Intestinal barrier disorders and metabolic endotoxemia in obesity: Current knowledge

Data from the World Health Organization (WHO) indicate that excessive weight is a global problem that affects over 1.9 billion people, and almost 650 million suffer from obesity. Excessive body weight was ranked as the fifth most significant cause of death – nearly 2.8 million adults die every year due to excessive weight or obesity [1]. The pathogenesis of these diseases is influenced by genetic predisposition, environmental factors, and lifestyle. In recent years, attention has been paid to another factor involved in the development of obesity: increased intestinal permeability, which may be additionally accompanied by a disturbance in intestinal microbiota homeostasis. Dysbiosis in the composition of the intestinal microbiota leads to a disruption of the integrity of the intestinal barrier. This can lead to the development of low-grade inflammation, which may worsen the metabolic state and cause further disturbances in the composition of the gut microbiota, leading to a vicious cycle of obesity, inflammation, and intestinal dysbiosis [2].

Due to the vast contact surface with the external environment, the gastrointestinal epithelium is vulnerable to harmful external factors. To provide effective protection against the penetration of toxins and antigens through the mucosa, but at the same time to enable the absorption of water and nutrients, a selective intestinal barrier exists [3]. The complete intestinal barrier is composed of such elements as the intestinal microenvironment, mucus layer, intestinal epithelium, and cells of the immune system located in the intestine [4]. The intestinal microenvironment consists of elements such as gastric juice with a low pH, proteolytic enzymes present in the intestinal juice, lysozyme and other antibacterial substances (defensins, lactoferrin), and the composition of the natural intestinal microbiota. Some of the bacteria that are part of the physiological ecosystem of the digestive tract release substances with an antimicrobial character: bacteriocins, organic acids, and hydrogen peroxide. Bacteria support the proper functioning of the organism by the synthesis of short-chain fatty acids (SCFA) and polyamines that provide the necessary nutritional substrates for the intestinal epithelial cells and stimulate the secretion of mucin, a substance that is a component of the intestinal mucus which seals the epithelium.

The next line of defense against the adherence and penetration of pathogens into the intestinal barrier is the mucus layer, which consists of two characteristic parts:

The outer layer, rich in antibacterial peptides released by Paneth cells and immunoglobulin A, produced in plasma cells; in this part of the intestinal barrier there are also numerous intestinal microorganisms, which are the most active elements of the barrier;

The main role of the mucus layer is to irrigate, regenerate, and protect the epithelium against the action of digestive enzymes. The most important element is the glycocalyx, synthesized by goblet cells, whose function is to limit the penetration of antigens to the lamina propria of the mucosa [5].

The tight intestinal mucosa provides selective contact between the intestinal lumen and the cells of the immune system, which is particularly important in inducing immunity against commensal bacteria and food-derived antigens [6]. The next element of the intestinal barrier is a single layer of epithelial cells, formed almost 80% by enterocytes, which takes part in the absorption of nutrients and influences the development of immune activity by mediating the release of cytokines and the expression of receptors involved in generating the immune response [7]. Tight junctions (TJ) are present at the top of the cell membrane to ensure the integrity of the epithelium. These are the most significant elements responsible for the control of selective intestinal permeability, conditioning the passive transport of water-soluble molecules [8]. TJ are made of multiprotein complexes composed of transmembrane proteins: occludins, claudins, junctional adhesion molecules (JAM), and tricellulins. There are two subcategories of claudins with the opposite effect. The first are proteins, such as claudin-2, which create paracellular channels and thus increase permeability. The second group, which includes claudin-1, -3, -4, -5, is associated with tightening the gut barrier [9]. As a result of interactions between the intracellular domains of transmembrane proteins and zonula occludens (ZO-1, ZO-2, ZO-3), that is, cytosolic proteins linked to actin filaments of the enterocyte cytoskeleton, the integrity of the intestinal barrier is disrupted. Myosin light-chain kinase (MLCK) and Rho-associated kinase (ROCK) induce MLC phosphorylation, which causes actomyosin rings to disengage the TJ, allowing paracellular transport [10]. Therefore, zonulin is a physiological modulator of TJ in the intestines. It is responsible for the transepithelial transport of substances between the intestinal lumen and the bloodstream, and also the regulation of the permeability of the intestinal barrier. It has been observed that cells lacking ZO-1 do not lose the ability to form tight connections, which ensures the proper tightness of the intestinal barrier [11, 12]. Epithelial cell junctions are also formed by adherent and GAP junctions. The former are made of desmosomes and cadherins connected to the cytoskeleton, which guarantees the high mechanical strength of these junctions. GAP connections, in turn, are formed by 6 transmembrane proteins, so-called connexins, involved in the maturation and differentiation of epithelial cells, ensuring the proper functioning of the intestinal barrier [13].

Another important component of the intestinal barrier is the lymphoid tissue associated with the intestinal mucosa – gut-associated lymphoid tissue (GALT), which fulfils an important role in both local and general organism immunity. The GALT cells are dispersed in the intestinal epithelium where they form endothelial lymphocytes (IEL) or within the lamina propria in the form of lymph nodes, Peyer's patches, and mesenteric lymph nodes [4]. The intestinal barrier's schematic structure is demonstrated in Figure 1.

Fig. 1

As a result of dysbiosis, caused by a variety of factors, including a high-fat, high-protein, and low-fiber diet, the integrity of the intestinal barrier is disengaged. The highly selective barrier permits the passage of many toxic substances through it, where lipopolysaccharide (LPS) is particularly harmful [14]. LPS is a heteropolymer composed of hydrophobic lipid A and a hydrophilic sugar chain. Lipid A binds tightly to other components of the lipid bilayer, making LPS an integral part of the outer cell membrane of Gram-negative bacteria and cyanobacteria that live in the gastrointestinal tract, constituting up to 70% of the human intestinal microbiota [15]. There are three different regions usually identified in LPS: lipid A, the core oligosaccharide, and the O-specific chain (so-called antigen-O) [16]. Lipid A is a highly conserved region of LPS that anchors this molecule to the outer cell wall membrane, and the specific structure of this fragment plays an important role in the biological activity of the LPS molecule, and it is therefore called a toxicity center. The core LPS fragment is divided into an inner and an outer part (hexose region), and the complete core oligosaccharide probably modulates the properties of the lipid part of the LPS, which is considered to be its active center. The outer part of the cortical region of the LPS joins its O-specific part, which consists of heteropolysaccharides composed of a repeating oligosaccharide unit typically comprised of two to six sugars. This LPS fragment is characterized by a high variability of its composition, which makes it specific for a various bacterial strain [16, 17]. LPS is a major component of the outer leaflet of the outer membrane of gram-negative bacteria, providing a protective barrier for the cytoplasmic membrane against the action of the active complement of the immune system and hindering the penetration of antibiotics into bacterial cells [15]. The release of LPS from the bacterial cell wall, which occurs as a result of the death or lysis of the bacterial cell, enhances the toxic effect of LPS [18].

The hypothesis that increased chronic exposure to LPS in the blood causes systemic inflammation and weight gain is inconsistent with the fact that the major type of Gram-negative bacteria, Bacteroidetes, has been reported to decrease during obesity. This discrepancy may be attributed to the fact that the endotoxic activity of LPS is not the same for all bacteria. LPS derived from Bacteroidetes is thought to be less harmful than that derived from Proteobacteria group microorganisms [19]. Depending on the structure, LPS may have a different endotoxic potential. The presence of C14-20H fatty acid in lipid A elicits a stronger inflammatory response in the host than in its absence. For example, the presence of this fatty acid in the lipid A of Fusobacterium bacteria indicates their harmful characteristics, whereas Prophyromonas and Provotella, in which this compound does not appear, have less deleterious activity [20]. Although LPS is not a component of the Gram-positive bacteria structure, it can also be involved in developing endotoxemia by the enhancement of intestinal permeability and thereby facilitating the passage of LPS from the intestinal lumen into the bloodstream. Figure 2 shows the structure of Gram-positive and Gram-negative bacterial cell walls, with an emphasis on the structure of lipopolysaccharide.

Fig. 2

The interaction between the gut microbiota and the host is essential for the development and proper functioning of the immune system. LPS in the intestinal lumen may influence the maturation of regulatory T cells and facilitate the destruction of pathogenic organisms. Furthermore, a high level of LPS in the blood can lead to septic shock, which is a life-threatening condition. Studies show that a moderately elevated level of LPS in the blood is associated with obesity and the development of metabolic diseases such as type 2 diabetes, insulin resistance, atherosclerosis, and cardiovascular disease. The host's reaction to the presence of LPS depends on the location and concentration of this compound [16]. LPS naturally occurs in microorganisms present in the intestine and it is believed to be continuously released into the lumen of the gut by the lysis of Gram-negative commensal bacteria. Defensive secretions such as antimicrobial peptides and bile acids provide natural protection against excessive bacterial growth, leading to the release of LPS from the bacterial cell walls. Small amounts of LPS are also released during bacterial division. It is estimated that approximately 1g of unbound LPS is found in the intestinal lumen of healthy individuals [16].

Endotoxins can enter the bloodstream through a local or systemic infection caused by exogenous Gram-negative bacteria, through paracellular absorption in the intestines, and through intercellular transport – as a component of chylomicrons [21]. During lipid digestion, LPS translocation is escalated due to chylomicron formation. High-fat diets contribute to developing endotoxemia by favoring the colonization of Gram-negative bacteria and by the increased formation of chylomicrons, which bind with LPS and bring it into the bloodstream. Not all types of fatty acids induce endotoxemia with similar effects. Unlike long-chain fatty acids, short and medium-chain fatty acids are absorbed into the bloodstream directly, without the formation of chylomicrons, therefore this does not lead to the development of endotoxemia. It proves that not only the amount but also the quality of consumed fat affects endotoxin translocation [22]. Another factor which contributes to the development of endotoxemia is chronic stress, which causes the activation of the hypothalamic–pituitary–adrenal (HPA) axis. Elevation in the activity of the HPA axis stimulates cortisol secretion, and therefore increases the permeability of the intestinal barrier [23]. Increased mucosal permeability is caused not only by abnormal paracellular transport across the TJ but also through gaps caused by the abnormal damage or death of epithelial cells [3].

The endocannabinoid system regulates a variety of gastrointestinal functions including food intake and fat storage. It also affects the metabolism of lipids and glucose [35]. In the large intestine it is proposed to interact with intestinal microbiota and regulate epithelial barrier permeability [36]. Cannabinoid type 1 receptors (CB1Rs) decrease the expression of TJ proteins, occludin, and ZO-1, which results in increased permeability. On the other hand, the activation of CB1Rs in enteroendocrine cells in the small intestine inhibits the release of cholecystokinin (CCK). CCK causes postprandial satiation by stimulating the vagus nerve [36]. A low concentration of this compound is associated with craving food intake and the preference for high-fat products. Interestingly, endocannabinoid signaling in the jejunum is triggered not only by fasting but also by tasting dietary fats. The consumption of tasty high-fat products increases the appetite for such products, which increases the intake of fat and calories and at the same time increases intestinal permeability. All of these factors create ideal conditions for the translocation of LPS into the bloodstream. According to Muccioli et al., the activation of CB1Rs increased the circulating levels of LPS in mice [37].

In a murine study, where metabolic endotoxemia was induced by a 4-week-long subcutaneous infusion of LPS, inflammation, an increase in total body and liver weight, an increase in plasma glucose and triglycerides, and the emergence of insulin resistance were observed [38]. Animals with artificially induced endotoxemia had the same phenotype as those fed a high-fat diet, which is obesity, insulin resistance, diabetes, and hepatic steatosis [38].

A nine-week dietary intervention, which included whole grain products and prebiotics, decreased endotoxin-producing bacteria belonging to Enterobacteriaceae and Desulfovibrionaceae taxa. At the same time, it favored the growth of Bifidobacteriaceae bacteria, which are considered to have a beneficial effect on the intestinal barrier. Parallel to these observations, a decrease in intestinal permeability in the lactulose/mannitol test was also observed, along with lower levels of LPS binding protein and other inflammatory markers like IL-6 and TNF-alfa [39]. Feeding the mouse a diet enriched in oligofructose caused a reduction in plasma LPS and proinflammatory cytokine levels, which was associated with an improvement in intestinal permeability and TJ integrity. The authors indicate that the beneficial effect of prebiotics results from the fact that probiotics changed the gut micro-biota. After the intervention, the total amount of bacteria and selected bacteria –Bifidobacterium, Lactobacillus, Eubacterium rectale – increased, and as a result, endogenous glucagon-like peptide-2 (GLP-2) production increased [40]. Another study involving the administration of oligofructose concludes that pre-biotic feeding formalized Akkermansia muciniphila abundance, which correlated with an improved metabolic profile. Additionally, A. muciniphila intake reversed metabolic disorders induced by a high-fat diet, including metabolic endotoxemia, adipose tissue inflammation, insulin resistance, and fat-mass gain [41]. In a randomized, double-blind, placebo-controlled trial with human volunteers, supplementation with a probiotic (A. muciniphila) improved the gut barrier function and thus reduced the plasma level [42].

Bariatric surgery (BS), the most effective therapy for weight loss, significantly modifies the composition and function of intestinal microbiota, including a decrease in metabolic endotoxemia, which may have a significant impact on weight loss and an improvement in metabolic status [43, 44]. BS alters the gut microbiota not only through anatomical changes in the digestive tract, changing the food flow, but also by modifying the secretions of the stomach, pancreas, and liver, as well as modifying intestinal pH [45]. As a result of BS, the amount of calories consumed drops drastically, which lowers the ratio of Firmicutes to Bacteroides [46]. Moreover, a decrease in fat intake reduces bacterial translocation, which reduces the LPS level [47]. Increased intestinal pH favors the presence of Gram-positive bacteria (Akkermansia muciniphila, Escherichia coli, Bacteroides) or bacteria characteristic for the oral cavity [43]. Postoperative increased oxygen availability leads to an increase in the amount of aerobic and facultative anaerobic bacteria [47]. What is more, altered bile flow after Roux-en-Y gastric bypass (RYGB) favors the growth of bile-tolerant bacteria such as Alistipes, Bilophila, and Bacteroides [48, 49]. According to research conducted by Casselbrant [9], after RYGB changes appear in the mucosal barrier function. Claudin-3 and −4 proteins were upregulated, while the occludin and ZO-1 expressions were decreased. It was found that the intestinal epithelial electrical resistance (REP) was elevated after RYGB and correlated with the claudin-3 expression. This may suggest that intestinal integrity was improved by strengthening the TJ function [9].

In animal models, RYGB-operated rats not only consumed less food and weighed less, but also had lower LPS and fasting plasma insulin levels in comparison to sham-operated rats [50]. Similar effects on LPS plasma levels were noted in a study involving ZDF (Zuker Diabetic Fatty) rats, in which obesity and its complications were genetically induced. Ten weeks after RYGB, lower concentrations of LPS in plasma and the inflammatory cytokines IL-1β and TNF-α in both serum and adipose tissue were observed [45].

This effect was also noticed in human studies. Monte et al. showed that 6 months after RYGB, in patients with type 2 diabetes mellitus (DM2), there was a significant reduction in the LPS level in plasma, associated with weight loss and a simultaneous decrease in the mRNA expression of CD14 (cluster of differentiation) and TLR-4 (Toll-like receptor 14) along with the plasma CRP level [51]. Another group of scientists examined the influence of bariatric procedures on LBP [52], which is considered to be blood marker of endotoxin [53]. LBP is an acute-phase protein, which is synthesized primarily by the hepatocytes. It is involved in the development of the systemic response to LPS. LBP, through binding with and presenting LPS to CD14, enhances the cellular response to LPS. Several recent studies have shown a relationship between elevated LBP levels and metabolic diseases like obesity and insulin resistance [54]. Yang et al. found that LBP was increased in patients with obesity and its level decreased one year after bariatric surgery, including 4 different bariatric procedures: sleeve gastrectomy (SG), one-anastomosis gastric bypass (OAGB), RYGB, and adjustable gastric band (AGB). Additionally, researchers indicate that there was a relationship between the LBP concentration and CRP and BMI before bariatric surgery. However, after the surgical procedure, the LBP level was correlated only with BMI. Although the authors emphasize that the mechanisms which result in a reduction of endotoxemia after bariatric surgery are not fully understood, they indicate that one of the main possible causes of this phenomenon is altered intestinal microbiota after bariatric surgery [52]. Another study in which a significant decrease in LPS was observed in people with obesity after bariatric surgery was the study by Trøseid et al. in 2013. At baseline, before introducing dietary intervention following surgery, the plasma LPS and soluble CD14 levels were significantly higher in patients with obesity compared with a control group. Additionally, pre-operative LPS levels were positively correlated with adipose tissue and glycated hemoglobin as well as several cardiometabolic risk factors like fasting triglycerides (TG), systolic blood pressure and BMI, and negatively correlated with HDL cholesterol. A year after bariatric surgery there was a close correlation between a reduced LPS level and improved glycemic control (HbA1c) and fasting TG [39]. The study also showed a greater correlation of LPS with visceral compared to subcutaneous adipose tissue. On this basis, the authors hypothesized that the fatty tissue located near the intestines, due to bacterial translocation through the intestinal walls, would have a greater quantity of bacterial DNA. Indeed, the amount of bacterial DNA did decline in that order: mesenteric, omental, and subcutaneous adipose tissue [55]. This may indicate that the improvement of metabolic parameters, including improved glycemic control in bariatric patients, may be caused by reduced bacterial translocation.

It seems that postoperative changes in LPS and LBP levels differ depending on the type of surgery performed and the time elapsed following the operation. It appears that the more radical the operation is, the more time the body needs to regenerate tissues and the longer the levels of acute phase proteins, including LBP, are elevated. Clemente-Postigo et al. compared changes in LPS and LBP in the short term (<3 months) after two different bariatric procedures: SG and biliopancreatic diversion (BPD). The authors reported that SG has a greater effect on LBP and LPS plasma concentrations than BPD in a short period after surgery. In addition, 90 days after surgery, patients with impaired glucose control had lower LPS levels, which was not observed 15 days after surgery [54]. Similar results were obtained from the analysis of LBP plasma concentrations after VBG and AGB. Up to 6 months after surgery, no significant changes were noticed, but 12 and 24 months after bariatric interventions, LBP was significantly reduced compared to the baseline results [56].

In a study on the Chinese population, significant changes in LPS levels following bariatric surgery were noticed after one month, including a decrease in inflammatory markers (IL-6, IL-8, CRP) and an improvement in metabolic status [57]. On the other hand, in the murine model one month after SG, different results were observed. SG diminished both paracellular and transcellular permeability ex vivo in jejunum, along with a greater mRNA expression of TJ proteins, JAM A proteins and occluding junctions, as expected. While in the distal colon, both permeabilities were increased without alteration in the expression of cell junction proteins. In vivo, the paracellular permeability was also enhanced, along with the plasma LPS level and mRNA levels of hepatic and adipose inflammatory markers 3 weeks after BS [58].

Restoring the balance of the gut microbiota can strengthen the intestinal barrier function and reduce metabolic impairments. BS, the most effective therapy for permanent weight loss, significantly modifies the composition and function of intestinal microbiota, which may have a great impact on weight loss and on improving metabolic status, including a decrease in metabolic endotoxemia [43, 44]. Table 1 presents the results of particular studies examining changes in LPS and LBP levels after BS.

A high-fat diet and the dysbiosis caused by it lead to an increase in the concentration of lipopolysaccharide in plasma, which is termed “metabolic endotoxemia.” Considering the importance of inflammation and oxidative stress in the etiology of cardiovascular and other diseases, and metabolic disorders associated with overweight and obesity, it has been suggested that endotoxemia may be an important mediator of metabolic disorders occurring in overweight and obesity. Although bariatric surgery itself is an effective treatment in the therapy against obesity and its consequences, including metabolic endotoxemia, recent studies show that the modulation of microbiota after bariatric surgery can bring tangible benefits. The improper composition of intestinal microorganisms can affect the tightness of the intestinal barrier, and thus promote pro-inflammatory processes and the development of oxidative stress, which, in consequence, can lead to many health disorders. Although interest in the microbiome and its importance in obesity and metabolic disorders has increased significantly in recent years, there are few scientific studies examining the effects of bariatric surgery on endotoxemia. Therefore more research is needed to fully understand this mechanism.