Incretin hormone glucagon-like peptide 1 and gut microbiota

Tian-ru JIN

(1.Division of Advanced Diagnostics,Toronto General Research Institute,University Health Network, Toronto,Ontario,Canada;2.Banting and Best Diabetes Centre,Faculty of Medicine, 3.Department of Physiology,University of Toronto,Toronto,Ontario,Canada)

Incretin hormone glucagon-like peptide 1 and gut microbiota

Tian-ru JIN1,2,3

(1.Division of Advanced Diagnostics,Toronto General Research Institute,University Health Network, Toronto,Ontario,Canada;2.Banting and Best Diabetes Centre,Faculty of Medicine, 3.Department of Physiology,University of Toronto,Toronto,Ontario,Canada)

There are more than two dozens of peptide hormones that are produced and released from the gastrointestinal(GI)tract.Among them,the incretin hormone glucagon-like peptide 1(GLP-1) has received the most intensive attention for the past 30 years.Functional studies on GLP-1 and anoth⁃er gut incretin hormone glucose-dependent insulinotropic peptide(GIP)have led to the development of novel diabetes therapeutic agents known as GLP-1 receptor agonists and DPP-Ⅵinhibitors.Instead of forming endocrine glands,the gut hormone producing endocrine cells are widely spread throughout the entire GI tract,permitting vital interactions with the″external″environment.Here a brief introduction on GLP-1 and how nutritional components regulate its secretion were made,followed by reviewing some key development on how gut environment affects the production and secretion of GLP-1,including the contribution of gut microbiota.

glucagon-like peptide-1,incretins;L cell models;microbiota,gut;short-chain fatty acids

1GUTPEPTIDEHORMONESAND INCRETIN HORMONE GLUCAGON-LIKE PEPTIDE 1（GLP-1）

Many hormones,including steroids and peptides or amino acid derivatives,are produced by designated endocrine glands,including pineal gland,pituitary gland,thyroid and parathyroid glands,adrenal gland,pancreatic islets,and gonads.In the gastrointestinal(GI)tract,however, many different types of endocrine cells are broadly spread within the non-endocrine epithelia.These endocrine cells produce more than two dozens of peptide hormones,exerting various regulatory functions on the GI tract and elsewhere.The production and function of these hormones can be directly regulated by nutritional and other environmental changes.Among them,two gut hormones,known asglucagon-like peptide 1 (GLP-1)and glucose-dependentinsulinotropic peptide(GIP),have received intensive attention during the past 30 years.They are known as incretins,defined as gut produced hormones that can stimulate insulin secretion from pancreatic islet β-cells in glucose concentration dependent manners.Physiologically,incretin secretion is mainly a postprandial event,i.e.occurring after food ingestion[1].

The concept of incretin was established in 1902 by two British scientists,Bayliss and Starling[2],even before insulin was successfully extracted from dog pancreatic islets in 1921 by Canadian scientists Frederick Banting and Charles Best.The first incretin GIP was isolated by Brown and Dryburgh[3]from intestinal mucosa in 1971.GIP is produced by the enteroendocrine K cells,mainly located within the duodenum and jejunum.Fourteen years later,the isolation of mammalian cDNAs that encodes the pre-hormone proglucagon led to the recognition of the secondincretin GLP-1[4-5].

The proglucagon gene(Gcg)is expressed in pancreatic islet α cell,gut endocrine L cells and certain neuronal cells in the brain stem.Gcg Encodes the pre-hormone namely proglucagon with 180 amino acid residues.Post-translational processing,which occurs in tissue-or cell typespecific manners,leads to the production of active hormone glucagon in the pancreas,GLP-1 and GLP-2(a growth factor for small intestinal epithelia)in the gut and the brain stem[4,6].Since clinical studies have reproducibly shown that GLP-1 is able to facilitate insulin secretion and normalize blood glucose levels in diabetes patients when it is administered at supra-physiological dosages,while GIP could not,more attention has been made to develop novel therapeutic agents based on studies of GLP-1.It is worth mentioning that brain GLP-1 signaling controls satiety,and the body mass lowering effect of GLP-1 based drugs are also appreciable.For other extra-pancreatic function of GLP-1,please see review articles elsewhere[5,7-9].

Mainly due to its short half-life,native GLP-1 itself cannot be utilized as the diabetes therapeutic agent.Scientists have overcome the challenge with two different strategies;each of them has led to the development of a category of new drugs.The first category is called GLP-1 analogues or GLP-1 receptor(GLP-1R)agonists,which mimic the function of GLP-1 in activating GLP-1R but have prolonged half-life.In 1992,Eng,et al[10-11]isolated the peptide hormone exendin-4 from the salivary glands of the gila monster(Heloderma suspectum),which mimics the function of GLP-1 in stimulating insulin secretion.The cDNA that en⁃codes exendin-4 was later isolated by Chen and Drucker[12].The first GLP-1R agonist,commercially known as exentide(Byetta),is the synthesized version of exendin-4,produced by Eli Lilly&Co. The laterones are modified human GLP-1 molecules.The second category is called dipeptidyl peptidase-4(DPP-4)inhibitors.DPP-4 is considered as a major enzyme that inactivates GLP-1 and GIP,while DPP-4 inhibition in preventing their degradation will raise the plasma levels of native GLP-1 and GIP.The first FDA approved DPP-4 inhibitor is sitagliptin,the product of Merck&Co., commercially known as Januvia.

Although GLP-1 based drugs can improve the glycemic control in diabetes patients,they do not mimic the physiological meal-regulated GLP-1 elevation and fall. Thus,investigations on mechanisms underlying endogenousGLP-1 secretion and the modulation of the development of incretin-producing cells may lead to novel drug development.Recently,Petersen,et al[13]demon⁃strated that in a high fat diet(HFD)fed mouse model,NOTCH signaling pathway inhibition with the chemical inhibitor dibenzazepine increased L cell numbers in the gut,associated with improved glucose tolerance.

2 NUTRITIONALCOMPONENTSON GLP-1 SECRETION

During the development of GLP-1 based drugs for treating diabetes and its complications, cell models have also been developed for exploring mechanisms and dissecting signaling cascades that underline GLP-1 secretion as well as the biology of GLP-1-producing gut endocrine L cells[14-18].Tab.1 shows the available endocrine L cell and GLP-1 producing cell models.

Among these cellular models,the mouse GLUTag cell line was utilized the most,due to the convenience as well as the fact that signaling cascades that regulate endogenous proglucagon gene expression,GLP-1 production,and GLP-1 secretion can be recaptured in this cell line.The GLUTag cell line was established by Drucker,et al[14,19]with the single-cell sub-cloning method against the large bowl tumor in the proglucagon-SV40 large T antigen transgenic mouse line in 1994.

In the intestinal L cells,GLP-1 is stored in the secretory granules and released upon the stimu⁃lation.Neuronal factors,hormones,as well as directnutritionalcontactcan triggerGLP-1 secretion[20].Here I will briefly summarize thestudies on the three major nutritional components on GLP-1 secretion.For detailed mechanisms underlying GLP-1 secretion in response to nutri⁃tional,neuronal,and hormonal factors,please see excellent reviews elsewhere[21-23].

Tab.1 GLP-1 producing intestinal cell models

Plasma GLP-1 level during the fasting status ranges from 2-10 pmmol·L-1,and increases about 2-3 folds after the meal.The postprandial peak of GLP-1 level appears 20-30 min after the meal[1,20].Nutritional components that stimulate GLP-1 secretion include carbohydrates,amino acids and proteins,fatty acids and other lipids. Therefore,GLP-1 secretion can be influenced by the meal nutritional composition as well as the digestion process of the macronutrients.Since the nutritional composition of the meal can influence endogenous GLP-1 secretion,great efforts have been made to work towards providing nutritional alternatives in the prevention of obesity,diabetes and other metabolic disorders[24-26].

Glucose is the known stimulus of GLP-1 secretion as oral glucose ingestion in humans and mouse increases plasma GLP-1 levels more effectively than intravenous glucose injection[27]. It has been suggested that glucose be up-taken by the endocrine L cells via the sodium-glucose transporter 1(SGLT1),leading to the activation of the voltage-gated Ca2+channels,the rising of intracellular Ca2+concentration and the activa⁃tion of the exocytotic machinery[28-29].Intracel⁃lular metabolism of glucose may also contribute to the stimulation of GLP-1 secretion,involving the closure of ATP-sensitive potassium(KATP)channel, the reduction of K+efflux,membrane depolarization and the influx of Ca2+[18,30-31].A number of human subject studies have assessed the effect of fructose,isomaltulose(palatinose),xylitol and erythritol on promoting GLP-1 secretion[32-35].

In 2009,Greenfield,et al[36]demonstrated that glutamine can enhance GLP-1 secretion, accompanied with increased insulin levels,in both healthy but over body mass and diabetic subjects.Detailed mechanisms underlying the stimulation of GLP-1 release by glutamine needsfurther investigations,although it appears involving the elevation of intracellular Ca2+influx[37-38].Meat hydrolysate and essentialamino acid were shown to stimulate GLP-1 secretion in GLUTag and other cellular models,as well as in isolated perfused rat intestines[39-40].Although detailed underlying mechanisms need further exploration, it is likely that proton coupled transport through peptide transporter-1(PEPT1)and the activation ofthe calcium-sensing receptor(CaSR)are implicated[41].

Fatty acids can serve as natural ligands of various G-protein coupled receptors(GPCRs). Both saturated and unsaturated long-chain fatty acid(LCFA)canactivateGPR40andGPR120[42-43], followed by the stimulation of phospholipase C (PLC),the activation of inositol-1,4,5-triphosphate (IP3)-mediated Ca2+release and GLP-1 secretion[44-45]. Short-chain fatty acids(SCFAs)can serve as the ligandsoftwootherGPCRs,GPR41and GPR43,which use both PLC dependent and independent signaling cascades to accelerate GLP-1 secretion[46-47].Recently,the cannabinoid receptor GPR119 has received intensive attention. In the gut endocrine L cells,GPR119 activation results in the elevation of cAMP level and elevated GLP-1 secretion[48].Very recently,Chepurny,et al[49]have shown that the GPR119 agonist AS1269574 can stimulate GLP-1 secretion in cultured GLP-1 producing cells via activating the TRPA1 cation channels.Indeed,attempts have been made to use GPR119 and GPR40 agonists as therapies in diabetic animal models[50-52].SCFAs can be the fermentation productsofgutmicrobiota, which will be further discussed in below.

3A BRIEFINTRODUCTIONOFGUT MICROBIOTA

The human body contains more than 100 trillion bacteria and other microorganisms in the GI tract,a number that 10 times greater than the total number of human cells in the body[53-54].The functional activities exerted by these microorganisms resemble those of an organ,making some scientiststo suggestthatthisbe indeed a″forgotten″organ.It has been estimated that human gut microflora have around a hundred times as many genes in aggregate as there are in the human genome[54-57],making the human genome only a very small portion of the″meta-genome″of the human body.Many physiological functions of our human body can be influenced by gut microflora and its alterations may facilitate the development and progression of obesity, cardiovascular disease(CVD),diabetes and nonalcoholic fatty liver disease(NAFLD)[58-60].

On one hand,gut bacteria may produce toxins, such as endotoxin and other harmful materials. On the other hand,bacteria in our gut fulfill the host a large profile of useful functions,including the digestion of unutilized energy substrates,the stimulation of cell growth,the repression of the growth of harmful microorganisms,the training of the immune system to respond only to pathogens, and the defense against infections and other categories of diseases[53,61-62].As mentioned above, gut bacteria are involved in the production of SCFAsby the degradation ofdietary fiber. SCFAs Possess multiple beneficialeffects, including providing energy for gut mucosal cells, promoting mucosal cell differentiation and reducing the risk of carcinogenesis.Importantly,SCFAs utilized GPR41 and GPR43 as their mediators in facilitating GLP-1 secretion.

4 GUT MICROBIOTA AND GLP-1 SECRETION

It has been mentioned in the beginning that endocrine cells are widely spread within the GI tract,allowing them to be directly influenced by the ″external″environment,including both the nutritional components and the gut microbiota. GLP-1 Producing endocrine L cells are mainly located within the distal ileum and colon regions, although they can be located in other parts of the small intestine.The studies on how the gut micro⁃biota can directly and indirectly affect GLP-1 secretion,or its function,from the following threeaspects were summaried.

4.1Gutmicrobiotafermentationproducts SCFAs affect GLP-1 secretion

We have learned for about three decades that non-digestible carbohydrates can be fermented by gut microbiota.On one hand,this process can affect the gut microbiota composition;while on the other hand,the fermentation products affect energy metabolism of the host[63].

The major fermentation products of nondigestible carbohydrate by the gut bacteria are SCFAs,consisting of acetate,propionate,butyrate, lactate and succinate[64].Back to 1987,Goodlad,et al[65]described the first time that fermentable carbohydrates are involved in glucose and energy homeostasis in experimental rats.They found that inert bulk fiber was unable to stimulate colonic epithelial cell proliferation in their rat model,while the fermentation processes on the non-digested carbohydrateswererequired to activate cell prolif⁃eration in the rat colon,linked with increased plasma GLP-1 level(known as enteroglucagon at that time)[65].Four years later,Longo,et al[66]demonstrated in anex vivorabbit distal colon model that SCFAs were able to stimulate the production of PYY,another anorexigenic peptide hormone produced in the ileum and colon.A large number of follow up studies have been conducted since then,in determining the role of fiber diet on GLP-1 secretion in various animal models[67-72].Notably,gut microbiota fermentation may also lead to reduced levels of plasma ghrelin, a″hunger hormone″that is produced in the gut as well[69].In 2013,Yadav,et al[73]tested in several mouse models that the administration of probiotics VSL#3(product of Tau Sigma,live bacteria) promoted GLP-1 secretion,associated with reduced food intake and improved glucose disposal. These changes are accompanied with the increase in the feces butyrate levels,whilein vitrostudy indicated that butyrate could stimulate GLP-1 secretion in the GLP-1 producing endocrine cell line.

A number of human studies have also provided evidence that links gut microbiota fermentation and GLP-1 secretion,at least with the long-term fiber diet intervention.In 1996,in testing the hypothesis that colonic fermentation products of carbohydrates affect proximal gut motility and gastric tone,Ropert,et al[74]tested the effect of lactulose administration in healthy volunteers. They observed the decrease in gastric tone after oral lactulose administration or intracolonic infusion of lactose and SCFAs,but could not detect the changes on GLP-1 secretion.Increased GLP-1 secretion,however,was observed by Piche,et al[75]in 2003 in the oraladministration of fructooligosaccharides in patients with gastro⁃esophagealreflux disease.Cani,et al[76]have then demonstrated that in human subjects that 2 week administration of inulin-type fructans repressed appetite sensations,which was correlated with increased plasma GLP-1 and PYY levels. Parnell and Reimer[77]found that a 12 week inter⁃vention with oligofructose decreased visceral fat mass,associated with reduced circulating ghrelin and increased circulating PYY.No significant difference,however,was observed for plasma GLP-1 levels.In 2013,Nilsson,et al[78]found that an evening meal with Swedish brown beans, in comparison with white wheat bread,lowered the plasma blood glucose level and increased insulin sensitivity.These changes were associated with increased satiety hormones PYY and suppressedhungerhormoneghrelininnext morning.No difference was observed for GLP-1,although increased plasma GLP-2 (which is encoded by the same proglucagon gene)was observed.As GLP-2 is co-produced and co-released with GLP-1,the failure in detecting GLP-1 elevation could be due to a technical challenge,due to its relative short half-life.Bodinham,et al[79]reported that dietary fiber consumption in healthy male subjects did not acutely increase endogenous GLP-1 concentrations.They have suggested thatfurtherfiberfeeding studies be needed to clarify whether plasma GLP-1 level increases following a l onger-term dietary consumption[79].

It is worth mentioning that a study performed by Cani,et al[80]with the Wistar rat model suggestedthat carbohydrates fermented in the gut may promote L-cell differentiation.Thus,long-term chronic fiber dietary uptake may indirectly affect gut GLP-1 signaling via increasing the gut endocrine L cell mass.In addition,Everard,et al[81]found that in the diabeticob/obmouse model,prebiotic feeding can increase L cell number,associated with improved glucose tolerance.

4.2Certain dietary plant polyphenols can stimulate GLP-1 secretionin vitro

We have learned for decades that many dietary plant polyphenol(PP)compounds,such as the curry compound curcumin,resveratrol and antho⁃cyanin,can function as therapeutic or even prevention agents for obesity,diabetes,and other metabolic diseases[82-85].Investigations have been conducted asking whether some of the metabolic beneficialeffects can be attributed to the improvement of GLP-1 production,or secretion, or signaling.

Turmeric has been a medicine in a number of Asian countries for nearly 3000 years in the treatment of inflammatory diseases,while curcumin is the major component of Turmeric.Intensive animal studies have demonstrated the capability of curcumin in the treatment of various inflammatory and other diseases,including type 2 diabetes mellitus(T2DM)and obesity[82-83,86-88].In 2013,a team led by Tsuda[89]reported that in the mouse GLUTag cell line,curcumin treatment stimulated GLP-1 secretion.The same team reported more recently that anthocyanin can also induce GLP-1 secretion in this GLUTag cell model[90].The signif⁃icance of these findings needs to be validated with the properin vivoapproaches.The absorption rates for dietary PP are very low[91-92].Usually, the plasma polyphenol concentrations are more than 100 fold lower than the effective concentrations demonstrated in thein vitrocell culture studies[93], raising the possibility thatnotthe parental polyphenols but their gut metabolites exerting the metabolic beneficial effects.It is also worth mentioning that Antonyan,et al[94]have demon⁃strated the inhibitory effect of curcumin on DPP-4, raising the possibilities that curcumin intervention can raise plasma endogenous active GLP-1 and GIP levels and that curcumin-based DPP-4 inhibitors can be developed as novel diabetes drugs.

4.3Gut microbiota may produce yet to be identified products that affect GLP-1 secretion or mimic function of GLP-1 or other metabolic hormones

A very recent investigation conducted by Breton,et al[95]revealed that gut bacteria can influence host appetite and metabolism from a novel angle.In this study,they aimed to ask a big question:can nutrient-induced bacterial growth affects host metabolism.Regular nutrient provision for 20 min can stabilize exponential growthoftheharmlesscommensalbacteriaEscherichia coli(E.coli),accompanied with the change in bacterial proteomes.When they infused the stationary phase proteins from theE.coliinto the rat gut,they saw elevated plasma PYY levels. When rats received intraperitoneal injection of these proteins,food intake was repressed acutely, accompanied with neuron c-Fos activation in the hypothalamic POMC area,indicating the activation of the brain satiety pathway.The authors have also assessed the effect ofE.coliprotein colonic infusion on GLP-1 secretion.The exponential phaseE.coliprotein infusion but not thestationaryphaseE.coliprotein infusion resulted in increased levels of plasma GLP-1[95].Thus,E.colimay produce yet to be determined factors that can acutely facilitate GLP-1 secretion.

Breton,et al[96]have then verified the effect of a bacterial protein namely caseinolytic protease B(CIpB)in the activation of hypothalamic POMC neurons.It appears that CIpB is the mimetic of α-melanocyte-stimulating hormone(α-MSH),a POMC-derived anorexigenic neuron peptide in mammals.Thus,bacteria can produce a protein which mimics the brain GLP-1 signaling cascade.

5 SUMMARY

Fig.1 summarizes our current understanding on the effect of gut microbiota on GLP-1 secretion, with the participation of dietary plant polyphenols.

Fig.1 Summary of our current understanding on effect of nutrients and gut microbiota on GLP-1 secretion.

Nutrient components including sugar,amino acids/peptides and lipids are known to regulate GLP-1 secretion.Selected dietary PP,or their gut metabolites may affect GLP-1 secretion or the biology(proliferation and protection)of GLP-1 producing endocrine L cells.Gut microbiota may exert their regulatory effect on Gcg transcription, or GLP-1 production,or GLP-1 secretion via its own products,or the fermentation products of diet fibers(i.e.SCFAs),or the PP metabolites. Importantly,dietary PP can bring the prebiotic effect,″re-shaping″gut microbiota,generating chronic beneficial effects,including the influence on the production and secretion of GLP-1 and other gut metabolic hormones.

It has been generally accepted that gut microbiota can convert diet fibers(via the fermen⁃tation process)into SCFAs,which affect the secretion of GLP-1 and othergutmetabolic hormones,although thein vivoeffect could be masked due to technical challenge.The fermen⁃tation process may affect the proliferation and other features of gut hormone producing cells including the GLP-1 producing endocrine L cells, by yet to be explored mechanisms.We are convinced by thein vivobeneficial effects of dietary PP intervention and are aware of thein vitrostimulatory effect of at least two polyphenols (curcumin and anthocyanin)on GLP-1 secretion. The extremely low bioavailability of these PP in the circulation made us to wonder whether the acute stimulatory effect by the two PP on GLP-1 secretion indeed occursin vivo.Alternatively, this can be achieved by yet to be identified gut metabolites of the parental PP;or by combined effect with yet to be identified host or gut microbiota product(s).Nevertheless,the long-term metabolic beneficial effect of curcumin and other PP can be attributed to the stimulation of gut endocrine cell proliferation and the protection of these cells from the damage,due to the interaction between these PP and gut microbiota in re-shaping the gut microbiota.It needs to be investigated.We anticipate that following the recent work presented by Breton,et al[95],efforts will be made to determine whetherE.coliand other members of the gut microbiota can produce effective products in regulating the secretion of GLP-1 and other gut hormones,in addition to the production of host mimetic molecules,such as CIpB,to participate host metabolic regulation.

ACKNOWLEDGEMENTS:The author thanks the Canadian Diabetes Association and Canadian Institutes of Health Research for supporting his GLP-1 related research.

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胰高血糖素样肽1和肠道菌群

金天如1,2,3

(1.Division of Advanced Diagnostics,Toronto General Research Institute,University Health Network, Toronto,Ontario,Canada;2.Banting and Best Diabetes Centre,Faculty of Medicine, 3.Department of Physiology,University of Toronto,Toronto,Ontario,Canada)

胃肠道生产20多种肽类激素。其中，胰高血糖素样肽1（GLP-1）在过去的30年里受到最多关注。人们对GLP-1以及另一肠道激素葡萄糖依赖性促胰岛素肽（GIP）功能的研究已导致了两类新的糖尿病治疗药物的开发，分别称为GLP-1R激动剂和DPP-Ⅳ抑制剂。肠道的这些内分泌细胞不是聚集在内分泌腺体中，而是广泛分布在整个胃肠道中，从而与“外部”环境包括食物以及肠道菌群充分接触。本文简要介绍了GLP-1以及营养成分如何调节其分泌，并重点讨论了肠道环境如何影响GLP-1的产生和分泌，包括肠道菌群的贡献。

胰高血糖素样肽1；肠促胰岛素肽；L细胞模型；肠道菌群；短链脂肪酸

2016-05-17 接受日期：2016-06-14）

金天如，Tel：+1（416）581-7670,E-mail：tianru.jin@utoronto.ca

R335

:A

:1000-3002-(2016)06-0691-12

10.3867/j.issn.1000-3002.2016.06.008

（本文编辑：乔 虹）

Tian-ru JIN,Tel:+1(416)581-7670，

E-mail:tianru.jin@utoronto.ca