- Email: [email protected]
DNA methylation suppresses liver Hamp expression in response to iron deficiency after bariatric surgery
Surgery for Obesity and Related Diseases - (2019) 1–10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
Original article
DNA methylation suppresses liver Hamp expression in response to iron deficiency after bariatric surgery Yeping Huanga, Hong Zhanga, Chen Wangb, Jian Zhoua, Yao Lic, Cheng Hua,*
Q1 Q13 a
Shanghai Diabetes Institute, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China b Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China c Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China Received 2 July 2019; accepted 5 October 2019
Q12
Abstract
Background: Iron deficiency is extremely common after bariatric surgery. HEPCIDIN, encoded by Hamp, is a hormone that negatively regulates iron homeostasis. Objectives: We aimed to investigate the alteration of Hamp expression and related regulatory factors to explore the probable role of DNA methylation in modulating Hamp expression in the context of iron deficiency after bariatric surgery. Setting: Laboratories of Diabetes Institute. Methods: RNA-seq was performed using rat liver tissue after either Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy surgery to identify differentially expressed genes between the bariatric surgery and sham group. Hamp expression were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The DNA methylation level was determined using MassARRAY EpiTYPER. Iron status, erythrocyte parameters, and inflammation factors were assessed. Results: RNA-seq data showed that liver Hamp expression changed most dramatically in RYGBoperated rats. Both the mRNA expression of Hamp and the abundance of its protein product HEPCIDIN-25 decreased markedly after bariatric surgery compared with sham, while sleeve gastrectomy–operated rats showed marginally higher Hamp expression than RYGB-operated rats. The DNA methylation level of the Hamp promoter region was significant higher in RYGBoperated rats than sham, while sleeve gastrectomy rats increased slightly in DNA methylation. Consistent with the change of HEPCIDIN-25, serum iron was significantly lower for both bariatric groups than sham and particularly low in RYGB. Conclusions: Our data demonstrate that elevated DNA methylation of the Hamp promoter region suppresses its expression, this epigenetic modification likely occurs in reaction to iron deficiency after bariatric surgery, helping to maintain system iron homeostasis. (Surg Obes Relat Dis 2019;-:1–10.) Ó 2019 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.
Key words:
DNA methylation; Hamp; Iron deficiency; Roux-en-Y gastric bypass; Sleeve gastrectomy
The present study was supported by grants from National Key R&D Program of China (2016 YFC1304801), the Outstanding Academic Leaders of Shanghai Health System program (2017 BR008), the Yangtze River Scholar program, the National Natural Science Foundation of China (81800708), and the Shanghai Sixth People’s Hospital Grant (YNLC201725).
* Correspondence: Cheng Hu, Ph.D., Shanghai Diabetes Institute, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai, 200233, China. E-mail address: [email protected] (C. Hu).
https://doi.org/10.1016/j.soard.2019.10.005 1550-7289/Ó 2019 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
2
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Obesity and type 2 diabetes (T2D) are increasing in prevalence, and they rank among the most devastating health problems worldwide [1,2]. Bariatric/metabolic surgery is currently considered the most effective treatment for morbid obesity and an effective therapy for obese T2D patients [3,4]. Since 2014, sleeve gastrectomy (SG) has become the leading bariatric procedure in the world for its effectiveness in weight loss mainly through its effect on food intake restriction and gastric emptying promotion, follow by Rouxen-Y gastric bypass (RYGB), and one-anastomosis gastric bypass procedures [5,6]. Despite the prominent beneficial effects of bariatric surgery on weight loss and glycemic control, it is worth noting that nonnegligible nutritional complications usually occur after bariatric procedures. Among the numerous nutritional complications, iron deficiency (with or without anemia) is extremely common after bariatric surgery, especially RYGB, and the extent of iron depletion after bariatric surgery ranges from 30% to 60% [7]. HEPCIDIN is a 25-amino-acid peptide hormone critical in regulating iron homeostasis [8]. There are 3 isoforms for HEPCIDIN, numbered 20, 22, 25, of which HEPCIDIN-25 is the active form and the most extensively studied of the 3 [9]. Hepatocytes are the predominant producers of HEPCIDIN and 1 of the storage cells for iron. HEPCIDIN negatively regulates the main iron flows that enter the plasma compartment from macrophages and hepatocytes by degrading the iron exporter ferroportin-1, limits the absorption of dietary iron in the duodenum, reducing iron bioavailability. In turn, the synthesis of HEPCIDIN is feedback regulated by iron, such that hepatocytes will produce more HEPCIDIN when iron is abundant, limiting further iron absorption and release of stored iron [10]. Apart from iron, HEPCIDIN synthesis by hepatocytes is inhibited by erythropoietic activity and promoted by inflammation mainly through interleukin-6 (IL-6) and other mediators [8]. Epigenetics principally concerns mechanisms for modifying gene expression at the transcriptional level and affecting gene function without alterations in DNA sequences [11]. DNA methylation is a vitally important epigenetic factor influencing gene activities; this modification occurs almost exclusively at cytosine residues linked to guanine residues (CpG sites), where a methyl radical (CH3) is covalently added to the cytosine base [12]. DNA methylation in different genomic regions may exert different impacts on gene activities, while methylation in the promoter region is typically associated with stable gene silencing [13]. In recent years, some evidence has suggested that bariatric surgery can change DNA methylation patterns [14]. Multiple studies have revealed the effects of environmental exposure on the epigenome. One study has shown that hypermethylation of the ACSL3 gene is correlated with increased maternal polyaromatic hydrocarbon exposure [15]. Furthermore, emerging evidence suggests that environmental factors (nutrition, diet, residence/workplace, pharmacologic treatments) affect epigenetic patterns;
however, it must be acknowledged that the link between environment and epigenetics largely remains to be explored [16]. Iron deficiency is well recognized as a nutritional complication that can result from bariatric surgery. Nevertheless, the underlying mechanism through which system ironregulating elements react system-wide to this accumulating environment change remains poorly understood. We hypothesized that DNA methylation may be involved in modulating the expression of iron-regulated genes and helping to maintain iron homeostasis. We aimed to investigate whether DNA methylation plays a role in Hamp expression and investigate the changes of related regulatory factors of HEPCIDIN, the protein product of Hamp. Therefore, the expression of iron-regulating genes and their related or possible regulatory factors, including DNA methylation, iron status, erythrocyte parameters, and inflammation, were investigated; we expected to determine the potential mechanism by which the system adapts to environmental change—in this case, iron deficiency after bariatric surgery.
Methods Rats All animals were maintained and used in accordance with the guidelines of the Animal Care and Use Committee of Shanghai Sixth People’s Hospital. Eight-week-old male Sprague-Dawley rats were purchased from Shanghai Sipper-BK Lab Animal Co. Ltd. (Shanghai, China) and maintained under specific pathogen-free conditions (20 C–24 C, 12/12-hr light-dark cycle) with free access to food and water. Rats were fed either a chow diet or a high-fat diet (D12492; Research Diets, New Brunswick, NJ, USA) for 8 weeks, the latter of which induced obesity. The rats were then given a single low dose of streptozotocin (Sigma, Saint Louis, MO, USA) to induce T2D. Rats with random blood glucose levels 16.7 mM were considered successful models of T2D. The obese diabetic rats were randomly allocated to the sham, RYGB, or SG groups and then underwent sham, RYGB, or SG surgery 2 weeks after streptozotocin injection. RYGB, SG, and sham surgery were performed according to procedures described by Yan et al. [17] and Patrikakos et al. [18]. Eight weeks after surgery, rats were euthanized with sodium pentobarbital (.05 mg/g), and the liver tissue was removed, snap-frozen, and stored in liquid nitrogen before use. Four independent batches of obese diabetic rats underwent bariatric surgery. The first batch included sham (n 5 4) and RYGB (n 5 5; 2 rats died after surgery and were excluded); the second batch included sham (n 5 5) and RYGB (n 5 5; 1 rat died after surgery and was excluded); the third batch included sham (n 5 5) and RYGB (n 5 6; 1 rat died after surgery and was excluded); and the fourth
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
batch included sham (n 5 4), RYGB (n 5 6; 2 rats died and were excluded), and SG (n 5 4). Surgical procedures
Q2
For RYGB surgery, a 3.5- to 4-cm midline incision was made, the stomach was transected approximately 5 mm below the gastroesophageal junction from the lesser to the greater curvature, and the proximal and distal stomach was closed with 5-0 silk sutures (Ningbo Medical Needle, City, China) using an interrupted suture technique. Approximately 20% of the stomach was retained. Two ligations were placed on the jejunum 10 cm below the Treitz ligament using 5-0 silk sutures, and the intestine was transected between the 2 ligatures. A gastrojejunostomy was created between the distal jejunum and the anterior wall of the gastric pouch by performing a 7-mm side-to-side anastomosis. The proximal jejunum was connected to the jejunum 15 cm distal to the gastrojejunal anastomosis with a 7-mm sideto-side jejunojejunostomy. For SG, in brief, the greater curvature of the proximal from the antrum to the fundus and the distal stomach was incised, approximately 70% to 80% gastric volume was removed, including 80% to 90% of the proximal stomach and 70% of the distal stomach. The abdominal wall and skin were closed with continuous 4-0 silk sutures (Ningbo Medical Needle). For sham-operated rats, a 3.5- to 4-cm middle incision was made. Laparotomy was performed to expose the esophagus, stomach, and small intestine. No other procedure was carried out. Muscle layers and skin were closed using continuous 4-0 silk sutures (Ningbo Medical Needle) in the same manner as in RYGB and SG groups. Postoperative care After operation, 20 mL/kg (weight) saline solution were injected subcutaneously to hydrate the rats. The rats were fasted on the day of surgery, had free access to water the day after, were given 10% Ensure (Abbott, Lake Bluff, IL, USA) for 1 week from the third day after surgery, and then were maintained on 60% high-fat diet. The weight was measured at baseline before surgery and weekly after surgery after a 12-hour overnight fast. Metabolic analyses
Q3
Q4
The weight was monitored at baseline and weekly after bariatric surgery. For glucose tolerance test, rats were fasted overnight for approximately 16 hours and then injected intraperitoneally with glucose solution at 1 g/kg. For insulin tolerance test, rats were fasted for 4 hours and injected intraperitoneally with insulin (novolin; Novo Nordisk, city, Denmark) solution at .5 IU/kg. Blood glucose was measured before and after either glucose or insulin injection with glucometer (Accu-CheK; Roche, city, state, country).
3
RNA-seq The liver tissue from first batch (sham n 5 4, RYGB n 5 3) and the fourth batch (sham n 5 4, SG n 5 4) of rats underwent bariatric surgery were used to perform RNA-seq. Total RNA was extracted from rat liver tissue using TRIzol (Life Technologies, Carlsbad, CA, USA). RNA quality and quantity were detected by agarose gel electrophoresis and Qubit Fluorometric Quantitation (Life Technologies Corporation), respectively. PolyA-tailed cDNA libraries were constructed using the TruSeq RNA LT Sample Prep Kit v2 (Illumina, Inc., San Diego, CA, USA). Sample preparation followed the manufacturer’s protocol with a workflow, including isolation of polyadenylated RNA molecules, RNA fragmentation, cDNA synthesis, ligation of adapters, and PCR amplification. Sequencing of 2 ! 150-bp paired-end reads was performed with an Illumina HiSeq 3000. RSeQC (version 2.6.4) was applied to evaluate the quality of RNA-seq data. Then, the clean reads were aligned to the index file of the rn6 genome generated by HISAT2 (version 2.0.5). Read counts for each gene were generated by HTSeq-count, and the normalization and differentially expressed genes (DEGs) were determined by the DESeq2 package in R. DEGs were identified using cutoff values of 2-fold change between the sham and bariatric surgery groups and an adjusted P , .05.
DNA methylation determination The liver tissue from the third and fourth batches of rats that underwent bariatric surgery were used to analyze DNA methylation level of Hamp promoter region (sham n 5 9, RYGB n 5 9, SG n 5 4). Genomic DNA was isolated from rat liver tissue using a QIAamp Fast DNA Tissue Kit (Qiagen, Hilden, Germany). Bisulfite conversion of genomic DNA was performed using the EpiTect Fast Bisulfite Conversion Kit (Qiagen). The promoter region, defined as the region covering 1500 bp upstream and 500 bp downstream of the transcription start site, was selected to analyze the CpG methylation level. Target-specific primer pairs to amplify bisulfite-treated genomic DNA were designed using the EpiDesigner tool (Agena Bioscience, Inc., San Diego, CA, USA). Primers were synthesized by Invitrogen, and each reverse primer had a T7 promoter tag (50 -CAGTAATACGACTCACTATAGGGAGAAGGCT-30 ) for transcription. The forward primer was tagged with a 10-mer (50 AGGAAGAGAG-30 ) to balance the melting temperature. The polymerase chain reaction (PCR)-amplified products were treated with shrimp alkaline phosphatase, and in vitro transcription and base-specific cleavage were performed simultaneously. The resulting DNA fragments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and EpiTYPER Q5 (manufacturer, city, state, country) was used for the quantification of CpG methylation level.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348
4
349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408
Q6
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Quantitative PCR
Statistical analysis
The liver tissue (sham n 5 4, RYGB n 5 4, SG n 5 4) from the fourth batch of rats underwent bariatric surgery were used to validate the RNA-seq results. Total RNA was isolated from rat liver tissue using TRIzol (Life Technologies). One microgram of RNA was transcribed to cDNA using a PrimeScrip RT Reagent Kit (manufacturer, city, state, country). Gene-specific primers were synthesized by Sangon Biotech (Shanghai, China). FastStart Universal SYBR Green Master (ROX; Roche, Mannheim, Germany) was used for quantitative PCR on an ABI 7900 HT Fast RealTime PCR System (Applied Biosystems, Foster, CA, USA). Samples were analyzed in triplicate. Gene expression was expressed as 2(2DDCT) and normalized to the housekeeping gene b-Actin. Primer sequences are listed in the Supplementary Table.
All data are presented as the mean 6 SEM. Statistical significance (*P , .05, **P , .01, ***P , .001) was assessed with unpaired t tests or Mann-Whitney U tests (2-sided in both cases).
Erythrocyte parameters, serum HEPCIDIN-25, and iron status determination
Q7
All the serum samples from the 4 batches of rats underwent bariatric surgery were used to measure the serum HEPCIDIN-25 concentration and iron status (sham n 5 18, RYGB n 5 16, SG n 5 4). The serum samples from the fourth batch were used to determine the erythrocyte parameters (sham n 5 4, RYGB n 5 4, SG n 5 4). Whole blood or serum was collected 8 weeks after bariatric surgery. Blood samples were obtained after a 12-hour overnight fast. Erythrocyte parameters, including red blood cell, hemoglobin, hematocrit, and mean corpuscular volume, were determined using an automatic hematology analyzer (XN-350; Sysmex, city, state, country) with whole-blood samples. Serum HEPCIDIN-25 levels were detected using a rat serum HEPCIDIN-25 kit (Peninsula Laboratories International, Inc., San Carlos, CA, USA). Serum iron, ferritin, and unsaturated iron-binding capacity were measured using an automatic biochemical analyzer (Mindray BS-800, Shenzhen, China). Anemia is defined as hemoglobin 120 g/L, serum iron ,10 mmol/L, total iron-binding capacity (TIBC) .60 mmol/L.
Rat serum inflammatory cytokine detection
Q8
All the serum samples (sham n 5 18, RYGB n 5 16, SG n 5 4) from the 4 batches of rats underwent bariatric surgery were used to determine inflammatory factors. IL-6, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor (TNF-a) concentrations were quantified using a Rat Metabolic Hormone Panel Milliplex kit (Millipore, St. Charles, MO, USA) and a Luminex instrument (manufacturer, city, state, country) following the manufacturer’s specifications. The sensitivity for the Milliplex Kit (pg/mL) was 38 (IL-6), 34 (MCP-1), and 2 (TNF-a).
Results RNA-seq data demonstrated that liver Hamp mRNA expression changed most significantly after RYGB or SG surgery Both RYGB- and SG-operated rats achieved significant weight loss compared with preoperative status and improved glycemic control compared with sham-operated rats, and this benefit effect was more remarkable for RYGB-operated rats (Figs. S1 and S2). To characterize the transcriptomic differences between sham and bariatric surgery, including RYGB and SG, gene expression profiles were analyzed. Thresholds of jlog2 FoldChangej 1 and false discovery rate ,.05 were set to determine significantly different expression genes. For RYGB, 149 downregulated genes and 71 upregulated genes were identified compared with the sham group (Fig. 1A). For SG, 160 downregulated genes and 218 upregulated genes were detected compared with the sham group (Fig. 1B). Both the top 10 upregulated and downregulated genes for RYGB and the corresponding expression change levels of these genes in SG were exhibited in the heatmap (Fig. 1C). Among the top DEGs, Hamp ranked first in RYGB and had the maximum change in gene expression. A significant change in Hamp expression was also observed in SG. Liver Hamp expression changed dramatically after RYGB or SG surgery as confirmed by quantitative PCR To validate the RNA-seq results, several genes, including Hamp, Nox4, Litaf, Abca8, Npas2, Arntl, Abcg5, and Grem2, were analyzed by quantitative PCR, and the results were consistent with those of RNA-seq (Fig. 2). As showed by quantitative PCR (Fig. 2 the upper graph), the mRNA expression of Hamp was relatively low after RYGB surgery compared with sham-operated rats, while SG-operated rats showed marginally higher expression of Hamp than RYGB-operated rats. Consistent with quantitative PCR results, RNA-seq data (Fig. 2 the lower table) manifested larger fold change for RYGB than SG when both compared with sham-operated rats. The abundance of Hamp protein product HEPCIDIN-25 is markedly decreased after bariatric surgery Because the mRNA expression of Hamp changed the most drastically with the greatest significance, the serum protein level of HEPCIDIN-25, the active form of HEPCIDIN, was measured. The results showed that the serum
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468
469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528
5
web 4C=FPO
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Fig. 1. Hamp mRNA expression changed most significantly after RYGB and SG surgery. (A) Volcano plots of RNA-seq data showing significant differentially expressed genes between RYGB and sham. Each gene (dot) is plotted based on its expression level in log2(fold change) and –log10(false discovery rate). Most genes cluster around 0, indicating no change in expression. Blue dots indicate downregulated genes (log2(fold change) 1, false discovery rate ,.05), and red dots indicate upregulated genes (log2(fold change) 1, false discovery rate ,.05). The genes of interest with the most significant changes are indicated. (B) Volcano plots of RNA-seq data showing significant differentially expressed genes between sleeve gastrectomy and sham. (C) Heatmap representing the top 10 differentially expressed genes (base mean .1000) between the bariatric surgery group and the sham group. Colors ranging from blue to red on the heatmap reflect degrees of expression ranging from low to high, as shown on the scale in the upper right corner of the figure.
HEPCIDIN-25 level of rats after RYGB and SG surgery declined dramatically compared with sham, while the value of SG was slightly higher than RYGB, which is consistent with the quantitative PCR results (Fig. 3). DNA methylation of Hamp promoter region increased significantly after RYGB surgery DNA methylation is a major epigenetic factor influencing gene transcription and activities. It is speculated that DNA methylation possibly contributes to changes in Hamp gene
expression. With this assumption, the DNA methylation level of the promoter region of Hamp was determined. The results demonstrated that 5 of 12 CpG sites showed a significant increase in methylation level in RYGB compared with sham, while another 7 CpG sites showed statistically nonsignificant increase. For SG, among 12 CpG sites, 1 site showed a significant increase in CpG methylation level compared with the sham group, while 6 CpG sites showed slight increases that were not statistically significant (Fig. 4). Specific information for each CpG site is shown in Table 1.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588
6
589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Fig. 2. Quantitative polymerase chain reaction analysis to validate the RNA-seq results. Data were normalized to b-Actin mRNA expression. Data represent 2 independent experiments. Data are presented as the mean 6 standard error of the mean. Statistical differences were analyzed with the unpaired t test (n 5 4 for each group). *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between sleeve gastrectomy and sham. The lower table shows the results from RNA-seq data between the 2 bariatric surgery groups and the sham group.
RYGB-operated rats developed anemia while SG-operated rats showed iron deficiency HEPCIDIN is the principal regulator of extracellular iron homeostasis in health and disease. In addition, HEPCIDIN is feedback regulated by the substance whose concentration it controls: iron. Additionally, bariatric surgeries, including RYGB and SG, are reported to affect iron status to some extent; therefore, the serum iron status of rats after bariatric surgery was investigated. As shown in Fig. 5A, serum iron levels declined sharply 8 weeks after RYGB surgery, while rats after SG surgery demonstrated a significant decrease in serum iron levels compared with sham rats but a slight increase compared with RYGB rats. In addition, both the RYGB and SG groups showed significantly higher levels of unsaturated iron-binding capacity and TIBC than the sham group, while higher TIBC levels indicated iron deficiency status after RYGB and SG surgery (Figs. 5C–D). No significant changes were observed for ferritin in either RYGB or SG rats compared with sham-operated rats (Fig. 5B). Consistent with the severe iron-deficiency status, anemia is quite serious for RYGB-operated rats, as indicated by significant lower level of red blood cells, hemoglobin,
Fig. 3. Serum HEPCIDIN-25 concentration declined dramatically after Roux-en-Y gastric bypass and sleeve gastrectomy surgery. Data are presented as the mean 6 standard error of the mean for the preoperative (n 5 17), sham (n 5 18), Roux-en-Y gastric bypass (n 5 16), and sleeve gastrectomy rats (n 5 4). *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between sleeve gastrectomy and sham.
hematocrit, and mean corpuscular volume compared with sham-operated rats. In contrast to RYGB-operated rats, those underwent SG did not seem to develop anemia despite for observable decrease of serum iron compared with shamoperated rats (Figs. 5E–F). Inflammation profile showed minimal change after both bariatric surgery Apart from serum iron, inflammation factor also regulates HEPCIDIN synthesis, among which IL-6 is the most extensively studied known to regulate HEPCIDIN transcription in hepatocytes. The results demonstrated that the proinflammatory factors IL-6, MCP-1, and TNF-a did not show significant changes, despite profound effect of weight loss and remission of T2D, especially for RYGB-operated rats (Supplementary Figs. S1–S3). Discussion In this study, RNA-seq data demonstrated that liver Hamp expression was reduced most remarkably after RYGB surgery compared with sham, while a significant change in Hamp was also observed after SG surgery. The consensus finding is that HEPCIDIN-25, the only isoform of HEPCIDIN known to be associated with iron regulation, is dramatically decreased after both bariatric surgeries. Similar observations were reported that serum HEPCIDIN levels after bariatric procedures (gastric banding, SG, or RYGB) were significantly lower
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
web 4C=FPO
709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768
7
Fig. 4. Methylation status for each CpG site of the Hamp promoter in bariatric surgery and sham. Values are presented as the mean 6 standard error of the mean, sham (n 5 9), Roux-en-Y gastric bypass (n 5 9), and sleeve gastrectomy (n 5 4). The lower schematic diagram exhibits CpG sites in the Hamp promoter. CpG sites are indicated with lollipop-shaped markers. The binding sites for the forward and reverse primers are shown with arrows below the diagram. The number of lollipop-shaped markers indicates the number of detected CpG sites. *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between sleeve gastrectomy and sham.
in obese individuals compared with baseline [19–21]. It is noteworthy in our study that the methylation level of the Hamp promoter region significantly increased after RYGB surgery, while the SG group increased slightly in the methylation level, indicating that significantly elevated methylation levels after bariatric surgery likely
contribute to the decline in Hamp expression and consequently decrease serum HEPCIDIN-25 levels. Reduction in HEPCIDIN-25 likely allowed for enhanced dietary absorption, which is relatively critical for helping the system adapt to its iron-depleted status postoperatively.
Table 1 Specific information on each CpG site CpG site
Position
DMethylation (%) (RYGB–sham)
DMethylation (%) (SG–sham)
P value (RYGB versus sham)
P value (SG versus sham)
CpG_1 CpG_2 CpG_3 CpG_4 CpG_5 CpG_6 CpG_7 CpG_8 CpG_9 CpG_10 CpG_11
Chr1:89371083 Chr1:89371077 Chr1:89371027 Chr1:89370496 Chr1:89370475 Chr1:89370454/89370451 Chr1:89370399 Chr1:89369928 Chr1:89369878 Chr1:89369865 Chr1:89369708
12.6 7.4 4.9 12.0 11.4 13.3 6.6 15.7 4.3 6.8 3.0
-3.2 5.1 -1.1 8.1 4.8 7.5 3.6 4.7 3.8 3.7 -1.3
.002 .002 .1 .016 .057 .058 .2 .07 .046 .2 .006
.09 .003 .2 .5 .1 .3 .7 .5 .2 .8 .056
CpG site 5 representing each CpG site indicated in the main text; Position 5 the position of each CpG site on the chromosome; DMethylation 5 difference of methylation level between 2 comparing groups; RYGB 5 Roux-en-Y gastric bypass; Sham 5 sham operation; SG 5 sleeve gastrectomy; RYGB–sham 5 the difference of DNA methylation level between RYGB and sham group; SG–sham 5 the difference of DNA methylation level between SG and sham group; RYGB versus sham 5 the P value between RYGB and sham group; SG versus sham 5 the P value between SG and sham group.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828
8
829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Fig. 5. Iron status and erythrocyte parameters of rats underwent bariatric surgery or sham operation. Data are presented as the mean 6 standard error of the mean. (A–D) Sham (n 5 18), Roux-en-Y gastric bypass (n 5 16), and sleeve gastrectomy (n 5 4); (E–H) sham (n 5 4), Roux-en-Y gastric bypass (n 5 4), and sleeve gastrectomy (n 5 4). *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between SG and sham.
It is generally known that HEPCIDIN, which regulates system iron homeostasis, can be feedback modulated by iron. The present study demonstrated obvious iron deficiency with anemia in RYGB-operated rats, characterized by significantly reduced serum iron, elevated TIBC, poor functional iron status (hemoglobin, hematocrit, mean corpuscular volume). By contrast, despite an observable decrease of serum iron, iron deficiency–induced anemia did not occur for SG-operated rats, indicated by nearly unchanged erythrocyte parameters. It is worth noting that the serum iron of the SG group was slightly higher than that of the RYGB group, which is consistent with the observation that Hamp expression and the HEPCIDIN level of the SG group were marginally higher than those of the RYGB group, although this difference was not statistically significant. The differences in serum iron and Hamp expression between the RYGB and SG groups could be ascribed to the fact that RYGB surgery limits both food intake and nutrient absorption, while SG is perceived as a restrictive surgical approach that primarily limits food intake [22]. In particular, RYGB surgery involves gut manipulation where, excluding the duodenum, iron uptake happens from the alimentary tract resulting in iron malabsorption. Therefore, iron deficiency status due to poorer nutrition in the RYGB group is more common and more severe than in the SG group, which is confirmed by the observation that RYGBoperated rats were in poor functional iron status, while SG-operated rats did not show anemia despite for observable iron deficiency just as demonstrated in this study. To respond to lower serum iron, HEPCIDIN synthesis is downregulated to maintain iron homeostasis by increasing CpG site methylation of the Hamp promoter. In addition, inflammation can stimulate HEPCIDIN synthesis. Upon analyzing changes in inflammation after
bariatric surgery, no significant change in IL-6 and MCP-1 concentrations 8 weeks after surgery in the case of either RYGB or SG was found. Obesity is considered a chronic low-grade inflammatory state [5], which is thought to be involved in the development of obesity and T2D. Bariatric surgery is reported to have weight loss of .50% of weight [23]. Along with substantial weight loss, the inflammatory state is mitigated, and C-reactive protein, MCP-1, and IL6 decrease after bariatric surgery [19,24]. However, in line with this study, numerous studies have shown that IL-6 remains unchanged after bariatric surgery, despite metabolic improvement [25–27]. Little change of inflammatory profile in this study was likely account for exist of postoperative inflammation, which commonly occur after bariatric surgery. In contrast to IL-6 and TNF-a, C-reactive protein seems to change uniformly, undergoing a marked decline after bariatric surgery [19,20,27]. It is important to note that IL-6 is the key inflammation factor reported to promote Hamp expression; nevertheless, the effect of IL-6 on Hamp transcription seems to be limited, as no change in its serum level was observed in the present study. Furthermore, in addition to being an important iron stores marker, ferritin is an acute-phase reactant, which is supposed to increase under system inflammation status, even in irondeficient conditions [28]. Just as shown in present study, serum ferritin level 8 weeks after both RYGB and SG surgery remains nearly unchanged. Minimal change of inflammatory profile and ferritin level indicate that inflammatory signal may not have a major impact on HEPCIDIN production. Previous studies reported the association of serum HEPCIDIN level with diabetes [29], although the results were inconsistent. We used an obese diabetic rat model in present study. To rule out the potential impact of diabetic status on
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008
Q9
HEPCIDIN level, we set up an appropriate control—sham operation; those rats that underwent bariatric surgery were in the same background, and both were obese diabetic rats. Therefore, it is scientific and reasonable to conclude that the changes of HEPCIDIN level were result from implementation of surgical operations but not other potential factors. It is of great importance to improve and advance the indicators of bariatric/metabolic surgery to obtain maximum benefits and minimize potential complications. Here, we assume that the baseline serum HEPCIDIN level may be used as one of the indicators in a future scenario of customized procedures for patients underwent bariatric/metabolic surgery. This assumption is based on 2 facts as follows: (1) the function of HEPCIDIN is to negatively regulate iron from entering the plasma from the stores and limit iron uptake from diet, therefore the serum HEPCIDIN level can indicate the system iron regulatory status; and (2) different surgical techniques for bariatric/metabolic surgery result in different reduction in Hamp expression, as demonstrated in our study, the room for reduction may reflect the capacity of system to regulate iron status. However, it should be prudent, rather than using serum HEPCIDIN level solely to assess the iron status, to use a combination of serum HEPCIDIN level, biochemical markers (ferritin, serum iron, TIBC, soluble transferrin receptor), and erythrocyte parameters (hemoglobin, mean corpuscular volume); this could give more comprehensive insight into the iron and anemia status. Furthermore, due care should be taken when interpreting the data of serum HEPCIDIN level because HEPCIDIN synthesis can be influenced by inflammatory disorders; thus, examination of the inflammation status is also necessary. Simultaneously, a standard range of HEPCIDIN level for healthy population should be set up, which can provide reference for evaluating serum HEPCIDIN level. Taking the 4 elements (serum HEPCIDIN level, biochemical markers of iron status, erythrocyte parameters, and inflammatory status) into consideration, a relative comprehensive understanding of iron metabolism will be obtained and is likely to help in choosing the superior surgical techniques. For instance, for patients with higher baseline serum HEPCIDIN level and without iron deficiency/anemia, surgical techniques (such as RYGB, one-anastomosis gastric bypass procedures), which can lead to more effective weight loss and/or metabolic improvement, would be more appropriate. In contrast, for patients with lower baseline serum HEPCIDIN level and with iron deficiency/anemia, a surgical technique having lower risk of anemia, such as SG, could be a better choice. Our study had strengths and limitations. First, although it has been confirmed in the present study that iron deficiency occurred after bariatric surgery, no direct determination of iron absorption or iron transporter ferroportin-1 expression at the duodenal enterocyte was evaluated; this awaits further investigation. Second, it is reported here that elevated DNA methylation at Hamp promoter region likely contributes to
9
significantly decrease Hamp expression in response to iron deficiency after bariatric surgery; however, the underlying mechanism of how the changing internal environment (iron deficiency) affects gene methylation status and eventually impacts gene function warrants further study to confirm our findings. Finally, due to the unavailability of human tissue samples after bariatric surgery, the methylation status of Hamp in individuals who underwent bariatric surgery could not be verified. Conclusions In summary, we reported first that DNA hypermethylation in the Hamp promoter region may contribute to remarkable suppression of Hamp expression, resulting in reduced HEPCIDIN levels in response to iron deficiency after bariatric surgery, especially for RYGB, helping to maintain iron homeostasis. Our study provides a potential basis for modulating iron homeostasis by DNA methylation of ironregulating elements after bariatric surgery. The epigenetic influences on iron homeostasis after bariatric surgery may be closely linked, and the underlying mechanism warrants further investigation. Acknowledgments Yeping Huang and Hong Zhang contributed equally to this work. Disclosures The authors have no commercial associations that might be a conflict of interest in relation to this article. Supplementary materials Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/ j.soard.2019.10.005. References
Q10
[1] Obesity and overweight. Fact sheet. Geneva: World Health Organization; c2019 [updated 2018 Feb 16; cited yr mo d]. Available from: https://www. who.int/news-room/fact-sheets/detail/obesity-and-overweight. Q11 [2] Ogurtsova K, da Rocha Fernandes JD, Huang Y, et al. IDF Diabetes Atlas: global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract 2017;128:40–50. [3] Malin SK, Kashyap SR. Differences in weight loss and gut hormones: Roux-en-Y gastric bypass and sleeve gastrectomy surgery. Curr Obes Rep 2015;4(2):279–86. [4] Mingrone G, Panunzi S, De Gaetano A, et al. Bariatric surgery versus conventional medical therapy for type 2 diabetes. N Engl J Med 2012;366(17):1577–85. [5] Batterham RL, Cummings DE. Mechanisms of diabetes improvement following bariatric/metabolic surgery. Diabetes Care 2016;39(6):893– 901. [6] Welbourn R, Hollyman M, Kinsman R, et al. Bariatric surgery worldwide: baseline demographic description and one-year outcomes from
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068
10
1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128
[7]
[8] [9]
[10] [11]
[12] [13] [14]
[15]
[16] [17]
[18]
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10 the Fourth IFSO Global Registry Report 2018. Obes Surg 2019;29(3):782–95. Mu~ noz M, Botella-Romero F, Gomez-Ramırez S Campos A, GarcıaErce JA. Iron deficiency and anaemia in bariatric surgical patients: causes, diagnosis and proper management. Nutr Hosp 2009;24(6):640–54. Ganz T, Nemeth E. HEPCIDIN and iron homeostasis. Biochim Biophys Acta 2012;1823(9):1434–43. Addo L, Ikuta K, Tanaka H, et al. The three isoforms of HEPCIDIN in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS). Int J Hematol 2016;103(1):34–43. Ganz T, Nemeth E. HEPCIDIN and disorders of iron metabolism. Annu Rev Med 2011;62(1):347. Dupont Catherine, Armant D, Brenner C. Epigenetics: definition, mechanisms and clinical perspective. Semin Reprod Med 2009;27(5):351–7. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16(1):6–21. Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology 2013;38(1):23–38. Nicoletti CF, Nonino CB, de Oliveria BA, et al. DNA methylation and hydroxymethylation levels in relation to two weight loss strategies: energy-restricted diet or bariatric surgery. Obes Surg 2015;26(3):1–9. Perera F, Tang WY, Herbstman J, et al. Relation of DNA methylation of 50 -CpG island of ACSL3 to transplacental exposure to airborne polycyclic aromatic hydrocarbons and childhood asthma. PLoS One 2009;4(2):e4488. Feil R. Environmental and nutritional effects on the epigenetic regulation of genes. Mutat Res 2006;600(1-2):46–57. Yan Y, Zhou Z, Kong F, et al. Roux-en-Y Gastric bypass surgery suppresses hepatic gluconeogenesis and increases intestinal gluconeogenesis in a type 2 diabetes rat model. Obes Surg 2016;26(11):2683–90. Patrikakos P, Toutouzas KG, Perrea D, et al. A surgical rat model of sleeve gastrectomy with staple technique: long-term weight loss results. Obes Surg 2009;19(11):1586–90.
[19] Tussinghumphreys LM, Nemeth E, Fantuzzi G, et al. Decreased serum HEPCIDIN and improved functional iron status 6 months after restrictive bariatric surgery. Obesity 2010;18(10):2010–6. [20] Cepedalopez AC, Allende-Labastida J, Melse-Boonstra A, et al. The effects of fat loss after bariatric surgery on inflammation, serum HEPCIDIN, and iron absorption: a prospective 6-mo iron stable isotope study. Am J Clin Nutr 2016;104(4):1030. [21] Gesquiere I, Foulon V, Augustijns P, et al. Micronutrient intake, from diet and supplements, and association with status markers in pre- and post-RYGB patients. Clin Nutr 2016;36(4):1175–81. [22] Sala PC, Torrinhas RS, Giannella-Neto D, Waitzberg DL. Relationship between gut hormones and glucose homeostasis after bariatric surgery. Diabetol Metab Syndr 2014;6(1):87. [23] Shoelson SE, Herrero L, Naaz A. Obesity, inflammation, and insulin resistance. Gastroenterology 2014;132(6):2169–80. [24] Buchwald H, Estok R, Fahrbach K, et al. Weight and type 2 diabetes after bariatric surgery: systematic review and meta-analysis. Am J Med 2009;122(3):248–56. [25] Monte SV, Caruana JA, Ghanim H, et al. Reduction in endotoxemia, oxidative and inflammatory stress, and insulin resistance after Rouxen-Y gastric bypass surgery in patients with morbid obesity and type 2 diabetes mellitus. Surgery 2012;151(4):587–93. [26] Netto BD, Bettini SC, Clemente AP, et al. Roux-en-Y gastric bypass decreases pro-inflammatory and thrombotic biomarkers in individuals with extreme obesity. Obes Surg 2015;25(6):1010–8. [27] Miller GD, Nicklas BJ, Fernandez A. Serial changes in inflammatory biomarkers after Roux-en-Y gastric bypass surgery. Surg Obes Relat Dis 2011;7(5):618–24. [28] Baynes R, Bezwoda W, Bothwell T, Khan Q, Mansoor N. The nonimmune inflammatory response: serial changes in plasma iron, ironbinding capacity, lactoferrin, ferritin and C-reactive protein. Scand J Clin Lab Invest 1986;46(7):695–704. [29] Aregbesola A, Voutilainen S, Virtanen JK, Aregbesola A, Tuomainen T. Serum hepcidin concentrations and type 2 diabetes. World J Diabetes 2015;6(7):978–82.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
Original article
DNA methylation suppresses liver Hamp expression in response to iron deficiency after bariatric surgery Yeping Huanga, Hong Zhanga, Chen Wangb, Jian Zhoua, Yao Lic, Cheng Hua,*
Q1 Q13 a
Shanghai Diabetes Institute, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China b Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China c Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China Received 2 July 2019; accepted 5 October 2019
Q12
Abstract
Background: Iron deficiency is extremely common after bariatric surgery. HEPCIDIN, encoded by Hamp, is a hormone that negatively regulates iron homeostasis. Objectives: We aimed to investigate the alteration of Hamp expression and related regulatory factors to explore the probable role of DNA methylation in modulating Hamp expression in the context of iron deficiency after bariatric surgery. Setting: Laboratories of Diabetes Institute. Methods: RNA-seq was performed using rat liver tissue after either Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy surgery to identify differentially expressed genes between the bariatric surgery and sham group. Hamp expression were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The DNA methylation level was determined using MassARRAY EpiTYPER. Iron status, erythrocyte parameters, and inflammation factors were assessed. Results: RNA-seq data showed that liver Hamp expression changed most dramatically in RYGBoperated rats. Both the mRNA expression of Hamp and the abundance of its protein product HEPCIDIN-25 decreased markedly after bariatric surgery compared with sham, while sleeve gastrectomy–operated rats showed marginally higher Hamp expression than RYGB-operated rats. The DNA methylation level of the Hamp promoter region was significant higher in RYGBoperated rats than sham, while sleeve gastrectomy rats increased slightly in DNA methylation. Consistent with the change of HEPCIDIN-25, serum iron was significantly lower for both bariatric groups than sham and particularly low in RYGB. Conclusions: Our data demonstrate that elevated DNA methylation of the Hamp promoter region suppresses its expression, this epigenetic modification likely occurs in reaction to iron deficiency after bariatric surgery, helping to maintain system iron homeostasis. (Surg Obes Relat Dis 2019;-:1–10.) Ó 2019 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.
Key words:
DNA methylation; Hamp; Iron deficiency; Roux-en-Y gastric bypass; Sleeve gastrectomy
The present study was supported by grants from National Key R&D Program of China (2016 YFC1304801), the Outstanding Academic Leaders of Shanghai Health System program (2017 BR008), the Yangtze River Scholar program, the National Natural Science Foundation of China (81800708), and the Shanghai Sixth People’s Hospital Grant (YNLC201725).
* Correspondence: Cheng Hu, Ph.D., Shanghai Diabetes Institute, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai, 200233, China. E-mail address: [email protected] (C. Hu).
https://doi.org/10.1016/j.soard.2019.10.005 1550-7289/Ó 2019 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
2
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Obesity and type 2 diabetes (T2D) are increasing in prevalence, and they rank among the most devastating health problems worldwide [1,2]. Bariatric/metabolic surgery is currently considered the most effective treatment for morbid obesity and an effective therapy for obese T2D patients [3,4]. Since 2014, sleeve gastrectomy (SG) has become the leading bariatric procedure in the world for its effectiveness in weight loss mainly through its effect on food intake restriction and gastric emptying promotion, follow by Rouxen-Y gastric bypass (RYGB), and one-anastomosis gastric bypass procedures [5,6]. Despite the prominent beneficial effects of bariatric surgery on weight loss and glycemic control, it is worth noting that nonnegligible nutritional complications usually occur after bariatric procedures. Among the numerous nutritional complications, iron deficiency (with or without anemia) is extremely common after bariatric surgery, especially RYGB, and the extent of iron depletion after bariatric surgery ranges from 30% to 60% [7]. HEPCIDIN is a 25-amino-acid peptide hormone critical in regulating iron homeostasis [8]. There are 3 isoforms for HEPCIDIN, numbered 20, 22, 25, of which HEPCIDIN-25 is the active form and the most extensively studied of the 3 [9]. Hepatocytes are the predominant producers of HEPCIDIN and 1 of the storage cells for iron. HEPCIDIN negatively regulates the main iron flows that enter the plasma compartment from macrophages and hepatocytes by degrading the iron exporter ferroportin-1, limits the absorption of dietary iron in the duodenum, reducing iron bioavailability. In turn, the synthesis of HEPCIDIN is feedback regulated by iron, such that hepatocytes will produce more HEPCIDIN when iron is abundant, limiting further iron absorption and release of stored iron [10]. Apart from iron, HEPCIDIN synthesis by hepatocytes is inhibited by erythropoietic activity and promoted by inflammation mainly through interleukin-6 (IL-6) and other mediators [8]. Epigenetics principally concerns mechanisms for modifying gene expression at the transcriptional level and affecting gene function without alterations in DNA sequences [11]. DNA methylation is a vitally important epigenetic factor influencing gene activities; this modification occurs almost exclusively at cytosine residues linked to guanine residues (CpG sites), where a methyl radical (CH3) is covalently added to the cytosine base [12]. DNA methylation in different genomic regions may exert different impacts on gene activities, while methylation in the promoter region is typically associated with stable gene silencing [13]. In recent years, some evidence has suggested that bariatric surgery can change DNA methylation patterns [14]. Multiple studies have revealed the effects of environmental exposure on the epigenome. One study has shown that hypermethylation of the ACSL3 gene is correlated with increased maternal polyaromatic hydrocarbon exposure [15]. Furthermore, emerging evidence suggests that environmental factors (nutrition, diet, residence/workplace, pharmacologic treatments) affect epigenetic patterns;
however, it must be acknowledged that the link between environment and epigenetics largely remains to be explored [16]. Iron deficiency is well recognized as a nutritional complication that can result from bariatric surgery. Nevertheless, the underlying mechanism through which system ironregulating elements react system-wide to this accumulating environment change remains poorly understood. We hypothesized that DNA methylation may be involved in modulating the expression of iron-regulated genes and helping to maintain iron homeostasis. We aimed to investigate whether DNA methylation plays a role in Hamp expression and investigate the changes of related regulatory factors of HEPCIDIN, the protein product of Hamp. Therefore, the expression of iron-regulating genes and their related or possible regulatory factors, including DNA methylation, iron status, erythrocyte parameters, and inflammation, were investigated; we expected to determine the potential mechanism by which the system adapts to environmental change—in this case, iron deficiency after bariatric surgery.
Methods Rats All animals were maintained and used in accordance with the guidelines of the Animal Care and Use Committee of Shanghai Sixth People’s Hospital. Eight-week-old male Sprague-Dawley rats were purchased from Shanghai Sipper-BK Lab Animal Co. Ltd. (Shanghai, China) and maintained under specific pathogen-free conditions (20 C–24 C, 12/12-hr light-dark cycle) with free access to food and water. Rats were fed either a chow diet or a high-fat diet (D12492; Research Diets, New Brunswick, NJ, USA) for 8 weeks, the latter of which induced obesity. The rats were then given a single low dose of streptozotocin (Sigma, Saint Louis, MO, USA) to induce T2D. Rats with random blood glucose levels 16.7 mM were considered successful models of T2D. The obese diabetic rats were randomly allocated to the sham, RYGB, or SG groups and then underwent sham, RYGB, or SG surgery 2 weeks after streptozotocin injection. RYGB, SG, and sham surgery were performed according to procedures described by Yan et al. [17] and Patrikakos et al. [18]. Eight weeks after surgery, rats were euthanized with sodium pentobarbital (.05 mg/g), and the liver tissue was removed, snap-frozen, and stored in liquid nitrogen before use. Four independent batches of obese diabetic rats underwent bariatric surgery. The first batch included sham (n 5 4) and RYGB (n 5 5; 2 rats died after surgery and were excluded); the second batch included sham (n 5 5) and RYGB (n 5 5; 1 rat died after surgery and was excluded); the third batch included sham (n 5 5) and RYGB (n 5 6; 1 rat died after surgery and was excluded); and the fourth
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
batch included sham (n 5 4), RYGB (n 5 6; 2 rats died and were excluded), and SG (n 5 4). Surgical procedures
Q2
For RYGB surgery, a 3.5- to 4-cm midline incision was made, the stomach was transected approximately 5 mm below the gastroesophageal junction from the lesser to the greater curvature, and the proximal and distal stomach was closed with 5-0 silk sutures (Ningbo Medical Needle, City, China) using an interrupted suture technique. Approximately 20% of the stomach was retained. Two ligations were placed on the jejunum 10 cm below the Treitz ligament using 5-0 silk sutures, and the intestine was transected between the 2 ligatures. A gastrojejunostomy was created between the distal jejunum and the anterior wall of the gastric pouch by performing a 7-mm side-to-side anastomosis. The proximal jejunum was connected to the jejunum 15 cm distal to the gastrojejunal anastomosis with a 7-mm sideto-side jejunojejunostomy. For SG, in brief, the greater curvature of the proximal from the antrum to the fundus and the distal stomach was incised, approximately 70% to 80% gastric volume was removed, including 80% to 90% of the proximal stomach and 70% of the distal stomach. The abdominal wall and skin were closed with continuous 4-0 silk sutures (Ningbo Medical Needle). For sham-operated rats, a 3.5- to 4-cm middle incision was made. Laparotomy was performed to expose the esophagus, stomach, and small intestine. No other procedure was carried out. Muscle layers and skin were closed using continuous 4-0 silk sutures (Ningbo Medical Needle) in the same manner as in RYGB and SG groups. Postoperative care After operation, 20 mL/kg (weight) saline solution were injected subcutaneously to hydrate the rats. The rats were fasted on the day of surgery, had free access to water the day after, were given 10% Ensure (Abbott, Lake Bluff, IL, USA) for 1 week from the third day after surgery, and then were maintained on 60% high-fat diet. The weight was measured at baseline before surgery and weekly after surgery after a 12-hour overnight fast. Metabolic analyses
Q3
Q4
The weight was monitored at baseline and weekly after bariatric surgery. For glucose tolerance test, rats were fasted overnight for approximately 16 hours and then injected intraperitoneally with glucose solution at 1 g/kg. For insulin tolerance test, rats were fasted for 4 hours and injected intraperitoneally with insulin (novolin; Novo Nordisk, city, Denmark) solution at .5 IU/kg. Blood glucose was measured before and after either glucose or insulin injection with glucometer (Accu-CheK; Roche, city, state, country).
3
RNA-seq The liver tissue from first batch (sham n 5 4, RYGB n 5 3) and the fourth batch (sham n 5 4, SG n 5 4) of rats underwent bariatric surgery were used to perform RNA-seq. Total RNA was extracted from rat liver tissue using TRIzol (Life Technologies, Carlsbad, CA, USA). RNA quality and quantity were detected by agarose gel electrophoresis and Qubit Fluorometric Quantitation (Life Technologies Corporation), respectively. PolyA-tailed cDNA libraries were constructed using the TruSeq RNA LT Sample Prep Kit v2 (Illumina, Inc., San Diego, CA, USA). Sample preparation followed the manufacturer’s protocol with a workflow, including isolation of polyadenylated RNA molecules, RNA fragmentation, cDNA synthesis, ligation of adapters, and PCR amplification. Sequencing of 2 ! 150-bp paired-end reads was performed with an Illumina HiSeq 3000. RSeQC (version 2.6.4) was applied to evaluate the quality of RNA-seq data. Then, the clean reads were aligned to the index file of the rn6 genome generated by HISAT2 (version 2.0.5). Read counts for each gene were generated by HTSeq-count, and the normalization and differentially expressed genes (DEGs) were determined by the DESeq2 package in R. DEGs were identified using cutoff values of 2-fold change between the sham and bariatric surgery groups and an adjusted P , .05.
DNA methylation determination The liver tissue from the third and fourth batches of rats that underwent bariatric surgery were used to analyze DNA methylation level of Hamp promoter region (sham n 5 9, RYGB n 5 9, SG n 5 4). Genomic DNA was isolated from rat liver tissue using a QIAamp Fast DNA Tissue Kit (Qiagen, Hilden, Germany). Bisulfite conversion of genomic DNA was performed using the EpiTect Fast Bisulfite Conversion Kit (Qiagen). The promoter region, defined as the region covering 1500 bp upstream and 500 bp downstream of the transcription start site, was selected to analyze the CpG methylation level. Target-specific primer pairs to amplify bisulfite-treated genomic DNA were designed using the EpiDesigner tool (Agena Bioscience, Inc., San Diego, CA, USA). Primers were synthesized by Invitrogen, and each reverse primer had a T7 promoter tag (50 -CAGTAATACGACTCACTATAGGGAGAAGGCT-30 ) for transcription. The forward primer was tagged with a 10-mer (50 AGGAAGAGAG-30 ) to balance the melting temperature. The polymerase chain reaction (PCR)-amplified products were treated with shrimp alkaline phosphatase, and in vitro transcription and base-specific cleavage were performed simultaneously. The resulting DNA fragments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and EpiTYPER Q5 (manufacturer, city, state, country) was used for the quantification of CpG methylation level.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348
4
349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408
Q6
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Quantitative PCR
Statistical analysis
The liver tissue (sham n 5 4, RYGB n 5 4, SG n 5 4) from the fourth batch of rats underwent bariatric surgery were used to validate the RNA-seq results. Total RNA was isolated from rat liver tissue using TRIzol (Life Technologies). One microgram of RNA was transcribed to cDNA using a PrimeScrip RT Reagent Kit (manufacturer, city, state, country). Gene-specific primers were synthesized by Sangon Biotech (Shanghai, China). FastStart Universal SYBR Green Master (ROX; Roche, Mannheim, Germany) was used for quantitative PCR on an ABI 7900 HT Fast RealTime PCR System (Applied Biosystems, Foster, CA, USA). Samples were analyzed in triplicate. Gene expression was expressed as 2(2DDCT) and normalized to the housekeeping gene b-Actin. Primer sequences are listed in the Supplementary Table.
All data are presented as the mean 6 SEM. Statistical significance (*P , .05, **P , .01, ***P , .001) was assessed with unpaired t tests or Mann-Whitney U tests (2-sided in both cases).
Erythrocyte parameters, serum HEPCIDIN-25, and iron status determination
Q7
All the serum samples from the 4 batches of rats underwent bariatric surgery were used to measure the serum HEPCIDIN-25 concentration and iron status (sham n 5 18, RYGB n 5 16, SG n 5 4). The serum samples from the fourth batch were used to determine the erythrocyte parameters (sham n 5 4, RYGB n 5 4, SG n 5 4). Whole blood or serum was collected 8 weeks after bariatric surgery. Blood samples were obtained after a 12-hour overnight fast. Erythrocyte parameters, including red blood cell, hemoglobin, hematocrit, and mean corpuscular volume, were determined using an automatic hematology analyzer (XN-350; Sysmex, city, state, country) with whole-blood samples. Serum HEPCIDIN-25 levels were detected using a rat serum HEPCIDIN-25 kit (Peninsula Laboratories International, Inc., San Carlos, CA, USA). Serum iron, ferritin, and unsaturated iron-binding capacity were measured using an automatic biochemical analyzer (Mindray BS-800, Shenzhen, China). Anemia is defined as hemoglobin 120 g/L, serum iron ,10 mmol/L, total iron-binding capacity (TIBC) .60 mmol/L.
Rat serum inflammatory cytokine detection
Q8
All the serum samples (sham n 5 18, RYGB n 5 16, SG n 5 4) from the 4 batches of rats underwent bariatric surgery were used to determine inflammatory factors. IL-6, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor (TNF-a) concentrations were quantified using a Rat Metabolic Hormone Panel Milliplex kit (Millipore, St. Charles, MO, USA) and a Luminex instrument (manufacturer, city, state, country) following the manufacturer’s specifications. The sensitivity for the Milliplex Kit (pg/mL) was 38 (IL-6), 34 (MCP-1), and 2 (TNF-a).
Results RNA-seq data demonstrated that liver Hamp mRNA expression changed most significantly after RYGB or SG surgery Both RYGB- and SG-operated rats achieved significant weight loss compared with preoperative status and improved glycemic control compared with sham-operated rats, and this benefit effect was more remarkable for RYGB-operated rats (Figs. S1 and S2). To characterize the transcriptomic differences between sham and bariatric surgery, including RYGB and SG, gene expression profiles were analyzed. Thresholds of jlog2 FoldChangej 1 and false discovery rate ,.05 were set to determine significantly different expression genes. For RYGB, 149 downregulated genes and 71 upregulated genes were identified compared with the sham group (Fig. 1A). For SG, 160 downregulated genes and 218 upregulated genes were detected compared with the sham group (Fig. 1B). Both the top 10 upregulated and downregulated genes for RYGB and the corresponding expression change levels of these genes in SG were exhibited in the heatmap (Fig. 1C). Among the top DEGs, Hamp ranked first in RYGB and had the maximum change in gene expression. A significant change in Hamp expression was also observed in SG. Liver Hamp expression changed dramatically after RYGB or SG surgery as confirmed by quantitative PCR To validate the RNA-seq results, several genes, including Hamp, Nox4, Litaf, Abca8, Npas2, Arntl, Abcg5, and Grem2, were analyzed by quantitative PCR, and the results were consistent with those of RNA-seq (Fig. 2). As showed by quantitative PCR (Fig. 2 the upper graph), the mRNA expression of Hamp was relatively low after RYGB surgery compared with sham-operated rats, while SG-operated rats showed marginally higher expression of Hamp than RYGB-operated rats. Consistent with quantitative PCR results, RNA-seq data (Fig. 2 the lower table) manifested larger fold change for RYGB than SG when both compared with sham-operated rats. The abundance of Hamp protein product HEPCIDIN-25 is markedly decreased after bariatric surgery Because the mRNA expression of Hamp changed the most drastically with the greatest significance, the serum protein level of HEPCIDIN-25, the active form of HEPCIDIN, was measured. The results showed that the serum
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468
469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528
5
web 4C=FPO
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Fig. 1. Hamp mRNA expression changed most significantly after RYGB and SG surgery. (A) Volcano plots of RNA-seq data showing significant differentially expressed genes between RYGB and sham. Each gene (dot) is plotted based on its expression level in log2(fold change) and –log10(false discovery rate). Most genes cluster around 0, indicating no change in expression. Blue dots indicate downregulated genes (log2(fold change) 1, false discovery rate ,.05), and red dots indicate upregulated genes (log2(fold change) 1, false discovery rate ,.05). The genes of interest with the most significant changes are indicated. (B) Volcano plots of RNA-seq data showing significant differentially expressed genes between sleeve gastrectomy and sham. (C) Heatmap representing the top 10 differentially expressed genes (base mean .1000) between the bariatric surgery group and the sham group. Colors ranging from blue to red on the heatmap reflect degrees of expression ranging from low to high, as shown on the scale in the upper right corner of the figure.
HEPCIDIN-25 level of rats after RYGB and SG surgery declined dramatically compared with sham, while the value of SG was slightly higher than RYGB, which is consistent with the quantitative PCR results (Fig. 3). DNA methylation of Hamp promoter region increased significantly after RYGB surgery DNA methylation is a major epigenetic factor influencing gene transcription and activities. It is speculated that DNA methylation possibly contributes to changes in Hamp gene
expression. With this assumption, the DNA methylation level of the promoter region of Hamp was determined. The results demonstrated that 5 of 12 CpG sites showed a significant increase in methylation level in RYGB compared with sham, while another 7 CpG sites showed statistically nonsignificant increase. For SG, among 12 CpG sites, 1 site showed a significant increase in CpG methylation level compared with the sham group, while 6 CpG sites showed slight increases that were not statistically significant (Fig. 4). Specific information for each CpG site is shown in Table 1.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588
6
589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Fig. 2. Quantitative polymerase chain reaction analysis to validate the RNA-seq results. Data were normalized to b-Actin mRNA expression. Data represent 2 independent experiments. Data are presented as the mean 6 standard error of the mean. Statistical differences were analyzed with the unpaired t test (n 5 4 for each group). *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between sleeve gastrectomy and sham. The lower table shows the results from RNA-seq data between the 2 bariatric surgery groups and the sham group.
RYGB-operated rats developed anemia while SG-operated rats showed iron deficiency HEPCIDIN is the principal regulator of extracellular iron homeostasis in health and disease. In addition, HEPCIDIN is feedback regulated by the substance whose concentration it controls: iron. Additionally, bariatric surgeries, including RYGB and SG, are reported to affect iron status to some extent; therefore, the serum iron status of rats after bariatric surgery was investigated. As shown in Fig. 5A, serum iron levels declined sharply 8 weeks after RYGB surgery, while rats after SG surgery demonstrated a significant decrease in serum iron levels compared with sham rats but a slight increase compared with RYGB rats. In addition, both the RYGB and SG groups showed significantly higher levels of unsaturated iron-binding capacity and TIBC than the sham group, while higher TIBC levels indicated iron deficiency status after RYGB and SG surgery (Figs. 5C–D). No significant changes were observed for ferritin in either RYGB or SG rats compared with sham-operated rats (Fig. 5B). Consistent with the severe iron-deficiency status, anemia is quite serious for RYGB-operated rats, as indicated by significant lower level of red blood cells, hemoglobin,
Fig. 3. Serum HEPCIDIN-25 concentration declined dramatically after Roux-en-Y gastric bypass and sleeve gastrectomy surgery. Data are presented as the mean 6 standard error of the mean for the preoperative (n 5 17), sham (n 5 18), Roux-en-Y gastric bypass (n 5 16), and sleeve gastrectomy rats (n 5 4). *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between sleeve gastrectomy and sham.
hematocrit, and mean corpuscular volume compared with sham-operated rats. In contrast to RYGB-operated rats, those underwent SG did not seem to develop anemia despite for observable decrease of serum iron compared with shamoperated rats (Figs. 5E–F). Inflammation profile showed minimal change after both bariatric surgery Apart from serum iron, inflammation factor also regulates HEPCIDIN synthesis, among which IL-6 is the most extensively studied known to regulate HEPCIDIN transcription in hepatocytes. The results demonstrated that the proinflammatory factors IL-6, MCP-1, and TNF-a did not show significant changes, despite profound effect of weight loss and remission of T2D, especially for RYGB-operated rats (Supplementary Figs. S1–S3). Discussion In this study, RNA-seq data demonstrated that liver Hamp expression was reduced most remarkably after RYGB surgery compared with sham, while a significant change in Hamp was also observed after SG surgery. The consensus finding is that HEPCIDIN-25, the only isoform of HEPCIDIN known to be associated with iron regulation, is dramatically decreased after both bariatric surgeries. Similar observations were reported that serum HEPCIDIN levels after bariatric procedures (gastric banding, SG, or RYGB) were significantly lower
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
web 4C=FPO
709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768
7
Fig. 4. Methylation status for each CpG site of the Hamp promoter in bariatric surgery and sham. Values are presented as the mean 6 standard error of the mean, sham (n 5 9), Roux-en-Y gastric bypass (n 5 9), and sleeve gastrectomy (n 5 4). The lower schematic diagram exhibits CpG sites in the Hamp promoter. CpG sites are indicated with lollipop-shaped markers. The binding sites for the forward and reverse primers are shown with arrows below the diagram. The number of lollipop-shaped markers indicates the number of detected CpG sites. *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between sleeve gastrectomy and sham.
in obese individuals compared with baseline [19–21]. It is noteworthy in our study that the methylation level of the Hamp promoter region significantly increased after RYGB surgery, while the SG group increased slightly in the methylation level, indicating that significantly elevated methylation levels after bariatric surgery likely
contribute to the decline in Hamp expression and consequently decrease serum HEPCIDIN-25 levels. Reduction in HEPCIDIN-25 likely allowed for enhanced dietary absorption, which is relatively critical for helping the system adapt to its iron-depleted status postoperatively.
Table 1 Specific information on each CpG site CpG site
Position
DMethylation (%) (RYGB–sham)
DMethylation (%) (SG–sham)
P value (RYGB versus sham)
P value (SG versus sham)
CpG_1 CpG_2 CpG_3 CpG_4 CpG_5 CpG_6 CpG_7 CpG_8 CpG_9 CpG_10 CpG_11
Chr1:89371083 Chr1:89371077 Chr1:89371027 Chr1:89370496 Chr1:89370475 Chr1:89370454/89370451 Chr1:89370399 Chr1:89369928 Chr1:89369878 Chr1:89369865 Chr1:89369708
12.6 7.4 4.9 12.0 11.4 13.3 6.6 15.7 4.3 6.8 3.0
-3.2 5.1 -1.1 8.1 4.8 7.5 3.6 4.7 3.8 3.7 -1.3
.002 .002 .1 .016 .057 .058 .2 .07 .046 .2 .006
.09 .003 .2 .5 .1 .3 .7 .5 .2 .8 .056
CpG site 5 representing each CpG site indicated in the main text; Position 5 the position of each CpG site on the chromosome; DMethylation 5 difference of methylation level between 2 comparing groups; RYGB 5 Roux-en-Y gastric bypass; Sham 5 sham operation; SG 5 sleeve gastrectomy; RYGB–sham 5 the difference of DNA methylation level between RYGB and sham group; SG–sham 5 the difference of DNA methylation level between SG and sham group; RYGB versus sham 5 the P value between RYGB and sham group; SG versus sham 5 the P value between SG and sham group.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828
8
829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
Fig. 5. Iron status and erythrocyte parameters of rats underwent bariatric surgery or sham operation. Data are presented as the mean 6 standard error of the mean. (A–D) Sham (n 5 18), Roux-en-Y gastric bypass (n 5 16), and sleeve gastrectomy (n 5 4); (E–H) sham (n 5 4), Roux-en-Y gastric bypass (n 5 4), and sleeve gastrectomy (n 5 4). *P value for comparison between Roux-en-Y gastric bypass and sham; #P value for comparison between SG and sham.
It is generally known that HEPCIDIN, which regulates system iron homeostasis, can be feedback modulated by iron. The present study demonstrated obvious iron deficiency with anemia in RYGB-operated rats, characterized by significantly reduced serum iron, elevated TIBC, poor functional iron status (hemoglobin, hematocrit, mean corpuscular volume). By contrast, despite an observable decrease of serum iron, iron deficiency–induced anemia did not occur for SG-operated rats, indicated by nearly unchanged erythrocyte parameters. It is worth noting that the serum iron of the SG group was slightly higher than that of the RYGB group, which is consistent with the observation that Hamp expression and the HEPCIDIN level of the SG group were marginally higher than those of the RYGB group, although this difference was not statistically significant. The differences in serum iron and Hamp expression between the RYGB and SG groups could be ascribed to the fact that RYGB surgery limits both food intake and nutrient absorption, while SG is perceived as a restrictive surgical approach that primarily limits food intake [22]. In particular, RYGB surgery involves gut manipulation where, excluding the duodenum, iron uptake happens from the alimentary tract resulting in iron malabsorption. Therefore, iron deficiency status due to poorer nutrition in the RYGB group is more common and more severe than in the SG group, which is confirmed by the observation that RYGBoperated rats were in poor functional iron status, while SG-operated rats did not show anemia despite for observable iron deficiency just as demonstrated in this study. To respond to lower serum iron, HEPCIDIN synthesis is downregulated to maintain iron homeostasis by increasing CpG site methylation of the Hamp promoter. In addition, inflammation can stimulate HEPCIDIN synthesis. Upon analyzing changes in inflammation after
bariatric surgery, no significant change in IL-6 and MCP-1 concentrations 8 weeks after surgery in the case of either RYGB or SG was found. Obesity is considered a chronic low-grade inflammatory state [5], which is thought to be involved in the development of obesity and T2D. Bariatric surgery is reported to have weight loss of .50% of weight [23]. Along with substantial weight loss, the inflammatory state is mitigated, and C-reactive protein, MCP-1, and IL6 decrease after bariatric surgery [19,24]. However, in line with this study, numerous studies have shown that IL-6 remains unchanged after bariatric surgery, despite metabolic improvement [25–27]. Little change of inflammatory profile in this study was likely account for exist of postoperative inflammation, which commonly occur after bariatric surgery. In contrast to IL-6 and TNF-a, C-reactive protein seems to change uniformly, undergoing a marked decline after bariatric surgery [19,20,27]. It is important to note that IL-6 is the key inflammation factor reported to promote Hamp expression; nevertheless, the effect of IL-6 on Hamp transcription seems to be limited, as no change in its serum level was observed in the present study. Furthermore, in addition to being an important iron stores marker, ferritin is an acute-phase reactant, which is supposed to increase under system inflammation status, even in irondeficient conditions [28]. Just as shown in present study, serum ferritin level 8 weeks after both RYGB and SG surgery remains nearly unchanged. Minimal change of inflammatory profile and ferritin level indicate that inflammatory signal may not have a major impact on HEPCIDIN production. Previous studies reported the association of serum HEPCIDIN level with diabetes [29], although the results were inconsistent. We used an obese diabetic rat model in present study. To rule out the potential impact of diabetic status on
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10
949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008
Q9
HEPCIDIN level, we set up an appropriate control—sham operation; those rats that underwent bariatric surgery were in the same background, and both were obese diabetic rats. Therefore, it is scientific and reasonable to conclude that the changes of HEPCIDIN level were result from implementation of surgical operations but not other potential factors. It is of great importance to improve and advance the indicators of bariatric/metabolic surgery to obtain maximum benefits and minimize potential complications. Here, we assume that the baseline serum HEPCIDIN level may be used as one of the indicators in a future scenario of customized procedures for patients underwent bariatric/metabolic surgery. This assumption is based on 2 facts as follows: (1) the function of HEPCIDIN is to negatively regulate iron from entering the plasma from the stores and limit iron uptake from diet, therefore the serum HEPCIDIN level can indicate the system iron regulatory status; and (2) different surgical techniques for bariatric/metabolic surgery result in different reduction in Hamp expression, as demonstrated in our study, the room for reduction may reflect the capacity of system to regulate iron status. However, it should be prudent, rather than using serum HEPCIDIN level solely to assess the iron status, to use a combination of serum HEPCIDIN level, biochemical markers (ferritin, serum iron, TIBC, soluble transferrin receptor), and erythrocyte parameters (hemoglobin, mean corpuscular volume); this could give more comprehensive insight into the iron and anemia status. Furthermore, due care should be taken when interpreting the data of serum HEPCIDIN level because HEPCIDIN synthesis can be influenced by inflammatory disorders; thus, examination of the inflammation status is also necessary. Simultaneously, a standard range of HEPCIDIN level for healthy population should be set up, which can provide reference for evaluating serum HEPCIDIN level. Taking the 4 elements (serum HEPCIDIN level, biochemical markers of iron status, erythrocyte parameters, and inflammatory status) into consideration, a relative comprehensive understanding of iron metabolism will be obtained and is likely to help in choosing the superior surgical techniques. For instance, for patients with higher baseline serum HEPCIDIN level and without iron deficiency/anemia, surgical techniques (such as RYGB, one-anastomosis gastric bypass procedures), which can lead to more effective weight loss and/or metabolic improvement, would be more appropriate. In contrast, for patients with lower baseline serum HEPCIDIN level and with iron deficiency/anemia, a surgical technique having lower risk of anemia, such as SG, could be a better choice. Our study had strengths and limitations. First, although it has been confirmed in the present study that iron deficiency occurred after bariatric surgery, no direct determination of iron absorption or iron transporter ferroportin-1 expression at the duodenal enterocyte was evaluated; this awaits further investigation. Second, it is reported here that elevated DNA methylation at Hamp promoter region likely contributes to
9
significantly decrease Hamp expression in response to iron deficiency after bariatric surgery; however, the underlying mechanism of how the changing internal environment (iron deficiency) affects gene methylation status and eventually impacts gene function warrants further study to confirm our findings. Finally, due to the unavailability of human tissue samples after bariatric surgery, the methylation status of Hamp in individuals who underwent bariatric surgery could not be verified. Conclusions In summary, we reported first that DNA hypermethylation in the Hamp promoter region may contribute to remarkable suppression of Hamp expression, resulting in reduced HEPCIDIN levels in response to iron deficiency after bariatric surgery, especially for RYGB, helping to maintain iron homeostasis. Our study provides a potential basis for modulating iron homeostasis by DNA methylation of ironregulating elements after bariatric surgery. The epigenetic influences on iron homeostasis after bariatric surgery may be closely linked, and the underlying mechanism warrants further investigation. Acknowledgments Yeping Huang and Hong Zhang contributed equally to this work. Disclosures The authors have no commercial associations that might be a conflict of interest in relation to this article. Supplementary materials Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/ j.soard.2019.10.005. References
Q10
[1] Obesity and overweight. Fact sheet. Geneva: World Health Organization; c2019 [updated 2018 Feb 16; cited yr mo d]. Available from: https://www. who.int/news-room/fact-sheets/detail/obesity-and-overweight. Q11 [2] Ogurtsova K, da Rocha Fernandes JD, Huang Y, et al. IDF Diabetes Atlas: global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes Res Clin Pract 2017;128:40–50. [3] Malin SK, Kashyap SR. Differences in weight loss and gut hormones: Roux-en-Y gastric bypass and sleeve gastrectomy surgery. Curr Obes Rep 2015;4(2):279–86. [4] Mingrone G, Panunzi S, De Gaetano A, et al. Bariatric surgery versus conventional medical therapy for type 2 diabetes. N Engl J Med 2012;366(17):1577–85. [5] Batterham RL, Cummings DE. Mechanisms of diabetes improvement following bariatric/metabolic surgery. Diabetes Care 2016;39(6):893– 901. [6] Welbourn R, Hollyman M, Kinsman R, et al. Bariatric surgery worldwide: baseline demographic description and one-year outcomes from
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068
10
1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128
[7]
[8] [9]
[10] [11]
[12] [13] [14]
[15]
[16] [17]
[18]
Yeping Huang et al. / Surgery for Obesity and Related Diseases - (2019) 1–10 the Fourth IFSO Global Registry Report 2018. Obes Surg 2019;29(3):782–95. Mu~ noz M, Botella-Romero F, Gomez-Ramırez S Campos A, GarcıaErce JA. Iron deficiency and anaemia in bariatric surgical patients: causes, diagnosis and proper management. Nutr Hosp 2009;24(6):640–54. Ganz T, Nemeth E. HEPCIDIN and iron homeostasis. Biochim Biophys Acta 2012;1823(9):1434–43. Addo L, Ikuta K, Tanaka H, et al. The three isoforms of HEPCIDIN in human serum and their processing determined by liquid chromatography-tandem mass spectrometry (LC-tandem MS). Int J Hematol 2016;103(1):34–43. Ganz T, Nemeth E. HEPCIDIN and disorders of iron metabolism. Annu Rev Med 2011;62(1):347. Dupont Catherine, Armant D, Brenner C. Epigenetics: definition, mechanisms and clinical perspective. Semin Reprod Med 2009;27(5):351–7. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16(1):6–21. Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology 2013;38(1):23–38. Nicoletti CF, Nonino CB, de Oliveria BA, et al. DNA methylation and hydroxymethylation levels in relation to two weight loss strategies: energy-restricted diet or bariatric surgery. Obes Surg 2015;26(3):1–9. Perera F, Tang WY, Herbstman J, et al. Relation of DNA methylation of 50 -CpG island of ACSL3 to transplacental exposure to airborne polycyclic aromatic hydrocarbons and childhood asthma. PLoS One 2009;4(2):e4488. Feil R. Environmental and nutritional effects on the epigenetic regulation of genes. Mutat Res 2006;600(1-2):46–57. Yan Y, Zhou Z, Kong F, et al. Roux-en-Y Gastric bypass surgery suppresses hepatic gluconeogenesis and increases intestinal gluconeogenesis in a type 2 diabetes rat model. Obes Surg 2016;26(11):2683–90. Patrikakos P, Toutouzas KG, Perrea D, et al. A surgical rat model of sleeve gastrectomy with staple technique: long-term weight loss results. Obes Surg 2009;19(11):1586–90.
[19] Tussinghumphreys LM, Nemeth E, Fantuzzi G, et al. Decreased serum HEPCIDIN and improved functional iron status 6 months after restrictive bariatric surgery. Obesity 2010;18(10):2010–6. [20] Cepedalopez AC, Allende-Labastida J, Melse-Boonstra A, et al. The effects of fat loss after bariatric surgery on inflammation, serum HEPCIDIN, and iron absorption: a prospective 6-mo iron stable isotope study. Am J Clin Nutr 2016;104(4):1030. [21] Gesquiere I, Foulon V, Augustijns P, et al. Micronutrient intake, from diet and supplements, and association with status markers in pre- and post-RYGB patients. Clin Nutr 2016;36(4):1175–81. [22] Sala PC, Torrinhas RS, Giannella-Neto D, Waitzberg DL. Relationship between gut hormones and glucose homeostasis after bariatric surgery. Diabetol Metab Syndr 2014;6(1):87. [23] Shoelson SE, Herrero L, Naaz A. Obesity, inflammation, and insulin resistance. Gastroenterology 2014;132(6):2169–80. [24] Buchwald H, Estok R, Fahrbach K, et al. Weight and type 2 diabetes after bariatric surgery: systematic review and meta-analysis. Am J Med 2009;122(3):248–56. [25] Monte SV, Caruana JA, Ghanim H, et al. Reduction in endotoxemia, oxidative and inflammatory stress, and insulin resistance after Rouxen-Y gastric bypass surgery in patients with morbid obesity and type 2 diabetes mellitus. Surgery 2012;151(4):587–93. [26] Netto BD, Bettini SC, Clemente AP, et al. Roux-en-Y gastric bypass decreases pro-inflammatory and thrombotic biomarkers in individuals with extreme obesity. Obes Surg 2015;25(6):1010–8. [27] Miller GD, Nicklas BJ, Fernandez A. Serial changes in inflammatory biomarkers after Roux-en-Y gastric bypass surgery. Surg Obes Relat Dis 2011;7(5):618–24. [28] Baynes R, Bezwoda W, Bothwell T, Khan Q, Mansoor N. The nonimmune inflammatory response: serial changes in plasma iron, ironbinding capacity, lactoferrin, ferritin and C-reactive protein. Scand J Clin Lab Invest 1986;46(7):695–704. [29] Aregbesola A, Voutilainen S, Virtanen JK, Aregbesola A, Tuomainen T. Serum hepcidin concentrations and type 2 diabetes. World J Diabetes 2015;6(7):978–82.
FLA 5.6.0 DTD SOARD3943_proof 2 November 2019 1:33 pm ce
1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188