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Short chain fatty acids and GPR43 in human health and disease

Periodic Report Summary - GPR43 AND OBESITY (Short chain fatty acids and GPR43 in human health and disease)


Objectives:

Obesity and its metabolic consequences are a major public health issue, and new tools to fight against this epidemic on a population scale are urgently needed. The colonic microbiota ferment carbohydrates to produce short chain fatty acids (SCFAs) which stimulate the release of the anorectic gut hormones peptide YY (PYY) and glucagon like peptide-1 (GLP-1) from rodent enteroendocrine L-cells. We hypothesised that colonic delivery of the SCFA propionate would also increase PYY and GLP-1 secretion and reduce energy intake in humans. Furthermore, recent evidence suggests that acetate and propionate are natural ligands of the previously called orphan G-protein coupled free fatty acid receptor (FFAR) 2, expressed on enteroendocrine L-cells, adipocytes, immune cells, pancreatic beta-cells and possibly in the brain. It has been shown that the release of gut peptides is induced in rodents by SCFA stimulation of FFAR2 on enteroendocrine L-cells of the colon. Increasing colonic propionate could therefore be an attractive target for appetite modulation. The challenge is that SCFAs are likely to mediate their effects on appetite by acting in the colon, but orally administered SCFAs are rapidly absorbed in the small intestine where L-cells are sparse, and thus do not stimulate gut hormone release or reduce appetite. It is thus necessary to target SCFA delivery to the colon in order to exploit their appetite-inhibiting effects.

Activities / techniques:

In order to achieve targeted colonic delivery of propionate, we have developed a novel carrier molecule in collaboration with University of Glasgow, whereby propionate is chemically bound to inulin, a natural polymer composed mainly of fructose, through an ester bond. The majority of propionate chemically bound to inulin should only be released when the inulin polymer is fermented by the colonic microbiota, thus providing targeted colonic delivery. Our pharmacological studies including 13C labelled propionate ester confirmed that 80 % of propionate is released only in the colon, and that the delivered propionate gets systemically available by increasing plasma and urine concentrations.

Clinical studies were planned in four stages:

1.) an initial 'esterification study' to determine the optimal amount of propionate which has to be bound to the inulin carrier molecule to achieve greatest effects;
2.) a 'dose escalation study' to determine the optimal dose needed of the best propionate ester as determined in 1;
3.) a 'dose confirmation study' (n = 20) to assess the effects on appetite scores (VAS), food intake, gut peptides and glucose homeostasis using the optimal dose as determined in 2;
4.) and finally, a large double-blind randomised controlled trial (RCT) to assess the effects on body weight, body composition and glucose homeostasis after long term (6 month) administration of propionate ester to 80 subjects (40 each group).

Appetite was assessed by visual analogue scales (VAS). Food intake was assessed by an ad libitum excess buffet meal seven hours after the intake of the propionate ester. Glucose homeostasis was assessed by IVGTT and hyperinsulinaemic-euglycaemic clamp. PYY and GLP-1 were measured during the whole study visit, from intake of the supplement until the buffet meal (seven hours) using in-house radioimmunoassays.

For animal studies, we imported FFAR2-KO mice on a C57BL6 background from Sydney, and we performed ipGTT and ipITT for metabolic phenotyping. Concomitant injection of 500 mg/kg propionate during the ipGTT tested any possible effect of propionate on beta-cell function. Food intake was measured in both strains, and the effect of ip propionate was assessed. Finally, high fat feeding experiments were started supplementing the feed with 7.5 % wt/wt inulin or cellulose as a control, and the effect on weight gain and food intake was assessed in WT and KO animals.

Results:

Esterification study:
Threetypes of esters with a wt/wt ratio of 0 %,10 %, 20 % and 30 % were produced and tested in 10 health volunteers in a cross-over design by assessing food intake. All supplements were equally well tolerated, with the highest esterified molecule being the most effective in reducing food intake, showing a dose dependent linear trend.

Dose escalation study:
The 30 % wt/wt esterified propionate inulin was tested in increasing doses, starting from 0 g/day and increasing up to 20 g/day. Again, a linear trend was seen with increasing doses on effect of appetite suppression, but here it could be shown that a decrease in palatability with the higher doses would lead to a compromise between effectivity and palatability, and therefore adherence. An optimal dose of 10 g/day could be identified to be used in the RCT.

Dose confirmation study:
10 g/day of the 30 % wt/wt inulin propionate ester was tested in 20 healthy volunteers, compared to the carrier inulin alone, and effects on food intake, appetite ratings, gut peptides and glucose / insulin was assessed. This supplement significantly reduced food intake during the ad libitum buffet meal, and consistently increased PYY and GLP-1 levels. Appetite ratings and glucose / insulin levels were not changed by the propionate ester.

Double-blind randomised controlled trial:
This study has started in October 2011 aiming at 80 volunteers being at increased risk for type 2 diabetes, with 40 subjects in each group either receiving propionate ester or inulin as control. So far, 48 subjects have been randomised into the study, and the final assessment 6 months after start of the supplement of all subjects is expected by end of 2012. AV has been a key driver in developing this study protocol and continues to be an active part of the study team at Imperial College, and will be responsible for final data collection and statistical analysis.

Animal studies:
FFAR2KO animals did not have an obvious phenotype from observation having identical body weight and sise. However, at week 16 they showed an impaired glucose tolerance and an abnormal insulin tolerance suggesting insulin resistance. Intraperitoneal injection of propionate before the GTT resulted in increased insulin levels and lower glucose levels in WT animals but not KO animals, suggesting a stimulating effect of propionate on beta-cells which is FFAR2-dependent. Intraperitoneal injection of propionate alone suppressed food intake in WT but not KO animals, also suggesting that FFAR2 could be involved in central appetite regulation. Finally, WT mice on high fat diet supplemented with insulin showed a partial protection from diet induced weight gain, whereas in KO animals the protective effect of insulin was completely lost. This suggested that FFAR2 is essential for mediating the beneficial effects of fibre, either through regulation of gut peptides or via central appetite regulating effects or both.

Conclusions:

Propionate ester, as a colonic delivery tool of propionate, has demonstrated in all our clinical studies that it effectively increases gut peptides PYY and GLP-1 and suppresses appetite.

Our ongoing RCT will demonstrate in near future whether long term treatment with this supplement has any beneficial effects on weight loss, insulin sensitivity, beta-cell function and body composition. Furthermore, our animal studies using the FFAR2-KO mouse has shed light on the possible underlying mechanism, suggesting that this receptor plays an important role in glucose homeostasis and appetite regulation.

Socio-economic impact:

The promising results from these clinical studies suggest that colonic delivery of propionate may be a promising tool to fight against the obesity epidemic at a population level, by using it as a food supplement in a wide range of products. The quantities of the supplement needed lies in a range where it seems to be feasible to be incorporated into food processing. From the clinical side, it has been demonstrated repeatedly that fighting against the obesity epidemic by encouraging life-style changes, diet and exercise are mostly frustrating, with only modest and short lived effects, and also being very resource intense and expensive. However, tackling this epidemic from the side of food supply, by simply adding a supplement, which mimics the beneficial effects of dietary fibres in very high concentrations, could clearly have a significant protective effect on a whole population.

The findings from our animal research also opens the perspective to develop and use a specific FFAR2 agonist as a new potential treatment target against obesity and type 2 diabetes. Given orally, this agonist could be designed to be either quickly absorbed to be transported to the pancreas and the brain, or to be encapsulated in order to be delivered directly to the enteroendocrine L-cells in the colon to stimulate gut hormone release.

Ongoing collaborative research activities for the next years will hopefully lead to further strong evidence supporting our preliminary findings and to important advances in this field.

Wider societal implications:

The world has observed an obesity epidemic developing in the last 20 years, and still no effective treatment is available for most affected subjects. Prevention programs have started in many countries, but its success has yet to be demonstrated. This project has the potential to demonstrate that the administration of SCFA has beneficial effects on metabolism and body weight, and may be suitable to use as a nutrition supplement to prevent or treat obesity. The newly developed molecule together with the tested novel delivery method could set the stage to develop a nutrition supplement, which could be applied to a large number of affected subjects to fight against the obesity epidemic.

This research project was novel in many ways. This project revealed novel mechanisms in the pathogenesis of obesity, and could identify new potential targets for a pharmacological approach to fight against obesity.

Prior to this project, there were no data available on the effect of SCFA administration in humans. The use of propionate-esters was novel, and overcomes the limitations to raise SCFA concentrations high enough to achieve significant effects. The gained results from this human studies have significant advantages against data reported from animal studies, where the preliminary promising data cannot necessarily be transferred to the human setting.

So far this project suggests that SCFA-GPR43 signalling has a positive impact on appetite, body composition, beta-cell function, adipose tissue biology and the immune system, and we believe that this receptor is a novel important link between immunity, metabolism and the hypothalamus. These data therefore provide valuable information for future studies, investigating the effects of SCFA on appetite regulation, metabolism and inflammation in obesity.

Advances achieved in this field will definitely contribute to the scientific excellence of the European Union (EU) and enhance EU competitiveness.