- Email: [email protected]
Bile acid elevation after Roux-en-Y gastric bypass is associated with cardio-protective effect in Zucker Diabetic Fatty rats
International Journal of Surgery 24 (2015) 70–74
Contents lists available at ScienceDirect
International Journal of Surgery j o u r n a l h o m e p a g e : w w w. j o u r n a l - s u r g e r y. n e t
Original research
Bile acid elevation after Roux-en-Y gastric bypass is associated with cardio-protective effect in Zucker Diabetic Fatty rats Sunil Kumar a, Raymond Lau b,c, Christopher Hall a, Thomas Palaia a, Collin E. Brathwaite b,d, Louis Ragolia a,d,* a
Department of Biomedical Research, Winthrop University Hospital, Mineola, NY 11501, USA Department of Surgery, Winthrop University Hospital, Mineola, NY 11501, USA c Department of Endocrinology, Winthrop University Hospital, Mineola, NY 11501, USA d Department of Medicine, Stony Brook University School of Medicine, Stony Brook, NY 11794, USA b
H I G H L I G H T S
• • • •
RYGB ameliorated cardiometabolic risk through alteration of bile acid levels. Bile acid levels significantly increased 14 week post RYGB. L-PGDS levels were found significantly elevated after 14 week. RYGB in ZDF rodents led to a significant decrease in aortic wall thickness.
A R T I C L E
I N F O
Article history: Received 4 September 2015 Received in revised form 26 October 2015 Accepted 5 November 2015 Available online 10 November 2015 Keywords: RYGB Cardiovascular ZDF rats Bile acid L-PGDS Aortic wall thickness
A B S T R A C T
Background: Roux-en-Y gastric bypass (RYGB) may improve cardiometabolic risk through alteration of bile acids and L-PGDS levels. Objective: The objective of this study was to investigate the effect of RYGB on aortic wall thickness, in relation to bile acid and L-PGDS metabolism. Methods: Zucker diabetic fatty (ZDF) rats were divided into two groups, ad lib (n = 4), and RYGB (n = 6). Bile acid and L-PGDS were measured presurgery and fourteen weeks post-surgery. Results: Elevation of bile acid levels following RYGB in Zucker Diabetic Fatty (ZDF) rodents was observed, as compared to ad lib. RYGB in ZDF rodents led to a significantly decreased aortic wall thickness (25%) as compared to ad lib control. Although bile acid metabolism is implicated in these alterations, other mediators are likely involved. Our laboratory has demonstrated lipocalin prostaglandin D2 synthase (L-PGDS) is a kno n cardiometabolic modulator that also functions as a bile acid binding protein. Therefore, L-PGDS levels were measured and a significant elevation was observed with RYGB compared to ad lib control. Conclusion: Based on these findings, RYGB showed beneficial effect on aortic wall thickness, possibly through bile acids and L-PGDS elevation in a severely obese and diabetic rodent model. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cardiovascular disease (CVD) is the leading cause of death and accounts for 36% of all deaths in the United States [1]. The estimated economic burden due to CVD in 2009 was $475 billion, which highlights the need for the development of novel therapies [2]. Recently, bile acid metabolism has been considered as a potential target
* Corresponding author. Stony Brook University School of Medicine, Biomedical Research, Winthrop University Hospital, 222 Station Plaza North, Suite 505-B, Mineola, NY, 11501, USA. E-mail address: [email protected] (L. Ragolia).
in the field of cardiovascular research due to its beneficial effects [3]. Bile acids have a number of essential biological functions including facilitation of digestion through the solubilization of dietary fats, prevention of cholesterol deposition, and regulation of cholesterol homeostasis [4–6]. Emerging evidences also demonstrated its impact on glucose and lipid metabolism through a G-coupled bile acid receptor (TGR5/GPBAR1) and the farnesoid X receptor (FXR) [6]. The farnesoid X receptor (FXR) is a ligand-activated transcription factor belonging to the nuclear receptor super family. FXR is mainly expressed in liver and small intestine, where it plays a key role in bile acid, lipid, and glucose metabolism [7]. FXR-deficient (FXR(−/−)) mice have shown elevated triglyceride levels [5]. Similarly, usage of synthetic FXR agonists have demonstrated reduced aortic
http://dx.doi.org/10.1016/j.ijsu.2015.11.010 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
plaque formation in atherosclerotic rodent models [8]. Therefore, FXR signaling has been considered as a potential target of study in ameliorating cardiovascular risk and disease. We also believe that there is an additional factor beyond bile acid metabolism which may have a cardioprotective effect that is Lipocalin-type prostaglandin D2 synthase (L-PGDS). L-PGDS is a multifunctional protein which is known to transport retinoids, biliverdin, and bilirubin [9]. However, L-PGDS has also demonstrated to have a significant role in cardio-protection in animal models [10–12], and there is growing evidence of its beneficial effects in humans [13]. Because of its role as a bile acid transport protein, L-PGDS levels were measured to elucidate its possible role in the improvement of cardiovascular disease after RYGB surgery. 2. Materials and methods 2.1. Animals and diets Male Zucker diabetic fatty (ZDF) rats weighing (250–300 g) at 8 weeks old were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed individually in wire-mesh cages at a constant temperature of 21–23 °C with a 12-h light–dark cycle (lights on 07:00, off at 19:00). Rats were given access for 3 weeks to a diet consisting of Purina 5008 (fat 15%, carbohydrate 56%, protein 26%). After surgery, only Ensure (Fat 9%, carbohydrate 14%, protein 18%) a liquid meal supplement was available for the first 6 days, and then switched back to the Purina 5008 diet on the 7th day. All the protocols involved in this study were approved by the institutional animal care and use committee in accordance with guidelines established by the National Institutes of Health. ZDF rodents underwent RYGB (n = 6) and sham surgery (n = 4). Numbers of animals were calculated based on our previous studies. Blood samples were collected at initial and the 14th week following the surgery. 2.2. Preoperative care and anesthesia ZDF rats were fasted for 12 h prior to surgery and housed in suspended wire mesh caging to allow feces and urine to fall through the rack and to prevent the rats from copography. All the procedures were performed approximately at 10 am EST in the operating room located in the animal care facility. The rats were anesthetized with isoflurane using a calibrated vaporizer equipped with a device for properly scavenging waste anesthetic gas. An induction chamber was used with an initial vaporizer flow rate of 3–5%. After induction, animals were removed from the chamber and anesthesia was maintained using a rodent-specific nose cone apparatus at the same flow rate. Ceftriaxone (25 mg/kg) and Ketoprofen (5 mg/kg) were administered subcutaneously. The abdomen was shaved in a remote location prior to transfer to the operative field. The abdomen was prepped and draped aseptically. Heating pads were used throughout the operation. The isoflurane vaporizer flow rate was reduced to 1–2% after the abdominal incision was made. Rats were monitored for signs of either pain or respiratory depression and the flow rate adjusted accordingly. Sham operations were conducted with identical preoperative care and operative conditions. Abdominal contents were mobilized and manipulated to parallel the RYGB procedure. The abdominal incision was kept open until the mean anesthesia time for RYGB was reached. Abdominal wound closure and normal saline administration was consistent with RYGB. 2.3. Roux-en-Y gastric bypass RYGB procedure was performed using ZDF rodents. A 4 cm midline incision was made and subsequently, peritoneal attach-
71
ments to the stomach were divided, with particular attention to the attachment between the posterior stomach and the caudate lobe. The stomach was divided using a linear stapler (Endo GIA 45-2.5; Covidien, New Haven, CT) to create a small gastric pouch (20% of stomach). Care was taken to avoid injury or outflow obstruction to the esophagus or duodenum. The proximal jejunum was divided 10 cm distal to the Ligament of Treitz (corresponding to 15% of total jejunal-ileal length). A 6 mm gastrostomy was made in the anterior surface of the pouch to gently empty any luminal contents onto gauze. An end-to-side gastrojejunostomy is constructed using 6-0 Polypropylene sutures in a continuous manner. The jejunojejunostomy was constructed 10 cm distal to the gastrojejunostomy with interrupted 6-0 Polypropylene. Handling of the abdominal structures was minimized by use of sterile cotton applicators instead of surgical forceps when possible. Saline-soaked gauze was used to keep the abdominal viscera moist throughout the procedure. 5 ml of 0.9% normal saline was administered intraperitoneally prior to closing the incision. The abdominal incision was closed with two layers of continuous 4-0 sutures. An additional 5 ml of 0.9% normal saline was injected subcutaneously. Isoflurane was turned off once the surgery was over. The rats regained consciousness within 3–5 min and monitored until no residual anesthetic effects were observed.
2.4. Measurement of fasting bile acids levels Bile acids were measured using the Total Bile Acids Assay Kit (Calorimetric; BQ Kits, San Diego, CA) according to the manufacturer’s instructions [14]. Briefly, all the contents supplied in the kit were pre-warmed at the room temperature before reconstitution. Diaphorase was reconstituted with the phosphate buffer which is stable for 1 week at 4 °C after reconstitution. 150 μl of Diaphorase and 20 μl of sample or standards were mixed and incubated at 37 °C for 4 min. After 4 min incubation, 30 μl of 3-α-HSD was added, mixed well and read immediately at 540 nm as A1. Samples were again incubated for 5 min and read the absorbance again at 540 nm as A2. Values were calculated by subtracting the change in absorbance A1from A2. Total bile acid concentrations were calculated using the equation below:
ΔAbsorbance 540 (sample) ΔAbsorbance 540 (standard) × standard (35 μm mole L )
2.5. Measurement of fasting L-PGDS levels L-PGDS level was determined using ELISA kit supplied by MyBiosource. Assay procedure was followed as directed in the kit. Briefly, all reagents were brought at room temperature prior to the assay. 100 μl of standard or sample were added into the wells, covered with the adhesive strip and incubated for 2 h at 37 °C. After 2 h, liquid was removed (Note: Do not wash) and 100 μl of Biotinantibody was added into each well, covered with the adhesive strip and incubated again for 1 h at 37 °C. Solution in each well was mixed gently until solution appeared uniform. All the wells washed three times using 200 μl wash buffer. After the last wash, plate was inverted and blotted against clean paper towels. 100 μl of HRPavidin was added to each well and covered the microtiter plate with a new adhesive strip and again incubated for 1 h at 37 °C. After 1 h, wells were washed 6 times; 90 μl of TMB Substrate was added to each well and incubated for 15–30 min at 37 °C. (Note: Protect from light). Reaction was stopped using 50 μl of stop solution. The optical density was determined within 5 min, using a microplate reader at 450 nm and 540 nm. Wavelength was subtracted readings at 570 nm from the readings at 450 nm. L-PGDS concentration was calculated using the standard curve and plotted.
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
2.6. Measurement of aortic wall thickness
3. Results
Initial Final
20
10
B G
lib
0
d
All the data were analyzed using t-test and one-way ANOVA analysis of variance with the correction of Bonferroni post hoc test for multiple comparisons where appropriate. Data was considered statistically significant with p-values *P < 0.05. Statistical analysis was performed using Graph pad Prism 5.0 a (Inc, La Jolla, San Diego, CA, USA).
*
A
2.7. Statistical analysis
30 L-PGDS levels (ng/ml)
Animal were sacrificed and tissues were collected for further analysis. Aortic tissues were fixed in 10% formalin, processed for paraffin sections and stained with H&E. Aortas were observed with 4× objective on Nikon eclipse Ti microscope utilizing Nikon Elements morphometric software, Melville, NY. Aortic wall thickness was measured and presented as a pixel2.
L-PGDS levels (ZDF rats)
RY
72
Fig. 1. Bile acid levels were measured in Zucker Diabetic Fatty Rodents prior to RYGB surgery and fourteen weeks post surgery. Levels are compared to ad lib sham surgery controls. Bile acid levels in RYGB groups were significantly increased compared to sham control with p < 0.05.
3.1. RYGB increased bile acid levels In order to determine whether RYGB impacts bile acid levels, we performed RYGB on diabetic obese rats (n = 6). ad lib groups underwent a sham surgery (n = 4). We found significant elevation in bile acid levels of RYGB group from 25.40 ± 6.709 μmol/l to 94.68 ± 34.20 μmol/l compared to the ad lib control group where initial value was 19 ± 11.6 μmol/l and reached only to 39 ± 7.8 μmol/l by the fourteen-weeks post-surgery with p < 0.05. 3.2. RYGB increased L-PGDS levels In order to determine whether RYGB have any effect on L-PGDS levels, we performed RYGB on diabetic obese rats (n = 6). Again, ad lib groups underwent sham surgery (n = 4). We found significant elevation in L-PGDS levels of RYGB groups starting from 13.15 ± 1.5 ng/ml to 27.33 ± 4 ng/ml compared to the ad lib control group where values ranged from 13.75 ± 3.1 ng/ml to 13.17 ± 0.8 ng/ ml at fourteen-weeks post-surgery with p < 0.05. 3.3. RYGB decreased the aortic wall thickness In order to determine whether RYGB have any effect on aortic wall thickness, we performed the RYGB on diabetic obese rats (n = 6) and sham surgery on ad lib groups (n = 4). Interestingly, we found a 25% decrease in aortic wall thickness in RYGB group compared to ad lib group with p < 0.05 at fourteen-week post-surgery. Average aortic wall thickness of RYGB group was found to be 224.18 ± 26 pixel2 compared to ad lib group which was 298.40 ± 78 pixel2 (Fig. 3A). This finding clearly demonstrated a cardioprotective effect of RYGB in a diabetic obese rodent model. 4. Discussion Bile acid metabolism and associated signaling pathways appear to have a significant role in the development of cardiovascular diseases. Therefore, the present study was designed to investigate if there is a correlation between bile acid levels and its relative impact on arterial wall thickness after RYGB in a Zucker Diabetic Fatty (ZDF) rodent model. The ZDF rodent model was chosen because they develop progressive glucose intolerance, hyperlipidemia, hypertension and cardiovascular disease [15] which is similar to the metabolic syndrome seen in humans [16], and therefore may have direct clinical significance for future studies. Thickened arterial wall denotes either an increase in the vascular tunica media or intimal tissue layers. Smooth muscle cell
accumulation within the intima is the characteristic change that often leads to atherosclerosis and arterial stenosis [17]. Interestingly, bile acid levels were significantly elevated post-operatively after RYGB and its elevation can be explained by altered anatomy and therefore enhanced enterohepatic circulation. The decrease in cardiovascular events seen with bariatric surgery also does not have as strong a correlation with weight loss as previously thought [18]. We also believe that bile acid metabolism has beneficial cardiovascular effects independent of body weight. Growing clinical evidences have also demonstrated the beneficial cardiovascular effects independent to weight [18–21]. In our study, fasting bile acid levels were increased after RYGB (Fig. 1) compared to ad lib group, which may have the involvement of bile acid metabolism after RYGB [22]. It is of great interest to note what happens to gene knockouts of the FXR receptor that undergo another metabolically similar bariatric surgery, the gastric sleeve (GS). These rodents demonstrate an inability to achieve the optimal glucose benefit seen in wild type rodents that undergo GS [23]. Therefore, one may hypothesize that while bile acids are the key to the cardiometabolic improvements observed after bariatric surgery, an additional unidentified beneficial factor may exist. We propose an additional factor, lipocalin-type prostaglandin D2 synthase (L-PGDS) as being responsible for the cardioprotective effect post RYGB. L-PGDS is the first reported enzyme among members of the lipocalin family [24], and interestingly, it has ability to bind with lipophilic ligands such as bile acids, retinoids, thyroid hormones [9,25]. Our laboratory has demonstrated L-PGDS as an advantageous cardiometabolic modulator. L-PGDS genetic knockouts demonstrated accelerated atherosclerosis as well as glucose intolerance [10]. Its elevation has been implicated as anti-thrombogenic and cardio-protective [26]. Present study results have also shown significantly elevated L-PGDS levels fourteen weeks post-surgery compared to the ad lib group as shown (Fig. 2). Since, L-PGDS is a bile acid transport protein [9], therefore the concomitant elevation with bile acid levels after RYGB may not be unexpected. In totality, bile acids and L-PGDS in combination may be responsible for the cardiometabolic improvements. Measuring aortic wall thickness in the ZDF rats that undergo RYGB may provide further insight into the cardiovascular mechanisms of improvement. Therefore, we measured aortic wall thickness and found significantly decreased the aortic wall thickness by 25% in RYGB group compared to ad lib group. Aortic wall thickness was calculated by measuring the total area bordered by inner layer of the tunica intima (luminal side) subtracted from total area bordered by
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
Bile Acid levels (ZDF rats) Bile Acid levels (µmole/l)
150
Initial Final
*
100
50
G B RY
A
d
lib
0
Fig. 2. L-PGDS levels were measured in Zucker Diabetic Fatty Rodents fourteen week post RYGB surgery and sham controls. L-PGDS levels in RYGB groups were significantly increased compared to sham control with p < 0.05.
tunica media (adventitia side). As per Fig. 3B, changes were only observed in tunica media layer. Precisely, in the tunica media, there was a fragmentation of elastic lamellae with the widening of interlamellar spaces. In conclusion, RYGB resulted in elevated bile acids levels with a significant reduction in aortic wall thickness. L-PGDS was also
73
shown to be elevated after RYGB and may have a significant role in the cardiovascular improvement as well. Bile acid metabolism is at least partially responsible for the cardiovascular improvement observed after RYGB. Further studies need to be performed to investigate the precise mechanism of action of how L-PGDS and bile acid elevation post RYGB leads to cardioprotective effects in rodents. Collectively, based on the obtained results, aortic wall thickness of RYGB group was significantly reduced compared to ad lib group which could be the result of significant elevation of bile acid and L-PGDS affording cardio-protection. Our L-PGDS knockout models should offer further insights into the cardio-protective effects of bile acid and L-PGDS. Ethical approval IACUC approval LR#13. Funding This project was funded by American Heart Association grant (#15GRNT22420001). Author contribution Louis Ragolia- Experimental Design, data analysis and manuscript writing. Sunil Kumar- Experimental Design, data analysis and manuscript writing. Raymond Lau- Data analysis. Collin Brathwaite- Data analysis. Christopher Hall- Data collection. Thomas Palaia- Data collection. Conflicts of interest None. Guarantor Louis Ragolia. Sunil Kumar. Acknowledgments The authors wish to thank Drs. Drew A. Rideout, MD and Keneth Hall, MD for their contributions to this manuscript. Appendix A: Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ijsu.2015.11.010. References
Fig. 3. A. Aortic wall thickness was measured in Zucker Diabetic Fatty Rodents fourteen weeks post surgery in RYGB (n = 6) and sham (n = 4) control groups. Aortic wall thickness in RYGB groups were significantly decreased compared to sham control with p < 0.05. B. Aortic wall thickness was measured in Zucker Diabetic Fatty Rodents fourteen weeks post surgery in RYGB (n = 6) and sham (n = 4) control groups. Aortic wall thickness in RYGB groups were significantly decreased compared to sham control with p < 0.05.
[1] L.J. Shaw, et al., Postmenopausal women with a history of irregular menses and elevated androgen measurements at high risk for worsening cardiovascular event-free survival: results from the National Institutes of Health–National Heart, Lung, and Blood Institute sponsored Women’s Ischemia Syndrome Evaluation, J. Clin. Endocrinol. Metab. 93 (4) (2008) 1276–1284. [2] C.A. Manueddu, et al., Infective iliopsoas bursitis. A case report, Int. Orthop. 15 (2) (1991) 135–137. [3] G. Porez, et al., Bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease, J. Lipid Res. 53 (9) (2012) 1723–1737. [4] W. Insull Jr., Clinical utility of bile acid sequestrants in the treatment of dyslipidemia: a scientific review, South Med. J. 99 (3) (2006) 257–273. [5] P. Lefebvre, et al., Role of bile acids and bile acid receptors in metabolic regulation, Physiol. Rev. 89 (1) (2009) 147–191. [6] J. Hageman, et al., A role of the bile salt receptor FXR in atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 30 (8) (2010) 1519–1528.
74
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
[7] X. Zhang, et al., Farnesoid X receptor (FXR) gene deficiency impairs urine concentration in mice, Proc. Natl. Acad. Sci. U. S. A. 111 (6) (2014) 2277–2282. [8] H.B. Hartman, et al., Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice, J. Lipid Res. 50 (6) (2009) 1090–1100. [9] C.T. Beuckmann, et al., Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase, Biochemistry 38 (25) (1999) 8006–8013. [10] L. Ragolia, et al., Accelerated glucose intolerance, nephropathy, and atherosclerosis in prostaglandin D2 synthase knock-out mice, J. Biol. Chem. 280 (33) (2005) 29946–29955. [11] R. Tanaka, et al., Knockout of the l-pgds gene aggravates obesity and atherosclerosis in mice, Biochem. Biophys. Res. Commun. 378 (4) (2009) 851–856. [12] S. Tokudome, et al., Glucocorticoid protects rodent hearts from ischemia/ reperfusion injury by activating lipocalin-type prostaglandin D synthase-derived PGD2 biosynthesis, J. Clin. Invest. 119 (6) (2009) 1477–1488. [13] C.L. Cheung, et al., Reduced serum beta-trace protein is associated with metabolic syndrome, Atherosclerosis 227 (2) (2013) 404–407. [14] X. Wu, et al., Dual actions of fibroblast growth factor 19 on lipid metabolism, J. Lipid Res. 54 (2) (2013) 325–332. [15] M. Shiota, R.L. Printz, Diabetes in Zucker diabetic fatty rat, Methods Mol. Biol. 933 (2012) 103–123. [16] J. Siwy, et al., Evaluation of the Zucker diabetic fatty (ZDF) rat as a model for human disease based on urinary peptidomic profiles, PLoS One 7 (12) (2012) e51334. [17] H. Hao, G. Gabbiani, M.L. Bochaton-Piallat, Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development, Arterioscler. Thromb. Vasc. Biol. 23 (9) (2003) 1510–1520.
[18] L. Sjostrom, et al., Bariatric surgery and long-term cardiovascular events, JAMA 307 (1) (2012) 56–65. [19] A. Mor, P. Omotosho, A. Torquati, Cardiovascular risk in obese diabetic patients is significantly reduced one year after gastric bypass compared to one year of diabetes support and education, Surg. Endosc. 28 (10) (2014) 2815– 2820. [20] P. Pujante, et al., Modification of cardiometabolic profile in obese diabetic patients after bariatric surgery: changes in cardiovascular risk, Rev. Esp. Cardiol. (Engl. Ed.) 66 (10) (2013) 812–818. [21] P. Sanchis, et al., Cardiovascular risk profile in Mediterranean patients submitted to bariatric surgery and intensive lifestyle Intervention: impact of both interventions after 1 year of follow-up, Obes. Surg 25 (1) (2015 Jan) 97–108. [22] R. Kohli, et al., Weight loss induced by Roux-en-Y gastric bypass but not laparoscopic adjustable gastric banding increases circulating bile acids, J. Clin. Endocrinol. Metab. 98 (4) (2013) E708–E712. [23] K.K. Ryan, et al., FXR is a molecular target for the effects of vertical sleeve gastrectomy, Nature (2014). [24] A. Nagata, et al., Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic-ligand carrier proteins, Proc. Natl. Acad. Sci. U. S. A. 88 (9) (1991) 4020–4024. [25] T. Tanaka, et al., Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter, J. Biol. Chem. 272 (25) (1997) 15789– 15795. [26] T. Inoue, et al., Serum prostaglandin D synthase level after coronary angioplasty may predict occurrence of restenosis, Thromb. Haemost. 85 (1) (2001) 165– 170.
Contents lists available at ScienceDirect
International Journal of Surgery j o u r n a l h o m e p a g e : w w w. j o u r n a l - s u r g e r y. n e t
Original research
Bile acid elevation after Roux-en-Y gastric bypass is associated with cardio-protective effect in Zucker Diabetic Fatty rats Sunil Kumar a, Raymond Lau b,c, Christopher Hall a, Thomas Palaia a, Collin E. Brathwaite b,d, Louis Ragolia a,d,* a
Department of Biomedical Research, Winthrop University Hospital, Mineola, NY 11501, USA Department of Surgery, Winthrop University Hospital, Mineola, NY 11501, USA c Department of Endocrinology, Winthrop University Hospital, Mineola, NY 11501, USA d Department of Medicine, Stony Brook University School of Medicine, Stony Brook, NY 11794, USA b
H I G H L I G H T S
• • • •
RYGB ameliorated cardiometabolic risk through alteration of bile acid levels. Bile acid levels significantly increased 14 week post RYGB. L-PGDS levels were found significantly elevated after 14 week. RYGB in ZDF rodents led to a significant decrease in aortic wall thickness.
A R T I C L E
I N F O
Article history: Received 4 September 2015 Received in revised form 26 October 2015 Accepted 5 November 2015 Available online 10 November 2015 Keywords: RYGB Cardiovascular ZDF rats Bile acid L-PGDS Aortic wall thickness
A B S T R A C T
Background: Roux-en-Y gastric bypass (RYGB) may improve cardiometabolic risk through alteration of bile acids and L-PGDS levels. Objective: The objective of this study was to investigate the effect of RYGB on aortic wall thickness, in relation to bile acid and L-PGDS metabolism. Methods: Zucker diabetic fatty (ZDF) rats were divided into two groups, ad lib (n = 4), and RYGB (n = 6). Bile acid and L-PGDS were measured presurgery and fourteen weeks post-surgery. Results: Elevation of bile acid levels following RYGB in Zucker Diabetic Fatty (ZDF) rodents was observed, as compared to ad lib. RYGB in ZDF rodents led to a significantly decreased aortic wall thickness (25%) as compared to ad lib control. Although bile acid metabolism is implicated in these alterations, other mediators are likely involved. Our laboratory has demonstrated lipocalin prostaglandin D2 synthase (L-PGDS) is a kno n cardiometabolic modulator that also functions as a bile acid binding protein. Therefore, L-PGDS levels were measured and a significant elevation was observed with RYGB compared to ad lib control. Conclusion: Based on these findings, RYGB showed beneficial effect on aortic wall thickness, possibly through bile acids and L-PGDS elevation in a severely obese and diabetic rodent model. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cardiovascular disease (CVD) is the leading cause of death and accounts for 36% of all deaths in the United States [1]. The estimated economic burden due to CVD in 2009 was $475 billion, which highlights the need for the development of novel therapies [2]. Recently, bile acid metabolism has been considered as a potential target
* Corresponding author. Stony Brook University School of Medicine, Biomedical Research, Winthrop University Hospital, 222 Station Plaza North, Suite 505-B, Mineola, NY, 11501, USA. E-mail address: [email protected] (L. Ragolia).
in the field of cardiovascular research due to its beneficial effects [3]. Bile acids have a number of essential biological functions including facilitation of digestion through the solubilization of dietary fats, prevention of cholesterol deposition, and regulation of cholesterol homeostasis [4–6]. Emerging evidences also demonstrated its impact on glucose and lipid metabolism through a G-coupled bile acid receptor (TGR5/GPBAR1) and the farnesoid X receptor (FXR) [6]. The farnesoid X receptor (FXR) is a ligand-activated transcription factor belonging to the nuclear receptor super family. FXR is mainly expressed in liver and small intestine, where it plays a key role in bile acid, lipid, and glucose metabolism [7]. FXR-deficient (FXR(−/−)) mice have shown elevated triglyceride levels [5]. Similarly, usage of synthetic FXR agonists have demonstrated reduced aortic
http://dx.doi.org/10.1016/j.ijsu.2015.11.010 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
plaque formation in atherosclerotic rodent models [8]. Therefore, FXR signaling has been considered as a potential target of study in ameliorating cardiovascular risk and disease. We also believe that there is an additional factor beyond bile acid metabolism which may have a cardioprotective effect that is Lipocalin-type prostaglandin D2 synthase (L-PGDS). L-PGDS is a multifunctional protein which is known to transport retinoids, biliverdin, and bilirubin [9]. However, L-PGDS has also demonstrated to have a significant role in cardio-protection in animal models [10–12], and there is growing evidence of its beneficial effects in humans [13]. Because of its role as a bile acid transport protein, L-PGDS levels were measured to elucidate its possible role in the improvement of cardiovascular disease after RYGB surgery. 2. Materials and methods 2.1. Animals and diets Male Zucker diabetic fatty (ZDF) rats weighing (250–300 g) at 8 weeks old were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed individually in wire-mesh cages at a constant temperature of 21–23 °C with a 12-h light–dark cycle (lights on 07:00, off at 19:00). Rats were given access for 3 weeks to a diet consisting of Purina 5008 (fat 15%, carbohydrate 56%, protein 26%). After surgery, only Ensure (Fat 9%, carbohydrate 14%, protein 18%) a liquid meal supplement was available for the first 6 days, and then switched back to the Purina 5008 diet on the 7th day. All the protocols involved in this study were approved by the institutional animal care and use committee in accordance with guidelines established by the National Institutes of Health. ZDF rodents underwent RYGB (n = 6) and sham surgery (n = 4). Numbers of animals were calculated based on our previous studies. Blood samples were collected at initial and the 14th week following the surgery. 2.2. Preoperative care and anesthesia ZDF rats were fasted for 12 h prior to surgery and housed in suspended wire mesh caging to allow feces and urine to fall through the rack and to prevent the rats from copography. All the procedures were performed approximately at 10 am EST in the operating room located in the animal care facility. The rats were anesthetized with isoflurane using a calibrated vaporizer equipped with a device for properly scavenging waste anesthetic gas. An induction chamber was used with an initial vaporizer flow rate of 3–5%. After induction, animals were removed from the chamber and anesthesia was maintained using a rodent-specific nose cone apparatus at the same flow rate. Ceftriaxone (25 mg/kg) and Ketoprofen (5 mg/kg) were administered subcutaneously. The abdomen was shaved in a remote location prior to transfer to the operative field. The abdomen was prepped and draped aseptically. Heating pads were used throughout the operation. The isoflurane vaporizer flow rate was reduced to 1–2% after the abdominal incision was made. Rats were monitored for signs of either pain or respiratory depression and the flow rate adjusted accordingly. Sham operations were conducted with identical preoperative care and operative conditions. Abdominal contents were mobilized and manipulated to parallel the RYGB procedure. The abdominal incision was kept open until the mean anesthesia time for RYGB was reached. Abdominal wound closure and normal saline administration was consistent with RYGB. 2.3. Roux-en-Y gastric bypass RYGB procedure was performed using ZDF rodents. A 4 cm midline incision was made and subsequently, peritoneal attach-
71
ments to the stomach were divided, with particular attention to the attachment between the posterior stomach and the caudate lobe. The stomach was divided using a linear stapler (Endo GIA 45-2.5; Covidien, New Haven, CT) to create a small gastric pouch (20% of stomach). Care was taken to avoid injury or outflow obstruction to the esophagus or duodenum. The proximal jejunum was divided 10 cm distal to the Ligament of Treitz (corresponding to 15% of total jejunal-ileal length). A 6 mm gastrostomy was made in the anterior surface of the pouch to gently empty any luminal contents onto gauze. An end-to-side gastrojejunostomy is constructed using 6-0 Polypropylene sutures in a continuous manner. The jejunojejunostomy was constructed 10 cm distal to the gastrojejunostomy with interrupted 6-0 Polypropylene. Handling of the abdominal structures was minimized by use of sterile cotton applicators instead of surgical forceps when possible. Saline-soaked gauze was used to keep the abdominal viscera moist throughout the procedure. 5 ml of 0.9% normal saline was administered intraperitoneally prior to closing the incision. The abdominal incision was closed with two layers of continuous 4-0 sutures. An additional 5 ml of 0.9% normal saline was injected subcutaneously. Isoflurane was turned off once the surgery was over. The rats regained consciousness within 3–5 min and monitored until no residual anesthetic effects were observed.
2.4. Measurement of fasting bile acids levels Bile acids were measured using the Total Bile Acids Assay Kit (Calorimetric; BQ Kits, San Diego, CA) according to the manufacturer’s instructions [14]. Briefly, all the contents supplied in the kit were pre-warmed at the room temperature before reconstitution. Diaphorase was reconstituted with the phosphate buffer which is stable for 1 week at 4 °C after reconstitution. 150 μl of Diaphorase and 20 μl of sample or standards were mixed and incubated at 37 °C for 4 min. After 4 min incubation, 30 μl of 3-α-HSD was added, mixed well and read immediately at 540 nm as A1. Samples were again incubated for 5 min and read the absorbance again at 540 nm as A2. Values were calculated by subtracting the change in absorbance A1from A2. Total bile acid concentrations were calculated using the equation below:
ΔAbsorbance 540 (sample) ΔAbsorbance 540 (standard) × standard (35 μm mole L )
2.5. Measurement of fasting L-PGDS levels L-PGDS level was determined using ELISA kit supplied by MyBiosource. Assay procedure was followed as directed in the kit. Briefly, all reagents were brought at room temperature prior to the assay. 100 μl of standard or sample were added into the wells, covered with the adhesive strip and incubated for 2 h at 37 °C. After 2 h, liquid was removed (Note: Do not wash) and 100 μl of Biotinantibody was added into each well, covered with the adhesive strip and incubated again for 1 h at 37 °C. Solution in each well was mixed gently until solution appeared uniform. All the wells washed three times using 200 μl wash buffer. After the last wash, plate was inverted and blotted against clean paper towels. 100 μl of HRPavidin was added to each well and covered the microtiter plate with a new adhesive strip and again incubated for 1 h at 37 °C. After 1 h, wells were washed 6 times; 90 μl of TMB Substrate was added to each well and incubated for 15–30 min at 37 °C. (Note: Protect from light). Reaction was stopped using 50 μl of stop solution. The optical density was determined within 5 min, using a microplate reader at 450 nm and 540 nm. Wavelength was subtracted readings at 570 nm from the readings at 450 nm. L-PGDS concentration was calculated using the standard curve and plotted.
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
2.6. Measurement of aortic wall thickness
3. Results
Initial Final
20
10
B G
lib
0
d
All the data were analyzed using t-test and one-way ANOVA analysis of variance with the correction of Bonferroni post hoc test for multiple comparisons where appropriate. Data was considered statistically significant with p-values *P < 0.05. Statistical analysis was performed using Graph pad Prism 5.0 a (Inc, La Jolla, San Diego, CA, USA).
*
A
2.7. Statistical analysis
30 L-PGDS levels (ng/ml)
Animal were sacrificed and tissues were collected for further analysis. Aortic tissues were fixed in 10% formalin, processed for paraffin sections and stained with H&E. Aortas were observed with 4× objective on Nikon eclipse Ti microscope utilizing Nikon Elements morphometric software, Melville, NY. Aortic wall thickness was measured and presented as a pixel2.
L-PGDS levels (ZDF rats)
RY
72
Fig. 1. Bile acid levels were measured in Zucker Diabetic Fatty Rodents prior to RYGB surgery and fourteen weeks post surgery. Levels are compared to ad lib sham surgery controls. Bile acid levels in RYGB groups were significantly increased compared to sham control with p < 0.05.
3.1. RYGB increased bile acid levels In order to determine whether RYGB impacts bile acid levels, we performed RYGB on diabetic obese rats (n = 6). ad lib groups underwent a sham surgery (n = 4). We found significant elevation in bile acid levels of RYGB group from 25.40 ± 6.709 μmol/l to 94.68 ± 34.20 μmol/l compared to the ad lib control group where initial value was 19 ± 11.6 μmol/l and reached only to 39 ± 7.8 μmol/l by the fourteen-weeks post-surgery with p < 0.05. 3.2. RYGB increased L-PGDS levels In order to determine whether RYGB have any effect on L-PGDS levels, we performed RYGB on diabetic obese rats (n = 6). Again, ad lib groups underwent sham surgery (n = 4). We found significant elevation in L-PGDS levels of RYGB groups starting from 13.15 ± 1.5 ng/ml to 27.33 ± 4 ng/ml compared to the ad lib control group where values ranged from 13.75 ± 3.1 ng/ml to 13.17 ± 0.8 ng/ ml at fourteen-weeks post-surgery with p < 0.05. 3.3. RYGB decreased the aortic wall thickness In order to determine whether RYGB have any effect on aortic wall thickness, we performed the RYGB on diabetic obese rats (n = 6) and sham surgery on ad lib groups (n = 4). Interestingly, we found a 25% decrease in aortic wall thickness in RYGB group compared to ad lib group with p < 0.05 at fourteen-week post-surgery. Average aortic wall thickness of RYGB group was found to be 224.18 ± 26 pixel2 compared to ad lib group which was 298.40 ± 78 pixel2 (Fig. 3A). This finding clearly demonstrated a cardioprotective effect of RYGB in a diabetic obese rodent model. 4. Discussion Bile acid metabolism and associated signaling pathways appear to have a significant role in the development of cardiovascular diseases. Therefore, the present study was designed to investigate if there is a correlation between bile acid levels and its relative impact on arterial wall thickness after RYGB in a Zucker Diabetic Fatty (ZDF) rodent model. The ZDF rodent model was chosen because they develop progressive glucose intolerance, hyperlipidemia, hypertension and cardiovascular disease [15] which is similar to the metabolic syndrome seen in humans [16], and therefore may have direct clinical significance for future studies. Thickened arterial wall denotes either an increase in the vascular tunica media or intimal tissue layers. Smooth muscle cell
accumulation within the intima is the characteristic change that often leads to atherosclerosis and arterial stenosis [17]. Interestingly, bile acid levels were significantly elevated post-operatively after RYGB and its elevation can be explained by altered anatomy and therefore enhanced enterohepatic circulation. The decrease in cardiovascular events seen with bariatric surgery also does not have as strong a correlation with weight loss as previously thought [18]. We also believe that bile acid metabolism has beneficial cardiovascular effects independent of body weight. Growing clinical evidences have also demonstrated the beneficial cardiovascular effects independent to weight [18–21]. In our study, fasting bile acid levels were increased after RYGB (Fig. 1) compared to ad lib group, which may have the involvement of bile acid metabolism after RYGB [22]. It is of great interest to note what happens to gene knockouts of the FXR receptor that undergo another metabolically similar bariatric surgery, the gastric sleeve (GS). These rodents demonstrate an inability to achieve the optimal glucose benefit seen in wild type rodents that undergo GS [23]. Therefore, one may hypothesize that while bile acids are the key to the cardiometabolic improvements observed after bariatric surgery, an additional unidentified beneficial factor may exist. We propose an additional factor, lipocalin-type prostaglandin D2 synthase (L-PGDS) as being responsible for the cardioprotective effect post RYGB. L-PGDS is the first reported enzyme among members of the lipocalin family [24], and interestingly, it has ability to bind with lipophilic ligands such as bile acids, retinoids, thyroid hormones [9,25]. Our laboratory has demonstrated L-PGDS as an advantageous cardiometabolic modulator. L-PGDS genetic knockouts demonstrated accelerated atherosclerosis as well as glucose intolerance [10]. Its elevation has been implicated as anti-thrombogenic and cardio-protective [26]. Present study results have also shown significantly elevated L-PGDS levels fourteen weeks post-surgery compared to the ad lib group as shown (Fig. 2). Since, L-PGDS is a bile acid transport protein [9], therefore the concomitant elevation with bile acid levels after RYGB may not be unexpected. In totality, bile acids and L-PGDS in combination may be responsible for the cardiometabolic improvements. Measuring aortic wall thickness in the ZDF rats that undergo RYGB may provide further insight into the cardiovascular mechanisms of improvement. Therefore, we measured aortic wall thickness and found significantly decreased the aortic wall thickness by 25% in RYGB group compared to ad lib group. Aortic wall thickness was calculated by measuring the total area bordered by inner layer of the tunica intima (luminal side) subtracted from total area bordered by
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
Bile Acid levels (ZDF rats) Bile Acid levels (µmole/l)
150
Initial Final
*
100
50
G B RY
A
d
lib
0
Fig. 2. L-PGDS levels were measured in Zucker Diabetic Fatty Rodents fourteen week post RYGB surgery and sham controls. L-PGDS levels in RYGB groups were significantly increased compared to sham control with p < 0.05.
tunica media (adventitia side). As per Fig. 3B, changes were only observed in tunica media layer. Precisely, in the tunica media, there was a fragmentation of elastic lamellae with the widening of interlamellar spaces. In conclusion, RYGB resulted in elevated bile acids levels with a significant reduction in aortic wall thickness. L-PGDS was also
73
shown to be elevated after RYGB and may have a significant role in the cardiovascular improvement as well. Bile acid metabolism is at least partially responsible for the cardiovascular improvement observed after RYGB. Further studies need to be performed to investigate the precise mechanism of action of how L-PGDS and bile acid elevation post RYGB leads to cardioprotective effects in rodents. Collectively, based on the obtained results, aortic wall thickness of RYGB group was significantly reduced compared to ad lib group which could be the result of significant elevation of bile acid and L-PGDS affording cardio-protection. Our L-PGDS knockout models should offer further insights into the cardio-protective effects of bile acid and L-PGDS. Ethical approval IACUC approval LR#13. Funding This project was funded by American Heart Association grant (#15GRNT22420001). Author contribution Louis Ragolia- Experimental Design, data analysis and manuscript writing. Sunil Kumar- Experimental Design, data analysis and manuscript writing. Raymond Lau- Data analysis. Collin Brathwaite- Data analysis. Christopher Hall- Data collection. Thomas Palaia- Data collection. Conflicts of interest None. Guarantor Louis Ragolia. Sunil Kumar. Acknowledgments The authors wish to thank Drs. Drew A. Rideout, MD and Keneth Hall, MD for their contributions to this manuscript. Appendix A: Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ijsu.2015.11.010. References
Fig. 3. A. Aortic wall thickness was measured in Zucker Diabetic Fatty Rodents fourteen weeks post surgery in RYGB (n = 6) and sham (n = 4) control groups. Aortic wall thickness in RYGB groups were significantly decreased compared to sham control with p < 0.05. B. Aortic wall thickness was measured in Zucker Diabetic Fatty Rodents fourteen weeks post surgery in RYGB (n = 6) and sham (n = 4) control groups. Aortic wall thickness in RYGB groups were significantly decreased compared to sham control with p < 0.05.
[1] L.J. Shaw, et al., Postmenopausal women with a history of irregular menses and elevated androgen measurements at high risk for worsening cardiovascular event-free survival: results from the National Institutes of Health–National Heart, Lung, and Blood Institute sponsored Women’s Ischemia Syndrome Evaluation, J. Clin. Endocrinol. Metab. 93 (4) (2008) 1276–1284. [2] C.A. Manueddu, et al., Infective iliopsoas bursitis. A case report, Int. Orthop. 15 (2) (1991) 135–137. [3] G. Porez, et al., Bile acid receptors as targets for the treatment of dyslipidemia and cardiovascular disease, J. Lipid Res. 53 (9) (2012) 1723–1737. [4] W. Insull Jr., Clinical utility of bile acid sequestrants in the treatment of dyslipidemia: a scientific review, South Med. J. 99 (3) (2006) 257–273. [5] P. Lefebvre, et al., Role of bile acids and bile acid receptors in metabolic regulation, Physiol. Rev. 89 (1) (2009) 147–191. [6] J. Hageman, et al., A role of the bile salt receptor FXR in atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 30 (8) (2010) 1519–1528.
74
S. Kumar et al./International Journal of Surgery 24 (2015) 70–74
[7] X. Zhang, et al., Farnesoid X receptor (FXR) gene deficiency impairs urine concentration in mice, Proc. Natl. Acad. Sci. U. S. A. 111 (6) (2014) 2277–2282. [8] H.B. Hartman, et al., Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice, J. Lipid Res. 50 (6) (2009) 1090–1100. [9] C.T. Beuckmann, et al., Binding of biliverdin, bilirubin, and thyroid hormones to lipocalin-type prostaglandin D synthase, Biochemistry 38 (25) (1999) 8006–8013. [10] L. Ragolia, et al., Accelerated glucose intolerance, nephropathy, and atherosclerosis in prostaglandin D2 synthase knock-out mice, J. Biol. Chem. 280 (33) (2005) 29946–29955. [11] R. Tanaka, et al., Knockout of the l-pgds gene aggravates obesity and atherosclerosis in mice, Biochem. Biophys. Res. Commun. 378 (4) (2009) 851–856. [12] S. Tokudome, et al., Glucocorticoid protects rodent hearts from ischemia/ reperfusion injury by activating lipocalin-type prostaglandin D synthase-derived PGD2 biosynthesis, J. Clin. Invest. 119 (6) (2009) 1477–1488. [13] C.L. Cheung, et al., Reduced serum beta-trace protein is associated with metabolic syndrome, Atherosclerosis 227 (2) (2013) 404–407. [14] X. Wu, et al., Dual actions of fibroblast growth factor 19 on lipid metabolism, J. Lipid Res. 54 (2) (2013) 325–332. [15] M. Shiota, R.L. Printz, Diabetes in Zucker diabetic fatty rat, Methods Mol. Biol. 933 (2012) 103–123. [16] J. Siwy, et al., Evaluation of the Zucker diabetic fatty (ZDF) rat as a model for human disease based on urinary peptidomic profiles, PLoS One 7 (12) (2012) e51334. [17] H. Hao, G. Gabbiani, M.L. Bochaton-Piallat, Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development, Arterioscler. Thromb. Vasc. Biol. 23 (9) (2003) 1510–1520.
[18] L. Sjostrom, et al., Bariatric surgery and long-term cardiovascular events, JAMA 307 (1) (2012) 56–65. [19] A. Mor, P. Omotosho, A. Torquati, Cardiovascular risk in obese diabetic patients is significantly reduced one year after gastric bypass compared to one year of diabetes support and education, Surg. Endosc. 28 (10) (2014) 2815– 2820. [20] P. Pujante, et al., Modification of cardiometabolic profile in obese diabetic patients after bariatric surgery: changes in cardiovascular risk, Rev. Esp. Cardiol. (Engl. Ed.) 66 (10) (2013) 812–818. [21] P. Sanchis, et al., Cardiovascular risk profile in Mediterranean patients submitted to bariatric surgery and intensive lifestyle Intervention: impact of both interventions after 1 year of follow-up, Obes. Surg 25 (1) (2015 Jan) 97–108. [22] R. Kohli, et al., Weight loss induced by Roux-en-Y gastric bypass but not laparoscopic adjustable gastric banding increases circulating bile acids, J. Clin. Endocrinol. Metab. 98 (4) (2013) E708–E712. [23] K.K. Ryan, et al., FXR is a molecular target for the effects of vertical sleeve gastrectomy, Nature (2014). [24] A. Nagata, et al., Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic-ligand carrier proteins, Proc. Natl. Acad. Sci. U. S. A. 88 (9) (1991) 4020–4024. [25] T. Tanaka, et al., Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter, J. Biol. Chem. 272 (25) (1997) 15789– 15795. [26] T. Inoue, et al., Serum prostaglandin D synthase level after coronary angioplasty may predict occurrence of restenosis, Thromb. Haemost. 85 (1) (2001) 165– 170.