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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: J Surg Res. 2022 Jan 24;273:161–171. doi: 10.1016/j.jss.2021.12.027

Gastric bypass regulates early inflammatory responses in high-fat diet-induced obese mice

Rui Xu 1,*, Chenyu Zhu 1,*, Yuxin Li 2, Michael Andrade 2, Deng Ping Yin 2
PMCID: PMC8960359  NIHMSID: NIHMS1774440  PMID: 35085943

Abstract

Background.

Obesity and diabetes are characterized by chronic inflammatory responses. Roux-en-Y gastric bypass (RYGB) is increasingly regarded as an effective approach for the improvement of glucose homeostasis. In this study, we examined the effects of RYGB on regulation of early inflammatory responses in the liver and adipose tissue in high-fat diet (HFD)-induced obese (DIO) mice.

Materials and Methods.

RYGB was performed in DIO mice followed by analyses of adiposity, insulin sensitivity, plasma and tissue cytokines and adipokines, tissue NF-κB and JNK/c-Jun activation and tissue macrophage and T-cell subsets.

Results.

We found that RYGB resulted in sustained improvement of adiposity and insulin sensitivity. Plasma insulin and leptin levels were increased in untreated DIO mice and reduced in RYGB mice. RYGB maintained plasma adiponectin levels and inhibited MCP-1 and IL-6 in white adipose tissue (WAT) and liver. RYGB inhibited NF-κB activation in WAT and muscle, but not in the liver. However, RYGB attenuated the JNK/c-Jun signaling pathway in the liver and WAT at one week after surgery, suggesting that RYGB regulates the tissue specific inflammatory pathway. RYGB reduced M1-like (F4/80+/CD11c+) differentiation and enhanced M2-like population (F4/80+/CD206c+). RYGB also regulated CD4+ and CD8+ T-cell infiltration and increased Treg cells in the liver and WAT at the same time-point.

Conclusions.

Our findings demonstrate that RYGB improves obesity and insulin resistance which are associated with the regulation of early inflammatory reactions in the liver and WAT.

Keywords: Gastric bypass, inflammation, insulin resistance, mice

Introduction

Obesity is recognized as an epidemic linked to increased incidence of Type 2 Diabetes (T2D), atherosclerotic cardiovascular diseases, various forms of liver disease and specific forms of cancer1, 2. There is increasing evidence that obesity is strongly linked to chronic low grade inflammation, which has been known to play a critical role in the development of insulin resistance and T2D3, 4. Macrophage activation in obesity represents a common pathogenic mechanism underlying associated insulin resistance. This is promoted by a transition in macrophage polarization from an alternative M2-like activation state to a classic M1-like differentiation to produce pro-inflammatory and anti-inflammatory cytokines5–7. T-cell subsets such as CD4+, CD8+ and regulatory T-cells (CD4+/FoxP3+) have emerged as mediators promoting and regulating inflammatory reactions8, 9. These inflammatory reactions lead to activation of the Jun NH2-terminal kinase (JNK)/activator protein 1 (AP-1) signaling pathway that triggers transcription of pro-inflammatory cytokines, as well as the phosphorylation of serine in insulin receptor substrate (IRS) proteins10.

Gastric bypass is currently considered the most effective and sustainable treatment for obesity and has been shown to result in resolution of T2D as well as many of the associated comorbidities11–15. Initial experiments support the perception that the resolution of the obesity related comorbidities is due to a variety of factors including caloric restriction, weight loss, changes in intestinal hormones and regulation of inflammatory responses (reviewed in16). Serial changes in inflammatory biomarkers are found following gastric bypass17, and improved inflammation is also observed in the later phase (6–24 months)18, 19. However, whether improved metabolism is associated with early regulation of inflammatory responses remains to be investigated.

We have developed the Roux-en-Y gastric bypass (RYGB) mouse model, in which gastric bypass significantly improves obesity and insulin resistance in HFD-induced obese mice, and the different gastric pouch size does not impact metabolic benefits20–22. In the current study, we tested whether gastric bypass improves early inflammatory responses in the liver and WAT responsible for improved metabolic phenotypes using the RYGB mouse model in HFD-induced obese mice.

Materials and Methods

Mice and surgery

Wild-type (WT) C57BL/6 (H-2b) mice were purchased from Jackson Lab. NF-κB-luc mice, on a C57BL/6 background and expressing luciferase under the control of the NF-κB promoter, were kindly provided by Dr. Timothy Blackwell at Vanderbilt University Medical Center23. In the current study, male mice were used and housed at 23°C on a 07:00–19:00 light cycle. DIO mice were generated by feeding a HFD (60% Kcal fat, Bio-Serv), starting at six weeks of age, for 12 weeks (18 weeks old). DIO was defined when body weight exceeded 40.0 g with a blood glucose level ≥ 350 mg/dl. Low-fat diet (LFD)-fed C57BL/6 mice were used as control.

The surgical procedure was performed under general anesthesia (isoflurane plus O2 through a cone placed around the nose). The animals’ vital signs were monitored throughout surgery, including respiratory rate, response to noxious stimuli and assessment of spontaneous movements. RYGB was performed in DIO mice as previously described20–22. We have compared different mouse gastric bypass models, including gastric bypass with a small gastric pouch, a large gastric pouch and no gastric pouch. Gastric bypass with or without gastric pouch resulted in comparable effects in improving obesity and insulin resistance21. However, gastric bypass without gastric pouch shows a higher success rate than other models and was performed in the current study. For the sham surgical procedures, the stomach, duodenum and jejunum were mobilized, and the esophagus, stomach and jejunum were clamped without incision followed by closure of the abdomen. After recovery, mice were maintained on the same HFD after surgery through the endpoint. SHAM-PF mice were fed the same amount of HFD consumed by RYGB mice. Epididymal WAT tissues and liver tissues were collected for H&E staining and for various assays. Unless described otherwise, week 8 was defined as the endpoint of this study. All protocols were approved by Institutional Animal Care and Use Committee (IACUC) at the Three Gorges University and the University of Chicago.

Body weight and composition

Body weight and diet consumption were analyzed every day through the endpoint (TSE Systems, Chesterfield, MO)24. Body mass was measured using mq10 NMR analyzer (Bruker Optics Inc., Billerica, MA) following 2 hrs of fasting20. Fat was calculated as grams of total mass.

Intraperitoneal glucose tolerance tests (IPGTT)

Mice were fasted for 4 hrs prior to IPGTT. Blood was sampled from the tail vein before and at 30, 60, 90 and 120 min after an intraperitoneal injection of dextrose (20%) at 2.0 mg/g body weight. Blood glucose levels (mg/dl) were measured using a blood glucose meter (SureStep, LifeScan, Inc.). The AUC (area under the curve) was calculated using the trapezoidal rule as previously described20.

Tissue cytokine and chemokine analyses

Liver and epididymal WAT samples were subjected to total RNA extraction using Trizol reagent (Invitrogen, Grand Island, NY). After RNA quantification, 2 μg of total RNA were digested with RNase-free DNase I (Roche Diagnostics, Indianapolis, IN), followed by reverse transcription using Omniscript RT Kit (Qiagen). Quantitative expressions of IL-6 and monocyte chemoattractant protein-1 (MCP-1) were determined using SYBR GreenER qPCR Supermix (Invitrogen) on a qRT-PCR system (Bio-Rad) with Integrated DNA Technologies (Coralville, IA) primers and probes, as described previously20. Standard curves for IL-6, MCP-1 and β-actin were generated and the efficiency of qRT-PCR for each gene was calculated. All data were normalized to the house-keeping gene, β-actin, and a comparative threshold cycle (Ct) method, 2−ΔΔCt, was used to compare the relative expression levels.

Circulating insulin, leptin and adiponectin were analyzed by MilliPlex map kit using Luminex 200 System analyzer (Millipore, Co., Billerica, MA) and RIA kit.

Tissue luciferase assay

Tissue luciferase activity was detected by the Bradford Luciferase Reporter Assay (Promega, Madison, WI)25. The liver, epididymal WAT and muscle were collected from NF-κB-luc mice at 7 days post-surgery. 100 μl of freshly reconstituted luciferase assay buffer was added into 20 μl of the tissue homogenate ground in reporter lysis buffer. Luciferase activity was quantified as relative light units (RLU) using a luminometer (BD Biosciences).

Western blot for JNK and c-Jun analyses

The liver, epididymal WAT and muscle were homogenized in lysis buffer and pelleted. Protein concentrations of the supernatant were analyzed, and equivalent amounts of protein were loaded onto the gel. Phospho-SAPK/JNK, SAPK/JNK, and phospho-c-Jun and c-Jun and secondary antibodies were purchased from Cell Signaling. The first antibody was diluted 1:1000 in blocking buffer and incubated at room temperature for 1 hr. The second antibody was diluted 1:4000 and incubated in blocking buffer for 1 hr with shaking. The blot was dried with a piece of towel and exposed to X-Omat film.

Analyses of macrophages and T-cell subsets in the liver and epididymal WAT

Liver non-parenchymal cells (NPCs) and epididymal WAT monocytes were isolated, as described previously26. The liver was perfused and minced into small pieces with surgical scissors and forced gently through a 200 μm gauge stainless steel mesh. The suspension was centrifuged at 500 r.p.m. (60 g) for 1 minute and the pellet was obtained. The resulting pellet was suspended in 10 ml PBS digestion buffer containing collagenase IV (0.2g/L, Worthington Biochemical Corp., Lakewood, NJ), DNase (0.02g/L) and 0.5% FBS in a water bath at 37°C with shaking for 40 min. Red blood cells were lysed with red blood cell lysing buffer, and the pellet was suspended in DPBS (with 0.5% FBS). Approximately 5×106 to 10×106 NPCs were obtained from one mouse liver. For WAT monocyte collection, epididymal adipose tissue was minced into fine pieces. After digestion with collagenase, type II (Sigma-Aldrich), red blood cells were lysed with lysing buffer. 1×106 cells were labeled with antibodies conjugated with FITC, PE and APC, including CD11b (M1/70), CD11c (NC418), CD206 (MR5D3), F4/80 (BM8), CD3 (2C11), CD4 (GK1.5) and FoxP3 (FJK16). These antibodies were purchased from BioLegend. FITC-, PE and APC-conjugated IgG isotypes were used as controls. 1×105 cells were analyzed by flow cytometry (3-laser BD LSRII system).

Statistics

Data from this study are presented as mean±SD. Sample sizes of 4 to 10 or more animals per experiment were chosen to ensure adequate power. P-value of < 0.05 was considered significant. When the same control groups were presented across multiple figures, the outcomes achieved were concatenated from multiple experiments performed over the course of the entire study and demonstrating similar outcomes. To calculate differences between experimental animals, we used Kruskal-Wallis test with Dunn’s post hoc test for pairwise multiple comparisons and Mann-Whitney unpaired t test.

Results

Gastric bypass improved obesity

HFD consumption in untreated DIO mice was 1.8 ± 0.5 g/day at 18 weeks of age (time 0 week) and increased to 2.6 ± 0.3 g/day at 26 weeks of age (8 weeks post-surgery). HFD consumption in RYGB DIO mice decreased by 56% (0.8 ± 0.2 g/day) at day 1 and by 50% (0.9 ± 0.5g/day) at one week post-surgery; the consumption returned to 75% of preoperative levels (1.35 ± 0.6 g/day) at 2 weeks post-surgery and returned to preoperative intake thoughout the 4–8 weeks post-surgery (Figure 1A).

Figure 1.

Figure 1.

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Diet consumption (panel a), body weight (panel b) and body composition (panel c) and glucose tolerance tests (panels d-g). (A) Diet induced obesity (DIO) was achieved by a HFD (60%) starting at 6 weeks of age and lasting for 12 weeks. The graphs denote DIO mice with food and water ad libitum; DIO mice with sham surgery (SHAM), and RYGB. *: RYGB vs. DIO, p < 0.01 from 3 days to 8 weeks post-surgery. (B) The changes in body weight following sham sugery, pair-fed DIO mice with sham surgery and DIO mice with RYGB. *: RYGB vs. DIO, p < 0.01 at 2, 4, 6 and 8 week spost-surgery. (C) The changes of fat mass and lean mass as measured by NMR (Bruker Optics Inc.), described in the Methods section. Fat mass SHAM-PF and RYGB vs. DIO, p < 0.01; and RYGB vs. SHAM-PF, p < 0.05 at 1, 2, 4 and 8 weeks (This study was repeated for 2–3 times, and n = 8 in all groups).

To test the effects of caloric restriction and surgical stress on weight loss, RYGB DIO mice were compared to sham surgery mice fed HFD ad libitum (SHAM ad lib) and sham surgery mice with pair-feeding (SHAM-PF). RYGB induced sustained weight loss and fat mass reduction. SHAM ad lib mice showed unsustained weight loss and SHAM-PF mice showed modest but reduced losses in body weight and fat mass when compared to RYGB mice (Figures 1B and 1C).

Gastric bypass increases circulating insulin level and improved glucose tolerance

Circulating insulin levels were significantly (p < 0.001) increased in untreated naive DIO mice (n = 4) compared to lean (n = 4), consistent with previous reports27, 28. RYGB resulted in significant reduction in plasma insulin levels at 1 and 4 weeks after surgery (p < 0.001, Figure 2A).

Figure 2.

Figure 2.

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RYGB enhances insulin sensitivity. (A) Fasting plasma insulin levels were significantly increased in DIO mice and inhibited by RYGB. *: DIO and SHAM-PF vs. Lean, p < 0.001; and #: RYGB vs. DIO and SHAM-PF, p < 0.01, at 1 and 4 weeks after surgery; and were reduced at 1 and 4 weeks in mice. *RYGB vs. SHAM-PF and DIO, p < 0.001 (n = 5–7 in each group). (B) Area under curves (AUC) of intraperitoneal glucose tolerance tests (IPGTT) obtained at 1, 2, 4 and 8 weeks after surgery. *: DIO vs. Lean, p < 0.01 at 1, 2, 4 and 8 weeks; **: RYGB (from 1 week to 8 weeks) and SHAM-PF (at 1 week) vs. DIO, p < 0.01; #: SHAM-PF vs. DIO, p < 0.05 at 2 weeks post-surgery, and $: RYGB vs. SHAM-PF, p < 0.01 at 4 and 8 weeks post-surgery (n = 8–12 in each group). (C) Blood glucose curves of IPGTT at the first week. (D) Blood glucose curves of IPGTT at the second week. (E) Blood glucose curves of IPGTT at the four weeks. (F) Blood glucose curves of IPGTT at the eight weeks. (G) Plasma leptin levels were significantly increased in DIO mice, compared to lean mice (**: DIO vs Lean, p < 0.01), and were reduced in RYGB mice from at the first week (*: RYGB vs. DIO and SHAM-PF, p < 0.05) and 4 weeks post-surgery. (**: RYGB vs. DIO and SHAM-PF, p < 0.001). (H) Plasma adiponectin levels were decreased in DIO mice and were increased in RYGB mice. *: RYGB vs. DIO and SHAM-PF, p < 0.01. The results in Figure 2 G and H are representative of 4 experiments in each group.

IPGTT was assessed at 1, 2, 4 and 8 weeks after surgery (Figures 2B–2H). RYGB significantly improved glucose tolerance persistently, whereas SHAM-PF, whereas SHAM-PF improved glucose tolerance in the first two weeks, and there was no statistical difference compared with untreated DIO mice after two weeks of surgery. On the other hand, RYGB resulted in sustained improvement of glucose tolerance starting at week 1 to the endpoint (8 weeks).

HFD triggers increased circulating leptin levels (DIO vs. Lean, p < 0.01, Figure 2G). SHAM-PF resulted in significant but transient reductions in plasma leptin levels at one week after surgery, while RYGB resulted in sustained reductions (p < 0.01). Adiponectin was reduced in DIO mice and elevated in RYGB mice, but not in SHAM-PF mice (RYGB vs. DIO and SHAM-PF, p < 0.01, Figure 2H).

Gastric bypass regulated tissue specific NF-κB activation and JNK and c-Jun phosphorylation

Mice expressing luciferase under the control of the NF-κB promoter, referred to as NF-κB-luc mice, allow direct visualization of NF-κB activation using bioluminescence imaging (BLI)20, 29. We assessed tissue specific NF-κB activation in the liver, epididymal WAT and muscle tissues obtained at one week after surgery using tissue luciferase assay. HFD increased luciferase activity in epididymal WAT and muscle (DIO vs. Lean, p < 0.001), but not in liver (DIO vs. Lean, p > 0.05). RYGB and SHAM-PF inhibited DIO-induced luciferase activity in epididymal WAT and muscle (Figure 3A).

Figure 3.

Figure 3.

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RYGB regulates tissue NF-κB activity and the JNK/c-Jun signaling. The liver, epididymal white adipose tissue (WAT) and skeletal muscle (hind limb) were collected from NF-κB-luc mice at one week after surgery and subjected to tissue luciferase activity tests and Western blot. (A) The luciferase activity was increased in WAT and muscle and inhibited by RYGB and SHAM-PF. *: DIO vs. Lean, p < 0.001; #SHAM-PF and RYGB vs. DIO, p < 0.001. There was no statistical difference of liver luciferase activity among the groups (p > 0.05). (B) Western blot analysis of JNK and c-Jun phosphorylation in the liver, WAT and muscle. The lower left panel, JNK phosphorylation. *: SHAM-PF and RYGB vs. DIO, p < 0.01. The lower right panel, c-Jun phosphorylation. *: SHAM-PF liver and RYGB liver and WAT vs. DIO, p < 0.01; and **: SHAM-PF WAT vs. DIO, p < 0.05. The results in Figure 3 are representative of 3 – 4 experiments in each group (n = 2 in each group).

Phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in WAT, liver and muscle were analyzed at one week after surgery. Figure 3B in the upper and lower panels show enhanced phosphorylation of JNK and c-Jun in the DIO liver and WAT, but not in muscle. Phosphorylation of both JNK and c-Jun was inhibited by RYGB and SHAM-PF.

Gastric bypass inhibited MCP-1 and IL-6 production in the liver and adipose tissue

The results in Figures 4A and 4B show that both MCP-1 and IL-6 mRNAs in the liver and epididymal WAT were increased in untreated DIO mice relative to lean mice. Sham pair feeding inhibited IL-6 mRNAs in the liver and WAT and this inhibition was further accentuated by RYGB (compared to DIO, p < 0.01 and compared to SHAM-PF group, p < 0.01). RYGB, but not SHAM-PF, significantly inhibited MCP-1 mRNAs in the liver and WAT (RYGB vs. DIO and SHAM-PF, p < 0.01) at one week after surgery.

Figure 4.

Figure 4.

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TNFα, IL-1β, IL-6 and MCP-1 mRNAs in the liver and epididymal WAT. (A) TNFα, IL-1β, IL-6 and MCP-1 mRNAs in liver tissue. (B) TNFα, IL-1β, IL-6 and MCP-1 mRNAs in epididymal WAT. *: TNFα, RYGB vs. DIO and SHAM-PF, p < 0.01; #: IL-1β, RYGB vs. DIO and SHAM-PF, p < 0.01; $: IL-6, RYGB vs. DIO and SHAM-PF, p < 0.01; @: MCP-1, RYGB vs. DIO and SHAM-PF, p < 0.01; &: IL-1β, SHAM-PF vs. DIO, p < 0.01; and !: SHAM-PF vs. DIO, p < 0.01.(qRT-PCR was repeated for 3 times, and n = 4 – 6 in each group.

Gastric bypass modulated macrophage subsets in the liver and adipose tissue

The liver and epididymal WAT were collected from lean C57BL/6 mice (Lean), untreated DIO, SHAM-PF and RYGB mice at one week after surgery. Immune cells were isolated and labeled with F4/80 (macrophages), CD11c and CD206 FITC, PE and APC conjugated mAbs. F4/80+ cells in the liver were gated followed by analyses of F4/80+/CD11c+ (M1-like) subset and F4/80+/CD206c+ (M2-like) population (Figure 5A). Compared to lean, we observed increased M1 and reduced M2 populations in DIO liver and WAT (Figures 5A and 5B). these findings are consistent with previous reports in obese human subjects3. SHAM-PF exhibited reduction of M1-like population in WAT (p < 0.05, Figure 5B); however, SHAM-PF neither regulated M1-like in liver nor M2-like in both WAT and liver. RYGB, but not SHAM-PF, significantly inhibited M1-like and maintained M2-like subsets in both the liver and WAT (RYGB vs. DIO, p < 0.01 for both F4/80+/CD11c+ and F4/80+/CD206c+, Figures 5A and 5B).

Figure 5.

Figure 5.

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Flow cytometry analysis of macrophage subsets in the liver and epididymal WAT at one week after surgery. (A) F4/80+ cells were gated followed by analysis of CD11c+ (F4/80+/CD11c+) and CD206+ (F4/80+/CD206+) cells. (B) F4/80+/CD11c+ and F4/80+/CD206+ macrophages in the liver. *: DIO vs. Lean, p < 0.001 in both F4/80+/CD11c+ cells and F4/80+/CD206+ cells; #: F4/80+/CD11c+ cells, RYGB vs. DIO, p < 0.05, and $: F4/80+/CD206+ cells, RYGB vs. DIO, p < 0.001. (C) F4/80+/CD11c+ and F4/80+/CD206+ macrophages in epididymal WAT. *: DIO vs. Lean, p < 0.001 in both F4/80+/CD11c+ cells and F4/80+/CD206+ cells; **: SHAM-PF vs. DIO, p < 0.01; #: RYGB vs. DIO and SHAM-PF, p < 0.01, and $: RYGB vs. DIO and SHAM-PF, p < 0.01. The flow cytometry analysis was repeated on four separate occasions (n = 2 in each groups).

Gastric bypass influenced tissue specific T-cell subsets in the liver and WAT

Both, CD4+ (CD3+/CD8−) and CD8+ (CD3+/CD4−) T-cells were significantly increased in DIO liver and WAT at one-week post-surgery (Figures 6A and 6B). Interestingly, RYGB resulted in near normalization of both CD4+ and CD8+ T-cells and the changes were highly significant in epididymal WAT and liver. In contrast, the changes in liver and epididymal WAT CD8+ and CD4+ T-cells were not significantly different between DIO and SHAM-PF mice (Figures 6A and 6B).

Figure 6.

Figure 6.

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Flow cytometry analysis of CD4+ and CD8+ T-cell subsets in the liver and epididymal WAT at one week after surgery. CD3+/CD4+ cells were defined as CD4+ T-cells and CD3+/CD4− cells as CD8+ T-cells. The lower left panel, CD3+/CD4+ T cells. *: DIO and SHAM-PF vs. Lean, p < 0.01; #RYGB vs. DIO and SHAM-PF in the liver and WAT, p < 0.01. The lower right panel, CD3+/CD8+ T-cells. *: DIO and SHAM-PF vs. Lean, p < 0.01; #: RYGB vs. DIO and SHAM-PF in the liver and WAT, p < 0.01. The flow cytometry analysis was repeated on four separate occasions (n = 2–3 in each groups).

Figure 7 shows significantly reduced percentage of Treg cell subsets (CD4+/FoxP3+) in the liver and WAT in DIO and SHAM-PF mice. However, RYGB maintained a higher percentage of CD4+/FoxP3+ T-cells in the liver (p < 0.05) and in the epididymal WAT (p < 0.01).

Figure 7.

Figure 7.

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Flow cytometry analysis of CD4+/FoxP3+ T-cells in the liver and epididymal WAT at one week after surgery. CD3+ cells were gated followed by analysis of CD4+/FoxP3+ cells. *: RYGB vs. DIO and SHAM-PF, p < 0.05; and **: RYGB vs. DIO and SHAM-PF, p < 0.01. The flow cytometry analysis was repeated on four separate occasions (n = 2 in each groups).

Discussion

There is increasing evidence that obesity is associated with a generalized activation of the immune system leading to the development of a chronic inflammatory state, insulin resistance and ultimately to significant morbidities such as cardiovascular diseases and T2D8, 9. It is well accepted that gastric bypass is the most effective therapeutic intervention for the amelioration of insulin resistance and for the reversal of T2D13, 14. There is little information, however, related to the effect of gastric bypass on the altered innate and adaptive immune responses associated with obesity in the early phase after surgery. Our results show that RYGB results in significant improvement in body weight, fat mass and glucose tolerance in DIO mice. The improved metabolic phenotype is associated with improved innate and adaptive immune responses.

NF-κB has the pleiotropic function involving the upregulation of multiple inflammatory and immune genes. Once activated, infiltrated macrophages release inflammatory cytokines further eliciting the recruitment of additional macrophages to adipose tissue30. Using a tissue luciferase assay, we also show that NF-κB activation is elevated in DIO WAT and muscle, with RYGB being the most effective in inhibiting NF-κB activation in both tissues. However, the hepatic findings related to NF-κB activation are intriguing; there is no significant change of hepatic NF-κB activation in HFD-fed mice. Neither RYGB, nor caloric restriction and sham surgery have any effect on NF-κB activation, suggesting the existence of yet undetermined hepatic driving force eliciting the hepatic NF-κB signaling pathway in DIO mice.

JNK is a member of mitogen-activated protein kinases. Increased expression of wild type JNK in the liver decreases insulin sensitivity, whereas suppression of the JNK pathway results in an improvement of insulin resistance31, 32. A major target of the JNK signaling pathway is activation of the AP-1 transcription factor that is mediated by the phosphorylation of c-Jun33, 34. Our study shows that hepatic insulin resistance associated with HFD is mediated by activation of the hepatic JNK/AP-1 signaling pathway rather than NF-κB activation that mediates liver fibrosis and nonalcoholic steatohepatitis (NASH)35. RYGB results in inhibition of phosphorylation of liver JNK, which may contribute to the improved glycemic control.

Obesity is associated with an increase in macrophage infiltration in WAT36 particularly M1 macrophages3, 37, 38. This increased infiltration results in high expression of pro-inflammatory genes such as TNF-α, inducible nitric oxide synthase and IL-6, all contributing to a state of insulin resistance39. Our findings in the DIO mice are confirmatory and associate the inflammatory changes with increased plasma leptin, TNFα, IL-1β, MCP-1 and IL-6 in WAT and liver, as well as with decreased plasma adiponectin. RYGB results in reduction in MCP-1 that plays an essential role in macrophage recruitment to adipose tissue. RYGB has also inhibited M1-like differentiation (F4/80+/CD11c+) associated with increased M2-like populations (F4/80+/CD206+) in the DIO liver and WAT. These findings are consistent with those previously observed in humans showing that RYGB resulted in decreased expression of pro-inflammatory markers in human WAT and plasma40. Interestingly, these effects were transiently mimicked, albeit to a lesser degree, by caloric restriction and sham pair-feeding, indicating that regulation of inflammation by RYGB mediates subsequent improvement in insulin resistance, and that RYGB’s improvement in insulin resistance is highly related to the de-activation of the inflammatory pathways.

The role of cellular immune responses in the mediation of obesity and insulin resistance is not yet clearly defined. We show that high-fat feeding results in an increase in both CD4+ and CD8+ T-cells in the liver and WAT, which are associated with macrophage activation and perpetuation of local inflammatory reactions as well as with the development of insulin resistance as was previously observed in ob/ob mice41. In addition, HFD-induced obesity is associated with depletion of Treg cells associated with enhancement of insulin resistance. Induction of Treg cells decreases adipose inflammation consistent with previous reports31. RYGB inhibits HFD-induced CD4+ and CD8+ cell infiltration and promotes Treg cells in the liver and WAT. These effects are not reproduced by caloric restriction.

Leptin and adiponectin are true adipokines produced exclusively by adipocytes and mediate inflammation and anti-inflammatory responses, respectively. Leptin negatively regulates Treg cell proliferation42, whereas adiponectin inhibits NF-κB activation43 and promotes M2 macrophage polarization which in turn enhances Treg populations44. RYGB, but not SHAM-PF, results in sustained decrease in plasma leptin and increase in plasma adiponectin, thereby contributing to the regulation of cellular immune responses. One major limitation of our findings relates the evidence that human and mouse obesity and immune responses are markedly different. Consequently, the extrapolation of early immune responses post-gastric bypass related results in mice must be carefully interpreted concerning human implications. In addition, biological variables, such as sex, weight and underlying nutrition conditions, are often critical factors resulting in different immune response and affecting health or disease. Given the fact that females are relatively resistant to HFD-induced obesity45, age- and sex-matched male C57BL/6 background mice are used in this study.

In conclusion, DIO mice display significant inflammatory reactions in insulin targeted tissues, including enhanced NF-κB activation in WAT and muscle and with activated JNK/AP-1 signaling pathway in the liver and WAT. RYGB significantly improves glucose tolerance associated with the tissue specific regulation of NF-κB activation and of the JNK/c-Jun signaling pathway. RYGB also inhibits M1-like and enhanced M2-like polarization, reduced CD4+ and CD8+ T-cell infiltration and promotes Treg cell generation in the liver and WAT. While our studies are suggestive of a direct relationship between improved metabolic phenotype and inflammatory regulation by RYGB, it is also important to note the potential contribution, albeit to a lesser degree, of caloric restriction (as exemplified by sham pair-feeding) to these processes.

Acknowledgements

This work was supported in part by the Department of Surgery at University of Chicago to the Animal Microsurgery Center and by the National Institution of Health (NIH) grant DK020595 to the Metabolic Core of the University of Chicago and the Tissue and Cell Models Core of the University of Chicago DDRCC (P30 DK42086).

Abbreviations:

AP-1

activator protein 1

DIO

high-fat diet-induced obesity

HFD

high-fat diet

IPGTT

intraperitoneal glucose tolerance test

IRS

insulin receptor substrate

RYGB

Roux-en-Y gastric bypass

SHAM-PF

sham surgery and pair-feeding

T2D

type 2 diabetes

WAT

white adipose tissue

Footnotes

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Conflict of interest statement: The authors have declared that no conflict of interest exists.

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