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TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats
Biochemical and Biophysical Research Communications xxx (2017) 1e7
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TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats Xiaoyan Sun a, b, Ming Song a, Hui Wang a, Huimin Zhou a, Feng Wang a, Ya Li a, Yun Zhang a, Wei Zhang a, Ming Zhong a, Yun Ti a, * a
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, China b Department of Cardiology, Heze Municipal Hospital, Heze, Shandong, China
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a b s t r a c t
Article history: Received 22 April 2017 Accepted 30 April 2017 Available online xxx
Our previous study had suggested Tribbles homolog 3 (TRB3) might be involved in metabolic syndrome via adipose tissue. Given prior studies, we sought to determine whether TRB3 plays a major role in adipocytes and adipose tissue with beneficial metabolic effects in obese and diabetic rats. Fully differentiated 3T3-L1 adipocytes were incubated to induce insulin resistant adipocytes. Forty male Sprague eDawley rats were all fed high-fat (HF) diet. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ). Compared with control group, in insulin resistant adipocytes, protein levels of insulin receptor substrate-1(IRS-1), glucose transporter 4(GLUT4) and phosphorylated-AMPactivated protein kinase (p-AMPK)were reduced, TRB3 protein level and triglyceride level were significantly increased, glucose uptake was markedly decreased. TRB3 silencing alleviated adipocytes insulin resistance. With TRB3 gene silencing, protein levels of IRS-1, GLUT4 and p-AMPK were significantly increased in adipocytes. TRB3 gene silencing decreased blood glucose, ameliorated insulin sensitivity and adipose tissue remodeling in diabetic rats. TRB3 silencing decreased triglyceride, increased glycogen simultaneously in diabetic epididymal and brown adipose tissues (BAT). Consistently, p-AMPK levels were increased in diabetic epididymal adipose tissue, and BAT after TRB3-siRNA treatment. TRB3silencing increased phosphorylation of Akt in liver, and improved liver insulin resistance. © 2017 Published by Elsevier Inc.
Keywords: TRB3 AMPK Insulin resistance Obesity Diabetes
1. Introduction Obesity and diabetes are increasing at an alarming rate worldwide, but the strategies for the prevention and treatment of these disorders remain inadequate. Type 2 diabetes mellitus (T2DM) is closely related to obesity. Its genesis is linked to insulin resistance (pre-diabetes condition). Diabetes and obesity are often accompanied by chronic inflammation and adipose tissue dysfunction [1]. Hyperglycemic damage in adipose tissue is characterized by oxidative stress and increased abundance of macrophage infiltration which further exaggerates inflammation in adipose tissue [2].
* Corresponding author. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, 250012, China. E-mail address: [email protected] (Y. Ti).
In patients with obesity, chronic low-grade inflammation in adipose tissue triggers adipocyte lipolysis, leading to increased free fatty acid (FFA) release into circulation. FFA and its metabolites impair insulin signaling, decrease glucose tolerance, and cause ectopic lipid accumulation [3]. Dysfunctional adipocytes contribute directly and indirectly (through insulin resistance) to the development of vascular risk factors and vascular disease [4]. Insulin resistance (IR) is assumed to trigger the developments of T2DM [5]. TRB3 has been demonstrated to be involved in lipid metabolism [6] and in impairment of insulin exocytosis [7]. Epidemiological studies recognized that TRB3 was increased in obese humans with IR [8] and in patients with T2DM [9]. Our previous study showed that TRB3 was increased in adipose tissue of fructose-fed rats compared with the controls [10]. Therefore, TRB3 could be a promising candidate for investigating IR and related clinical disorders. The role of TRB3 in adipose tissue dysfunction has not been studied and therefore remains to be elucidated. TRB3 is an important suppressor of AMPK via directly binding to
http://dx.doi.org/10.1016/j.bbrc.2017.04.154 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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X. Sun et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
it [11]. What is clear is that the consequences of dysfunctional AMPK include an increased risk of insulin resistance, hypertension and cardiovascular disease (CVD) and possibly a predilection to certain cancers [12e14]. However, the TRB3/AMPK signaling pathway has not been investigated in vitro on adipocytes and in vivo on adipose tissues directly. Thus, we hypothesized that TRB3/AMPK signaling pathway was an attractive strategy for the treatment of obesity and type 2 DM. First, we inducted IR adipocytes and type 2 DM model. TRB3 and its gene polymorphism are associated with insulin resistance, a vital pathophysiologic characteristic of type 2 diabetes. To further elucidate the role of TRB3/AMPK signaling pathway in obesity and DM, we used TRB3 gene silencing in vitro and in vivo to explore the mechanisms of TRB3/AMPK signaling pathway in obesity and DM as a potential target for treatment. 2. Materials and methods 2.1. Cell culture and induction of insulin resistance (IR) Murine 3T3-L1 pre-adipocytes, purchased from American Type Culture Collection (ATCC), were cultured in DMEM containing 10% FBS, and differentiated in a cocktail containing insulin, 3-isobutyl1-methylxanthine (IBMX) and dexamethason based on a standard protocol. The fully differentiated 3T3-L1 adipocytes were preincubated for 24 h in serum-free and low-glucose DMEM, and randomly divided into: control group (Control), insulin resistance (IR) group, and free fatty acids (FFA) group, control group was incubated with low-glucose DMEM containing 5.5 mM glucose and 10%FBS, IR was induced by incubating differentiated 3T3-L1 adipocytes in high-glucose DMEM containing 25 mM glucose, 10%FBS and 10 nM insulin for 24 h, FFA group was incubated with medium containing low-glucose DMEM, FBS, BSA and 0.75 mMpalmitate(Sigma) for 24 h as previously [15]. 2.2. Induction of diabetes Forty male SD rats were all fed high-fat (HF) diet, and randomly divided into four groups: HF þ Vehicle, HF þ TRB3siRNA, diabetes(DM)þVehicle and DM þ TRB3siRNA. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ) as previously [16]. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee. 2.3. Oil red O staining Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15min. Then, cells were washed with PBS and stained for 30 min with Oil Red O solution (60% isopropanol, 40% water). Excess stain was removed by washing with PBS, and then microscopic images were recorded. 2.4. Triglyceride assay Cells triglyceride were extracted, and measured using a Triglyceride Quantification Kit (Abcam, Cambridge,MA), following the manufacturer's instructions for colorimetric assay. 2.5. Glucose uptake For experiments, all culture medium was removed from each well and replaced with low-glucose DMEM containing 10% FBS, 1% 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 1 nM insulin for 30min. Removed the medium, cells
were washed and resuspended with PBS quickly, and then cells were moved at 1 104/well to 96-well plates. Fluorescence retained was measured using a microplate reader set at an excitation wave-length of 485 nm and an emission wavelength of 535 nm. 2.6. Histology and morphometric analysis Paraformaldehyde (4%)-fixed adipose tissue was bisected transversely, embedded in paraffin, and cut into 5-mm sections. A single adipocyte cell was measured with images captured from hematoxylin and eosinestained sections. The adipocyte crosssectional area was assessed under 3400 magnification within the adipose tissue, and a mean was obtained by quantitative morphometry with automated image analysis (Image-Pro Plus, Version 5.0; Media Cybernatics, Houston, TX). Liver was treated the same as adipose tissue. Liver frozen sections (5 mm) were stained with Oil Red O (Sigma) for 10 min, washed, and then counterstained with hematoxylin for 30 s. A Nikon microscope (Nikon, Melville, NY) was used to capture the Oil Red Oestained tissue sections. 2.7. Blood analyses After rats fasted overnight, we collected jugular blood. Total cholesterol, triglyceride levels, FBG and Fasting insulin level was measured as previously [16]. ISI was calculated. 2.8. Quantitative real-time PCR for TRB3 Total RNA was isolated from differentiated 3T3-L1 cells with the TRIzol reagent (Invitrogen). Total RNA was quantified by spectrophotometry and reverse-transcribed with the M-MLV Reverse Tran-scriptase System and oligo(dT) primers. The mRNA levels for TRB3 were determined by SYBR green real-time PCR, following the manufacturer's instructions. The PCR primers for TRB3 were 50 TCAAGCTGCGTCGCTTTGTC-30 (for-ward)and 50 -AGCTGAGTATCTCTG GTCCCACGTA-3’ (reverse); those for GAPDH were 50 TCAAGCTGCGTCGCTTTGTC-3’ (forward) and 50 - GATGCAGGGATGATGTTC -3’ (reverse). All values obtained were normalized to mouse GAPDH. 2.9. Western blot analysis Cells and tissue were harvested in ice-cold lysis buffer, Protein concentrations were determined with the Bradford method. Western blot analysis was as described previously [17]. We used antibodies against TRB3 (Calbiochem), GLUT4 (Abcam), and pAMPK/AMPK, IRS-1 (Cell Signaling Technology), Akt (Cell Signaling Technology), followed by anti-IgG horseradish peroxidaseeconjugated secondary antibody. TRB3, GLUT4, and IRS-1 protein levels were normalized to that of GAPDH/b-actin, as an internal control and phosphospecific proteins to that of total protein. 2.10. Gene silence of TRB3 The fully differentiated 3T3-L1 adipocytes were randomized to receive TRB3 small interfering RNA (siRNA) or vehicle treatment. Forty rats were randomized to receive TRB3 small interfering RNA (siRNA) or vehicle treatment as previously [16]. 2.11. Statistical analysis Values are presented as mean ± SEM, SPSS17.0 (SPSS, Chicago,
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
X. Sun et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
IL) was used for statistical analysis. Results were compared by oneway ANOVA, followed by Tukey-Kramer post hoc test and independent samples t-test. P < 0.05 was considered statistically significant.
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3.2. TRB3 gene silencing improved metabolism in vivo TRB3 gene silencing in this Type 2 DM model led to a significant decrease in water intake, urine volume, and FBG in DM þ TRB3siRNA group compared with DM þ Vehicle group (P < 0.05~P < 0.01). ISI was markedly increased in DM þ TRB3siRNA group compared with DM þ Vehicle group (P < 0.05) (Table 1).
3. Results 3.1. TRB3 gene silencing improved insulin resistance in vitro Lipid droplets in IR and FFA group adipocytes were larger in volume and quantities (Fig. 1A), and adipocytes in IR and FFA groups had significantly higher triglyceride level (P < 0.001) (Fig. 1C). Glucose uptake of adipocytes was significantly lower in IR and FFA group than in control group (P < 0.001) (Fig. 1B). Adipocytes in IR and FFA group had lower level of IRS-1, GLUT4 and pAMPK protein, had higher level of TRB3 protein correspondingly compared with control group (P < 0.001) (Fig. 1DeE). After TRB3 gene silencing, we detected adipocyte TRB3 expression by RT-PCR and western blot analysis. When compared with vehicle treatment, TRB3-siRNA treatment conferred downregulated TRB3 mRNA and protein expression of adipocyte. (Fig. 1FeH). Silence of TRB3 in IR and FFA adipocytes led to a significant increase of IRS-1 and GLUT4 protein level (P < 0.001). TRB3 gene silencing increased the phosphorylation of AMPK in TRB3siRNA groups compared with Vehicle. (P < 0.001) (Fig. 1GeH).
3.3. TRB3 gene silencing alleviated adipose tissue remodeling and improved triglyceride and glycogen metabolism in adipose tissue Silence of TRB3 decreased the adipocyte size of epididymal and subcutaneous adipose tissues in high-fat fed and diabetic rats, adipocyte aligned more evenly and orderly than control, TRB3 gene silencing alleviate the infiltration of white lipid droplet in brown adipose tissue(Fig. 2AeE). TRB3 gene silencing decreases triglyceride content level and increase glycogen content level in diabetic epididymal and brown adipose tissues in DM þ TRB3siRNA group(P < 0.05~P < 0.01) (Fig. 2FeH). 3.4. TRB3 gene silencing increased AMPK activity in adipose tissue Compared with vehicle treatment, TRB3-siRNA treatment conferred downregulated protein expression of adipose tissue TRB3. Silence of TRB3 significantly increased the phosphorylation of AMPK in epididymal adipose tissues, subcutaneous adipose tissues, and brown adipose tissues in HF þ TRB3siRNA group
Fig. 1. General characteristics of insulin resistant adipocytes. A: Adipocytes Stained with oil red O(scale bar: 50 mm). B: Glucose uptake of adipocytes. C: Triglyceride content in adipocytes. DeE: Western blot analysis of the protein expression and semi-quantification of IRS-1, GLUT-4, TRB3 and p-AMPK in adipocytes. Data are mean ± SEM. TRB3 gene silencing could improve insulin resistance in adipocytes. F: Silence of TRB3 significantly reduced relative TRB3 mRNA expression in adipocytes (scale bar: 50 mm). GeH: Western blot analysis of the protein expression and semiquantification of TRB3, IRS-1, GLUT4 and p-AMPK in adipocytes. Data are mean ± SEM.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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Table 1 Animal characteristics in four groups.
Body weight (g) Epididymal fat (g) Subcutaneous fat (g) Water intake (ml/d) Food intake (g/d) Urine volume(ml/d) FBG (mmol/l) FINS (mmol/l) ISI TC (mmol/l) TG (mmol/l)
HF þ Vehicle
HF þ TRB3siRNA
DM þ Vehicle
DM þ TRB3siRNA
575.12 ± 18.93 5.81 ± 0.61 4.02 ± 0.53 36.00 ± 3.11 18.67 ± 0.92 30.83 ± 1.68 10.45 ± 0.51 14.93 ± 1.56 5.01 ± 0.07 1.87 ± 0.12 1.51 ± 0.27
567.85 ± 16.79 4.77 ± 0.53 3.26 ± 0.40 32.00 ± 2.00 18.33 ± 1.02 30.00 ± 0.73 11.61 ± 1.11 12.63 ± 1.17 4.93 ± 0.11 1.80 ± 0.15 1.75 ± 0.20
533.50 ± 36.78 4.52 ± 0.72 3.49 ± 0.69 159.67 ± 4.33*** 27.50 ± 1.08*** 132.50 ± 5.08*** 25.69 ± 3.14** 13.01 ± 0.67 5.71 ± 0.15** 5.31 ± 1.76 8.30 ± 2.29
537.17 ± 17.29 3.93 ± 0.59 2.37 ± 0.57 146.00 ± 4.13y 26.50 ± 0.99 115.50 ± 2.22yy 15.54 ± 1.67y 11.31 ± 2.07 5.07 ± 0.13y 4.13 ± 1.49 7.44 ± 3.16
Data are mean ± SEM, n ¼ 6e9 per group. FBG, fasting blood glucose; FINS, fasting insulin; ISI, Insulin sensitivity index; TC, total cholesterol; TG, triglycercide. **P < 0.01, ***P < 0.001 vs. HF þ Vehicle; yP < 0.05, yyP < 0.01 vs. DM þ Vehicle.
compared with HF þ Vehicle group (P < 0.05~P < 0.001). The phosphorylation of AMPK in epididymal adipose tissues and brown adipose tissues increased in DM þ TRB3siRNA group compared with DM þ Vehicle group (P < 0.001) (Fig. 3AeC). 3.5. TRB3 gene silencing improved insulin resistance in vivo The protein expression of hepatic TRB3 was also downregulated after TRB3-siRNA treatment compared with vehicles. Diabetic rats had higher Oil Red Oestaining areas in liver than other groups, TRB3 gene silencing markedly decreased Oil Red Oestaining areas (P < 0.001) (Fig. 4AeB). Silence of TRB3 significantly increased phosphorylation of Akt in liver (P < 0.001) (Fig. 4C). 4. Discussion In present study we demonstrated that TRB3/AMPK signaling pathway was associated with insulin resistance in adipose tissue. TRB3 gene silencing could effectively improve glucose and lipid metabolism, and further improved insulin resistance by
modulating AMPK pathway in obesity and type 2 diabetes. TRB3 is implicated in dysfunction of adipose tissue of obesity and DM. In current study, the protein levels of IRS-1, GLUT4, pAMPK and glucose uptake in adipocytes were reduced in IR and FFA group, triglyceride level was significantly increased compared with control. The protein level of TRB3 was significantly increased in IR and FFA group. These findings indicate insulin resistance exists in our adipocytes in IR and FFA groups at the molecular level. Our previously study showed about 2-fold increase in TRB3 mRNA levels in adipose tissue of fructose-fed rats compared with those in adipose tissue of the controls [10]. In the present study, diabetic rats showed severe insulin resistance and increased TRB3 level in adipose tissue. Adipocyte size was significantly increased and arranged disorganized in the obesity and diabetic rats versus the TRB3-siRNA group. Even more white lipid droplets infiltrated in brown adipose tissue in the vehicle group. The aberrant lipids accumulated within liver in obesity and diabetic rats. Consistently, p-AMPK levels were significantly decreased in obesity and diabetic epididymal adipose tissues and BAT, except diabetic subcutaneous adipose tissue. These findings are in accordance with previous
Fig. 2. TRB3 gene silencing alleviated adipose tissue remodeling. A: Representative epididymaladipose tissues (EAT) stained with H&E(scale bar: 50 mm). B: Representative subcutaneousadipose tissue(SAT) stained with H&E. (scale bar: 50 mm) C: Representative brown adipose tissues (BAT) stained with H&E(scale bar: 50 mm). D: Representative scatter diagram of the epididymal adipocyte size; E: Representative scatter diagram of the subcutaneous adipocyte size. Effect of TRB3 gene silencing on the Triglyceride and glycogen in adipose tissue. FeH: Triglyceride content and glycogen content in each group epididymal, subcutaneous and brown adipose tissues.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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Fig. 3. Effect of TRB3 gene silencing on TRB3/AMPK signaling pathway in adipose tissue. A: Western blot analysis of the protein expression and semiquantification of TRB3/AMPK in epididymal adipose tissues (EAT). B: Western blot analysis of the protein expression and semiquantification of TRB3/AMPK in subcutaneous adipose tissues (SAT). C: Western blot analysis of the protein expression and semiquantification of TRB3/AMPK in brown adipose tissues (BAT). Data are mean ± SEM.
Fig. 4. TRB3 gene silencing could improve insulin resistance in liver. A: Representative H&E and Oil Red Oestained liver(scale bar: 50 mm). B: Semiquantification of Oil Red O staining. Data are mean ± SEM. C: Western blot analysis of the protein expression and semiquantification of TRB3/Akt in liver. Data are mean ± SEM.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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studies [18,19], which indicates TRB3 is implicated in dysfunction of adipose tissue of obesity and DM. It has been reported that TRB3 was an important modulating protein of AMPK [11,20,21]. AMPK activity is decreased and oxidative stress increased in subcutaneous, epiploic and omental adipose tissue in humans who are insulin-resistant [22]. We found TRB3 markedly upregulated and p-AMPK significantly downregulated in insulin resistant adipocyte and in rats with diabetes. Therefore, activation of TRB3 may contribute to the development and progression of DM, possibly mediated by the AMPK pathway. Effect of TRB3 silencing on adipocytes and adipose tissue mediated by AMPK pathway. In light of the pivotal role of TRB3 in dysfunction of adipose tissue of DM, we wondered whether downregulation of TRB3 could reverse the progression of DM. According to our previous study [16], we used TRB3-siRNA in vitro in adipocytes and in vivo in rats. TRB3-siRNA treatment produced no notable adverse effects and no deaths in adipocytes and rats. TRB3 mRNA levels were significantly decreased, and the protein expression was reduced by ~60% with TRB3-siRNA treatment in diabetic rats. Thus, global silencing of TRB3 was feasible and effective. With TRB3 silencing, the protein levels of IRS-1, GLUT4 and pAMPK were significantly increased in adipocytes. The impaired glucose tolerance and decreased insulin sensitivity were improved, and the inhibited activation of AMPK was restored in vivo. So TRB3 silencing improved insulin resistance, which was attributed to increased phosphorylation of AMPK in adipocyte, adipose tissue and phosphorylation of Akt in liver. In the present study, diabetic rats showed serious insulin resistance and increased TRB3 level in adipose tissues and liver. Akt activation in liver was inhibited markedly. These findings indicate insulin resistance exists in our type 2 diabetic rat model at the molecular level. In addition to improving metabolic disturbance, TRB3 silencing alleviated adipose tissue remodeling, adipocyte aligned more evenly and orderly, the infiltration of white lipid droplet in brown adipose tissue was decreased; the aberrant lipids accumulation within liver in obesity and diabetic rats was markedly reduced after TRB3 silencing. With TRB3 gene silencing, triglyceride content level was decreased and glycogen content level was increased in diabetic epididymal and brown adipose tissues (BAT). Correspondingly, p-AMPK levels were significantly increased in diabetic epididymal adipose tissues and BAT after TRB3-siRNA treatment. The inhibited phosphorylation of p-Akt in liver was restored after TRB3 silencing. Thus, TRB3 silencing alleviated metabolism disturbance and insulin resistance. There is no significantly change about triglyceride, glycogen content and p-AMPK level in diabetic subcutaneous adipose tissue after TRB3 gene silencing. So we can come to a conclusion that visceral and brown adipose tissues (BAT) are correlation with high incidence of insulin resistance, subcutaneous adipose tissue is correlation with low incidence of insulin resistance. These alterations resulted in overall improvement of insulin resistance. In accordance with these changes at the molecular level, TRB3 silencing attenuated the metabolic disturbance in obesity and type 2 diabetic rats. In summary, the present study indicates that, TRB3 is involved in dysfunction of adipose tissue in obesity and diabetes. TRB3 silencing activates AMPK with beneficial metabolic effects. Thus, TRB3/AMPK signaling pathway has a pivotal role in obesity and DM and could be an attractive drug target for treating obesity and type 2diabetes. Although our results suggest that blockage of TRB3 activity could be a therapeutic approach to increase phosphorylation of AMPK to improve insulin resistance in vitro and in vivo, TRB3 may have a different effect in various cell types, tissues and dosages, and
more detailed studies in different cell types is required to clarify the precise function of TRB3. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the research grants from the National Natural Science Foundation of China (81670411, 81471036, 81470560, 81570400, 81600633, 81300168, 81270352, 81270287, 81100605, 91439201, 81530014 and 81320108004),the National Basic Research Program of China (973 Program, Grant No. 2013CB530703), Key research and development program of Shandong Province (2015GSF118062), the Natural Science Foundation of Shandong Province (ZR2014HQ037), Medicine and Health Science Technology development program of Shandong Province (2016WS0091), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP 20130131120065) and Program of Introducing Talents of Discipline to Universities (B07035). References [1] N. Kawasaki, R. Asada, A. Saito, S. Kanemoto, K. Imaizumi, Obesity-induced endoplasmic reticulum stress cause chronic inflammation in adipose tissue, Sci. Rep. 2 (2012) 799. [2] Angelika Neuhofer, Maximilian Zeyda, Daniel Mascher, et al., Impaired local production of proresolving lipid mediators in obesity and 17-HDHA as a potential treatment for obesity-associated inflammation, Diabetes 62 (6) (2013; Jun) 1945e1956. [3] V.T. Samuel, G.I. Shulman, Mechanisms for insulin resistance: common threads and missing links, Cell 148 (5) (2012; Mar 2) 852e871. [4] C. Bleau, A.D. Karelis, D.H. St-Pierre, et al., Crosstalk between intestinal microbiota, adipose tissue and skeletal muscle as an early event in systemic low-grade inflammation and the development of obesity and diabetes, Diabetes Metab. Res. Rev. 31 (6) (2015; Sep) 545e561. [5] Q. Tang, X. Li, P. Song, et al., Optimal cut-off values for the homeostasis model assessment of insulin resistance (HOMA-IR) and pre-diabetes screening: developments in research and prospects for the future, Drug Discov. Ther. 9 (6) (2015; Dec) 380e385. [6] D. Steverson Jr., L. Tian, Y. Fu, et al., Tribbles homolog 3 promotes foam cell formation associated with decreased proinflammatory cytokine production in macrophages: evidence for reciprocal regulation of cholesterol uptake and inflammation, Metab. Syndr. Relat. Disord. 14 (1) (2016; Feb) 7e15. [7] C.W. Liew, J. Bochenski, D. Kawamori, et al., The pseudokinasetribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse beta cells, J. Clin. Invest 120 (8) (2010; Aug) 2876e2888. [8] H. Oberkofler, A. Pfeifenberger, S. Soyal, et al., Aberrant hepatic TRIB3 gene expression in insulin-resistant obese humans, Diabetologia 53 (9) (2010;Sep) 1971e1975. [9] J. Liu, X. Wu, J.L. Franklin, et al., Mammalian Tribbles homolog 3 impairs insulin action in skeletal muscle: role in glucose-induced insulin resistance, Am. J. Physiol. Endocrinol. Metab. 298 (3) (2010; Mar) E565eE576. [10] X.P. Bi, H.W. Tan, S.S. Xing, et al., Overexpression of TRB3 gene in adipose tissue of rats with high fructose-induced metabolic syndrome, Endocr. J. 55 (4) (2008 Aug) 747e752. [11] K. Li, Y. Xiao, J. Yu, et al., Liver-specific gene inactivation of the transcription factor ATF4 alleviates alcoholic liver steatosisin mice, J. Biol. Chem. 291 (35) (2016; Aug 26) 18536e18546. [12] W. Zhang, Q. Wang, Y. Wu, et al., Endothelial cell-specific liver kinase B1 deletion causes endothelial dysfunction and hypertension in mice in vivo, Circulation 129 (13) (2014 Apr 1) 1428e1439. [13] G.R. Steinberg, H.M. O'Neill, N.L. Dzamko, et al., Whole body deletion of AMPactivated protein kinase {beta}2 reduces muscle AMPK activity and exercise capacity, J. Biol. Chem. 285 (48) (2010; Nov 26) 37198e37209. [14] G. Zadra, J.L. Batista, M. Loda, Dissecting the dual role of AMPK in cancer: from experimental to human studies, Mol. Cancer Res. 13 (7) (2015; Jul) 1059e1072. [15] J.A. Chavez, T.A. Knotts, L.P. Wang, et al., A role for ceramide, but not diacylglycerol, in the antagonism of insulin signal transduction by saturated fatty acids, J. Biol. Chem. 278 (12) (2003; Mar21) 10297e10303. Epub 2003 Jan 13. [16] Y. Ti, G.L. Xie, Z.H. Wang, et al., TRB3 gene silencing alleviates diabetic cardiomyopathy in a type 2 diabetic rat model, Diabetes 60 (11) (2011; Nov) 2963e2974. [17] Z.H. Wang, Y.Y. Shang, S. Zhang, et al., Silence of TRIB3 suppresses atherosclerosis and stabilizes plaques in diabetic ApoE-/-/LDL receptor-/- mice,
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TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats Xiaoyan Sun a, b, Ming Song a, Hui Wang a, Huimin Zhou a, Feng Wang a, Ya Li a, Yun Zhang a, Wei Zhang a, Ming Zhong a, Yun Ti a, * a
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, China b Department of Cardiology, Heze Municipal Hospital, Heze, Shandong, China
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a b s t r a c t
Article history: Received 22 April 2017 Accepted 30 April 2017 Available online xxx
Our previous study had suggested Tribbles homolog 3 (TRB3) might be involved in metabolic syndrome via adipose tissue. Given prior studies, we sought to determine whether TRB3 plays a major role in adipocytes and adipose tissue with beneficial metabolic effects in obese and diabetic rats. Fully differentiated 3T3-L1 adipocytes were incubated to induce insulin resistant adipocytes. Forty male Sprague eDawley rats were all fed high-fat (HF) diet. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ). Compared with control group, in insulin resistant adipocytes, protein levels of insulin receptor substrate-1(IRS-1), glucose transporter 4(GLUT4) and phosphorylated-AMPactivated protein kinase (p-AMPK)were reduced, TRB3 protein level and triglyceride level were significantly increased, glucose uptake was markedly decreased. TRB3 silencing alleviated adipocytes insulin resistance. With TRB3 gene silencing, protein levels of IRS-1, GLUT4 and p-AMPK were significantly increased in adipocytes. TRB3 gene silencing decreased blood glucose, ameliorated insulin sensitivity and adipose tissue remodeling in diabetic rats. TRB3 silencing decreased triglyceride, increased glycogen simultaneously in diabetic epididymal and brown adipose tissues (BAT). Consistently, p-AMPK levels were increased in diabetic epididymal adipose tissue, and BAT after TRB3-siRNA treatment. TRB3silencing increased phosphorylation of Akt in liver, and improved liver insulin resistance. © 2017 Published by Elsevier Inc.
Keywords: TRB3 AMPK Insulin resistance Obesity Diabetes
1. Introduction Obesity and diabetes are increasing at an alarming rate worldwide, but the strategies for the prevention and treatment of these disorders remain inadequate. Type 2 diabetes mellitus (T2DM) is closely related to obesity. Its genesis is linked to insulin resistance (pre-diabetes condition). Diabetes and obesity are often accompanied by chronic inflammation and adipose tissue dysfunction [1]. Hyperglycemic damage in adipose tissue is characterized by oxidative stress and increased abundance of macrophage infiltration which further exaggerates inflammation in adipose tissue [2].
* Corresponding author. The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, 250012, China. E-mail address: [email protected] (Y. Ti).
In patients with obesity, chronic low-grade inflammation in adipose tissue triggers adipocyte lipolysis, leading to increased free fatty acid (FFA) release into circulation. FFA and its metabolites impair insulin signaling, decrease glucose tolerance, and cause ectopic lipid accumulation [3]. Dysfunctional adipocytes contribute directly and indirectly (through insulin resistance) to the development of vascular risk factors and vascular disease [4]. Insulin resistance (IR) is assumed to trigger the developments of T2DM [5]. TRB3 has been demonstrated to be involved in lipid metabolism [6] and in impairment of insulin exocytosis [7]. Epidemiological studies recognized that TRB3 was increased in obese humans with IR [8] and in patients with T2DM [9]. Our previous study showed that TRB3 was increased in adipose tissue of fructose-fed rats compared with the controls [10]. Therefore, TRB3 could be a promising candidate for investigating IR and related clinical disorders. The role of TRB3 in adipose tissue dysfunction has not been studied and therefore remains to be elucidated. TRB3 is an important suppressor of AMPK via directly binding to
http://dx.doi.org/10.1016/j.bbrc.2017.04.154 0006-291X/© 2017 Published by Elsevier Inc.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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it [11]. What is clear is that the consequences of dysfunctional AMPK include an increased risk of insulin resistance, hypertension and cardiovascular disease (CVD) and possibly a predilection to certain cancers [12e14]. However, the TRB3/AMPK signaling pathway has not been investigated in vitro on adipocytes and in vivo on adipose tissues directly. Thus, we hypothesized that TRB3/AMPK signaling pathway was an attractive strategy for the treatment of obesity and type 2 DM. First, we inducted IR adipocytes and type 2 DM model. TRB3 and its gene polymorphism are associated with insulin resistance, a vital pathophysiologic characteristic of type 2 diabetes. To further elucidate the role of TRB3/AMPK signaling pathway in obesity and DM, we used TRB3 gene silencing in vitro and in vivo to explore the mechanisms of TRB3/AMPK signaling pathway in obesity and DM as a potential target for treatment. 2. Materials and methods 2.1. Cell culture and induction of insulin resistance (IR) Murine 3T3-L1 pre-adipocytes, purchased from American Type Culture Collection (ATCC), were cultured in DMEM containing 10% FBS, and differentiated in a cocktail containing insulin, 3-isobutyl1-methylxanthine (IBMX) and dexamethason based on a standard protocol. The fully differentiated 3T3-L1 adipocytes were preincubated for 24 h in serum-free and low-glucose DMEM, and randomly divided into: control group (Control), insulin resistance (IR) group, and free fatty acids (FFA) group, control group was incubated with low-glucose DMEM containing 5.5 mM glucose and 10%FBS, IR was induced by incubating differentiated 3T3-L1 adipocytes in high-glucose DMEM containing 25 mM glucose, 10%FBS and 10 nM insulin for 24 h, FFA group was incubated with medium containing low-glucose DMEM, FBS, BSA and 0.75 mMpalmitate(Sigma) for 24 h as previously [15]. 2.2. Induction of diabetes Forty male SD rats were all fed high-fat (HF) diet, and randomly divided into four groups: HF þ Vehicle, HF þ TRB3siRNA, diabetes(DM)þVehicle and DM þ TRB3siRNA. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ) as previously [16]. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee. 2.3. Oil red O staining Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15min. Then, cells were washed with PBS and stained for 30 min with Oil Red O solution (60% isopropanol, 40% water). Excess stain was removed by washing with PBS, and then microscopic images were recorded. 2.4. Triglyceride assay Cells triglyceride were extracted, and measured using a Triglyceride Quantification Kit (Abcam, Cambridge,MA), following the manufacturer's instructions for colorimetric assay. 2.5. Glucose uptake For experiments, all culture medium was removed from each well and replaced with low-glucose DMEM containing 10% FBS, 1% 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 1 nM insulin for 30min. Removed the medium, cells
were washed and resuspended with PBS quickly, and then cells were moved at 1 104/well to 96-well plates. Fluorescence retained was measured using a microplate reader set at an excitation wave-length of 485 nm and an emission wavelength of 535 nm. 2.6. Histology and morphometric analysis Paraformaldehyde (4%)-fixed adipose tissue was bisected transversely, embedded in paraffin, and cut into 5-mm sections. A single adipocyte cell was measured with images captured from hematoxylin and eosinestained sections. The adipocyte crosssectional area was assessed under 3400 magnification within the adipose tissue, and a mean was obtained by quantitative morphometry with automated image analysis (Image-Pro Plus, Version 5.0; Media Cybernatics, Houston, TX). Liver was treated the same as adipose tissue. Liver frozen sections (5 mm) were stained with Oil Red O (Sigma) for 10 min, washed, and then counterstained with hematoxylin for 30 s. A Nikon microscope (Nikon, Melville, NY) was used to capture the Oil Red Oestained tissue sections. 2.7. Blood analyses After rats fasted overnight, we collected jugular blood. Total cholesterol, triglyceride levels, FBG and Fasting insulin level was measured as previously [16]. ISI was calculated. 2.8. Quantitative real-time PCR for TRB3 Total RNA was isolated from differentiated 3T3-L1 cells with the TRIzol reagent (Invitrogen). Total RNA was quantified by spectrophotometry and reverse-transcribed with the M-MLV Reverse Tran-scriptase System and oligo(dT) primers. The mRNA levels for TRB3 were determined by SYBR green real-time PCR, following the manufacturer's instructions. The PCR primers for TRB3 were 50 TCAAGCTGCGTCGCTTTGTC-30 (for-ward)and 50 -AGCTGAGTATCTCTG GTCCCACGTA-3’ (reverse); those for GAPDH were 50 TCAAGCTGCGTCGCTTTGTC-3’ (forward) and 50 - GATGCAGGGATGATGTTC -3’ (reverse). All values obtained were normalized to mouse GAPDH. 2.9. Western blot analysis Cells and tissue were harvested in ice-cold lysis buffer, Protein concentrations were determined with the Bradford method. Western blot analysis was as described previously [17]. We used antibodies against TRB3 (Calbiochem), GLUT4 (Abcam), and pAMPK/AMPK, IRS-1 (Cell Signaling Technology), Akt (Cell Signaling Technology), followed by anti-IgG horseradish peroxidaseeconjugated secondary antibody. TRB3, GLUT4, and IRS-1 protein levels were normalized to that of GAPDH/b-actin, as an internal control and phosphospecific proteins to that of total protein. 2.10. Gene silence of TRB3 The fully differentiated 3T3-L1 adipocytes were randomized to receive TRB3 small interfering RNA (siRNA) or vehicle treatment. Forty rats were randomized to receive TRB3 small interfering RNA (siRNA) or vehicle treatment as previously [16]. 2.11. Statistical analysis Values are presented as mean ± SEM, SPSS17.0 (SPSS, Chicago,
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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IL) was used for statistical analysis. Results were compared by oneway ANOVA, followed by Tukey-Kramer post hoc test and independent samples t-test. P < 0.05 was considered statistically significant.
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3.2. TRB3 gene silencing improved metabolism in vivo TRB3 gene silencing in this Type 2 DM model led to a significant decrease in water intake, urine volume, and FBG in DM þ TRB3siRNA group compared with DM þ Vehicle group (P < 0.05~P < 0.01). ISI was markedly increased in DM þ TRB3siRNA group compared with DM þ Vehicle group (P < 0.05) (Table 1).
3. Results 3.1. TRB3 gene silencing improved insulin resistance in vitro Lipid droplets in IR and FFA group adipocytes were larger in volume and quantities (Fig. 1A), and adipocytes in IR and FFA groups had significantly higher triglyceride level (P < 0.001) (Fig. 1C). Glucose uptake of adipocytes was significantly lower in IR and FFA group than in control group (P < 0.001) (Fig. 1B). Adipocytes in IR and FFA group had lower level of IRS-1, GLUT4 and pAMPK protein, had higher level of TRB3 protein correspondingly compared with control group (P < 0.001) (Fig. 1DeE). After TRB3 gene silencing, we detected adipocyte TRB3 expression by RT-PCR and western blot analysis. When compared with vehicle treatment, TRB3-siRNA treatment conferred downregulated TRB3 mRNA and protein expression of adipocyte. (Fig. 1FeH). Silence of TRB3 in IR and FFA adipocytes led to a significant increase of IRS-1 and GLUT4 protein level (P < 0.001). TRB3 gene silencing increased the phosphorylation of AMPK in TRB3siRNA groups compared with Vehicle. (P < 0.001) (Fig. 1GeH).
3.3. TRB3 gene silencing alleviated adipose tissue remodeling and improved triglyceride and glycogen metabolism in adipose tissue Silence of TRB3 decreased the adipocyte size of epididymal and subcutaneous adipose tissues in high-fat fed and diabetic rats, adipocyte aligned more evenly and orderly than control, TRB3 gene silencing alleviate the infiltration of white lipid droplet in brown adipose tissue(Fig. 2AeE). TRB3 gene silencing decreases triglyceride content level and increase glycogen content level in diabetic epididymal and brown adipose tissues in DM þ TRB3siRNA group(P < 0.05~P < 0.01) (Fig. 2FeH). 3.4. TRB3 gene silencing increased AMPK activity in adipose tissue Compared with vehicle treatment, TRB3-siRNA treatment conferred downregulated protein expression of adipose tissue TRB3. Silence of TRB3 significantly increased the phosphorylation of AMPK in epididymal adipose tissues, subcutaneous adipose tissues, and brown adipose tissues in HF þ TRB3siRNA group
Fig. 1. General characteristics of insulin resistant adipocytes. A: Adipocytes Stained with oil red O(scale bar: 50 mm). B: Glucose uptake of adipocytes. C: Triglyceride content in adipocytes. DeE: Western blot analysis of the protein expression and semi-quantification of IRS-1, GLUT-4, TRB3 and p-AMPK in adipocytes. Data are mean ± SEM. TRB3 gene silencing could improve insulin resistance in adipocytes. F: Silence of TRB3 significantly reduced relative TRB3 mRNA expression in adipocytes (scale bar: 50 mm). GeH: Western blot analysis of the protein expression and semiquantification of TRB3, IRS-1, GLUT4 and p-AMPK in adipocytes. Data are mean ± SEM.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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Table 1 Animal characteristics in four groups.
Body weight (g) Epididymal fat (g) Subcutaneous fat (g) Water intake (ml/d) Food intake (g/d) Urine volume(ml/d) FBG (mmol/l) FINS (mmol/l) ISI TC (mmol/l) TG (mmol/l)
HF þ Vehicle
HF þ TRB3siRNA
DM þ Vehicle
DM þ TRB3siRNA
575.12 ± 18.93 5.81 ± 0.61 4.02 ± 0.53 36.00 ± 3.11 18.67 ± 0.92 30.83 ± 1.68 10.45 ± 0.51 14.93 ± 1.56 5.01 ± 0.07 1.87 ± 0.12 1.51 ± 0.27
567.85 ± 16.79 4.77 ± 0.53 3.26 ± 0.40 32.00 ± 2.00 18.33 ± 1.02 30.00 ± 0.73 11.61 ± 1.11 12.63 ± 1.17 4.93 ± 0.11 1.80 ± 0.15 1.75 ± 0.20
533.50 ± 36.78 4.52 ± 0.72 3.49 ± 0.69 159.67 ± 4.33*** 27.50 ± 1.08*** 132.50 ± 5.08*** 25.69 ± 3.14** 13.01 ± 0.67 5.71 ± 0.15** 5.31 ± 1.76 8.30 ± 2.29
537.17 ± 17.29 3.93 ± 0.59 2.37 ± 0.57 146.00 ± 4.13y 26.50 ± 0.99 115.50 ± 2.22yy 15.54 ± 1.67y 11.31 ± 2.07 5.07 ± 0.13y 4.13 ± 1.49 7.44 ± 3.16
Data are mean ± SEM, n ¼ 6e9 per group. FBG, fasting blood glucose; FINS, fasting insulin; ISI, Insulin sensitivity index; TC, total cholesterol; TG, triglycercide. **P < 0.01, ***P < 0.001 vs. HF þ Vehicle; yP < 0.05, yyP < 0.01 vs. DM þ Vehicle.
compared with HF þ Vehicle group (P < 0.05~P < 0.001). The phosphorylation of AMPK in epididymal adipose tissues and brown adipose tissues increased in DM þ TRB3siRNA group compared with DM þ Vehicle group (P < 0.001) (Fig. 3AeC). 3.5. TRB3 gene silencing improved insulin resistance in vivo The protein expression of hepatic TRB3 was also downregulated after TRB3-siRNA treatment compared with vehicles. Diabetic rats had higher Oil Red Oestaining areas in liver than other groups, TRB3 gene silencing markedly decreased Oil Red Oestaining areas (P < 0.001) (Fig. 4AeB). Silence of TRB3 significantly increased phosphorylation of Akt in liver (P < 0.001) (Fig. 4C). 4. Discussion In present study we demonstrated that TRB3/AMPK signaling pathway was associated with insulin resistance in adipose tissue. TRB3 gene silencing could effectively improve glucose and lipid metabolism, and further improved insulin resistance by
modulating AMPK pathway in obesity and type 2 diabetes. TRB3 is implicated in dysfunction of adipose tissue of obesity and DM. In current study, the protein levels of IRS-1, GLUT4, pAMPK and glucose uptake in adipocytes were reduced in IR and FFA group, triglyceride level was significantly increased compared with control. The protein level of TRB3 was significantly increased in IR and FFA group. These findings indicate insulin resistance exists in our adipocytes in IR and FFA groups at the molecular level. Our previously study showed about 2-fold increase in TRB3 mRNA levels in adipose tissue of fructose-fed rats compared with those in adipose tissue of the controls [10]. In the present study, diabetic rats showed severe insulin resistance and increased TRB3 level in adipose tissue. Adipocyte size was significantly increased and arranged disorganized in the obesity and diabetic rats versus the TRB3-siRNA group. Even more white lipid droplets infiltrated in brown adipose tissue in the vehicle group. The aberrant lipids accumulated within liver in obesity and diabetic rats. Consistently, p-AMPK levels were significantly decreased in obesity and diabetic epididymal adipose tissues and BAT, except diabetic subcutaneous adipose tissue. These findings are in accordance with previous
Fig. 2. TRB3 gene silencing alleviated adipose tissue remodeling. A: Representative epididymaladipose tissues (EAT) stained with H&E(scale bar: 50 mm). B: Representative subcutaneousadipose tissue(SAT) stained with H&E. (scale bar: 50 mm) C: Representative brown adipose tissues (BAT) stained with H&E(scale bar: 50 mm). D: Representative scatter diagram of the epididymal adipocyte size; E: Representative scatter diagram of the subcutaneous adipocyte size. Effect of TRB3 gene silencing on the Triglyceride and glycogen in adipose tissue. FeH: Triglyceride content and glycogen content in each group epididymal, subcutaneous and brown adipose tissues.
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Fig. 3. Effect of TRB3 gene silencing on TRB3/AMPK signaling pathway in adipose tissue. A: Western blot analysis of the protein expression and semiquantification of TRB3/AMPK in epididymal adipose tissues (EAT). B: Western blot analysis of the protein expression and semiquantification of TRB3/AMPK in subcutaneous adipose tissues (SAT). C: Western blot analysis of the protein expression and semiquantification of TRB3/AMPK in brown adipose tissues (BAT). Data are mean ± SEM.
Fig. 4. TRB3 gene silencing could improve insulin resistance in liver. A: Representative H&E and Oil Red Oestained liver(scale bar: 50 mm). B: Semiquantification of Oil Red O staining. Data are mean ± SEM. C: Western blot analysis of the protein expression and semiquantification of TRB3/Akt in liver. Data are mean ± SEM.
Please cite this article in press as: X. Sun, et al., TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.04.154
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studies [18,19], which indicates TRB3 is implicated in dysfunction of adipose tissue of obesity and DM. It has been reported that TRB3 was an important modulating protein of AMPK [11,20,21]. AMPK activity is decreased and oxidative stress increased in subcutaneous, epiploic and omental adipose tissue in humans who are insulin-resistant [22]. We found TRB3 markedly upregulated and p-AMPK significantly downregulated in insulin resistant adipocyte and in rats with diabetes. Therefore, activation of TRB3 may contribute to the development and progression of DM, possibly mediated by the AMPK pathway. Effect of TRB3 silencing on adipocytes and adipose tissue mediated by AMPK pathway. In light of the pivotal role of TRB3 in dysfunction of adipose tissue of DM, we wondered whether downregulation of TRB3 could reverse the progression of DM. According to our previous study [16], we used TRB3-siRNA in vitro in adipocytes and in vivo in rats. TRB3-siRNA treatment produced no notable adverse effects and no deaths in adipocytes and rats. TRB3 mRNA levels were significantly decreased, and the protein expression was reduced by ~60% with TRB3-siRNA treatment in diabetic rats. Thus, global silencing of TRB3 was feasible and effective. With TRB3 silencing, the protein levels of IRS-1, GLUT4 and pAMPK were significantly increased in adipocytes. The impaired glucose tolerance and decreased insulin sensitivity were improved, and the inhibited activation of AMPK was restored in vivo. So TRB3 silencing improved insulin resistance, which was attributed to increased phosphorylation of AMPK in adipocyte, adipose tissue and phosphorylation of Akt in liver. In the present study, diabetic rats showed serious insulin resistance and increased TRB3 level in adipose tissues and liver. Akt activation in liver was inhibited markedly. These findings indicate insulin resistance exists in our type 2 diabetic rat model at the molecular level. In addition to improving metabolic disturbance, TRB3 silencing alleviated adipose tissue remodeling, adipocyte aligned more evenly and orderly, the infiltration of white lipid droplet in brown adipose tissue was decreased; the aberrant lipids accumulation within liver in obesity and diabetic rats was markedly reduced after TRB3 silencing. With TRB3 gene silencing, triglyceride content level was decreased and glycogen content level was increased in diabetic epididymal and brown adipose tissues (BAT). Correspondingly, p-AMPK levels were significantly increased in diabetic epididymal adipose tissues and BAT after TRB3-siRNA treatment. The inhibited phosphorylation of p-Akt in liver was restored after TRB3 silencing. Thus, TRB3 silencing alleviated metabolism disturbance and insulin resistance. There is no significantly change about triglyceride, glycogen content and p-AMPK level in diabetic subcutaneous adipose tissue after TRB3 gene silencing. So we can come to a conclusion that visceral and brown adipose tissues (BAT) are correlation with high incidence of insulin resistance, subcutaneous adipose tissue is correlation with low incidence of insulin resistance. These alterations resulted in overall improvement of insulin resistance. In accordance with these changes at the molecular level, TRB3 silencing attenuated the metabolic disturbance in obesity and type 2 diabetic rats. In summary, the present study indicates that, TRB3 is involved in dysfunction of adipose tissue in obesity and diabetes. TRB3 silencing activates AMPK with beneficial metabolic effects. Thus, TRB3/AMPK signaling pathway has a pivotal role in obesity and DM and could be an attractive drug target for treating obesity and type 2diabetes. Although our results suggest that blockage of TRB3 activity could be a therapeutic approach to increase phosphorylation of AMPK to improve insulin resistance in vitro and in vivo, TRB3 may have a different effect in various cell types, tissues and dosages, and
more detailed studies in different cell types is required to clarify the precise function of TRB3. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the research grants from the National Natural Science Foundation of China (81670411, 81471036, 81470560, 81570400, 81600633, 81300168, 81270352, 81270287, 81100605, 91439201, 81530014 and 81320108004),the National Basic Research Program of China (973 Program, Grant No. 2013CB530703), Key research and development program of Shandong Province (2015GSF118062), the Natural Science Foundation of Shandong Province (ZR2014HQ037), Medicine and Health Science Technology development program of Shandong Province (2016WS0091), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP 20130131120065) and Program of Introducing Talents of Discipline to Universities (B07035). References [1] N. Kawasaki, R. Asada, A. Saito, S. Kanemoto, K. Imaizumi, Obesity-induced endoplasmic reticulum stress cause chronic inflammation in adipose tissue, Sci. Rep. 2 (2012) 799. [2] Angelika Neuhofer, Maximilian Zeyda, Daniel Mascher, et al., Impaired local production of proresolving lipid mediators in obesity and 17-HDHA as a potential treatment for obesity-associated inflammation, Diabetes 62 (6) (2013; Jun) 1945e1956. [3] V.T. Samuel, G.I. Shulman, Mechanisms for insulin resistance: common threads and missing links, Cell 148 (5) (2012; Mar 2) 852e871. [4] C. Bleau, A.D. Karelis, D.H. St-Pierre, et al., Crosstalk between intestinal microbiota, adipose tissue and skeletal muscle as an early event in systemic low-grade inflammation and the development of obesity and diabetes, Diabetes Metab. Res. Rev. 31 (6) (2015; Sep) 545e561. [5] Q. Tang, X. Li, P. Song, et al., Optimal cut-off values for the homeostasis model assessment of insulin resistance (HOMA-IR) and pre-diabetes screening: developments in research and prospects for the future, Drug Discov. Ther. 9 (6) (2015; Dec) 380e385. [6] D. Steverson Jr., L. Tian, Y. Fu, et al., Tribbles homolog 3 promotes foam cell formation associated with decreased proinflammatory cytokine production in macrophages: evidence for reciprocal regulation of cholesterol uptake and inflammation, Metab. Syndr. Relat. Disord. 14 (1) (2016; Feb) 7e15. [7] C.W. Liew, J. Bochenski, D. Kawamori, et al., The pseudokinasetribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse beta cells, J. Clin. Invest 120 (8) (2010; Aug) 2876e2888. [8] H. Oberkofler, A. Pfeifenberger, S. Soyal, et al., Aberrant hepatic TRIB3 gene expression in insulin-resistant obese humans, Diabetologia 53 (9) (2010;Sep) 1971e1975. [9] J. Liu, X. Wu, J.L. Franklin, et al., Mammalian Tribbles homolog 3 impairs insulin action in skeletal muscle: role in glucose-induced insulin resistance, Am. J. Physiol. Endocrinol. Metab. 298 (3) (2010; Mar) E565eE576. [10] X.P. Bi, H.W. Tan, S.S. Xing, et al., Overexpression of TRB3 gene in adipose tissue of rats with high fructose-induced metabolic syndrome, Endocr. J. 55 (4) (2008 Aug) 747e752. [11] K. Li, Y. Xiao, J. Yu, et al., Liver-specific gene inactivation of the transcription factor ATF4 alleviates alcoholic liver steatosisin mice, J. Biol. Chem. 291 (35) (2016; Aug 26) 18536e18546. [12] W. Zhang, Q. Wang, Y. Wu, et al., Endothelial cell-specific liver kinase B1 deletion causes endothelial dysfunction and hypertension in mice in vivo, Circulation 129 (13) (2014 Apr 1) 1428e1439. [13] G.R. Steinberg, H.M. O'Neill, N.L. Dzamko, et al., Whole body deletion of AMPactivated protein kinase {beta}2 reduces muscle AMPK activity and exercise capacity, J. Biol. Chem. 285 (48) (2010; Nov 26) 37198e37209. [14] G. Zadra, J.L. Batista, M. Loda, Dissecting the dual role of AMPK in cancer: from experimental to human studies, Mol. Cancer Res. 13 (7) (2015; Jul) 1059e1072. [15] J.A. Chavez, T.A. Knotts, L.P. Wang, et al., A role for ceramide, but not diacylglycerol, in the antagonism of insulin signal transduction by saturated fatty acids, J. Biol. Chem. 278 (12) (2003; Mar21) 10297e10303. Epub 2003 Jan 13. [16] Y. Ti, G.L. Xie, Z.H. Wang, et al., TRB3 gene silencing alleviates diabetic cardiomyopathy in a type 2 diabetic rat model, Diabetes 60 (11) (2011; Nov) 2963e2974. [17] Z.H. Wang, Y.Y. Shang, S. Zhang, et al., Silence of TRIB3 suppresses atherosclerosis and stabilizes plaques in diabetic ApoE-/-/LDL receptor-/- mice,
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