Increased plasma lipopolysaccharide-binding protein and altered inflammatory mediators in overweight women suggest a state of subclinical endotoxemia

Chronic low-grade inflammation has been recognized as an underlying event linking obesity to cardiovascular disease (CVD). However, inflammatory alterations in individuals who are overweight remain understudied. To provide insight, we determined the levels of key circulating biomarkers of endotoxemia and inflammation, including lipopolysaccharide-binding protein (LBP), CRP, IL-6, leptin, and adiponectin in adult female subjects (n = 20) who were lean or overweight and had high cholesterol and/or high blood pressure - two important conventional risk factors for CVD. Plasma levels of LBP (a recognized marker of metabolic endotoxemia in obesity) were significantly higher in the overweight group compared with the lean group (P = 0.005). The levels of CRP, a general marker of inflammation, were also significantly higher in overweight subjects (P = 0.01), as were IL-6 (P = 0.02) and leptin (P = 0.002), pro-inflammatory mediators associated with cardiovascular risk. Levels of adiponectin, an adipokine with anti-inflammatory and anti-atherogenic functions, were significantly lower in the overweight group (P = 0.002). The leptin/adiponectin ratio, a preferential atherogenic marker was significantly increased in women who are overweight (P = 0.02). LBP, CRP, leptin, and adiponectin levels significantly correlated with BMI, but not with age. These results reveal the presence of subclinical endotoxemia and a pro-inflammatory state in overweight women and are of interest for further studies with the goal for improved understanding of women’s cardiovascular health.


Introduction
Obesity and the closely related metabolic syndrome are associated with an increased risk for cardiovascular disease (CVD) and other debilitating and lethal disorders (1)(2)(3)(4)(5)(6).Publicly available information on the World Health Organization (WHO) website states that in 2016 around 1.9 billion adults (people over 18 years of age) were overweight, and more than 600 million were obese and the expectations are that more than 2.16 billion people will be overweight and 1.12 billion will be obese by 2030.A major underlying factor driving the pathogenesis in obesity and metabolic syndrome is the presence of a chronic low-grade in ammation, which is characterized by increased circulating IL-6 and other cytokines, as well as altered levels of adipokines, such as leptin and adiponectin (3,(7)(8)(9)(10)(11).An important driver of the in ammatory state in obesity is metabolic endotoxemia, manifested by increased gut lipopolysaccharide (LPS)-containing microbiota and the consequent compromising of intestinal permeability that leads to increased circulatory LPS levels (12)(13)(14)(15).Obesity-associated metabolic endotoxemia and chronic in ammation promote metabolic derangements and are associated with increased cardiovascular risk (9,(15)(16)(17)(18)(19)(20)(21).
In addition to obesity, there is evidence that overweight individuals may be at increased risk for CVD and other diseases (22,23).CVD is the leading cause of death among women in the United States (6, 24).Increased cholesterol levels (hypercholesterolemia) and high blood pressure (hypertension) are important risk factors for CVD (6,25).Elevated total cholesterol, hypertension, and excessive body weight have been linked to age-dependent increases of coronary heart disease incidence and mortality in both men and women, but to a larger extent in women (26).However, the underlying explanation for sex-speci c differences in the CVD pathophysiology remain poorly understood (6).As recently summarized, "Cardiovascular disease in women remains understudied, under-recognized, underdiagnosed, and undertreated globally" (6).
While metabolic endotoxemia and in ammation have been documented in people with obesity and linked to CVD and other diseases, endotoxemia and in ammatory alterations in overweight individuals remain to be characterized.This is of speci c interest for improved understanding of women's cardiovascular health.To generate insight, we pro led a panel of plasma biomarkers of endotoxemia and in ammation, previously associated with CVD in obesity, in a cohort of women who were overweight compared to those who were lean.As dyslipidemia and hypertension are recognized leading traditional risk factors for CVD in women (6, 25), we enrolled subjects having high cholesterol and/or high blood pressure in both groups.
We observed that increased circulating levels of LBP, a marker of metabolic endotoxemia in parallel with elevated CRP, leptin, and IL-6 levels, and decreased adiponectin levels were found in overweight women.

Human subjects and samples
All methods were carried out in accordance with relevant guidelines and regulations.Frozen plasma was obtained from research subjects who participated in the Institutional Review Board (IRB)-approved Genotype and Phenotype (GaP) registry (http://www.gapregistry.org),a research program at the Feinstein Institutes for Medical research, Northwell Health.All research subjects completed an informed consent prior to study participation.The consent permits the use of specimens for future research.The study was approved by the Northwell Health IRB -IRB #09-081A.Participants gave random blood samples and were chosen based on gender, BMI (lean: 18-24.9kg/m 2 (N = 20) vs. overweight: 25-29.9kg/m 2 (N = 20)), age, demographic information, and health/medical information (Supplementary Table 1).Subjects in both groups were relatively healthy, except they had self-reported hypertension (systolic ≥ 130mm Hg and diastolic > 80 mm Hg) and/or high cholesterol (200 mg/dL) and minor conditions, including acne, eczema, gastroesophageal re ux disease (GERD), drug allergies, and other allergies, as well as osteoarthritis, osteopenia, osteoporosis.Excluded conditions were Lyme disease, cancer (solid and blood [leukemia, lymphoma, etc.]), anemia, pancreatitis, emphysema, asthma, chronic obstructive pulmonary disease (COPD), in ammatory bowel disease (ulcerative colitis, Crohn's), lupus, rheumatoid arthritis, valvular disease, heart failure, HIV, excess alcohol use, diabetes (types 1 and 2) and Alzheimer's disease and other neurological conditions that would impair the subjects' ability to consent, as well as those using steroids, insulin, metformin, or glyburide and those who smoke or vape.

Statistical analysis
Data were analyzed using GraphPad Prism 9.5.1 software and applying an unpaired Student's t test with Welch's correction; P < 0.05 was considered signi cant.The linear correlation for the data was analyzed using the Pearson's correlation coe cient (or Pearson's r).The strength of the correlations was assessed based on the r values and considered to be weak (0.2-0.39), moderate (0.40-0.59), or strong (0.6-0.79).
Graphical representations were created using GraphPad Prism 9.5.1.

Results
There was no statistically signi cant difference in subjects' age (Table 1) The BMI of the subjects in the overweight group was signi cantly higher compared with the BMI of the lean group (Table 1).LBP is an important mediator of LPS interactions with immune cells and the LPS-induced transcription of pro-in ammatory cytokines (27).Because of the documented di culties in measuring LPS in biological uids (28), the LBP levels have been proposed and widely used as a reliable marker of endotoxemia (29)(30)(31).Increased levels of circulating LBP have been determined in obesity and the metabolic syndrome and associated with increased circulating IL-6 levels, impaired insulin resistance and cardiovascular risk (31)(32)(33)(34).In the present study we observed signi cantly increased plasma LBP in the overweight group compared with the lean group (Fig. 1A).In addition, plasma levels of CRP, a general in ammatory marker, were signi cantly higher in the overweight women (Fig. 1B), as were the cytokine IL-6 and the adipokine leptin (Fig. 1C, D).In contrast, the levels of adiponectin were signi cantly lower in the overweight group (Fig. 1E).In addition, the leptin/adiponectin ratio values were signi cantly increased in the overweight compared with the lean group (Fig. 1F).
Additional data evaluation revealed that plasma in ammatory marker alterations correlated with BMI of the study subjects (Fig. 2).A moderate, but signi cant correlation was observed between plasma LBP and BMI and plasma CRP and BMI (Fig. 2A, B) while a weak, non-signi cant correlation was found between plasma IL-6 levels and BMI (Fig. 2C).A moderate and signi cant correlation was observed between plasma leptin and BMI (Fig. 2D) and between plasma adiponectin and BMI (Fig. 2E).
Of note, no signi cant correlations were observed between the plasma in ammatory analytes and the age of the study participants as shown in Fig. 3.
Together, these results indicate that overweight women exhibit a signi cant systemic pro-in ammatory phenotype when compared to matched lean controls.

Discussion
Here, in a small cohort of women matched for age and having self-declared hypercholesterolemia and/or hypertension, we show that overweight individuals exhibited higher plasma concentrations of LBP and a pro-in ammatory state, compared to lean controls.These differences are relatively small, but notable for the existence of a signi cant correlation with BMI and a state of subclinical endotoxemia.
Previously, the results of one large study evaluating 500 apparently healthy lean individuals, a majority being female, has shown that LBP is weakly, but highly signi cantly, correlated with BMI (35).Further, LBP was also signi cantly positively correlated with blood pressure, HDL and LDL cholesterol, and triglycerides.The current results conform to these ndings and together indicate that markers of endotoxemia and a pro-in ammatory state vary continuously with the BMI, suggesting that adiposity of any degree, even if considered to be normal, is associated with detectable levels of pro-in ammatory molecules.Whether these have clinical signi cance will require additional large, prospective clinical studies, but it stands to reason that a sustained low grade in ammatory milieu likely has clinical consequences.
Although there is a lack of data regarding the risk of subclinical endotoxemia in overweight individuals, previous studies in obese individuals have clearly indicated the presence of metabolic endotoxemia (based on LBP levels) and a chronic in ammatory state and their role in promoting further metabolic dysfunction and pathogenesis (3,(7)(8)(9)(36)(37)(38)(39)(40)(41)(42).Metabolic endotoxemia in obesity has been speci cally linked to the pathogenesis of CVD (15,21) and increased LBP levels have been directly associated with an increased risk of CVD (21).Endotoxemia increases the production of IL-6 and other cytokines and signi cantly contributes to a pro-in ammatory state.IL-6 and the adipokine leptin are key mediators of in ammation in obesity (3,43).IL-6 has been characterized as an important link between obesity and coronary heart disease (44).Importantly, in a large prospective study, increased IL-6 levels were associated with a higher risk of CVD, speci cally coronary heart disease, as strongly as major established risk factors, such as blood pressure and blood cholesterol levels (45) Leptin is an adipokine with an essential role in energy balance through a variety of functions, some of which are related to cardiovascular health (7,18,20).Increased leptin levels in obesity are associated with activation of proin ammatory signaling and increased thrombosis and arterial distensibility in obese patients (11,46,47).Elevated leptin levels arising from leptin resistance in obesity are associated with insulin resistance and CVD (18).In contrast, adiponectin is an adipokine with anti-in ammatory and antithrombotic properties (7,48).Decreased plasma adiponectin levels were associated with an increased risk of myocardial infarction (49).Plasma levels of CRP (high-sensitive C-reactive protein), a general marker of chronic subclinical in ammation, have been positively correlated with plasma leptin levels and inversely with plasma adiponectin (50)(51)(52).The leptin/adiponectin ratio is indicated as a more reliable marker in CVD assessment compared with individual leptin and adiponectin measures (53)(54)(55) and proposed as a better marker of a rst cardiovascular event in men than plasma leptin and adiponectin levels alone (56).
Until longitudinal data concerning subclinical endotoxemia are available, it may be prudent to institute proactive measures to monitor and reduce the circulating levels of LBP, as a surrogate biomarker for endotoxemia.As composition of the diet has been shown to be a critical driver of metabolic endotoxemia (reviewed in ( 57)), with high saturated fat ingestion causing postprandial endotoxemia with increases in IL-6 even in lean subjects (58, 59), dietary intervention would be a reasonable initial step.Other possibilities include pharmacological interventions, including the potential development of anti-LPS peptides which neutralize LPS signaling of immune system activation (57).
In addition to the small number of subjects assessed, there are several limitations of this study.One important limitation is that the degree of abdominal adiposity (in contrast to subcutaneous fat deposits) has been implicated in the pathogenesis of a systemic in ammatory response and correlates well with the production of pro-in ammatory cytokines (60).Unfortunately, as a single measure the BMI cannot fully capture this variable as it is insensitive to changes in regional body composition.That is, a smaller waist circumference for any given BMI is indicative of subcutaneous fat deposits, in contrast to an abdominal location in an individual possessing a larger waist circumference.A consensus has appeared that including the waist circumference as a measured variable provides information independent of the BMI, and when both are considered together the predictive accuracy of cardiometabolic risk is signi cantly increased (61).Additional limitations include other potentially contributing factors that were not assessed, including the existence of abnormal glucose tolerance, degree of sedentary behavior, and the con rmation and severity of self-reported hypercholesterolemia and hypertension, among others.Lastly, as dietary factors are a major driver of the appearance of LPS into the circulation, in future studies plasma samples should be obtained under standardized fasting conditions.

Conclusion
Our results indicate that despite the presence of hypertension and/or high cholesterol levels in two groups of women characterized using BMI as overweight versus lean, circulating biomarkers of in ammation, including LBP, CRP, IL-6, and leptin are increased and adiponectin is decreased in the overweight group.Although these differences seem to be of subclinical importance, more extensive alterations of these molecules observed in obese individuals have been linked to an increased cardiometabolic risk.While the number of subjects in this exploratory study was small, the presence of subclinical endotoxemia the degree of which varied continuously with BMI encourage designing and implementing a larger con rmatory study to better characterize individuals who are overweight, but not yet classi ed as obese.
These individuals may bene t from therapy to alleviate chronic, low-grade in ammation as an additional risk factor for the development of CVD and other disorders.

Declarations
Acknowledgements: The authors wish to thank Gila Klein of the Genotype and Phenotype Registry, and the participants who provided blood for this study, as well as Michael Ryan and Bibu Jacob and staff members of the Boas Center Biorepository at the Feinstein Institutes for isolating and storing the plasma samples.

Figure 1 Levels
Figure 1

Table 1
Study participants' age and BMI.