Leveraging the gut to treat metabolic disease

Abstract

Twenty-five years ago, the future of treating obesity and diabetes focused on end organs known to be involved in energy balance and glucose regulation, including the brain, muscle, adipose tissue and pancreas. Today, the most effective therapies are focused around the gut. This includes surgical options, such as vertical sleeve gastrectomy and Roux-en-Y gastric bypass, that can produce sustained weight loss and diabetes remission, but also extends to pharmacological treatments that simulate or amplify various signals that come from the gut. The purpose of this review is to discuss the wealth of approaches currently under development that seek to further leverage the gut as a source of novel therapeutic opportunities with the hope that we can achieve the effects of surgical interventions with less invasive and more scalable solutions.

eToC

While traditional approaches to treating obesity and type 2 diabetes have focused on end-organs involved in energy metabolism, the gut has emerged as a source of our most effective therapies. From bariatric surgical procedures to pharmacological agents, harnessing the gut has and will continue to provide important therapeutic options for the treatment of these epidemic-level diseases.

Introduction

Type 2 Diabetes Mellitus (T2DM) is a large and growing public health problem that affects almost 10% of the US population (National Diabetes Statistics Report, 2017). Combined with rapidly rising obesity rates, currently estimated at 40% of the US adult population (National Health and Nutrition Examination Survey, 2015–2016), there is potential for a future crisis of T2DM. These statistics are more alarming when one considers that T2DM and obesity are associated with a broad spectrum of metabolic dysfunctions, including high blood pressure, dyslipidemia, heart failure, and nonalcoholic steatohepatitis. While there are a growing number of pharmacological treatments for T2DM and obesity, fewer than 50% of T2DM patients achieve treatment goals (Garcia-Perez, Alvarez et al. 2013) and none of the currently available therapies halt the progression of diabetes treatment towards insulinfocused therapy. Furthermore, rising rates of T2DM will result in escalating monetary and human costs.

Both existing and future therapies for T2DM and obesity have targeted several organ systems, with most of the research focused on the effector organs. In the case of T2DM, targeting pancreas, adipose tissue, muscle and liver are sensible approaches borne from decades of research on blood glucose regulation. While several strategies have been deployed to increase insulin secretion by acting directly or indirectly on the pancreas, an alternative strategy aims to increase the action of insulin (insulin sensitizers) by targeting effector organs such as adipose tissue, muscle and/or liver to lower glucose levels. In the case of obesity, most approaches target the appetite regulating circuits within the central nervous system. However, an alternative strategy aims to target muscle and/or adipose tissue to increase energy expenditure, capitalizing on decades of research that links these tissues directly to the regulation of energy intake or energy expenditure.

Today, the most effective strategies to treat T2DM and obesity involve the gastrointestinal (GI) tract, or gut, an outcome that would have been almost impossible to predict based on the available research literature 25 years ago. The purpose of this review is to outline current treatments options based on manipulations of the GI tract and to discuss next-generation, gut-based therapies that have been or are currently under investigation. We will also lay out potential strategies to identify novel therapeutic strategies based on targeting the GI tract or mimicking signals from the GI tract.

Current gut-based therapies

Surgical interventions

Among the available treatments for obesity and T2DM, various bariatric surgical interventions are clearly the most effective. The two most common are Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) (Figure 1). The clinical effects of both procedures are impressive. In RYGB, the average patient loses 70% of their excess body weight in the first 12 months after surgery, with VSG showing similar weight loss (Demssie, Jawaheer et al. 2012). However, what truly sets surgical interventions apart from other weight loss programs is durability. In the case of RYGB, the Swedish Outcome Study finds that patients still had a 28% reduction in body weight 14 years after the procedure (Sjostrom 2013). This stands in contrast to lifestyle interventions that have little overall benefit on weight even 5 years after the intervention (Look, Wing et al. 2013, Sjostrom 2013).

Figure 1:

Traditionally, therapeutic approaches to obesity and type 2 diabetes have focused on altering the actions of key effector organs such as brain, liver, pancreas, adipose tissue and muscle. Surgical alterations of the GI tract by either RYGB or VSG result in sustained weight loss and potent improvements and even remission of type 2 diabetes. Targeting the GI tract for therapies, therefore, can leverage signals from the GI tract to regulate each of these effector organs.

Both RYGB and VSG also provide profound improvements in the regulation of glucose. In one randomized trial, RYGB or VSG surgically treated type 2 diabetic patients had a greater mean percentage reduction from baseline in percent glycated hemoglobin level than did patients who received medical therapy alone after 5 years (−2.1% vs. −0.3%, P = 0.003)(Schauer, Bhatt et al. 2017). This is a single study, but a wide variety of trial and observational data points towards surgical intervention as producing higher rates of remission for type 2 diabetes than can be achieved by current medical therapies (Tsilingiris, Koliaki et al. 2019). These clinical effects have resulted in referring to these procedures as “metabolic surgery” when used primarily to treat T2DM. This is encapsulated in a recent position statement that has urged metabolic surgeries being adopted as treatment options for type 2 diabetes (Rubino, Nathan et al. 2016). This leads to an important question: are these potent effects of T2DM a benefit that accrues primarily from the profound weight loss that occurs with these procedures? This is a complicated and controversial topic that falls outside the scope of this review. However, profound improvements in glucose regulation are observed in the first few days after surgical intervention and prior to the weight loss which typically peaks close to 1 year after surgery (Miras, Herring et al. 2017). Moreover, improved glucose regulation is not the only benefit, as a number of other obesity-related complications are reduced after surgical intervention, including cardiovascular risk factors, sleep apnea and even obesity-related cancers (Sjostrom 2013). Overall, the rearrangement of the GI tract from either RYGB or VSG produces profound physiological effects that result in successful weight loss and improved glucose regulation.

Mechanical restriction and caloric malabsorption are often used to explain the potent effects from metabolic surgery. Consequently, VSG is referred to as a “restrictive” procedure since it reduces the volume of the stomach by approximately 80%, and RYGB is considered “restrictive” since the functional volume of the stomach is drastically reduced. However, RYGB is also “malabsorptive” since rerouting of the intestine results in a bypass of the absorptive capacity of the upper portion of the small intestine. While mechanical explanations are likely playing a role, these do not provide a full account of post-surgical improvements in energy and glucose homeostasis. After surgery, patients clearly report being less hungry despite profound weight loss (Al-Najim, Docherty et al. 2018). If surgery exerted its effect by preventing ingestion and/or absorption of calories, patients would lose weight but would be hungrier (Seeley, Chambers et al. 2015). Hence, the notion that surgery works primarily by preventing the ingestion or absorption of calories simply does not square with available evidence.

Since mechanical explanations are inadequate to explain the benefits of metabolic surgery, an increased level of research has focused on other mechanisms, notably neuronal and hormonal signals from the gut. Though local innervation of the gut provides important signals back to the CNS that can alter both food intake and metabolism, direct evidence that such signals are the basis for the effects of surgery are relatively scant. Moreover, while the vagus nerve may play a role in alterations of food choice after bariatric surgery (Wilson-Perez, Chambers et al. 2013, Hankir, Seyfried et al. 2017), there is little evidence at present that such signals contribute to weight loss and improvements in glucose regulation. While neural signals from the gut seem less likely, it is very clear that both VSG and RYGB alter the secretion of numerous hormonal signals from the GI tract, including several validated therapeutic strategies for T2DM and obesity, and likely highlighting future therapeutics. These include traditional gut peptides like GLP-1 and less traditional hormones such as bile acids.

While specific gut peptides will be discussed in the next section, RYGB and VSG also result in weight-independent increases in levels and altered composition of bile acids in circulation. Bile acids can bind to and activate both a cell-surface receptor (TGR5) and a nuclear receptor (FXR). In turn, loss-of-function of FXR greatly reduces the ability of VSG to improve either body weight or glucose tolerance (Ryan, Tremaroli et al. 2014), and a number of other studies link bile acid signaling to the beneficial effects of surgery (McGavigan, Garibay et al. 2017, Albaugh, Banan et al. 2019). It is important to note that the source of these signals may not always be the host organism, as bariatric surgical procedures can greatly affect the composition of the bacteria in the gut. These gut bacteria are also capable of producing a wide number of both neural and hormonal signals that may also underlie the effects of surgery. In short, it is unlikely that a single factor or signal underlies the effect of metabolic surgery. Rather, it more likely that “polypharmacy” makes these surgical procedures more effective than currently available pharmacological treatments.

Despite the effectiveness of RYGB and VSG, such interventions for obesity and/or T2DM are limited to treating small populations. Only about 228,000 patients received bariatric surgery in 2017, representing 1% of the eligible population (Gasoyan, Tajeu et al. 2019). Put simply, there are not enough surgeons and operating rooms for surgery to be our main therapeutic option. Further, surgery is not without risk. As surgery involves permanent changes in the anatomy of the GI tract, there are no easy solutions for patients who experience adverse side effects, which can include malnutrition, frequent hypoglycemia and severe reflux in the case of VSG (Gletsu-Miller and Wright 2013, Kassem, Durda et al. 2017, Lim, Lee et al. 2019).

Gut peptide-based approaches

An emerging alternative to bariatric surgery is pharmacotherapy based on gut peptides (Figure 2). The gut is the source of numerous factors, primarily peptides, that act to regulate intestinal motility and nutrient absorption. But gut peptides also function to prime other organs for the availability of nutrients and to initiate counter regulatory responses such as satiety. Some of these peptides, particularly GLP-1, but also other glucagon-related peptides, such as GIP and oxyntomodulin, as well as PYY, have become important therapeutic targets for obesity and type 2 diabetes. The efficacies observed place them among the most attractive new therapeutic approaches in these areas. Understanding how these gut-derived hormones function and how they could act together to potentially mimic the effects of bariatric surgery is an exciting area of research that could potentially lead to additional therapies for diabetes and obesity.

Figure 2:

Major gut hormones that regulate metabolic function

Here, we highlight some major gut peptides in terms of site of secretion, site and effort organ of action, and functions with emphasis on molecules whose therapeutic potential have been explored in clinical studies.

Incretins: GLP-1 and GIP

The term “incretin effect” was coined in the 1960s to describe the observation that oral administration of glucose leads to more pronounced insulin secretion compared to intravenous administration. In turn, this led to the hypothesis that gut-derived factors, so-called “incretins”, contribute to postprandial stimulation of insulin secretion. Two gut peptides, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are now recognized to be responsible for most, if not all, of the incretin effect (Nauck and Meier 2016). These peptides are released from enteroendocrine cells in the gut in response to nutrients, and act directly on pancreatic beta cells to activate insulin secretion in a glucose-dependent manner (Drucker, Habener et al. 2017). In healthy individuals, GLP-1 and GIP act in an additive manner, with GIP responsible for the majority of the incretin effect (Gasbjerg, Helsted et al. 2019). In contrast, in individuals with T2DM, GIP has little effect on insulin secretion, and the efficacy of GLP-1 appears to be greatly decreased, resulting in significant defects in the incretin effect (Nauck, Heimesaat et al. 1993, Nauck, Kleine et al. 1993). However, administration of exogenous GLP-1 partially restores the incretin effect in individuals with T2DM, albeit at doses that result in supraphysiological GLP-1 serum levels (approximately 5–10-fold above normal) (Nauck, Heimesaat et al. 1993, Nauck, Kleine et al. 1993). Consequently, much of the initial work focused on GLP-1 as a potential target for T2DM.

GLP-1 receptor agonists

Native GLP-1, while useful to characterize the physiology and pharmacology of this incretin, is not suitable for therapeutic use due to its very short half-life. Exendin-4, a GLP-1 related peptide isolated from the saliva of the Gila monster, is more stable, allowing for therapeutic development, and exenatide, a synthetic version of exendin-4 administered via twice daily injection, was the first GLP-1 – based therapy for T2DM to reach the market (Knop, Bronden et al. 2017, Aroda 2018)(Table 1). Subsequently, long-acting GLP-1 receptor agonists suitable for once daily or once weekly injection, including an extended release formulation of exenatide, have been approved for clinical use (Table 1)(Aroda 2018).

Table 1:

Select gut-peptide based therapies currently on the market or in clinical development

Class	Key Molecules	Administration	Status	Indication	References
GLP-1 receptor agonists	exenatide	SC BID, SC QW	Marketed	T2DM	(Knop, Bronden et al. 2017)
	liraglutide	SC QD	Marketed	T2DM, Obesity	(Nauck, Meier et al. 2017, Chukir, Shukla et al. 2018)
	dulaglutide	SC QW	Marketed	T2DM	(Nauck, Meier et al. 2017)
	semaglutide	SC QW	Marketed	T2DM	(Nauck, Meier et al. 2017)
		Oral QD	Marketed	T2DM	(Hedrington and Davis 2019)
		SC QW	Phase 3	Obesity	(Nauck, Meier et al. 2017, Christou, Katsiki et al. 2019)
	efpeglenatide	SC QW	Phase 3	T2DM	NCT03684642; NCT03353350
Dual GIP/GLP-1 receptor agonists	Tirzepatide (LY3298176)	SC QW	Phase 3	T2DM	(Frias, Nauck et al. 2018)
	NNC0090–2746 (RG7697)	SC QD	Discontinued (Phase 1)	T2DM	(Frias, Bastyr et al. 2017, Schmitt, Portron et al. 2017)
	Cpd 21	SC QW	Discontinued (Phase 1)	T2DM	(Finan, Ma et al. 2013)
Dual Glucagon/ GLP-1 receptor agonists	cotadutide (MEDI0382)	SC QD	Phase 2	Obesity, NASH	(Ambery, Parker et al. 2018)
	HM-12525A	SC QW	Phase 2	T2DM, Obesity	NCT03235219
	OPK88003	SC QW	Phase 2	T2DM	NCT03406377
	SAR425899	SC QD	Discontinued (Phase 2)	T2DM	(Tillner, Posch et al. 2019)
	MK-8521	SC QD	Discontinued (Phase 2)	T2DM	NCT02492763 (results available on CT.gov)
	NN9277/NN6177	SC QW	Phase 1	Obesity, NASH	NCT02941042
	LY3305677	SC QW	Phase 1	T2DM	NCT03325387
	BI-456906	SC QW	Phase 1	Obesity	NCT03591718
Glucagon/GIP/ GLP-1 receptor triagonists	NN9423	SC QD	Phase 1	Obesity	NCT03661879
	HM15211	SC QW	Phase 1	Obesity, NASH	NCT03744182
	LY3437943	SC QW	Phase 1	T2DM	NCT04143802
PYY analogs	NN-9775	SC QW	Phase 1	Obesity	NCT03707990
	NN-9747	SC QW	Discontinued (Phase 1)	Obesity	NCT03574584

Molecules on the market or in active clinical development (global programs) with emphasis on molecules supported by preclinical or clinical publications. Select non-published molecules in active clinical development as well as discontinued molecules with public disclosures are also included.

Several important principles have emerged from clinical studies with GLP-1 receptor agonists. All molecules approved to date show potent glucose-lowering, driven partly by their direct effect on the beta cell to stimulate glucose-dependent insulin secretion, but also partly due to inhibition of glucagon secretion and, with some molecules, delayed gastric emptying (Nauck, Meier et al. 2017). These molecules cause weight loss, which has been primarily attributed to direct effects of GLP-1 on appetite-regulatory neurons in the hypothalamus (Secher, Jelsing et al. 2014); actions on the brainstem as well as vagal-mediated effects may also contribute under certain circumstances (Plamboeck, Veedfald et al. 2013, Holt, Richards et al. 2019). While short-acting GLP-1 receptor agonists show significant inhibition of gastric emptying, this effect can be attenuated in long-acting molecules (Knop, Bronden et al. 2017). Nausea and vomiting are the most common side effects and can limit dose and thus therapeutic efficacy. Molecules with lower peak-to-trough ratios appear to have fewer gastrointestinal side effects, and these side effects can be further managed through slow, step-wise dose escalation resulting in higher doses and better efficacy (Knop, Bronden et al. 2017, Aroda 2018, Nauck and Meier 2018). These insights have led to the development of the current generation of GLP-1 receptor agonists where molecules with optimized pharmacokinetic properties delivered via optimized dosing regimens achieve impressive glucose lowering and weight loss in patients with T2DM. Weight loss induced by these molecules is even more pronounced in non-diabetic individuals (Davies, Bergenstal et al. 2015, Pi-Sunyer, Astrup et al. 2015), leading to the development of some of these molecules as obesity therapies (Table 1).

Long-term cardiovascular outcome studies, a requirement for T2DM development programs, have revealed additional benefits of the GLP-1 class. The majority of GLP-1 receptor agonists shows a cardiovascular benefit, as evidenced by a decrease in the composite endpoint of myocardial infarction, stroke and cardiovascular death (Marso, Bain et al. 2016, Marso, Daniels et al. 2016, Nauck, Meier et al. 2017, Hernandez, Green et al. 2018, Gerstein, Colhoun et al. 2019). In addition, a decrease in albuminuria and a slower decline in glomerular filtration rate was observed in several studies, raising the possibility of renal benefits (Mann, Orsted et al. 2017, Luo, Lu et al. 2018, Tuttle, Lakshmanan et al. 2018, Gerstein, Colhoun et al. 2019). The mechanisms underlying these benefits are not clear. GLP-1 receptor agonists consistently show increases in heart rate and decreases in blood pressure, as well as small improvements in serum lipid profile, most notably decreased excursion of postprandial triglycerides and small improvements in LDL- and HDL-cholesterol, as well as anti-inflammatory effects, which could contribute to the observed cardiovascular and renal profiles (Nauck, Meier et al. 2017). Initial concerns about potential safety issues with GLP-1 receptor analogs, including medullary thyroid carcinoma and pancreatitis, have not manifested themselves as significant issues in the clinic (Nauck, Meier et al. 2017). Indeed, GLP-1 receptor agonists are now firmly established in the T2DM treatment paradigm and are the preferred therapy for patients with atherosclerotic cardiovascular disease, patients that would benefit from weight loss and individuals needing to achieve excellent glucose control without the risk of hypoglycemia (American Diabetes Association 2019). The next wave of innovation, now in progress, aims to develop orally bioavailable versions of GLP-1 receptor agonists to extend the benefits of GLP-1-based therapy to earlier stages of the diabetes treatment paradigm.

GIP and dual GIP/GLP-1 receptor agonists

Unlike GLP-1, very little work has been conducted to develop GIP as a stand-alone therapeutic (Finan, Muller et al. 2016, Holst 2019). In individuals with T2DM, GIP has little effect on insulin secretion, leading to the concept of diabetes as a GIP-resistant state (Nauck, Heimesaat et al. 1993). While the molecular basis for GIP resistance is not well understood, it was found to be closely linked to hyperglycemia (Vilsboll, Knop et al. 2003), and GIP-induced insulin secretion can be at least partially restored by treatments that normalize glucose levels such as insulin (Vilsboll, Knop et al. 2003, Hojberg, Vilsboll et al. 2009) or sulfonylureas (Meneilly, Bryer-Ash et al. 1993, Aaboe, Knop et al. 2009). Thus, combinations of GIP with other glucose-lowering agents could help unlock its therapeutic benefits. GIPinfusion studies have informed our understanding of GIP physiology and pharmacology. Most prominently, GIP is well known to increase circulating glucagon levels under conditions of hypoglycemia, likely reflecting direct effects of GIP on pancreatic alpha cells (Meier, Gallwitz et al. 2003, Christensen, Calanna et al. 2014). GIP activates adipose tissue blood flow and increases glucose and fatty acid uptake into adipose tissue in man, consistent with high level of GIP receptor expression in adipose tissue (Ahlqvist, Osmark et al. 2013, Asmar, Asmar et al. 2017). Unlike GLP-1, GIP does not affect gastric emptying, and no inhibition of food intake has been reported with peripherally administered GIP molecules either preclinically or in man (Finan, Muller et al. 2016, Holst 2019).

The ability of GIP to stimulate fatty acid uptake into adipose tissue, together with the observation of decreased weight gain in GIP or GIP receptor knock-out mice (Miyawaki, Yamada et al. 2002, Nasteska, Harada et al. 2014), has led to the notion that GIP may be an obesogenic molecule, prompting the development of GIP receptor antagonists as a therapeutic approach to obesity. Indeed, potent and selective GIP or GIP receptor antagonist antibodies decrease body weight in mice and non-human primates (Boylan, Glazebrook et al. 2015, Killion, Wang et al. 2018). On the other hand, long-acting GIP agonist peptides do not cause weight gain in preclinical models, and rather potentiate the weight loss induced by GLP-1, challenging the assumption of GIP as an obesogenic factor (Frias, Bastyr et al. 2017, Coskun, Sloop et al. 2018, Norregaard, Deryabina et al. 2018, Mroz, Finan et al. 2019).

The observation of synergistic weight loss with combined GIP and GLP-1 treatment in pre-clinical models prompted the development of dual GIP and GLP-1 receptor agonists (also termed “twincretins”) for the treatment of T2DM and obesity. The first molecules to be tested in man showed significant glucose lowering and weight loss compared to placebo, but did not differentiate itself significantly from the single GLP-1 receptor agonist liraglutide in early clinical studies (Finan, Ma et al. 2013, Frias, Bastyr et al. 2017). Recently, a novel dual GIP and GLP-1 receptor agonist, LY3298176 (now known as tirzepatide), demonstrated profound improvements in glucose control and body weight in a Phase 2b study when compared to dulaglutide, a single GLP-1 receptor agonist (Coskun, Sloop et al. 2018, Frias, Nauck et al. 2018). Tirzepatide achieved weight loss of >15% in roughly one fourth of patients, and normoglycemia (HbA1c of less than 5.7%) in roughly one third of patients, defining a new level of efficacy for T2DM therapy (Frias, Nauck et al. 2018). The mechanisms underlying the profound efficacy of tirzepatide is not fully understood. Compared to previous dual GIP/GLP-1 receptor agonists, tirzepatide has greater potency for the GIP receptor compared to the GLP-1 receptor, providing one possible explanation for its profound clinical benefit. Preclinical data demonstrate that both the GIP and the GLP-1 receptor contribute to the glucose lowering efficacy of tirzepatide (Coskun, Sloop et al. 2018). Clinical studies are ongoing to examine the mechanisms underlying the body weight and glucose lowering effects of tirzepatide in more detail. It will be interesting to see whether well-characterized GIP actions, such as effects on adipose tissue blood flow and nutrient uptake, contribute to the clinical efficacy of tirzepatide, and how the recently identified role of GIP in modulating appetite-regulatory circuits in mice (Adriaenssens, Biggs et al. 2019) could modulate body weight in the context of GLP-1 action. Overall, tirzepatide demonstrates the potential of dual incretin therapy and is sure to spark additional investigation into the actions of GIP. Tirzepatide recently advanced into Phase 3 clinical testing for T2DM with additional plans for testing in obesity and non-alcoholic steatohepatitis (NASH).

Oxyntomodulin, glucagon, and dual glucagon-GLP-1 receptor agonists

Oxyntomodulin (OXM), originally described as one of several gut-derived, glucagon-reactive peptides collective referred to as “enteroglucagon”, is a naturally occurring 37-amino acid product of the glucagon gene secreted by enteroendocrine L cells in response to a meal, with serum levels of < 2 pM and 10 pM in fasting versus postprandial state in humans (Pocai 2012, Cox, Berna et al. 2016, Lee, Chappell et al. 2016). Consistent with a common precursor and secretion from the same cell type, serum levels of OXM tightly correlate with circulating GLP-1 levels (Lee, Chappell et al. 2016). While no endogenous OXM receptor has been identified, OXM is a weak agonist to both the glucagon and the GLP-1 receptors with ~ 10- to 20-fold lower potency compared to glucagon or GLP-1 (Day, Ottaway et al. 2009, Kosinski, Hubert et al. 2012, Willard, Wootten et al. 2012). Due to its low serum levels, it is unlikely that OXM contributes significantly to GLP-1 or glucagon receptor signaling under normal conditions, and its physiological functions remain unknown. Interestingly, however, after bariatric surgery, postprandial OXM levels are increased up to 200 pM, reaching levels that might be expected to contribute to metabolic changes in this state (Wewer Albrechtsen, Hornburg et al. 2016).

Glucagon is well known as a hormone secreted by the alpha cells of the pancreas that acts on the liver to increase hepatic glucose output, thus raising glucose levels under conditions of hypoglycemia. Low levels of glucagon may also be secreted from the gut, as recently demonstrated in pancreatectomized individuals (Lund, Bagger et al. 2016), though the significance of this is unclear. Interestingly, glucagon has also been recognized as a weight regulatory hormone. Acute administration of glucagon inhibits food intake and increases energy expenditure in man (Penick and Hinkle 1961, Geary, Kissileff et al. 1992, Salem, Izzi-Engbeaya et al. 2016). In mice, administration of a long-acting glucagon analog decreases body weight primarily by increasing energy expenditure, an effect that appears to be mediated by the liver via both FGF21 and FXR (Habegger, Stemmer et al. 2013, Kim, Nason et al. 2018). In man, glucagon increases FGF21 serum levels and induces lipolysis, suggesting that some of the same pathways are engaged (Arafat, Kaczmarek et al. 2013, Habegger, Stemmer et al. 2013). Despite the promising effects of glucagon on body weight regulation, the potent hyperglycemic effects of glucagon in rodents and man (Habegger, Stemmer et al. 2013, Neylon, Moran et al. 2013) have prevented therapeutic development of glucagon analogs for obesity or T2DM.

Interestingly, coadministration of glucagon and GLP-1 in preclinical species results in weight loss beyond what can be achieved by GLP-1 alone, while maintaining the glucose-lowering effects of GLP-1 (Day, Ottaway et al. 2009, Pocai, Carrington et al. 2009, Kosinski, Hubert et al. 2012, Henderson, Konkar et al. 2016). In man, similar effects could be achieved by administering oxyntomodulin at doses expected to activate both the glucagon and the GLP-1 receptor (Cohen, Ellis et al. 2003, Wynne, Park et al. 2005, Wynne, Park et al. 2006, Tan, Behary et al. 2017, Shankar, Shankar et al. 2018). In both preclinical species and in man, glucagon increased energy expenditure, though modestly, even in the presence of GLP-1. Furthermore, in preclinical models, lipid profiles were profoundly improved compared to a selective GLP-1 receptor agonist, suggesting a potential benefit in the treatment of NASH. These findings sparked significant interest in exploring unimolecular combinations of glucagon and GLP-1 with several molecules progressing to clinical testing (Table 1). The most advanced of these molecules, SAR425899 and MEDI0382 (now known as cotadutide), show impressive weight loss in short term studies in obese or T2DM individuals as well as lowering of hepatic lipids (Ambery, Parker et al. 2018, Tillner, Posch et al. 2019). Glucose lowering was similar to what would be observed with single GLP-1 receptor agonists, suggesting that these molecules are able to balance GLP-1 and glucagon activity. It is important to note that no direct comparisons of dual glucagon/GLP-1 receptor agonists to single GLP-1 receptor agonists in man have been reported to date. While MEDI0382 has progressed into Phase 2b studies for T2DM, obesity and NASH, SAR425899 as well as another dual glucagon/GLP-1 receptor agonist, MRK-8521, have been discontinued, likely reflecting challenges in developing multifunctional glucagon/GLP-1 receptor agonists with a competitive profile, possibly due to suboptimal engagement of one of the two receptors (Table 1) or suboptimal pharmacokinetic profile.

Concurrent with the development of dual analogs, efforts are underway to combine the pharmacology of GLP-1, GIP and glucagon to achieve even more weight loss. In preclinical models, these combinations can normalize the body weight of very obese mice, and similar efficacy can be obtained with unimolecular GLP-1, GIP, and glucagon triagonists (Finan, Yang et al. 2015, Jall, Sachs et al. 2017). Several unimolecular triagonists are currently in development, with at least three molecules in early clinical testing (Table 1).

PYY

A gut-derived peptide that may play a complementary role to GLP-1 is peptide tyrosine tyrosine (PYY). PYY is a 36-amino acid peptide belonging to the pancreatic polypeptide family, which includes pancreatic polypeptide (PP) secreted from the pancreas and neuropeptide Y (NPY) produced by neurons in the central nervous system. While composed of similar hairpin-fold motif structures and sharing the same G-protein coupled NPY receptors (Y1, Y2, Y4, and Y5), PYY is predominantly secreted from L cells of the gut and functionally distinct from PP and NPY (Holzer, Reichmann et al. 2012).

PYY exists in two endogenous forms, PYY1–36 and PYY3–36, which differ in abundance in fasting versus postprandial states and in selectivity to NPY receptors. Similar to GLP-1, postprandial PYY concentrations increase and correlate with prolonged weight loss obtained by 8-week low calorie dieting followed by weight loss maintenance for 1 year (Iepsen, Lundgren et al. 2016). PYY3–36 is produced from PYY1–36 through the action of dipeptidyl peptidase IV (DPP4) and is the major postprandial circulating form of PYY (Adrian, Ferri et al. 1985, Adrian, Sagor et al. 1986, Medeiros and Turner 1994). Unlike GLP-1, the enzymatic cleavage of PYY1–36 by DPP4 does not reduce the affinity of PYY to a single receptor, rather it improves selectivity (Dumont, Fournier et al. 1995). Thus, while PYY1–36 binds all NPY receptors, PYY336 acts as a selective Y2 agonist whereby it exerts its unique action.

Initially discovered in the early 1980s, the first reports suggested a confined role of PYY in the gastrointestinal tract, including promoting gastric and pancreatic secretion and controlling gastrointestinal motility (Tatemoto and Mutt 1980, Lundberg, Tatemoto et al. 1982, Tatemoto 1982). However, the discovery that PYY3–36 plays a role in energy homeostasis via central action, demonstrated by experiments where exogenous administration of PYY3–36 reduces food intake in both rodents and humans and attenuates body weight gain in rodents, inspired interest in the Y2 receptor as a potential target in treating metabolic disease (Batterham, Cowley et al. 2002, Batterham, Cohen et al. 2003, Pittner, Moore et al. 2004). Moreover, further evidence suggests that PYY may play a role in insulin sensitivity and glucose uptake, likely via the Y2 receptor (Pittner, Moore et al. 2004, van den Hoek, Heijboer et al. 2004, Boey, Sainsbury et al. 2007). While these mechanisms are still being intensely investigated, the benefit of insulin sensitizing action for the treatment of metabolic diseases cannot be overstated.

PYY and GLP-1 have been demonstrated to be co-localized to the same L cells, same secretory vesicles, and shown to respond to the same stimuli, suggesting that PYY and GLP-1 play a complementary role (Habib, Richards et al. 2013). In support, co-infusions of PYY3–36 and GLP-1 additively / synergistically lower food intake in humans (Neary, Small et al. 2005, De Silva, Salem et al. 2011, Schmidt, Gregersen et al. 2014) and acute and chronic studies in rodents demonstrate additive / synergistic effects of PYY3–36 and GLP-1 on improvements in energy homeostasis (Neary, Small et al. 2005, Talsania, Anini et al. 2005, Kjaergaard, Salinas et al. 2019). While gastrointestinal side effects could limit use of PYY3–36 analogs / Y2 receptor agonists as a therapeutic target, especially when combined with GLP-1 analogs, a long-acting PYY analog reduced food intake in non-human primates with minimal emesis, even when coadministered with liraglutide which further reduced food intake (Rangwala, D’Aquino et al. 2019). Several long-acting PYY3–36 analogs / Y2 receptor agonists are being tested in early clinical development (Table 1), and it is anticipated that these molecules will ultimately be tested in combination with GLP-1 analogs. In addition, unimolecular approaches to PYY/GLP1 are being explored preclinically (Chepurny, Bonaccorso et al. 2018).

Other gut peptide-based approaches

Many additional gut-derived peptides have been discussed as candidates for new therapeutics for obesity and/or T2DM (Sun, Martin et al. 2018). However, to date, only a few have progressed to rigorous clinical testing. The reason for this is multi-fold, but concerns about efficacy, safety and/or tolerability, as well as lack of a differentiation, particularly when compared to current incretin-based therapies, are all likely contributing factors. For example, cholecystokinin (CCK), a hormone secreted by enteroendocrine I-cells, received significant attention following studies that demonstrated CCK-mediated inhibition of food intake in man (Miller and Desai 2016). Several CCK1-receptor agonists were generated and at least one molecule was tested in man in a robust Phase 2 study. However, while inhibition of gastric emptying and stimulation of gall bladder contraction were observed, body weight was not changed even in the face of increased gastrointestinal side effects, suggesting that this approach would not result in a viable therapy for weight loss (Jordan, Greenway et al. 2008, Miller and Desai 2016, Hornigold, Roth et al. 2018).

Ghrelin is a stomach-derived hormone that has been termed the “hunger hormone” due to its ability to stimulate appetite and increase food intake in preclinical studies and in man (Kojima, Hosoda et al. 1999, Schellekens, Dinan et al. 2010). Levels of ghrelin are highest in the fasting state and attenuate during nutrient consumption; caloric restriction that occurs during dieting leads to an increase in levels of circulating ghrelin and ghrelin levels are decreased in certain types of bariatric surgery (Geloneze, Tambascia et al. 2003, Lin, Gletsu et al. 2004, Schellekens, Dinan et al. 2010, Hernandez, Green et al. 2018). Numerous efforts aimed at identifying antagonists to ghrelin, its receptor and the enzyme responsible for ghrelin acylation, ghrelin-O-acyl transferase (GOAT), were conducted; however, molecules tested to date failed in early-stage clinical testing due to a variety of factors, including lack of clinically significant weight loss, poor selectivity, and side effect profiles (Schalla and Stengel 2019).

New and existing gut peptides continue to be explored with an emphasis on potential new therapeutic avenues. Secretin, a gut-derived peptide best known for regulating stomach pH and pancreatic bicarbonate secretion, was recently found to mediate thermogenesis and satiation via its action on brown adipose tissue, demonstrating that even supposedly well understood peptides can have unexplored functions (Li, Schnabl et al. 2018). Acute infusion of secretin activates glucose uptake into brown adipose tissue in man, suggesting a potential role as a mediator of postprandial thermogenesis. Unfortunately, chronic infusion of a secretin analog to mice failed to result in sustained body weight reductions, making it unlikely that secretin will become a therapeutic for weight loss (Li, Schnabl et al. 2018). Another interesting gut-derived peptide is neurotensin, an anorexigenic hormone whose circulating levels are upregulated by bariatric surgery (Ratner, Skov et al. 2016). Recently, long-acting neurotensin analogs were found to inhibit food intake in rodents and synergize with GLP-1 analogs to reduce body weight (Ratner, He et al. 2019). Neurotensin has a broad range of activities, including effects on the cardiovascular system, glucose metabolism, thermal regulation and pain that may limit its therapeutic usefulness; to date, no neurotensin analogues have been tested in man.

The ability to rapidly evaluate pharmacology and physiology in acute settings in man using peptide infusion, and the potential for added efficacy through combination therapy make gut peptides uniquely suited for evaluation as novel therapeutic approaches. The short-half-life and rapid renal clearance that have limited the therapeutic evaluation of gut peptides in the past can now be overcome by peptide engineering, allowing assessment of pharmacology in a chronic setting as well. These advances, together with increasingly sensitive methods for peptide detection and quantitation, make it likely that additional gut-derived therapies will emerge in the future.

What is the role of gut peptides in bariatric surgery?

Can gut peptide-based therapy mimic the effects of bariatric surgery?

During bariatric surgery, multiple gut peptides are dramatically regulated. Within a few days after bariatric surgery, postprandial levels of GLP-1, PYY and oxyntomodulin increase, in line with improvements in fasting glucose and insulin concentrations (le Roux, Welbourn et al. 2007, Falkén, Hellström et al. 2011, Wewer Albrechtsen, Hornburg et al. 2016). Further increases in GLP-1 up to 6-fold above pre-surgery levels are observed in the months following surgery (Korner, Inabnet et al. 2009, Yousseif, Emmanuel et al. 2014). Interestingly, no consistent changes in GIP levels are reported following bariatric surgery (Rao and Kini 2011), suggesting little effect of bariatric surgery on gut peptide secretion from the proximal small intestine, and ghrelin levels are decreased in some, but not all types of bariatric surgery, generally correlating with effects of surgery on the stomach rather than overall weight loss (Kalinowski, Paluszkiewicz et al. 2017). Importantly, in observational studies, elevations in GLP-1 and PYY correlate with the durability of weight loss in patients at 1, 10, and even 20 years after surgery, suggesting alterations in gut hormone secretion contribute to sustained weight loss and glucose control (Naslund, Gryback et al. 1997, Falkén, Hellström et al. 2011, Dar, Chapman et al. 2012).

Given the potent effect of bariatric surgery to increase GLP-1 levels and the effects of GLP-1 receptor agonists to affect both food intake and glucose regulation, it is reasonable to hypothesize that increased GLP-1 secretion is a primary driver of the effects of surgery. Yet, this topic remains highly controversial. In humans, short-term blockade of the GLP-1 receptor leads to a clear worsening of glucose tolerance in patients who have had RYGB (Salehi and D’Alessio 2016, Holst, Madsbad et al. 2018, Salehi, Gastaldelli et al. 2019). However, the limitation of these experiments include the short duration of these blockades as well as the impairment of glucose tolerance by pharmacological blockade of the GLP-1 receptor in patients without surgery. Hence, it is not easy to show an interaction between surgery-induced increases in GLP-1 levels and sensitivity to the blockade of GLP-1 signaling. In rodents, loss-of-function of the GLP-1 receptor does not reduce the effectiveness of VSG or RYGB to cause either weight loss or improve glucose regulation (Wilson-Perez, Chambers et al. 2013, Mokadem, Zechner et al. 2014), suggesting that GLP-1 receptor signaling is not required for these effects. Recently, β-cell specific knockouts of the GLP-1 receptor have been tested in a mouse model of VSG. Unfortunately, two reports come to different conclusions with one showing that such tissue-specific knockdown impairs the ability of VSG to increase insulin secretion and improve glucose tolerance (Garibay, Lou et al. 2018), while another shows no effect (Douros, Lewis et al. 2018). While these results may suggest that GLP-1 is not solely responsible for the metabolic effects of surgery, GLP-1 likely plays an important part of the cocktail of hormone changes that occurs after surgical interventions. Interestingly, antagonizing GLP-1 and PYY secretion with the somatostatin analog octreotide can increase food appeal post bariatric-surgery-induced weight loss (Goldstone, Miras et al. 2016), suggesting an important role for gut peptide combinations. However, in mice where both GLP-1 and PYY receptors are knocked out, RYGB is equally effective to cause weight loss and improve glucose control (Boland, Mumphrey et al. 2019).

An alternate approach has been to attempt to mimic the results of bariatric surgery with infusions of native gut peptides at concentrations similar to the ones observed after bariatric surgery. Acute studies that co-infused GLP-1, PYY and oxyntomodulin into obese volunteers showed promising results on food intake (Tan, Behary et al. 2017), prompting the initiation of chronic studies designed to examine the effects of these combinations on body weight (Behary, Tharakan et al. 2019) and reflective of efforts to create dual and triple agonists of these peptides as described above.

It is important to note that mechanisms other than gut peptides likely contribute to the effects of bariatric surgery. As we have already discussed, a wide range of evidence points to alterations in bile acids coming from the liver to contribute to the effects of bariatric surgery and there remains the possibility that neural signals from the gut contribute. In addition, bariatric surgery is associated with alterations in the gut (Murphy, Tsai et al. 2017), and some evidence from fecal transplants indicate a role for the changes in the gut microbiome in the effectiveness of surgery (Liou, Paziuk et al. 2013, Tremaroli, Karlsson et al. 2015, Murphy, Tsai et al. 2017). However, in none of these experiments does the effect of the transplant entirely recapitulate the potent effects of surgery. Whether this is a limitation of the fecal transplant methodology or a reflection of a limited role of the microbiome remains controversial. While combining either pre- or probiotic approaches with gut-peptide based therapies may ultimately achieve efficacy similar to what is seen with bariatric surgery, we should not underestimate the difficulties associated with manipulating the microbiome for therapeutic benefit. The current approaches such as, probiotics, prebiotics and fecal transplants are all very blunt tools that do alter the gut bacterial communities in numerous ways. Fecal transplants focus on using the bacteria from the distal colon. While the number of bacteria is much lower in the small intestine, there is evidence that these bacteria play a larger role in influencing host metabolism (Arora, Seyfried et al. 2017). Given that the composition in the ileum is quite different, we simply may not be transplanting the most helpful bacteria or insuring they colonize the appropriate location within the gastrointestinal tract. Despite a potentially important role, there is no guarantee that we can manipulate the gut microbiome with the precision necessary for therapeutic benefit.

Is the gut really the primary source of gut peptides?

Endogenous GLP-1 is rapidly processed by DPP4 in the gastrointestinal tract, resulting in very low circulating levels of active GLP-1. This raises the question of how gut-derived GLP-1 can sufficiently activate the GLP-1 receptor in distant tissues to achieve its physiological functions (Hjollund, Deacon et al. 2011, Mulvihill, Varin et al. 2017, Mulvihill 2018). The prohormone encoding GLP-1, preproglucagon, is also expressed in tissues outside of the gut. Recent work using tissue-specific reactivation of preproglucagon has allowed for a more careful examination of this issue. In the gut, reactivation of preproglucagon had little impact on overall glucose tolerance, while in the pancreas it resulted in a reversal of the phenotype observed in preproglucagon null mice. More importantly, the ability of GLP-1 receptor blockade to impair glucose tolerance was absent in both whole-body null and gut reactivated mice. However, when preproglucagon is reactivated in the pancreas, the ability to respond to GLP-1 receptor blockade is restored (Chambers, Sorrell et al. 2017). Along with other observations (Ellingsgaard, Hauselmann et al. 2011), such data indicate that a key source of endogenous GLP-1 is from the pancreas rather than from the gut. Despite the fact that the majority of circulating GLP-1 does largely originate in the gut, it is not the critical actor in at least some tissues. Additionally, recent data indicates that pancreatic glucagon can activate GLP-1 receptors to increase insulin secretion (Capozzi, Svendsen et al. 2019). This has two important implications. First, it implies that the effects of GLP-1 agonists may not be truly mimicking the effect of GLP-1 as a gut hormone. Rather, such agonists probably engage the system in a way that is quite distinct from circulating native GLP-1. Second, as discussed below, one strategy towards novel therapies is to identify ways to activate intestinal cells that make GLP-1 and cause them to increase their secretion. If gut-derived GLP-1 is not a critical factor, this strategy may be less effective than what was originally thought.

Other Gut-based Approaches

Inhibition of Gut Peptide Degradation as a Therapeutic Strategy: DPP4 Inhibitors

Gut peptides are typically short-acting hormones whose duration and site of action is limited by rapid degradation, suggesting inhibition of gut peptide degradation as an alternative therapeutic approach. It is estimated that >90% of GLP-1 secreted by the L cell is degraded before it reaches the portal circulation (Hjollund, Deacon et al. 2011, Mulvihill, Varin et al. 2017, Mulvihill 2018). DPP4, an enzyme present both on the surface of vascular endothelial cells and in circulation, has been identified as the major GLP-1 degrading enzyme (Kieffer, McIntosh et al. 1995), resulting in the development of DPP4 inhibitors as an alternate way to harness the therapeutic benefits of GLP-1.

Ten DPP4 inhibitors are currently approved for the treatment of T2DM with several additional molecules under development (Table 2)(Deacon and Lebovitz 2016). DPP4 inhibitors have become an important component of diabetes therapy and are recommended as first-line therapy for a subset of individuals with T2DM (American Diabetes 2019). The pharmacological properties of these molecules, their clinical efficacy, safety, and long-term outcomes are remarkably consistent (Deacon and Lebovitz 2016, Nauck, Meier et al. 2017, Yamada, Shojima et al. 2018). DPP4 inhibitors as a class have excellent tolerability, show modest glucose lowering, and are weight neutral. Large outcome studies, conducted for five DPP4 inhibitors to date, confirmed safety, but interestingly showed no significant cardiovascular benefit (Nauck, Meier et al. 2017, McGuire, Alexander et al. 2019). Furthermore, no signals suggesting a renal benefit were detected in a meta-analysis of outcomes studies conducted to date (Luo, Lu et al. 2018).

Table 2:

Small molecule approaches to pathways related to gut peptides.

Class	Key Molecules	Administration	Status	Indication	References
Gut peptide degradation inhibitors
DPP4 inhibitors	sitagliptin	oral QD	Marketed	T2DM	(Deacon and Lebovitz 2016)
	vildagliptin	oral QD	Marketed	T2DM	(Deacon and Lebovitz 2016)
	linagliptin	oral QD	Marketed	T2DM	(Deacon and Lebovitz 2016)
	saxagliptin	oral QD	Marketed	T2DM	(Deacon and Lebovitz 2016)
	alogliptin	oral QD	Marketed	T2DM	(Deacon and Lebovitz 2016)
Putative gut peptide secretagogues tested in the clinic
GPR119 agonists	DS-8500a	oral QD	Phase 2	T2DM	(Matsumoto, Yoshitomi et al. 2018, Yamada, Terauchi et al. 2018)
	GSK1292263	oral QD	Phase 2 (discontinued)	T2DM	(Nunez, Bush et al. 2014)
	BMS-903452	oral QD	Phase 1 (discontinued)	T2DM	(Wacker, Wang et al. 2014)
	JNJ-38431055	oral QD	Phase 1 (discontinued)	T2DM	(Katz, Gambale et al. 2011, Semple, Ren et al. 2011)
GPR40 agonists (partial)	SHR0534	oral QD	Phase 1	T2DM	(Li, Qiu et al. 2016)
	TAK-875 (Fasiglifam)	oral QD	Phase 3 (discontinued)	T2DM	(Kaku, Enya et al. 2016, Menon, Lincoff et al. 2018)
	LY2881835 LY2922083 LY2922470	oral QD	Phase 1 (discontinued)	T2DM	(Chen, Li et al. 2016, Hamdouchi, Kahl et al. 2016)
	MK-8666	oral QD	Phase 1 (discontinued)	T2DM	(Krug, Vaddady et al. 2017, Lu, Byrne et al. 2017)
Putative gut peptide secretagogues with preclinical data only
GPR40 full agonists	AMG-1638; AM-6226	NA	Preclinical	T2DM	(Brown, Dransfield et al. 2012, Luo, Swaminath et al. 2012)
	BMS-986118	NA	Preclinical	T2DM	(Shi, Gu et al. 2018)
	Cpd 12	NA	Preclinical	T2DM	(Meegalla, Huang et al. 2018)
	AP1; AP3	NA	Preclinical	T2DM	(Pachanski, Kirkland et al. 2017)
GPR120 agonists	TUG-891	NA	Preclinical	T2DM	(Schilperoort, van Dam et al. 2018)
	Cpd 4x	NA	Preclinical	T2DM	(Zhang, Cai et al. 2017)
	Cpd 34	NA	Preclinical	T2DM	(Azevedo, Watterson et al. 2016)
TGR5 agonists	INT-777	NA	Preclinical	T2DM	(Pellicciari, Gioiello et al. 2009, Li, Holmstrom et al. 2011)
	Cpd 18	NA	Preclinical	T2DM	(Briere, Ruan et al. 2015)
GPR142 agonists	Cpd 33; Cpd 23	NA	Preclinical	T2DM	(Toda, Hao et al. 2013, Yu, Lizarzaburu et al. 2013)
	Cpd 40	NA	Preclinical	T2DM	(Wilson, Kurukulasuriya et al. 2016)
GPR39 agonists	Cpd 3	NA	Preclinical	T2DM	(Peukert, Hughes et al. 2014)
SSTR5 antagonists	S5A1	NA	Preclinical	T2DM	(Farb, Adeva et al. 2017)
	Cpd 10	NA	Preclinical	T2DM	(Liu, Shao et al. 2018)
	Cpd 25a	NA	Preclinical	T2DM	(Hirose, Yamasaki et al. 2017)

Molecules on the market or in active clinical development; discontinued molecules included if published information available. Select preclinical molecules included for mechanisms that have not progressed to the clinic to date.

The therapeutic profile of DPP4 inhibitors differs significantly from GLP-1 receptor agonists. For one, glucose lowering mediated by DPP4 inhibitors is more modest than what is achievable with GLP-1 analogs. In addition, weight loss is not observed, gastrointestinal side effects, such as nausea and vomiting, are not found, and other on-target effects of GLP-1 receptor agonists, such as heart rate increases and inhibition of gastric emptying, are also not observed with DPP4 inhibitors (Nauck, Meier et al. 2017, Aroda 2018). A likely explanation is that the increase of GLP-1 by DPP4 inhibitors is limited by the total amount of GLP-1 secreted endogenously. In man, concentrations of DPP4 inhibitors that inhibit circulating DPP4 activity by >90% and achieve maximum therapeutic benefits, result in serum levels of intact, active GLP-1 of ~ 15–30 pM postprandially (Herman, Bergman et al. 2006, Addy, Tatosian et al. 2016). This is significantly lower than the ~200 pM of native GLP-1 required for maximal pharmacological effects in GLP-1 infusion studies (Nauck and Meier 2018) and the 50 pM amount of GLP-1 equivalents that has been estimated to be required to achieve pharmacological effects (Calara, Taylor et al. 2005, Kothare, Linnebjerg et al. 2008).

A large number of proteins and peptides have been identified as potential substrates for DPP4 (Nauck, Meier et al. 2017), raising the question of whether gut peptides other than GLP-1 may contribute to the efficacy of DPP4 inhibitors. Indeed, mice lacking GLP-1 (and other preproglucagon-derived peptides), are able to still respond to DPP4 inhibitors with glucose-lowering (Mulvihill 2018) and a study in T2DM subjects treated with a combination of a DPP4 inhibitor and the GLP-1 receptor antagonist exendin-9 suggested that as much as 50% of the glucose lowering observed with DPP4 inhibitors in man may be attributable to non-GLP-1 effects (Aulinger, Bedorf et al. 2014). Interestingly, DPP4 is also the major degrading enzyme for GIP (Kieffer, McIntosh et al. 1995), and DPP4 inhibitors increase the levels of circulating active GIP even more than circulating GLP-1 levels (up to 60 pM postprandially) (Yamada, Shojima et al. 2018). Additional peptides may contribute to the overall therapeutic profile of DPP4 inhibitors, although direct evidence is lacking. For example, processing of PYY1–36 to PYY3–36 by DPP4 changes the receptor selectivity profile, allowing it to activate the NPYR2 receptor to inhibit food intake (Mentlein, Dahms et al. 1993, Ostergaard, Kofoed et al. 2018). DPP4 inhibitors significantly reduce levels of circulating PYY3–36 (Aaboe, Knop et al. 2010), possibly limiting the effects of endogenous PYY on food intake. On the other hand, inhibition of intra-islet PYY processing via DPP4 has been linked to increased insulin secretion in vitro via the NPYR1 receptor, suggesting that PYY could contribute to glucose-lowering activities of DPP4 inhibitors (Guida, McCulloch et al. 2018). It is important to note, however, that definite proof for a role of PYY in the actions of DPP4 inhibitor is lacking. It is well established that DPP4 inhibitors decrease GLP-1 secretion through feedback inhibition on the L-cell, lowering the circulating concentration of total GLP-1 as well as other gut peptides. Overall, the mechanism of action of DPP4 inhibitors may be more complex than initially appreciated, reflecting its action on multiple peptides.

Activation of Nutrient Receptors in the Gut as a Therapeutic Strategy

Given that inhibition of gut peptide degradation resulted in DPP4 inhibitors as a new class of diabetes therapy, could increasing gut peptide secretion also open new therapeutic avenues? Furthermore, could the combination of both approaches allow even more powerful glucose lowering and weight loss? Numerous receptors – many of them highly druggable G protein coupled receptors – modulate gut peptide secretion in response to nutrients, such as fatty acids, amino acids, carbohydrates and other stimuli (Gribble and Reimann 2019). Despite a large amount of effort dedicated to these receptors, however, therapeutics have so far been elusive (see Table 2 for key clinical and preclinical efforts to date).

Receptors for bile acids

In addition to aiding the processing of dietary fats and fat-soluble nutrients, bile acids activate the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1, more commonly called TGR5. While FXR activation regulates bile acid synthesis and triglyceride metabolism (Ding, Yang et al. 2015), TGR5 activation results in the release of GLP-1 and PYY from L-cells and increases energy expenditure, at least in mice (Katsuma, Hirasawa et al. 2005, Watanabe, Houten et al. 2006). FXR agonists are currently under investigation for the treatment of NASH with promising results in clinical studies to date (Malhi and Camilleri 2017, Connolly, Ooka et al. 2018). While early studies with TGR5 agonists, such as the bile acid analog, INT-777, established strong effects on incretin secretion and glucose excursions in rodents (Pellicciari, Gioiello et al. 2009, Thomas, Gioiello et al. 2009), target-mediated side effects have so far prevented TGR5 activators from entering clinical evaluation. Specifically, activation of TGR5 leads to smooth muscle relaxation and stimulation of gallbladder filling, resulting in large increases in gall bladder size (Li, Holmstrom et al. 2011) that could not be separated from elevations in GLP-1 or glucose homeostasis (Briere, Ruan et al. 2015). Given the high expression of TGR5 in gallbladder (Keitel, Cupisti et al. 2009), current efforts are focused on reducing systemic exposure of a TGR5 agonist by creating gut-restricted compounds.

Receptors for long-chain fatty acids and related lipids

The G-protein coupled receptors GPR40, GPR119 and GPR120 share an affinity for long-chain fatty acids and related molecules. Interest in GPR119 increased significantly after its identification as a receptor that mediates the inhibitory effects of 2-monoacylglycerols, specifically oleoylethanolamide, on food intake (Overton, Babbs et al. 2006). In addition, GPR119 is highly expressed in enteroendocrine cells in the gut as well as in pancreatic islets. Numerous potent and selective GPR119 agonists have been identified and preclinical studies show a consistent picture: molecules induce glucose-dependent insulin secretion and modest increases in GLP-1 and GIP secretion, but have little or no effect on body weight (Ritter, Buning et al. 2016, Matsumoto, Yoshitomi et al. 2018). Clinical studies were conducted with more than 10 molecules. While glucose lowering was observed, the effect size was modest, impact on GLP-1 and GIP levels was minimal (<50%) and no significant weight loss was observed (Katz, Gambale et al. 2011, Semple, Ren et al. 2011, Katz, Gambale et al. 2012, Nunez, Bush et al. 2014, Wacker, Wang et al. 2014, Matsumoto, Yoshitomi et al. 2018, Yamada, Terauchi et al. 2018). These modest effects led to discontinuation of the vast majority of GPR119 agonist efforts.

GPR40 agonists were originally developed as glucose-dependent insulin secretagogues and later recognized to also have potential as GLP-1 secretagogues, reflecting expression of this receptor on pancreatic beta cells as well as enteroendocrine cells. First generation GPR40 agonists, such as fasiglifam (TAK-875), are partial agonists that showed significant glucose-lowering in the clinic (Araki, Hirayama et al. 2012), but did not increase circulating GLP-1 levels either preclinically or clinically (Luo, Swaminath et al. 2012, Chen, Li et al. 2016, Hamdouchi, Kahl et al. 2016); no effect on body weight was noted. While development of fasiglifam was discontinued due to concerns about drug-induced liver injury (Menon, Lincoff et al. 2018), several partial GPR40 agonists remain in clinical development. Recently, full agonists of the GPR40 receptor have been developed that bind to a distinct site and activate both G_αq and G_αs signaling (Brown, Dransfield et al. 2012, Lin, Guo et al. 2012, Luo, Swaminath et al. 2012). In preclinical studies, these molecules are among the strongest insulin and incretin secretagogues described, resulting in 3- to 5-fold increases in circulating GLP-1 levels as well as elevated levels of PYY and GIP, and strong increases of glucose-dependent insulin secretion. Despite significant efforts across the industry, to date, however, no full GPR40 agonists have entered clinical development.

GPR120 is a receptor for long-chain fatty acids expressed in multiple tissues, most prominently adipocytes, immune cells and enteroendocrine cells of the gut. Numerous companies as well as academic investigators have developed specific small molecule GPR120 agonists and demonstrated glucose lowering in rodents in acute and chronic settings (Oh, Walenta et al. 2014, Azevedo, Watterson et al. 2016, Cox, Chu et al. 2017, Sparks, Aquino et al. 2017, Sundstrom, Myhre et al. 2017, Zhang, Cai et al. 2017, Sheng, Yang et al. 2018, Winters, Sui et al. 2018). However, GLP-1 secretion has only been reported for a couple of these molecules with mixed results (Oh, Walenta et al. 2014, Sundstrom, Myhre et al. 2017), and development of potent and selective GPR120 molecules has been challenging. To date, no clinical studies with a GPR120 agonist have been reported.

Receptors for amino acids

A relatively recent nutrient receptor to be explored is GPR142, now recognized to be a receptor for aromatic amino acids that regulates secretion of incretins from the gut, as well as glucagon and insulin secretion from pancreatic islets (Rudenko, Shang et al. 2019). Potent and selective small molecule GPR142 agonists increase insulin and glucagon secretion from mouse and human islets, improve oral glucose tolerance upon acute administration in lean and obese mice, obese rats as well as in monkeys, and improve insulin sensitivity and basal glucose levels upon chronic administration to diet-induced obese mice (Toda, Hao et al. 2013, Yu, Lizarzaburu et al. 2013, Guo, Parker et al. 2016, Wilson, Kurukulasuriya et al. 2016, Rudenko, Shang et al. 2019). Circulating gut peptides, including GLP-1, GIP, and CCK are significantly increased in both rodents and non-human primates with GPR142 agonists with a particularly strong (^~ 5-fold) effect on GIP (Lin, Efanov et al. 2016, Rudenko, Shang et al. 2019). Interestingly, GPR142 agonists can induce both beta-cell proliferation as well as protect beta-cells from stress in isolated islets in vitro, suggesting a potential for increased durability of therapy (Lin, Wang et al. 2018). To date, no clinical studies with a GPR142 agonist have been reported.

Other gut receptors with potential therapeutic relevance

Many additional receptors are highly expressed on intestinal L and/or K cells, raising the possibility of novel therapeutic targets that could potentially mediate GLP-1 secretion under physiological or pharmacological conditions. Among these, the short-chain fatty acid receptors GPR41 (FFAR2) and GPR43 (FFAR3), as well as GPR39, an orphan GPCR highly expressed in various metabolically important tissues including enteroendocrine cells, pancreatic beta cells and hepatocytes, have received significant attention (Tolhurst, Heffron et al. 2012, Peukert, Hughes et al. 2014, Christiansen, Gabe et al. 2018). However, pharmacological data to date are mixed and no molecules have entered clinical development (Peukert, Hughes et al. 2014, Fjellstrom, Larsson et al. 2015, Frimurer, Mende et al. 2017).

Inhibiting the brake: Can somatostatin antagonists be useful for the treatment of diabetes?

Somatostatin (SST) is a hormone produced by delta cells in the duodenum and pyloric regions of the gut, the pancreas, and the brain (Patel and Reichlin 1978, Hauge-Evans, King et al. 2009). SST has two active forms, SST-14, secreted from the hypothalamus, gut, and pancreas, and SST-28, secreted almost exclusively by intestinal cells (Shen, Pictet et al. 1982, O’Carroll, Lolait et al. 1992, Patel 1999). In humans, the major circulating form is SST-28 (Polonsky, Shoelson et al. 1983) and SST-28 serum levels appear nutritionally regulated, increasing by a small amount after a fat challenge (D’Alessio and Ensinck 1990). SST isoforms bind to a family of five receptors (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) that are all G_αi-coupled receptors that signal via cAMP (Patel 1999). While SST-14 binds to all SSTRs similarly, SST-28 preferentially binds to SSTR5. Expression profiles of SSTRs differ from tissue to tissue; for a complete review see (Gunther, Tulipano et al. 2018).

Somatostatin is a potent inhibitor of intestinal gut hormone secretion, affecting GLP-1, GIP, PYY, ghrelin and other hormones (Vu, van Oostayen et al. 2001, Tan, Vanderpump et al. 2004, Rigamonti, Cella et al. 2011). In fact, a study in healthy volunteers showed complete suppression of the postprandial rise in GLP-1 and GIP serum levels upon treatment with the somatostatin analog pasireotide (Henry, Ciaraldi et al. 2013). Moreover, somatostatin analogs have been used clinically to reverse high levels of plasma GLP-1 and PYY observed after bariatric surgery (Goldstone, Miras et al. 2016). Of the five characterized somatostatin receptors, SSTR5 is highly expressed in enteroendocrine cells (Chisholm and Greenberg 2002) and the selectivity profile of the somatostatin analog pasireotide, as well as in vitro and in vivo studies in rodents, swine and sheep all point to activation of somatostatin receptor 5 (SSTR5) on intestinal L-cells as the most likely mechanism mediating its inhibitory effects on GLP-1 secretion (Martin and Faulkner 1996, Hansen, Hartmann et al. 2000, Farb, Adeva et al. 2017). An intriguing approach is to inhibit SSTR5 to relieve the inhibitory effect of endogenous somatostatin on gut peptide secretion. Studies in preclinical models suggest that this can lead to very large increases in GLP-1 while at the same time increasing insulin secretion due to relieving inhibition of SST-14 in human islets (Farb, Adeva et al. 2017). It will be interesting to see if somatostatin 5 receptor antagonists will show efficacy in the clinic.

Combination approaches

Can one combine different approaches to modulate gut peptide secretion and stability to achieve efficacy more similar to what is possible with combination gut peptide approaches? One proposed combination strategy aims to mimic the changes observed in bariatric surgery by simultaneously targeting three GLP-1 regulatory nodes: an agonist to a gut-localized GPCR (TGR5 or second generation GPR40 agonist) to promote GLP-1 secretion, a SSTR5 antagonist to blunt inhibition of GLP-1 release, and a DPP4 inhibitor to preserve active GLP-1 (Briere, Bueno et al. 2018). Preclinically, this approach results in levels of active GLP-1 (100–400 pM) that are higher than what is observed with bariatric surgery or with GLP-1 receptor agonists (Briere, Bueno et al. 2018, Sloop, Briere et al. 2018). Whether these increases translate into efficacy similar to what is observed with current combination peptide approaches in man remains to be determined

The future

There remain many further opportunities to reverse engineer the physiological effects of surgery to find less invasive and more scalable therapeutic strategies. Moreover, they may involve less invasive procedures, new pharmacotherapies or nutrition strategies that will make effective therapies available to more individuals. A wide range of new technologies will facilitate these activities. The ability to use TRAPseq and DROPseq in the GI tract will clearly provide for a much deeper understanding of gut function and provide clues as to how existing interventions alter critical signals coming from the gut. A deeper map of the entire gut will be available in the next several years that will make it possible to mine many more potential targets for intervention. In addition, the ability to visualize ongoing neural activity in vivo and the ability to manipulate specific populations of neurons using both DREADD and optogenetics are already revolutionizing our understanding of the vagus and its targets in the caudal brainstem (Williams, Chang et al. 2016). Such strategies can be applied to the enteric nervous system as well. It is hard to imagine that a better understanding of the major neural signals from the gut won’t lead to additional targets for intervention.

Finally, we would be remiss to not mention the possibility of combining gut-peptide based approaches with therapies based on circulating hormones derived from tissues other than the gut. For example, analogs of FGF21, a liver-derived circulating hormone, have been demonstrated to cause weight-loss, lipid lowering and insulin sensitizing effects in man, and have emerged as an attractive therapeutic approach for NASH (Sumida and Yoneda 2018, Kliewer and Mangelsdorf 2019). Preclinically, FGF21 analogs show synergistic effects on body weight when combined with GLP-1. Similarly, the beta-cell-derived hormone amylin has well characterized effects on food intake and glucose control, and long-acting amylin analogs as well as dual amylin-calcitonin receptor agonists are being evaluated for obesity and T2DM both alone and in combination with GLP-1 analogs (Srivastava and Apovian 2018). It is likely that in the future gut- and non-gut-based therapies will be combined with the goal of normalizing body weight and glucose control to a degree not possible with current therapies.

Perspective

We are amid a revolution that will rapidly increase our understanding of the complexities of gut function and its impact on a wide range of physiology. The challenge will be to turn these insights into safe and efficacious therapies to more effectively deal with the twin epidemics of obesity and type 2 diabetes. Gut function and the signals generated by the gut have already led to important advances in the treatments for obesity and diabetes. However, there is an urgent need for next generation therapies for obesity and T2DM that not only address weight loss and glucose control in the short term, but also provide durability of these effects. Ideally, some of these treatments could provide sustained benefits, resulting in diabetes remission similar to what is observed after bariatric surgery. It will be interesting to see whether the gut can live up to the challenge and help identify this next generation of therapies.