MAPK pathway was clearly activated in the obese mouse liver as evidenced by the increases in p-JNK, p-ERK and p-p38 MAPK (Fig. 6A and B), the increases in these protein levels were almost completely counteracted in Ad-BMP7 transduced mouse liver (Fig. 6A and B). We further measured MAPKs in HepG2 cells, and the results showed that palmitate treatment greatly stimulated the protein levels of p-JNK, p-ERK and p-p38 MAPK (Fig. 6C and D). Similar to the results in the liver, increased expression of BMP7 was found to block these stimulations induced by palmitate (Fig. 6C and D). Moreover, we also measured effects of BMP7 on MAPKs in AML12 cells. Our data showed that palmitate stimulated MAPKs, which was largely prevented by BMP7 (Fig. 6E and F). These results suggest that the inhibition of MAPKs may be responsible for the effects of BMP7 on insulin signal transduction.
BMP7 regulates insulin signal transduction via inhibition of MAPKs. (A) Mitogen-activated protein kinases (MAPKs) signaling pathway in the liver of obese mice was analyzed by Western blot. GAPDH was used as a loading control. Male C57BL/6J mice were fed with NCD or HFD diet for 14 weeks and then injected with Ad-BMP7 or Ad-GFP via the tail vein. At 14 days post-adenovirus injection, mice were killed and liver samples were collected. Total cell lysates were prepared and subjected to Western blot analysis. (B) Quantitative analysis of the blots as shown in (A). (C) BMP7 counteracts palmitate-stimulated MAPKs signaling pathway in HepG2 cells. HepG2 cells were transduced with Ad-GFP or Ad-BMP7 as indicated. 12 h post transduction, cells were incubated with or without 0.4 mM palmitate (PA) for 24 h. Insulin signal transduction was stimulated by 10 nM insulin for 5 min. Total cell lysates were prepared and subjected to Western blot analysis. GAPDH was used as a loading control. (D) Quantitative analysis of the blots as shown in (C). (E) BMP7 blocks MAPKs signaling pathway in palmitate-treated AML12 cells. Cell treatments were similar to the procedures described in (C). Protein levels were examined by Western blot and GAPDH was used as a loading control. (F) Quantitative analysis of the blots as shown in (E). NCD, normal chow diet; HFD, high-fat diet; Ad-GFP, adenovirus expressing GFP; Ad-BMP7, adenovirus expressing BMP7; INS, insulin; PA, palmitate. Data are presented as means ± s.e.m. from two (in vivo; n = 7) or three (in vitro; n = 3) independent experiments. *P < 0.05 vs NCD; # P < 0.05 vs HFD + Ad-GFP in (B); *P < 0.05 vs INS; # P < 0.05 vs INS + PA + Ad-GFP in (D).
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
MAPK inhibition is responsible for BMP7-mediated upregulation in insulin signal transduction
Our results strongly suggested the notion that BMP7 regulates insulin signal transduction by inhibiting MAPKs. To confirm this notion, we employed several chemical molecules to inhibit MAPKs and examined whether these treatments could recapitulate the effects of BMP7 on insulin signal transduction. U0126, SB203580 and SP600125 are potent inhibitors of the ERK, p38 MAPK and JNK signaling pathways, respectively. As aforementioned, palmitate treatment resulted in activation of MAPKs (Fig. 7A, B and C). Treatment of U0126 greatly blocked the activation of p-ERK induced by palmitate. Palmitate-induced increase in c-jun, a downstream target of ERK signaling pathway, was eliminated by U0126 (Fig. 7A). These data indicated that the treatment of U0126 successfully inhibited the ERK signaling pathway in HepG2 cells. Similar to U0126, application of SB203580 and SP600125 successfully blocked the p38 MAPK and JNK signaling pathways, respectively (Fig. 7B and C). Interestingly, inhibition of the ERK, p38 MAPK or JNK signaling pathways restored insulin signal transduction in the palmitate-treated cells (Fig. 7A, B and C). Next, we chose the p38 MAPK signaling pathway to further confirm the observed results. MKK6 is an upstream kinase of p38 MAPK, and MKK6Glu is a constitutively activated form of MKK6 (Raingeaud et al. 1996). Indeed, we found that transfection of MKK6Glu stimulated the p38 MAPK signaling pathway, as the protein levels of p-p38 MAPK and p-ATF2 were both increased (Fig. 7D). Activation of p38 MAPK induced by MKK6Glu completely eliminated the effects of BMP7 on insulin signal transduction in HepG2 cells.
Inhibition of the MAPKs pathway is responsible for BMP7-mediated upregulation in insulin signal transduction in HepG2 cells. The HepG2 cells were pre-treated with ERK inhibitor (U0126, 10 μM), or p38 inhibitor (SB203580, 10 μM) or JNK inhibitor SP600125 (10 μM) for 30 min and then 0.4 mM PA was added for additional 24 h. Insulin signal transduction was stimulated by 10 nM insulin for 5 min. (A) Effects of ERK inhibition on insulin signal transduction. The protein levels were analyzed by Western blot. GAPDH was used as a loading control. (B) Effects of p-P38 MAPK inhibition on insulin signal transduction. Protein levels were analyzed by Western blot. GAPDH was used as a loading control. (C) Effects of JNK inhibition on insulin signal transduction. Protein levels were analyzed by Western blot. GAPDH was used as a loading control. (D) p-P38 MAPK activation abolishes the stimulating effect of BMP7 on insulin signal transduction. HepG2 cells were transduced with Ad-BMP7 or Ad-GFP for 24 h, and then the cells were transfected with MKK6Glu. Palmitate (PA; 0.4 mM) was added for 24 h to induce insulin resistance. Insulin signal transduction was stimulated by 10 nM insulin for 5 min. Total cell lysates were prepared and subjected to Western blot analysis. GAPDH was used a loading control. INS, insulin; PA, palmitate; Ad-GFP, adenovirus expressing GFP; Ad-BMP7, adenovirus expressing BMP7. Data are expressed as mean ± s.e.m. from three independent experiments (n = 3). *P < 0.05 vs INS, # P < 0.05 vs INS + PA in (A, B and C). *P < 0.05 vs INS, # P < 0.05 vs INS + PA, $ P < 0.05 vs INS + PA + BMP7 in (D).
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
To further confirm the key role of p38 MAPK on BMP7-mediated insulin signal transduction, we activated p38 MAPK in the liver by adenovirus expressing MKK6Glu (Ad-MKK6Glu) via tail vein injection. First, we analyzed glucose tolerance, and the data showed that blood glucose clearance were greatly improved by BMP7 (Fig. 8A). Activation of p38 MAPK partially mitigated the improved glucose tolerance induced by BMP7 (Fig. 8A). Next, we examined insulin signal transduction in the liver of these obese mice. Increased expression of BMP7 decreased p-p38 MAPK, whereas MKK6Glu markedly stimulated p-p38 MAPK (Fig. 8B and C), indicating p38 MAPK pathway was successfully activated by MKK6Glu. BMP7 expression was not affected by MKK6Glu. As expected, the improved p-Akt and p-GSK3β by BMP7 were counteracted by MKK6Glu-mediated activation of p38 MAPK (Fig. 8B and C). Collectively, these data imply that MAPK inhibition is responsible for BMP7-mediated upregulation of insulin signal transduction in hepatocytes.
Activation of p-P38 MAPK counteracts the effects of BMP7 on insulin resistance in obese mice. (A) Activation of p-P38 MAPK by MKK6Glu mitigates the beneficial role of BMP7 on glucose tolerance in obese mice. HFD-induced obese mice were injected with adenovirus expressing GFP (Ad-GFP), BMP7 (Ad-BMP7) or MKK6Glu (Ad-MKK6Glu) as indicated. GTT was performed at 14 days post-viral injection. (B) Activation of p-P38 MAPK by MKK6Glu eliminates BMP7-induced insulin signal transduction in the liver of obese mice. At 21 days post-viral injection, mice were killed and livers were collected for preparing total cell lysates. Protein levels were analyzed by Western blot and GAPDH was used as a loading control. (C) Quantitative analysis of the blots as shown in (B). HFD, high-fat diet; Ad-GFP, adenovirus expressing GFP; Ad-BMP7, adenovirus expressing BMP7; Ad-MKK6Glu, adenovirus expressing MKK6Glu. Data are presented as means ± s.e.m. from two (n = 8) independent experiments. *P < 0.05, **P < 0.01 vs HFD; # P < 0.05 vs HFD + BMP7.
Citation: Journal of Endocrinology 243, 2; 10.1530/JOE-18-0693
Discussion
In this study, we found that BMP7 expression is decreased in the liver of obese mice and exogenous supplement of BMP7 greatly improves glucose homeostasis and insulin signal transduction in the liver and cultured hepatocytes. We further found that BMP7 represses MAPK signaling pathways involving JNK, p38 MAPK and ERK. Inhibition of these pathways recapitulates the effect of BMP7 on insulin signal transduction in HepG2 cells. In addition, we found that activation of p38 MAPK nearly completely counteracts the BMP7-induced increase in insulin signal transduction.
A recent study suggests that BMP7 is a potent inducer of brown adipose tissue from white adipose tissue (Tseng et al. 2008). Kajimura and colleagues showed that activated BAT dissipates energy through thermogenesis (Kajimura et al. 2015), implying that promoting BAT development is an attractive strategy for treating obesity. Thus, these studies establish BMP7 as a candidate target against obesity and obesity-related diseases such as T2D. Indeed, BMP7 has been shown to improve glucose tolerance and insulin tolerance ability in T2D model animals (Chattopadhyay et al. 2017); in agreement with this study, we found that BMP7 treatment promotes glucose clearance rate and improves insulin sensitivity in obese mice. It is worth emphasizing that while we increased BMP7 expression in liver by administering adenovirus expressing BMP7 via the tail vein, Chattopadhyay et al. treated the mice with recombinant BMP7; these two different approaches for improving BMP7 both resulted in similar consequences, that is promoting glucose clearance rate and improving insulin sensitivity in obese mice. These similar findings further strengthen the notion that BMP7 plays important euglycemic roles in T2D model mice.
To further determine the underlying molecular mechanisms, we measured MAPK signaling pathways in obese mouse liver and found that three main components involving JNK, p38 MAPK and ERK are robustly increased as compared to the lean control. Consistent with our results, it was shown that JNK activity is stimulated in the liver of high-fat diet-induced obese mice and ob/ob mice (Hirosumi et al. 2002). Treatment with a cell-permeable JNK-inhibitory peptide markedly improves insulin resistance and ameliorated glucose tolerance in diabetic mice (Kaneto et al. 2004). On the contrary, however, one study has shown that deficiency of JNK1 in hepatocytes exhibits glucose intolerance, insulin resistance and hepatic steatosis (Sabio et al. 2009). One explanation is that the kinase activity of JNK2 may be stimulated in the Jnk1−/− liver, although the authors have shown that both mRNA and protein levels of JNK2 were not altered by JNK1 deficiency (Sabio et al. 2009).
In addition, the ERK signaling pathway is activated in the liver of obese mice and inactivation of ERK in the liver greatly improves systemic insulin and glucose tolerance in obese mice (Jiao et al. 2013). Liver-specific deletion of MKP-1 activates p38 MAPK, which enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice (Lawan et al. 2015). Together, these previous reports suggest that inhibition of MAPKs is an efficient way for improving systemic glucose and insulin tolerance in obese mice. In this study, we found that increased expression of BMP7 reduces the JNK, p38 MAPK and ERK signaling pathways in the liver, suggesting that BMP7 likely achieves its euglycemic roles via inhibition of JNK, p38 MAPK and/or ERK pathways. To confirm this notion, we employed pharmacological intervention to inhibit these pathways separately, and our results showed that inhibition of JNK or p38 MAPK or ERK restore insulin signal transduction in HepG2 cells. Furthermore, we found that activation of p38 MAPK nearly completely abolished the effect of BMP7 on insulin signal transduction in cultured hepatocytes. Collectively, these findings imply that MAPKs are downstream targets of BMP7 for mediating its beneficial roles on glucose homeostasis.
In this study, we found that BMP7 treatment greatly inhibits MAPK signaling pathways that involve JNK, p38 MAPK and ERK. It is worth emphasizing that BMP7 was reported by others to affect these pathways differently, that is, BMP7 was found to activate JNK activity in mouse neuroblastoma cells and nephrogenic zone-derived cells (Blank et al. 2009, Podkowa et al. 2010); in cultured primary rat Schwann cells, BMP7 appears to have no effect on JNK activity (Liu et al. 2016); and several studies reported that BMP7 activates p38 MAPK in various cell lines (Tseng et al. 2008, Kobayashi et al. 2011, Liu et al. 2016). The difference is likely due to the differences in cell lines and/or dosage of BMP7. For ERK, deficiency of BMP7 receptor type 1A causes the activation of ERK in white adipose tissue, suggesting BMP7 might repress the ERK signaling pathway (Schulz et al. 2016), which is consistent with our observation that BMP7 inhibits ERK activity.
Notably, the inhibitory effects of BMP7 on MAPK signaling pathways occur in the insulin-resistant liver or HepG2 cells. The effects of BMP7 on these pathways under normal conditions remain unclear. Unlike many other studies (Tseng et al. 2008, Blank et al. 2009, Podkowa et al. 2010, Liu et al. 2016), which employed recombinant BMP7 to treat cells or animal, we increased BMP7 expression by adenovirus-mediated gene manipulation both in vivo and in vitro in this study. In the studies using recombinant BMP7, exogenous BMP7 must bind to its receptors to exhibit its biological functions. In our study, there is a strong possibility that BMP7 acts directly in cells and thus regulates the targeted substrates. Thus, a suitable way to further dissect the effects of BMP7 on MAPK signaling pathways is to generate of cells deficient of BMP7 receptors and transduce these cells with adenovirus expressing BMP7. Moreover, for BMP7-mediated blood glucose clearance, which is partially due to glucose uptake in hepatocytes via GLUT2/4. GLUT2 is a dominant glucose transporter in the liver and it works in an insulin-independent manner (Thorens 2015). Unlike GLUT2, GLUT4 facilitates glucose transport in hepatocytes in an insulin-dependent manner (Klip et al. 2019). As BMP7 improves insulin signal transduction in the liver, the observed blood glucose clearance induced by BMP7 is probably due to GLUT4-mediated glucose uptake in hepatocytes. Indeed, BMP7 application stimulated GLUT4 translocation in cell membranes. In addition to the liver, other peripheral organs such as skeletal muscle and white adipose tissues may play roles in glucose deposition. However, BMP7 expression in these peripheral tissues is not altered by the tail vein injection with adenovirus expressing BMP7. A crosstalk between the liver and these peripheral tissues might be involved for improving glucose uptake in peripheral organs. Euglycemic clamp technique together with isotope tracing will clarify this prediction.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
The current study is supported by the Jiangsu Provincial Six Talent Peaks for high-level talents (2016-WSN-098; SWYY-051); Nantong Municipal Science and Technology Project (HS2014036); and the Project of Preventive Medicine Association of Jiangsu Provincial Health and Family Planning Commission (Y2015070).
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