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1. Introduction

1. Background and search strategy

This project involves investigating the of role of short chain fatty acids in systemic inflammation. Most of the research has been done in a human monocytic cell line. A part of the research is based on a clinical trial in which 60 overweight individuals were either provided with an inulin control sachet or an inulin propionate ester sachet (to be mixed with food) over a period of 24 weeks. The blood samples from these individuals were used to determine the levels of different inflammatory markers. Through this project we aim to assess the effects of short chain fatty acids on systemic inflammation and understand the mechanistic pathways linked to it.

For retrieving research articles, OVID and google scholar were used. The keywords used for the search were- ‘obesity’, ‘inflammation’, ‘short chain fatty acids’ and ‘appetite’. The alternative terms like ‘overweight’, ‘obese’ and ‘high BMI’ were also used to capture as much literature as possible in the database. Combinations of the above words were used with ‘AND’ or ‘OR’ operators to find the specific papers related to the topic. A few resources other than the research articles like book chapters and public health websites were also used to gather relevant information on the statistics and current scenario of obesity.

2. Obesity –prevalence and risk factors

Obesity is a chronic disorder which is a result of the imbalance between energy intake and energy expenditure. The classification of obesity is based on BMI which has been represented in table 1.1 below for different classes. For children below 5 years, BMI z score or weight for height z score are used as indicators for obesity (Smith and Smith, 2016).

Table 1.1 Obesity classification

(Ng et al., 2014) conducted a systematic analysis of prevalence of obesity from 1980 to 2013 worldwide. They have reported an increase in prevalence in both developing countries as well as developed countries. The number of obese adults have increased by 27.5% from 1980-2013. More children are being affected by it as there has been a marked increase of 47.1% in the last three decades.

Results from the Health Survey of England (HSE) show that 62.9% of the adults in England are either overweight or obese. Obesity prevalence has increased by 12% from 1993 to 2015. The prevalence of obesity is similar in men and women. Men are more likely to be overweight, whereas women are more likely to have extreme BMI values. 28.2% of the children aged between 2 and 15 were classified as overweight or obese. The estimated direct cost of treating overweight and obese individuals by the NHS has increased from 479.3 million pounds in 1998 to 4.2 billion pounds in 2007(England). The prevalence of obesity in children between 2 and 15 and adults are shown in Figure 1.1 and Figure 1.2 respectively.

Figure 1.1- Obesity prevalence of children aged between 2 and 15 in England from 1995-2014.

Figure 1.2 –Obesity prevalence of adults in England from 1995-2014.

There are various risk factors in the development of obesity. (Smith and Smith, 2016) have assessed the risk factors of obesity in adults and children. They have reported genetic, socioeconomic, behavioural, medical and other novel factors in obesity. Genetic factors are evaluated based on the association of various genes with the BMI. 32 of the most common associated genetic variants account for just 1.5% of the inter-individual variation in BMI. A sedentary lifestyle with limited or no physical activity has a strong association with incidence of obesity. The availability of cheap convenience foods which are highly processed and calories rich have been implicated in weight gain. Previously, obesity was more prevalent in higher income group people. This has changed now with several other factors coming into play like availability of fast food chains, playground in the neighbourhood and level of education.

(Guh et al., 2009) used meta-analysis to investigate the co-morbidities associated with obesity taking into consideration a total of 89 studies.  They found significant associations between obesity prevalence and risk of cardiovascular diseases, type II diabetes, asthma, cancer (except prostrate and oesophageal), gall bladder disease and chronic back pain.

3. Inflammation and obesity

Inflammation is body’s natural response to infection and injury. It is mediated by different cell types like macrophages, dendritic cells, lymphocytes and mast cells. These cells are responsible for the secretion of soluble molecules like cytokines, chemokines, amines and eicosanoids which are responsible for removal of pathogen and tissue repair. The next step involves resolution of inflammation which is characterised by catabolism of pro-inflammatory mediators, PMN death and influx of monocyte derived macrophages. Chronic inflammation involves persistence of inflammatory trigger which leads to non-resolving inflammation in the long term shown in Figure 1.3(Fullerton and Gilroy, 2016).

Figure 1.3- Inflammation onset and resolution involving a range of different cells and molecules.

Obesity is complex as it is marked by a state of chronic low grade inflammation involving several inflammatory mediators and cell types (Lee and Pratley, 2005). Although there are several tissues and cell types, the adipose tissue is one of the key metabolic active sites which is involved in the secretion of hormones and cytokines shown in Figure 1.4.

Figure 1.4: The adipocytes in the adipose tissue secrete factors like M-CSF, MIF and MIP which attract and mature the monocytes in the blood to macrophages in the tissue. These macrophages along with the mature adipocytes secrete a variety of cytokines like IL-6, TNF- and resistin which induce a state of low grade systemic inflammation.  (Lee and Pratley, 2005)

The adipose tissue in response to the excess nutrients act via cytokine/toll like receptor and secrete cytokines which induce a pro-inflammatory state. When compared with a lean individual, the levels of TNF-, TGF-, IL-6, MCP-1 and several other molecules involved in inflammation are upregulated. Since, the inflammation seen in obesity is different from that of classical inflammation, metainflammation is used to describe the inflammatory characteristics in obesity. Metainflammation progresses through different pathways which are mainly regulated by kinases. The nutrient excess in obesity leads to the activation of three kinases in the cytoplasm-PKR, IKK and JNK shown in Figure 1.5. All the three kinases are responsible for the disruption of insulin signalling and hence cause insulin resistance which is common in obesity. The kinases also trigger a pro-inflammatory response by triggering transcription factors like AP-1 and NF-  leading to an upregulation of different pro-inflammatory cytokines observed in obesity. These cytokines then recruit macrophages and phagocytes which infiltrate the adipose tissue and produce further cytokines hence maintaining a state of low grade systemic inflammation(Gregor and Hotamisligil, 2011).

Figure 1.5: The effect of excess nutrients on several downstream proteins affecting the transcription of inflammatory mediators (Gregor and Hotamisligil, 2011).

1.4 Treatment options and new therapies

The field of medicine is continuously evolving and with better technologies available, the dynamics of obesity can be studied in more detail leading to a surge in treatment options. (Glenny et al., 1997) have looked into the treatment and prevention options available for obesity in children and adults using a systematic review approach.  Dietary, lifestyle, behavioural, pharmacological, surgical and combination therapies are available as treatment options for obesity. Although most of these therapies worked well during the intervention or a few months in the follow up, they were ineffective post follow up. Compliance during the intervention and post follow up is a major concern which limits the efficacy of these interventions. Weight regain after the completion of an intervention study is the most common outcome observed.

Since there are limitations to the current therapies for obesity, the scientific community is looking for alternative therapies which might help in the better management of the disease. The gut-brain axis is an important target for obesity management. It was observed that people who had undergone gastric-bypass had elevated levels of gut hormones and reduced food intake. The gut hormones GLP-1 and PYY play an important role in appetite regulation through their anorexigenic action (De Silva and Bloom, 2012). So, researchers are looking at different dietary approaches which might induce the gut hormones and regulate the appetite in the obese.

1.5 Role of SCFAs in appetite regulation

Dietary fibres are non-digestible carbohydrates which act as a substrate for intestinal bacteria and have physiological effects in humans by the production of short chain fatty acids. The consumption of dietary fibres has been linked to a decrease in risk of gastrointestinal diseases, cardiovascular diseases, liver diseases, type 2 diabetes and obesity (Dahl et al., 2017).

(Cummings et al., 1987) evaluated the SCFA contents in large intestine, portal, hepatic and venous blood in autopsy of patients with sudden death. The molar ratio of acetate, propionate and butyrate in the human gut was observed to be 57:22:21. The concentration of the SCFAs was found to be maximum in the caecum (1319 mmol/kg). There was a significant fall of 1/1000^th in the concentration of SCFAs in the portal blood which decreased continuously in the hepatic and the peripheral blood. So, the maximum concentration of SCFAs in blood us be found in the portal vein in the micromolar range with concentrations decreasing as they reach the tissues.

The SCFAs have an effect on brain either directly or via other chemical mediators. (Hu et al., 2016) have reported that only acetate is found in significant quantities in the blood (few hundred mol/l) and acts as an important energy source for astrocytes in the brain. It directly has an influence on the hypothalamus by increasing the expression of anorixigenic neuropeptides and decreasing the expression of orixigenic neuropeptides. The SCFA also stimulate the gut hormones which is one of the interesting avenues This approach is natural as it is a dietary intervention and has potential beneficial effects in other systems as well. In order to understand and better develop such strategies, a few important considerations like concentration response and effect size of the SCFA in different cell types and animal models need to be taken.

The average consumption of Non Digestible Carbohydrate (NDC) in UK is 15g/day which is much below the recommended intake set by the British Nutrition Foundation. At this level, the amounts of propionate produced per day is 0.4g. A dose escalation study recommended a dietary fibre intake of more than 35 g/day for increasing post-prandial PYY release and subsequent appetite suppression (Pedersen et al., 2013). (Chambers et al., 2015a) have discussed the challenges of increasing colonic production of propionate. They have mentioned that higher intake of dietary fibre is associated with gastrointestinal side effects. (Polyviou et al., 2016) in order to deliver higher amounts of propionate in the colon, developed a propionate-inulin ester with different weight percentages.  They performed in vivo and in vitro studies and found that the variant IPE-27 (27% loading of propionate) was the most efficient in releasing propionate in the gut. A 10 g/day supplementation of this variant is equivalent to a 2.5-fold increase in propionate levels. In a further extension to this study, (Chambers et al., 2015b) conducted a randomised controlled trial which involved the use of the inulin propionate ester in evaluating its effect on weight gain and energy intake in 60 overweight individuals. This trial was a first in man study conducted over a period of 24 weeks. The acute ingestion of this ester lead to a 14% decrease in food intake which if sustained over longer duration will lead to effective weight loss and management. Weight gain was significantly higher in the inulin control group than in the inulin propionate ester group. A reduced gain in intra-abdominal adipose tissue was also observed in the experimental group. Interestingly, there was no significant difference in the GLP-1 and PYY levels in the individuals after the long term supplementation of the inulin propionate ester. This suggests that there are other mechanisms which are important in appetite regulation mediated by short chain fatty acids. As it has been pointed out in earlier studies that obesity is characterised by low grade systemic inflammation, it would be interesting to see if propionate supplementation reduces systemic inflammation in the overweight individuals in the inulin propionate ester study. This way the benefits of consumption of the inulin propionate ester can be associated with the reduction in the systemic inflammation in the overweight individuals.

1.6 SCFA and modulation of inflammation

Fermentation of dietary fibres leads to the production of short chain fatty acids which have anti-inflammatory activity by modulating the mediators of inflammation at different levels (Vinolo et al., 2011).

1.6.1 SCFA and activation of inflammatory pathways

The mechanisms by which the SCFA act and modulate their action in different systems have been well studied. They activate the GPCRs (FFARs) and HATs further influencing the transcription of genes important in the inflammatory pathway (Vinolo et al., 2011). Figure 1.6 shows the effects of SCFAs at the genetic level as well as at the cellular level by modulating key proteins and enzymes. The proinflammatory cytokines are mainly mediated by the levels of transcription factors NF- and p38 MAPK.

Figure 1.6- A) SCFAs bind to the GPR43 receptors expressed on the surface of normal gut epithelium and inflammatory cells. They then interact with various proteins inside the cells like PLC, MAPK, adenylate cyclase and other molecules leading to the regulation of recruitment of other inflammatory cell types. B) SCFAs inhibit HDACs and regulate the expression of several genes involved in the process of inflammation.(Vinolo et al., 2011)

1.6.2 Role of transcription factors in inflammation

NF- is an important transcription factor responsible for the activation of a number of pro-inflammatory cytokines like IL-, TNF-, IL-6 and IL-8. In the cytoplasm, it is bound with certain inhibitory proteins called IB and is held in the inactive state. TNF-, IL-1and other cytokines are responsible for the activation of the transcription factor. When the macrophages receive any of these signals, the inhibitory protein associated with it breaks off and the transcription factor is translocated to the nucleus.  Inside the nucleus, it binds to the promotor regions of different genes and activates transcription (Tak and Firestein, 2001). The process of activation of NF- has been shown in Figure 1.7 below.

Fig:1.7 The metabolic stress and the inflammatory cytokines are responsible for the activation of the IKK which helps in the nuclear translocation of NF- which leads to the transcription of genes involved in inflammation(Tornatore et al., 2012).

(Libermann and Baltimore, 1990) demonstrated the activation of IL-6 gene by NF- in the presence of various inducers like LPS, TNF-, PHA and IFN-. They suggested that NF- mediates a central response in inflammation by the activation of a number of genes. This was also confirmed in a study by (Haugen and Drevon, 2007). They showed that adiponectin which is secreted by adipose tissue is involved in NF- activation. In their study, they found an increased expression of  IL-8 and TNF- upon stimulation by adiponectin associated with increased NF- activity.

(Ng et al., 2003) also conducted a study to assess the effects of different stimulants on IL-6 production in human intestinal smooth muscle and epithelial cells. At the basal conditions, both the cells produced detectable amounts of IL-6. They found that LPS and toxins from Clostridium difficle were unable to induce the production of IL-6.  TNF- and IL-1 increased the production of IL-6 several folds and the latter was found to be a better inducer of IL-6 expression.

1.6.3 SCFA modulates innate and adaptive immunity

SCFA are responsible for the regulation and modulation of recruitment of immune cells like macrophages, neutrophils and dendritic cells. They also regulate the activity of the components of the adaptive immune system like T cells and are involved in the reduction of pro-inflammatory cytokines such as IL-12 and TNF-  (Correa-Oliveira et al., 2016).

SCFAs have an effect on the immune function in a variety of ways. They play an important role in adaptive immunity. (Saemann et al., 2000) have demonstrated that butyrate inhibits IL-12 in human monocytes which is responsible for the activation of Th1 immune response. Sodium butyrate also increased the production of IL-10 which is a potent anti-inflammatory cytokine. SCFAs also interact with the cells of innate immunity and carry out a variety of functions. (Tedelind et al., 2007) have looked into the anti-inflammatory activity of SCFA in LPS induced neutrophils. All the three SCFAs inhibited LPS induced TNF- release. They have also demonstrated the inhibition of transcription factor NF- in the decreasing order of butyrate, propionate and acetate in human colon adenocarcinoma cell line.

1.6.4 SCFA in ex-vivo studies

There are several studies which have looked into the effect of SCFAs on human tissues. (Al-Lahham et al., 2012) showed the anti-inflammatory effects of propionic acid in human omental adipose tissue explants. 3mM propionic acid was used for the study and a significant decrease was observed in the levels of IL-4, G-CSF, MIP-1alpha, MIP-1beta and CCL5. They also showed an increase in expression of SREBP-1c upon propionic acid addition which led to an up regulation of GLUT-4 and LPL essential for glucose uptake and lipid homeostasis. (Al-Lahham et al., 2010) in their ex-vivo study with human subjects showed that propionic acid stimulated release of leptin in both omental adipose tissue and subcutaneous adipose tissue. It also decreased substantially the levels of resistin and had no apparent effect on adipokine secretion.

1.6.5 SCFA studies in animal models

Animal models are increasingly used to evaluate the role of SCFAs in modulating inflammation and thus affecting obesity. (Jakobsdottir et al., 2013) demonstrated that dietary fibres counteracted the effect of high fat diet in rats by reducing inflammation. The levels of MCP-1 decreased significantly when high fat diets were supplemented with different SCFAs. (Psichas et al., 2015) have demonstrated both in vivo in mice and in vitro in colonic crypt cultures that propionate stimulates the release of PYY and GLP-1 via the FFA2 receptor. The in vivo studies involved the use of FFA 2^-/- KO mice in which the propionate induced gut hormone release was attenuated. This opens up the avenue of establishing the role of other receptors in the gut involved in nutrient sensing. (Pivovarova et al., 2015) evaluated the expression of nutrition associated receptors in PBMC, isolated monocytes and monocytes derived macrophages from three healthy individuals. They found that all the three cell types expressed GPR 41 and GPR 43 which are important in SCFA signalling. These receptors are also expressed on dendritic cells, neutrophils and lymphocytes which have been previously reported. They also assessed the level of receptor expression in normal and obese subjects and they did not find any significant difference in the expression levels. Interestingly the GPR 43 expression in monocytes increased when the non-obese subjects switched from isocaloric high carbohydrate/low fat diet to low carbohydrate/high fat diet after six weeks.

1.6.6 Cell culture studies in human monocytes for studying inflammation

Cell culture studies are very helpful in studying the mechanistic links of different molecules in a physiological/metabolic pathway. Cell lines offer an interesting platform to work on and look deep into the cell and its products at the transcriptional & translational level. Although they are biased in the sense that they are of malignant origin and are grown outside the host in controlled conditions, they still can provide important information about the cellular state in isolation as in cell cultures(Chanput et al., 2015). Monocytes are cells of myeloid origin which differentiate into dendritic cells and macrophages in the tissue. Since, they are present in both systemic circulation and in tissues, they are versatile in studying inflammation. They are involved in a variety of functions inside the tissue like phagocytosis and antigen presentation. They secrete an array of different cytokines upon stimulation by different agents and hence are commonly used to study inflammation. THP-1 and U-937 cell lines are most commonly used to study inflammatory processes. THP-1 cells are of leukemic origin and are at a less mature stage than U-937 cells of tissue origin(Chanput et al., 2015).   U-937 is a human monocytic cell line derived from a patient having histiocytic lymphoma which was first characterised by (Sundström and Nilsson, 1976). It has been used extensively as a cellular model to study inflammatory processes. (Palacios et al., 1982) have shown that U-937 cells spontaneously produced IL-1 (increased in obesity) and its concentration was increased when stimulated by PMA. (Domsalla and Melzig, 2012) showed that there was a marked increase in expression of IL-6 from U-937 cells when a combination of PMA and thrombin was used.

PAF is a natural inflammatory mediator which amplifies the inflammatory and thrombotic response in different cell types. (Vlachogianni et al., 2013)investigated the effects of IL-1 stimulation on PAF in U-937 cells. They observed a biphasic response of PAF production upon stimulation at 3 hours and 12 hours suggesting that the early induction leads to a more pronounced effect later on due to signal amplification mediated by NF-. MCP-1 is a chemoattractant protein which is involved in the recruitment of macrophages and phagocytes. (Verouti et al., 2011) showed that PAF and IL-1 stimulated the release of MCP-1 with the latter causing a long term release.

1.7 Hypothesis and study objectives

Based on the knowledge from the previous studies, we hypothesise that short chain fatty acids reduce biological markers of systemic inflammation in overweight individuals. Experiments will be done on PBMCs extracted from overweight individuals who were on inulin propionate ester supplementation to evaluate the levels of markers of systemic inflammation. Cell culture studies on human U-937 blood monocytes will be conducted to assess the effects of different SCFA on the secretion of inflammatory markers. Mechanistic studies looking into the effect of SCFAs on transcription factors (NF- and p38 MAPK) will also be done.

Al-Lahham, S., Roelofsen, H., Rezaee, F., Weening, D., Hoek, A., Vonk, R. & Venema, K. 2012. Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects. European Journal of Clinical Investigation, 42, 357-64.

Al-Lahham, S. H., Roelofsen, H., Priebe, M., Weening, D., Dijkstra, M., Hoek, A., Rezaee, F., Venema, K. & Vonk, R. J. 2010. Regulation of adipokine production in human adipose tissue by propionic acid. European Journal of Clinical Investigation, 40, 401-7.

Chambers, E. S., Morrison, D. J., Tedford, M. C. & Frost, G. 2015a. A novel dietary strategy to increase colonic propionate production in humans and improve appetite regulation and bodyweight management. Nutrition Bulletin, 40, 227-230.

Chambers, E. S., Viardot, A., Psichas, A., Morrison, D. J., Murphy, K. G., Zac-Varghese, S. E., Macdougall, K., Preston, T., Tedford, C., Finlayson, G. S., Blundell, J. E., Bell, J. D., Thomas, E. L., Mt-Isa, S., Ashby, D., Gibson, G. R., Kolida, S., Dhillo, W. S., Bloom, S. R., Morley, W., Clegg, S. & Frost, G. 2015b. Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults. Gut, 64, 1744-54.

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