Lifestyle Changes Followed by Bariatric Surgery Lower Inflammatory Markers and the Cardiovascular Risk Factors C3 and C4

Abstract

Background:

Morbidly obese patients are at risk of developing insulin resistance and cardiovascular disease. Low-grade systemic inflammation is an important factor for this development. We evaluated the effect of bariatric surgery on markers of inflammation, coagulation and glucose metabolism.

Methods:

Ninety-seven morbidly obese patients and 17 lean subjects (control group) participated. Anthropometric measurements as well as fasting blood samples were obtained at first admission, prior to surgery, and 1 year after surgery.

Results:

At admission, the morbidly obese group had significantly elevated levels of the complement components C3 and C4 compared to the lean control group (P<0.0001). Levels of C3 and C4 dropped significantly in the morbidly obese group over time (P<0.0001), and, 1 year after the operation, levels were comparable to those of the control group. The same changes were seen for markers of inflammation (high-sensitivity C-reactive protein, tumor necrosis factor-α, interferon-γ, interleukin-1 receptor antagonist, IL-6, and IL-13), coagulation (fibrinogen and plasminogen activator inhibitor-1), and glucose metabolism (leptin and insulin). There was a positive correlation between changes in C3 and body mass index, weight, coagulation parameters, inflammatory parameters, and leptin, respectively.

Conclusions:

Bariatric surgery in morbidly obese patients reduced weight effectively. Even more importantly, the increased levels of several risk factors associated with diabetes and cardiovascular co-morbidity normalized 1 year after surgery.

Introduction

Obesity is now considered a worldwide pandemic.^1,2 Morbidly obese patients are at risk of developing cardiovascular complications such as hypertension and insulin resistance, as well as cerebral infarction and coronary disease. The mechanisms behind the development of co-morbidity are still not fully understood, but low-grade systemic inflammatory processes and visceral adiposity appear to play central roles.^3,4 Translocation of components of the gut microbiota to the circulation is considered to be a key factor in the initiation and maintenance of the low-grade inflammation.

Recently, we have shown that plasma levels of lipopolysaccharide (LPS) correlate closely with glycemic control and with intra-abdominal fat volume, and that the levels of LPS were reduced by bariatric surgery.⁵ LPS binds predominantly to Toll-like receptor 4, an important first-line component of the innate immune system. Another important component of innate immunity is the complement system. Complement components C3 and C4 are acute-phase proteins important in complement activation. C3 has been shown to be an important risk factor for the development of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD).⁶ The role of complement in heart disease has recently been reviewed.⁷ The synthesis of these proteins increases in response to inflammation and infections, and inflammation is associated with T2DM and CVD.^8

–11 Complement factor C3 is regulated by interleukin-6 (IL-6) and IL-1β and C4 is regulated by interferon-γ (IFN-γ).¹² Both complement factors are mainly synthesized in the liver, but adipose tissue can contribute.¹³ Moreover, fatty tissue is an important humoral organ that synthesizes and releases several proinflammatory cytokines that are important in the innate immunity system.^14
–16 Both fat cells and non-fat cells in the fatty tissue contribute in this respect.^17
–19 Here, we studied the impact of significant weight loss as a result of bariatric surgery on levels of C3 and C4 as well as several other markers of inflammation and hemostasis.

Methods

Study participants and experimental design

We prospectively studied 97 morbidly obese patients admitted to the regional center for treatment of morbid obesity, Nordland Hospital, Norway. Seventeen lean subjects served as a control group. The study was approved by the Regional Ethics Committee of Northern Norway and by the Norwegian Data Protection Authority, and complied with the Helsinki II declaration. Written informed consent was obtained from all participants included in the study.

Inclusion criteria for the morbidly obese group (MOG) were >18 years of age, body mass index (BMI) ≥40 kg/m², or BMI ≥35 with associated co-morbidity such as hypertension, T2DM, or sleep apnea. The patients were included consecutively, regardless of co-morbidity. The patients underwent lifestyle changes for a mean period of 3 months prior to bariatric surgery.

The control group (CG) consisted of nonobese subjects with a BMI <25 and without CVD or T2DM, scheduled to undergo elective laparoscopic cholecystectomy or laparoscopic fundoplication.

Waist circumference

In the morbidly obese group, waist circumference (WC) was measured in centimeters (cm) midway between the lateral lower ribs and the iliac crest with the patients in an upright position.

Blood sampling

Fasting blood samples were obtained by standard venipuncture on three occasions: At first admission, the day before surgery (after 3 months of lifestyle intervention), and 1 year after surgery. Routine blood analyses were performed on the day of sampling at the laboratory of Nordland Hospital. Serum, EDTA plasma, and citrate plasma were frozen in aliquots at −80°C and analyzed in batch at the end of the study. C3 and C4 in serum were determined with nephelometry (BN Pro Spec, Siemens, Marburg GmBH, Germany), D-dimer in citrate plasma with Liatest^® D-DI buffer, and Latex (STA-R Evolution, Diagnostica Stago S.A.S, Asnières sur Seine, France), and fibrinogen was analyzed in citrate plasma using STA-Fibrinogen-5 quantificative determination of fibrinogen (STA-R Evolution, Diagnostica Stago S.A.S, Asneières sur Seine, France). Cytokines, plasminogen activator inhibitor-1 (PAI-1), insulin, and leptin were analyzed in EDTA plasma using a magnetic bead-based multiplex assay (Bio-Rad Laboratories, Inc, Hercules, California, CA).

Lifestyle changes

The mandatory preoperative lifestyle change resulted in an average weight loss of 10%; the details of the program has been described previously.²⁰ Achieving this weight loss took on average 12 weeks.

Surgery

All operations were performed by two experienced bariatric surgeons, and two surgical methods were used: (1) Laparoscopic Roux-en-Y gastric bypass (LRYGB) used a standardized procedure by creating a small ventricular pouch of 30 mL, a bileopancreatic limb of 50 cm, and an alimentary limb of 100 cm.²¹ This method was used for patients with BMI ≤50. (2) Bileopancreatic diversion with duodenal switch (BPDDS) used a standardized laparotomy procedure. We created a gastric sleeve using a 32 French probe to measure the diameter of the gastric sleeve, then made an alimentary limb of 150 cm and a common channel of 100 cm.²¹ The latter method was chosen for patients with a BMI >50. One patient received a LRYGB despite a BMI of 51.7. Another patient received a BPDDS despite a BMI of 46.7. This patient had lost 14.7 kg prior to the first admission, so the initial BMI was 51.7.

Statistics

Numerical data are presented with mean and standard deviation (SD). An unpaired Student t-test was used to calculate the differences between the control group and the morbidly obese group. The Shapiro–Wilks test was used to test for normality distribution. A paired t-test was used to calculate the effect of lifestyle change between admission and prior to surgery (after 10% weight loss in the morbidly obese group). A repeated measures one-way analysis of variance (RM one-way ANOVA) was used to calculate the longitudinally effect of bariatric surgery on parameters of inflammation, coagulation, and glucose metabolism. Correlations were calculated with the parametric Pearson correlation test. All tests were two-tailed and results with a P<0.05 were considered statistically significant. Analyses were done using PRISM 6 (Graph Pad Software Inc, La Jolla, CA).

Results

Anthropometry

Baseline characteristics for both groups are shown in Table 1. The MOG had a mean preoperative weight loss of 14.5 kg (SD=6.8). One year after surgery, the mean total weight loss was 48.4 kg (SD=17.8) (Fig. 1A). Mean preoperative BMI reduction was 5.1 kg/m² (SD=2.3), and in total 16.8 kg/m² (SD=6.1) 1 year after surgery (Fig. 1B). The MOG had a mean WC of 137.0 cm (SD=14.3) at baseline, reduced to 101.2 cm (SD=11.8) 1 year after surgery (Fig.1C). The CG had a mean weight of 66.7 kg (SD±8.4) and a BMI of 23.0 (SD±1.4). WC measurements were not obtained for this group.

FIG. 1.

Anthropometry. Mean with standard deviation (SD) is indicated in upper bound for the morbidly obese group (MOG) and lower bound for the lean control group (CG). (***1) Difference in weight (A) and body mass index (BMI) (B) between the MOG and the CG at admission (unpaired t-test) P<0.0001. (***2) Changes over time in weight (A), BMI (B) [one-way analysis of variance (ANOVA) repeated measurements], and waist circumference (C) (unpaired t-test), P<0.0001, from admission to 1 year after surgery.

Table 1.

Characteristics at Admission

	Morbidly obese group		Control group
	n or mean	SD or range	n or mean	SD or range
LRYGBP	71	—	—	—
BPDDS	26	—	—	—
LCHOL	—	—	15	—
LFUNDO	—	—	2	—
Males	29	—	5	—
Females	68	—	12	—
Age (year)	44	26–61	42	17–70
Weight (kg)	135.5	25.4	66.7	8.4
BMI (kg/m²)	46.8	7.05	23	1.4
Waist (cm)	137	14.3	—	—
T2DM/non-T2DM	32/65	—	0/17	—
Hypertension/ non-hypertension	38/59	—	0/17	—

SD, standard deviation; LRYGBP, laparoscopic Roux-en-Y gastric bypass; BPDDS, bileopancreatic diversion with duodenal switch; LCHOL, laparoscopic cholecystectomy; LFUNDO, laparoscopic fundoplication; BMI, body mass index; T2DM, type 2 diabetes mellitus.

Inflammation

There was a significant difference between the MOG and CG at admission for high-sensitivity C-reactive protein (hsCRP), C3, and C4 (P<0.0001 for all). However, 1 year after bariatric surgery the MOG had levels comparable to the CG for all these parameters (Fig. 2).

FIG. 2.

Inflammation. Mean with standard deviation (SD) is indicated in upper bound for the morbidly obese group (MOG) and lower bound for the lean control group (CG). (***1) Difference in high-sensitivity C-reactive protein (hsCRP) (A), C3 (B), and C4 (C) between the MOG and the CG at admission (unpaired t-test) P<0.0001. (***2) Changes over time in hsCRP (A), C3 (B), and C4 (C) [one-way analysis of variance (ANOVA) repeated measurements], P<0.0001, from admission to 1 year after surgery.

For the MOG, there were statistically significant reductions in hsCRP and C3, but not C4, from admission to surgery (P<0.004 and P<0.0001, respectively) (Fig. 2). From admission to 1 year after surgery there were significant reductions for all three parameters (P<0.0001 for all) (Fig. 2). The differences between the MOG and the CG in IL-1 receptor antagonist (IL-1ra), TNF-α, IL-6, and IFN-γ at admission were borderline nonsignificant, with P=0.08 for IL-1ra and IL-6 and P=0.06 for TNF-α and IFN-γ (Table 2). One year postoperatively, there were no longer any obvious differences between the operated patients and the control group, except for TNF-α (Table 2).There were, however, significant changes over time in the MOG measuring the same inflammatory parameters (Table 2). The changes over time in C3 showed a significant correlation with changes over time in weight (r=0.359, P<0.0004), BMI (r=0.355, P<0.0005), hsCRP (r=0.412, P<0.0001), C4 (r=0.689, P<0.0001), fibrinogen (r=0.662, P<0.0001), PAI-1 (r=0.234, P<0.03), D-dimer (r=0.323, P<0.002), leptin (r=0.292, P<0.04), and glycated hemoglobin (HbA1c) (r=0.332, P<0.002).

Table 2.

Markers of Coagulation, Inflammation, and Glucose Metabolism: Effect over Time

	Control group (1)	Admission MOG (2)	Preoperatively MOG (3)	1 year postop. MOG (4)	1 vs. 2	1 vs. 3	1 vs. 4	2 vs. 3	2 vs. 3 vs. 4
PAI-1 (pg/mL)	1585 (828)	4170 (1561)	2575 (1363)	1690 (904)	^*	^*	NS	^*	^*
Fibrinogen (g/L)	2.98 (0.89)	3.96 (0.78)	3.91 (0.80)	3.33 (0.64)	^*	^*	NS	NS	^*
D-dimer (mg/L)	0.39 (0.13)	0.44 (0.33)	0.48 (0.48)	0.41 (0.26)	NS	NS	NS	NS	NS
TNF-α (pg/mL)	3.40 (4.76)	14.4 (22.48)	12.57 (22.84)	7.89 (14.39)	NS	^*	^*	NS	^*
IFN-γ (pg/mlL)	7.17 (10.00)	20.31 (27.47)	18.47 (29.42)	11.48 (18.66)	NS	^*	NS	NS	^*
IL-13 (pg/mL)	6.76 (8.31)	13.23 (10.70)	12.00 (10.55)	7.41 (6.69)	^*	^*	NS	NS	^*
IL-6 (pg/mL)	0.37 (0.99)	2.97 (5.86)	2.42 (5.44)	1.15 (3.42)	NS	^*	NS	NS	^*
IL-1ra (pg/ml)	15.21 (26.09)	81.87 (150.60)	119.30 (179.70)	31.93 (81.25)	NS	^*	NS	NS	^*
Leptin (pg/mL)	1307 (980)	10909 (5980)	5868 (4046)	2136 (1618)	^*	^*	^*	^*	^*
Insulin (pg/mL)	105.7 (77.4)	330.6 (234.3)	220.6 (198.7)	91.8 (64.1)	^*	^*	NS	^*	^*
HbA1c (%)	—	6.30 (1.06)	6.04 (1.00)	5.56 (0.40)	—	—	—	NS^a	^* ²

Values presented as mean with standard deviations. 1 vs. 2 refers to the difference between the control group (CG) and the morbidly obese group (MOG) at admission (unpaired t-test). 1 vs. 3 refers to the difference between the CG and the MOG preoperatively (after 10% weight loss in the MOG) (unpaired t-test).

1 vs. 4 refers to the difference between the CG and the MOG at 1 year postoperatively (MOG) (unpaired t-test). 2 vs. 3 refers to changes in the MOG from admission to surgery (after 10% weight loss) (paired t-test). 2 vs. 3 vs. 4 refers to the changes in the MOG from admission to 1 year postoperatively over three time points [one-way analysis of variance (ANOVA) repeated measures].

Because of missing glycated hemoglobin (HbA1c) values, unpaired t-test was used in this analyses.

P value <0.05.

PAI-1, plasminogen activator inhibitor-1; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL-13, interleukin-13; NS, nonsignificant.

Coagulation

There was a significant difference between the MOG and the CG for fibrinogen and PAI-1 (P<0.0001) at admission (Table 2). One year after surgery, these were no longer present (Table 2). There was a statistically significant difference in PAI-1 and fibrinogen over time (P<0.0001) both from admission to surgery and from admission to 1 year after surgery (Table 2). There were no significant differences in D-dimer, neither between MOG and CG nor over time (Table 2). Changes over time in fibrinogen were significantly correlated to changes in BMI (r=0.286, P<0.005), WC (r=0.215, P<0.04), hsCRP (r=0.456, P<0.0001), and C4 (r=0.558, P<0.0001).

Glucose metabolism

There was a significant difference between the MOG and the CG at admission concerning both insulin and leptin (P<0.0001 for both) (Table 2). There were statistically significant reductions in leptin between admission and preoperatively (P<0.0001) and between admission and 1 year after surgery (P<0.0001) (Table 2). Furthermore, there were statistically significant reductions in insulin between admission and preoperatively (P<0.0001) and between admission and 1 year after surgery (P<0.0001) (Table 2). Finally, there was a significant reduction of HbA1c from admission to 1 year after surgery (P<0.0001) in the MOG (Table 2). HbA1c was not analyzed in the CG.

In the MOG, RM two-way ANOVA was used to evaluate the outcome in the nondiabetic group versus the T2DM group for all parameters obtained. There were no significant differences between these two groups (data not shown).

Discussion

In the present study, we demonstrate how morbidly obese patients compared to lean controls have elevated levels of several serological markers known to be risk factors for CVD, such as complement factors 3 and 4, hsCRP, and PAI-1. Low-grade systemic inflammation is an important contributor to the development of co-morbidity (CVD and T2DM) in morbidly obese patients.^22

–27 It is also well documented that bariatric surgery has a positive effect not only on weight loss but also in reducing risk factors,^{20,25,28
–30} cardiovascular events,³¹ and improving or inducing resolution of T2DM.²⁴

We confirm these findings by showing how weight loss achieved by lifestyle changes and subsequent bariatric surgery induces a significant reduction in numerous well-established risk factors for CVD. The fact that 1 year after surgery the obese patients had levels of C3, C4, hsCRP, and PAI-1 comparable to the control group confirms the powerful effect of the intervention. Whether C3 has a causal effect in the development of CVD or whether there is just an association is at present unclear. Complement proteins can be synthesized in adipose tissue,¹³ and recent data demonstrate a close association between adipose tissue volume and C3 levels.¹² It has also been shown that C3 is independently associated with insulin resistance,³² and we have previously demonstrated that glycemic control correlates with plasma LPS levels and that there is a gradient of bacterial DNA from mesenteric via omental to subcutaneous fat.⁵ Thus, translocation of bacterial products from the gut microbiota to the fatty tissue and circulation may trigger synthesis of complement factors and subsequent insulin resistance. The effect of intervention may then be due both to a reduced adipose tissue volume, but also to a change in the bacterial composition in the intestine. The fact that C4 and hsCRP show a reduction similar to C3 strengthens our observation, and the findings are consistent with other studies.^12,33 Normalization of cardiovascular risk factors following bariatric surgery has been demonstrated previously.³⁴

To our knowledge this is the first study to compare levels of complement factors between morbidly obese patients and a lean control group. A similar comparison was also performed for a number of other inflammatory markers. For IL-1ra, IL-13, TNF-α, IL-6, and IFN-γ, no statistically significant differences were seen between the morbidly obese group and the control group at admission. However, there was a statistically significant reduction in the morbidly obese group following intervention for all these parameters, but as long as baseline values did not differ from the control group, the significance of this finding is unclear. For a number of other markers (IL-9, IL-10, IL-17, and IL-23), there were no differences between MOG and CG at admission, and no effect of intervention (data not shown). These results are consistent with other studies.³⁵

The concept of “immunothrombosis” is receiving increasing attention,³⁶ and the crosstalk between the hemostatic and inflammatory processes may prove to be a target for future therapeutic interventions in various diseases. PAI-1 is the main inhibitor of the fibrinolytic system. It is also a mediator in several other important inflammatory processes, including atherosclerosis, wound healing, and rheumatoid arthritis, through effects on inflammatory cell migration.³⁷ PAI-1 is produced in vascular endothelium, adipose tissue, and the liver. It is an important protein that is a major contributor to the development of both CVD and insulin resistance.^38
–40 The possible treatment of obesity and its co-morbidity using PAI-1 as a target has been discussed.⁴⁰ PAI-1 is important in the mechanisms of both inflammation and coagulation. Morbidly obese patients are at a higher risk of developing thromboembolism than lean subjects, and PAI-1 is therefore of particular interest when evaluating the effect of interventions.^40,41

We show that in the morbidly obese group the levels of fibrinogen and PAI-1 at admission are significantly different from the CG. Furthermore, the levels decreased significantly over time after intervention and were comparable to controls 1 year after surgery. D-dimer, however, did not differ between MOG and CG. D-dimer is a marker of ongoing fibrinolysis and not merely a marker of risk. The lack of difference between the two groups may be explained by the fact that none of the participants had known thromboembolic events during evaluation and follow-up.

The increasing number of patients suffering from T2DM is mainly caused by morbid obesity. Bariatric surgery is an effective approach in the morbidly obese patient, resulting in both loss of excessive weight and prevention⁴² and treatment²⁴ of T2DM. In our study, 32 out of 97 morbidly obese patients suffered from T2DM at admission, whereas 1 year after bariatric surgery only six received treatment for T2DM. In addition, seven patients had impaired glucose tolerance at admission. Leptin is a hormone responsible for hypothalamus-induced appetite reduction and increased energy expenditure.²⁵ Leptin is mainly produced in fatty tissue and has an indirect as well as a direct effect on blood pressure regulation through proliferation of endothelial cells leading to endothelial dysfunction. In morbidly obese patients, the leptin level is increased–an apparent contradiction when looking at the function. It is, however, known that in addition to insulin resistance, morbidly obese patients also develop leptin resistance.^43,44 Thus, the finding that leptin levels were reduced following bariatric surgery in the same manner as insulin and HbA1c is well in accordance with the theory of increased leptin sensitivity. Changes in leptin levels were significantly correlated to changes in weight, BMI, WC, C3, and PAI-1, indicating a close relationship between metabolism, inflammation and thrombosis.

Conclusion

Bariatric surgery is currently the most effective treatment of morbidly obese patients; however, there is considerable research ongoing to identify alternative approaches to resolve the development of obesity. Understanding the underlying processes is central in this respect. Gut microbiota, low-grade inflammation, and abdominal obesity are central factors. Massive weight loss and thereby reduction of abdominal obesity are the key factors in improvement or resolution of co-morbidity, such as CVD and T2DM. This prospective study shows that several established risk factors involved in the low-grade inflammation in morbidly obese subject are not only susceptible to change, but can in fact be normalized as the result of significant weight loss due to bariatric surgery.

Limitations

The study has limitations regarding the size of the control group (only 17 participants), as well as the fact that blood samples from the control were only obtained at admission because this group was not followed over time. Furthermore, two different surgical procedures were used in the morbidly obese group. Both procedures, however, led to a significant weight loss. Within the morbidly obese group, only 26 patients underwent BPDDS. The changes in weight, WC, and BMI were higher in this group, but the other parameters did not differ from the patients going through LRYGB, indicating that the type of surgical procedure was of no importance.

Footnotes

Acknowledgments

The skillful assistance by the nurses at the Regional Centre for treatment of Morbid Obesity and Department of Clinical Chemistry at Nordland Hospital is greatly acknowledged. This study was supported by Nordland Hospital and research grants from Odd Berg Research Foundation and the regional health authorities of Northern Norway.

T.K.N., E.W.N., and K.T.L. designed the study. T.K.N. interpreted the results of analyses, prepared figures and tables, and drafted the manuscript. E.W.N. and K.T.L. edited and revised the manuscript. J.K.L., H.F., A.L., and T.K.N. performed laboratory analyses. All authors approved the final version of manuscript.

Author Disclosure Statement

No competing financial interests exist.

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