The non-glycemic effects of incretin therapies on cardiovascular outcomes, cognitive function and bone health

Abstract

The incretin therapies, glucagon-like peptide-1 receptor agonists and dipeptidyl-peptidase-4 inhibitors, have been developed to lower blood glucose levels in patients with Type 2 diabetes. However, in addition to being a treatment strategy to improve metabolic control, incretin therapies have shown effects independent of glycemic control, including the potential to positively impact cardiovascular events, cognitive deficits and bone mineral density. This paper outlines the non-glycemic effects of incretin therapies on cardiovascular disease, cognitive function and bone health.

Keywords:

• bone health
• cardiovascular outcomes
• cognitive function
• dipeptidyl-peptidase-4
• DPP4 inhibitor
• GLP-1 receptor agonist
• glucagon-like peptide-1
• glucagon-like peptide-2
• glucose-dependent insulinotropic peptide
• Incretin

Incretins are gastrointestinal hormones that help to regulate carbohydrate metabolism in response to food intake [1,2]. The two main incretins are glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1), both secreted by intestinal epithelial cells. GIP was the first incretin hormone to be described. It circulates as a 42-amino acid peptide that is secreted from enteroendocrine K cells, mainly in the proximal small intestine (duodenum and jejunum), at low, basal levels during fasting and at increased levels after eating. GIP not only stimulates insulin secretion, it is also involved in fat metabolism and promotes beta-cell proliferation and cell survival [1,3].

GLP-1, the second incretin hormone to be described, is an endogenous 30-amino acid peptide secreted by enteroendocrine L cells throughout the small bowel and ascending colon. The actions of GLP-1 are mediated primarily through the GLP-1 receptor (GLP-1R) and are coupled to increases in intracellular cAMP. Binding of GLP-1 to this receptor facilitates glucose-dependent insulin secretion, inhibits gastric emptying and glucagon secretion, and slows the rate of endogenous glucose production, with the resultant effect of lowering blood glucose levels. In addition, it has been shown to stimulate beta cell proliferation and decrease beta cell apoptosis in experimental animals [1,3].

Both GLP-1 and GIP are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP4), also known as cluster of differentiation marker 26 [3]. DPP4 is widely expressed throughout the body on many cell types, including lymphocytes, epithelial cells and capillary endothelial cells. It is responsible for inactivating over 50% of GLP-1 before it can be widely circulated, resulting in a GLP-1 circulating half-life of less than two min; the half-life of GIP is 5–7 min [2,4]. By degrading the incretins, DPP4 acts to shorten their effect in vivo. After degradation, GLP-1 exists as the metabolite GLP-1(9–36), which binds weakly to GLP-1R, and is currently being investigated for additional clinical effects [3,5].

DPP4-resistant GLP-1R agonists have been developed in an attempt to extend the duration of action of the incretin hormones, resulting in prolonged effects on glucose homeostasis (Table 1). Clinically available GLP-1R agonists include exenatide, liraglutide, albiglutide and dulaglutide.

Table 1. Types of incretin therapy and their actions [5,87–89].

	GLP-1R agonists	DPP-4 inhibitors
Agents	Exenatide, liraglutide, and others	Alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin, and others
Administration	Injection	Oral
GLP-1 concentrations	Pharmacological	Physiological
Insulin secretion ↑	+++	+
Glucagon secretion ↓	++	+
Gastric emptying	Delayed	No effect
HbA1c ↓ (vs. placebo)	–1.02% (95% CI: –1.17 to –0.86%)	–0.69% (95% CI: –0.79 to –0.61%)
Weight effects	Weight loss	Weight neutral
Hypoglycemia	Low	Low

DPP-4: Dipeptidyl peptidase-4; GLP-1: Glucagon-like peptide-1; GLP-1R: Glucagon-like peptide-1 receptor; HbA1c: Hemoglobin A1c.

An alternative therapeutic strategy, developed to enhance circulating levels of GLP-1, is through inhibition of DPP4 (Table 1). Clinically available DPP4 inhibitors include saxagliptin, sitagliptin, alogliptin, vildagliptin and linagliptin. DPP4 inhibition with these agents has been shown to raise GIP and GLP-1 concentrations two- to threefold over 24 h [5,6]. The major difference between these agents is the route of elimination. Linagliptin is eliminated by nonrenal mechanisms; therefore, it does not require adjustment for patients with impaired renal function. However, saxagliptin, sitagliptin, vildagliptin and alogliptin all require dose reductions as renal function deteriorates [7].

Treatment for Type 2 diabetes with these GLP-1R agonists and DPP4 inhibitors is associated with improved glycemic control through increased insulin secretion in a glucose-dependent manner, suppression of glucagon secretion resulting in improved glycemic control, and low rates of hypoglycemia [2]. GLP-1R agonist use is associated with weight loss in most patients through an effect on satiety and slowing gastric emptying. In addition to these benefits, nonglycemic effects of these incretin therapies have been seen in cardiovascular disease, bone and in the CNS. This paper will outline the current knowledge regarding the effects of incretin therapies on these systems, independent of glycemic control.

Cardiovascular disease

Role of GLP-1 in cardiovascular biology

The GLP-1R has been found in multiple cell types within the heart, including the microvascular endothelium, endocardium, cardiomyocytes, and coronary smooth muscle cells [8]. Deletion of the GLP-1R gene in 2-month-old mice resulted in abnormal cardiac function, including left ventricular hypertrophy, reduced resting heart rate and increased left ventricular end-diastolic pressure. Mice that lacked a functional GLP-1R also showed an abnormal response to insulin, with an exaggerated increase in left ventricular end-diastolic pressure, which was not seen in control mice [9].

While deletion of the GLP-1R gene impaired cardiac function, infusion of recombinant GLP-1 or the GLP-1 (9–36) metabolite has been shown to improve cardiac function in preclinical animal models of heart failure and reperfusion, conferring cardiac protection [8,10,11]. In addition, using cardiomyocytes from primary cell cultures and cell lines, signaling pathways downstream of GLP-1R have been determined. These GLP-1R–dependent pathways have been shown to reduce apoptosis in cardiac myocytes [12]. GLP-1 may exert its effects through both receptor-dependent and receptor-independent mechanisms [3,12] and, based on animal studies, may potentially impact the development and/or progression of atherosclerotic plaque. However, data are not available on the long-term effects of incretin-based therapy on atherosclerosis-associated outcomes in patients with diabetes [12].

Cardiovascular effects of GLP-1R agonists & DPP4 inhibitors in preclinical studies

Exenatide, a clinically available GLP-1R agonist, has shown cardioprotective actions. In one preclinical trial, pigs were given an infusion of exenatide following a 75-min coronary occlusion. The infusion was able to reduce ischemia-reperfusion injury, decrease infarct size and prevent a decline in cardiac function. These results were associated with a reduction in oxidative stress and apoptosis [13]. However, the timing of treatment and the dosing regimen appear to be important in mediating the observed cardioprotection. Unlike the cardioprotective benefits of exenatide given after a coronary occlusion, an infusion of liraglutide given 3 days before a 40-min coronary occlusion showed no reduction in infarct size [14]. Of interest, liraglutide has a GLP-1 (9–36) metabolite, whereas exenatide does not [3].

DPP4 expression has been localized to endothelial and smooth muscle cells and its activity is increased in patients with diabetes [3,12]. Further study of the biological role of DPP4 in these cells is required to determine its function. DPP4 is also a circulating protein and may influence levels of GLP-1 and other vasoactive DPP4 substrates in the myocardium and vasculature [12].

As a treatment strategy, DPP4 inhibition is an area of intense research. Of interest, in normoglycemic and diabetic mouse models subjected to experimental myocardial infarction (MI), neither pharmacologic inhibition nor genetic deletion of DPP4 impaired cardiovascular function [15]. Therefore, DPP4 inhibitors may increase the activity of alternative substrates, which may impact the cardiovascular system. DPP4 cardioactive substrates include GLP-1, GIP, neuropeptide Y, peptide YY and B-type (brain) natriuretic peptide, among others [12]. One DPP4 substrate being investigated is stromal cell-derived factor-1 alpha (SDF-1α), which is an antiapoptotic and pro-angiogenic chemokine that promotes homing of endothelial progenitor cells to cellular injury sites and is speculated to mediate the biological response to DPP4 inhibition [12,16].

One recently published study hypothesized that DPP4 inhibition conferred benefit due to the role of enhanced SDF-1α availability versus potentiating GLP-1. This study compared the DPP4 inhibitor saxagliptin with the GLP-1R agonist liraglutide following experiments in a model of Type 1 diabetes, ensuring any effects would be glucose-independent. In this model, saxagliptin-mediated DPP4 inhibition improved ventricular systolic and diastolic function post MI. However, liraglutide-mediated GLP-1R agonism did not. These results suggest that SDF-1α potentiation may underlie the cardioprotective effects of DPP4 inhibition [16,17].

Clinical trials evaluating cardiovascular outcomes of GLP-1R agonists & DPP4 inhibitors

GLP-1 mediates many of its biological effects through activation of the GLP-1R. Ligand binding activates adenylate cyclase, which increases cAMP concentrations [8]. Increasing cAMP using pharmacological agents has been shown to have adverse effects in clinical trials of patients with heart failure [18]. Clinically, one manifestation of increased cAMP is an increase in heart rate, which has been observed consistently when patients are treated with GLP-1R agonists. For example, patients with Type 2 diabetes who were treated with liraglutide (control group sitagliptin) for 1 year, showed a two- to three-beat increase in heart rate [19]. A recent meta-analysis demonstrated that GLP-1R agonism increased heart rate by 1.86 beats per min compared with placebo [20]. Furthermore, the recent finding that myocytes within the sinoatrial node also express the GLP-1R suggests that there may be a direct effect on heart rate, over and above autonomic system modulation [21] although the significance of this finding remains unclear [22]. Importantly, heart rate has been shown to be an independent risk factor for adverse cardiac outcomes in patients with coronary artery disease and left-ventricular systolic dysfunction [23]; therefore, whether the small increase in heart rate observed will manifest as clinically important will be determined in a number of large-scale clinical trials designed to assess cardiovascular outcomes with GLP-1R agonists. In contrast to GLP-1R agonists, DPP4 inhibitors are neutral with respect to a chronotropic effect [24].

In addition to GLP-1R agonists increasing the heart rate, these agents also affect blood pressure (BP), which is important because hypertension remains a critical risk factor for the determination of cardiovascular disease [25,26]. Studies in rodents and humans have demonstrated that acute infusions of GLP-1R agonists increase BP [27–29]. However, chronic GLP-1R agonism appears to reduce systolic BP in hypertensive subjects with diabetes and obesity [30,31]. For example, the use of liraglutide in an obese, nondiabetic population saw a 4.6 mmHg reduction in systolic BP at the end of a 2-year treatment period [32], which was consistent with results from the LEAD-1 and LEAD-2 trials [33–35]. A decrease in systolic BP has also been shown in two other clinical trials with liraglutide [30,31] and a similar trend has been observed with exenatide [36]. In contrast, DPP4 inhibitors have demonstrated a neutral effect or small reduction in BP [24,37]. The mechanism behind the observed reduction in BP with chronic GLP-1R agonism remains multifactorial. However, a recent paper using a mouse model suggests that chronic GLP-1R activation in the atria promotes atrial natriuretic peptide secretion and a natriuretic effect resulting in a reduction in blood pressure [38]. Whether this mechanism is responsible for the observed effects in humans is unclear.

In early phase clinical studies, the GLP-1R agonist exenatide has shown beneficial effects on cardiac function and ischemia-reperfusion injury. In a cohort of patients with ST-segment elevation MI, the administration of exenatide 15 min prior to percutaneous coronary angioplasty was associated with a reduction in infarct size and larger myocardial salvage [39].

Meta-analyses of clinical trials have provided insight into the effects of GLP-1R agonists and DPP4 inhibitors on cardiovascular outcomes. In one meta-analysis of DPP4 inhibitors, Frederich and colleagues assessed the relative risk for cardiovascular events in eight randomized Phase II/III trials of saxagliptin treatment in patients with Type 2 diabetes [40]. The analysis included 3356 patients treated with saxagliptin versus 1251 patients treated with a comparator (i.e., placebo, metformin or uptitrated glyburide). Their results showed no increase in risk of cardiovascular death, MI or stroke, supporting a potential reduction in cardiovascular events with saxagliptin [40].

Another meta-analysis assessed whether DPP4 inhibitors reduced the incidence of major adverse cardiovascular events (MACE). Data were analyzed from 70 clinical trials involving 41,959 patients, and the results showed a reduction in cardiovascular events, particularly MI, and all-cause mortality in patients with Type 2 diabetes treated with a DPP4 inhibitor [41]. It is important to point out that both of these meta-analyses were limited by underreporting of information on cardiovascular events and by trial quality; therefore, the results must be interpreted cautiously [3].

Three meta-analyses of GLP-1R agonists have also been published [3,42–44]. One of the analyses determined the rates of MACE in 15 clinical trials that included 6638 patients, 4257 of whom were treated with liraglutide. There was no evidence that liraglutide increased the risk of cardiovascular events [3,42]. Two meta-analyses that examined cardiovascular outcomes in patients treated with exenatide showed that exenatide did not demonstrate any detrimental effects on cardiovascular outcomes [43,44]. However, similar to the DPP4 inhibitor studies, there are limitations to these meta-analyses. Clinical trials looking specifically at cardiovascular outcomes are underway, with two studies having results: SAVOR-TIMI 53 and EXAMINE [45,46].

In the SAVOR-TIMI 53 trial, 16,492 patients with Type 2 diabetes who had a history of, or were at risk for, cardiovascular events were randomly assigned to either the DPP4 inhibitor saxagliptin or placebo. After a median of 2.1 years, saxagliptin did not increase or decrease the rate of ischemic events compared with placebo [45]. In the EXAMINE trial, 5380 patients with Type 2 diabetes who had experienced either an acute MI or unstable angina, for which they were hospitalized in the previous 15–90 days, were randomly assigned the DPP4 inhibitor alogliptin or placebo (existing antihyperglycemic and cardiovascular drugs were continued). After a median of 18 months, the rates of MACE were not increased in patients taking alogliptin compared with those who received placebo [46].

While there was no increase in MACE in either EXAMINE or SAVOR-TIMI 53, there was an increase in the risk of hospitalization for heart failure in the SAVOR-TIMI 53 trial, a predefined, adjudicated end point (HR: 1.27 [1.07–1.51], p = 0.007) [45]. This finding was unexpected. In a post-hoc analysis of the SAVOR-TIMI 53 trial, the risk for heart failure hospitalization was highest in patients with a prior history of heart failure, chronic kidney disease and elevated N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) [47]. In comparison, a post-hoc analysis of the EXAMINE trial demonstrated that hospitalization for heart failure occurred in 3.1% of patients on alogliptin versus 2.9% on placebo (HR = 1.07, 95% CI: 0.79–1.46). Furthermore, hospitalization for heart failure was not increased in patients with a prior history of heart failure or elevated NT-proBNP [48]. Whether the increase in hospitalization due to heart failure leads to cardiovascular mortality is not clear from the preceding trials. In a recent population-based retrospective cohort study of 7620 patients with diabetes and pre-existing heart failure, patients treated with sitagliptin showed an increased risk of heart failure hospitalization (adjusted OR: 1.84; 95% CI: 1.16–2.92). However, sitagliptin use in this cohort was not associated with an increased risk of all-cause hospitalization or death [49]. Given the mixed signals in these clinical trials concerning hospitalization due to heart failure, a reasonable approach is to apply caution in prescribing DPP4 inhibitors to patients with prior heart failure, chronic kidney disease, and/or elevated NT-proBNP until further outcome studies involving DPP4 inhibitors with adjudicated heart failure end points are reported.

Ongoing, randomized trials with DPP4 inhibitors include two noninferiority trials: TECOS is investigating the time to first cardiovascular event with sitagliptin versus placebo and CAROLINA is comparing linagliptin with glimepiride in patients with Type 2 diabetes who are at elevated cardiovascular risk. The outcomes and reporting dates of these trials, and other DPP4 inhibitor Phase III and IV trials, are shown in Table 2. Ongoing, randomized clinical trials of GLP-1R agonists are shown in Table 3.

Table 2. Phase III and IV clinical trials with dipeptidyl peptidase-4 inhibitors looking at cardiovascular outcomes.

Trial	Drug	Design	Outcomes	Sample size	Date reporting	Ref.
Placebo control
TECOS NCT00790205	Sitagliptin	Phase III	Primary: time to first CV event	14,000	2014–15	[90]
CARMELINA NCT01897532	Linagliptin	Phase IV	Primary: time to first occurrence of CV death, nonfatal MI, nonfatal stroke, hospitalization for unstable angina pectoris	8300	2018	[91]
SAXATH NCT01552018	Saxagliptin	Phase IV	Primary: inflammatory biomarkers associated with atherosclerosis	12	2014	[92]
SAFEGUARD NCT01744236	Sitagliptin liraglutide exenatide	Phase IV	Primary: changes in resting HR variability, GFR, and pancreatic exocrine function; Secondary: effects on other CV parameters	70	2015	[93]
Active control
CAROLINA NCT01243424	Linagliptin vs glimepiride	Phase III	Primary: time to first CV death, nonfatal MI, nonfatal stroke, hospitalization for unstable angina pectoris	6000	2018	[94]
VAAST NCT01604213	Vildagliptin/metformin vs metformin alone	Phase IV	Primary: decrease in serum levels of IL-6; Secondary: improvement in other markers of atherosclerosis and inflammation	60	2015	[95]

CAD: Coronary artery disease; CV: Cardiovascular; GFR: Glomerular filtration rate; HR: Heart rate; IL: Interleukin; LV: Left ventricular; MI: Myocardial infarction.

Table 3. Phase III and IV placebo-controlled clinical trials with Glucagon-like peptide-1 receptor agonists looking at cardiovascular outcomes.

Trial	Drug	Design	Outcomes	Sample size	Date reporting	Ref.
EXSCEL NCT01144338	Exenatide	Phase IV	Primary: time to first occurrence of primary CV event: CV death, nonfatal MI, nonfatal stroke	14,000	2018	[96]
LEADER NCT01179048	Liraglutide	Phase III	Primary: time to first occurrence of primary CV event: CV death, nonfatal MI, nonfatal stroke	9340	2015	[97]
ELIXA NCT01147250	Lixisenatide	Phase III	Primary: time to first occurrence of primary CV event: CV death, nonfatal MI, nonfatal stroke, hospitalization for unstable angina	6000	2015	[98]
REWIND NCT01394952	Dulaglutide	Phase III	Primary: time to first occurrence of primary CV event: CV death, nonfatal MI, nonfatal stroke	9622	2019	[99]
FREEDOM CVO NCT01455896	ITCA 650	Phase III	Primary: time to first occurrence of primary CV event: CV death, nonfatal MI, nonfatal stroke, hospitalization for unstable angina	2000	2018	[100]
SAFEGUARD NCT01744236	Sitagliptin, liraglutide, exenatide	Phase IV	Primary: changes in resting HR variability, GFR, and pancreatic exocrine function; Secondary: effects on other CV parameters	70	2015	[93]
AddHope2 NCT01595789	Liraglutide/metformin	Phase IV	Primary: beta-cell function and LVEF changes	40	2015	[101]

CV: Cardiovascular; FDA: Food and Drug Administration; GFR: Glomerular filtration rate; LVEF: Left ventricular ejection fraction; MACE: Major adverse cardiac event; MI: Myocardial infarction.

Despite the positive preclinical and early clinical data on DPP4 inhibitors and GLP-1R agonists, the results of these large-scale clinical trials will determine their efficacy in preventing cardiovascular events in patients with Type 2 diabetes [3].

Cognitive function

Role of GLP-1 in central nervous system function

The GLP-1R has been shown to be localized to the cerebral cortex, hypothalamus, hippocampus, thalamus and brainstem [50–52]. In rat brains, the GLP-1R has been found in a number of areas, including the temporal cortex, caudal hippocampus and amygdala, areas that are associated with cognitive function [2,53].

Peripheral GLP-1 is capable of crossing the blood-brain barrier (BBB), as are the GLP-1R agonists exenatide, liraglutide, and lixisenatide [2,54,55]. However, GLP-1 is also produced in the CNS and functions as a neurotransmitter [5]. Studies have shown that a stable GLP-1 analog, [Ser8]-GLP-1, was able to cross the BBB by simple diffusion [55]. GLP-1 and its analogs have also been shown to enter the brain through organs that cross the BBB, such as the subfornical organ and the area postrema [2]. One hypothesized mechanism for GLP-1 to enter the brain is by transmitting information through the nucleus of the solitary tract via the vagal nerve and cranial nerves VII, IX and X, which alters neurons with GLP-1Rs [2,53]. The ability of GLP-1 and its analogs to cross the BBB, and the presence of the GLP-1R in different brain structures, provides evidence for a therapeutic role for incretin agents in the treatment of cognitive deficits.

Effects of GLP-1 & GLP-1R agonists on brain structure & function in preclinical investigations

Incretins have multiple effects on brain structure and function, including antiapoptotic and neuroprotective effects, as well as acting as potential cognitive enhancers. Using cultured neuronal cells, pretreatment with GLP-1 and exendin-4, a hormone that has biological properties similar to GLP-1, protected the cells from apoptosis caused by amyloid-beta peptide levels, which are a biomarker for Alzheimer’s disease, and reduced levels of amyloid precursor protein [56]. Treatment with GLP-1 also protected cells from methylglyoxal-induced apoptosis, which accumulates in the presence of excess glucose and is linked to the pathogenesis of cognitive disorders, including Alzheimer’s disease [57]. In addition, GLP-1 has been shown in vitro to prevent apoptosis caused by nerve growth factor deprivation [58].

In animal models, GLP-1 has been shown to reduce levels of amyloid-beta peptides and protect against oxidative stress [56,59]. There is also evidence in vivo suggesting that intracerebroventricular (icv) administration of GLP-1 can reduce levels of beta-amyloid precursor protein [56] and subcutaneous administration of exendin-4 reduces amyloid-beta and beta-amyloid precursor protein levels in a mouse model of Alzheimer’s disease [60].

Other effects of GLP-1 and exendin-4 include a role in enhancing proliferation and differentiation of neural stem/progenitor cells, which are significant in adult neurogenesis [2]. In addition, multiple studies with GLP-1R agonists have shown their potential for protecting long-term potentiation (LTP), a process that contributes to synaptic plasticity [2]. Liraglutide, when injected into the peritoneum of mice, prevented synapse loss and deterioration of synaptic plasticity in the hippocampus [61]. Following chronic icv and subcutaneous injection, liraglutide was shown to rescue hippocampal LTP from high-fat-diet-induced deficits [62]. Similar results were seen in mice that were fed a high-fat diet when they were treated with exendin-4 [63]. In healthy rats, liraglutide administration rapidly facilitated LTP [64], whereas mice with a deletion of the GLP-1R showed significant impairment of LTP [65].

In addition to impairment of LTP, GLP-1R knockout mice also showed impaired spatial learning and memory, and object recognition when compared with controls. However, GLP-1R knockout mice did not show any difference in exploratory behavior on an open-field assessment task. These results indicate that impairments are specific to cognitive processes implicated in memory, not to genetics [65].

In contrast, the administration of incretin analogs produced cognitive improvement in animal models (Table 4). In one animal study, mice on a high-fat diet were injected with liraglutide for 28 days. During object recognition testing, treated mice showed an increase in recognition index, which indicates enhanced learning and memory abilities [62]. In a similar study, mice fed on a high-fat diet and co-treated with exendin-4 also showed a marked increase in recognition index [63]. In a mouse model of Alzheimer’s disease, 8 weeks of liraglutide injections prevented memory impairments in object recognition and water maze tasks [61].

Table 4. Incretins as potential cognitive enhancers in animal models.

Agent	Model	Results	Ref.
GLP-1	Mouse; icv	Enhanced associative and spatial learning in Morris water maze task and passive avoidance task	[102]
	Rat: icv	Improved impairment of spontaneous alternation performance in Y-maze paradigm	[103]
	Mouse; GLP-1R-/-	GLP-1R -/- showed impaired learning in water maze task	[65]
Val^8-GLP-1	Rat; icv	Reduced impairment of spatial learning and memory observed by increased swim speed and decreased latencies on platform tests	[104]
Exendin-4	Rat; ip	Significantly reduced time to solve radial maze task and duration of immobility in forced swim test	[105]
	Rat; icv	Time spent in open arms of elevated plus maze was increased	[106]
	Mouse; sc	Marked increase in recognition index (improved learning and memory)	[63]
Liraglutide	Mouse; ip	60% increase in recognition index in object recognition tasks	[62]
		Prevented memory impairment in object recognition and water maze tasks	[61]

GLP-1: Glucagon-like peptide-1; GLP-1R: Glucagon-like peptide-1 receptor; icv: Intracerebroventricular; ip: Intraperitoneal; sc: Subcutaneous; Val: Valine.

Reproduced with permission from McIntyre RS, et al. [2] © Elsevier (2013).

GLP-1R agonists in clinical trials

At present, there are a number of clinical trials that include an investigation into the role of GLP-1R agonists on cognitive function (Table 5). One Phase III clinical trial [66] is comparing exenatide with placebo to determine the role of exenatide in weight loss in antipsychotic-associated obesity. As a secondary outcome, the study will look at cerebral end points, including the potential neuroprotective effects of exenatide.

Table 5. Glucagon-like peptide-1 receptor agonists in placebo-controlled clinical trials with cognitive outcomes.

Trial	Drug	Design	Outcomes	Sample size	Date reporting	Ref.
TAO NCT01794429	Exenatide	Phase III Antipsychotic-associated obesity	Primary: weight loss; Secondary: include potential neuroprotective effects	50	2014	[66]
NCT00845507	Exenatide	Phase IV Antipsychotic-associated obesity	Primary: change in weight from baseline; Secondary: include clinical global improvement of psychiatric symptoms, change in manic, depressive, and psychotic symptoms	60	2014	[67]
NCT01469351	Liraglutide	Phase N/A Alzheimer’s disease	Primary: intra-cerebral amyloid deposits in the CNS; Secondary: include neuropsychological tests	34	2013 completed	[107]
NCT01255163	Exenatide	Phase II Alzheimer’s disease	Primary: safety and tolerability; Secondary: include behavioral and cognitive performance measures, changes on structural and functional MRI and MRS, clinical dementia rating, Alzheimer’s disease assessment	100	2016	[108]
NCT01174810	Exenatide	Phase II Parkinson’s disease	Primary: UPDRS-off-medication motor subscore changes; Secondary: adverse event profile including MADRS score	40	2013 (status unknown)	[109]
NCT01550653	Liraglutide	Phase I Healthy patients	Primary: effects on memory in healthy patients	40	2014	[110]

CNS: Central nervous system; MADRS: Montgomery and Asberg Depression Rating Scale; MRI: Magnetic resonance imaging; MRS: Magnetic resonance spectroscopy; N/A: Not available; UPDRS: Unified Parkinson’s Disease Rating Scale.

The efficacy of exenatide in the treatment of weight gain associated with the use of olanzapine is being investigated in a Phase IV trial [67] in obese patients with bipolar disorder, major depressive disorder, schizophrenia or schizoaffective disorder. Secondary outcomes of this trial include measurement of clinical global improvement in psychiatric symptoms, and improvement in manic, depressive, or psychotic symptoms [2].

Research from preclinical in vitro cell culture studies and in vivo animal models of cognition have shown that GLP-1 and GLP-1R agonist therapy provide neuroprotective benefits, including antiapoptotic effects, protection from oxidative injury, and reduction in amyloid-beta levels. Mice treated with GLP-1 or GLP-1R agonists (e.g., exendin-4 or liraglutide) demonstrated enhanced memory, spatial learning and motor activity [2]. Clinical trials of patients with Parkinson’s or Alzheimer’s disease have started and GLP-1R agonists show promise in treating a range of neurodegenerative diseases [68]. Ongoing and upcoming clinical trials of GLP-1R agonists – measuring mood, cognitive and behavioral changes – will provide additional information about their role in the treatment of mood disorders.

Bone health

Role of GIP, GLP-1, & GLP-2 in bone remodeling

Bone remodeling is the continuous process throughout adult life of removing old, damaged bone and replacing it with new bone in order to maintain strength (i.e., both bone quantity and quality). Osteoclasts are the cells responsible for bone resorption and osteoblasts are the cells that lay down new bone at the site of previous resorption. In healthy individuals, these two processes are balanced in order to maintain bone homeostasis. Both osteoblasts and osteoclasts express receptors for GIP and the GLPs [69].

Activation of the GIP receptor (GIPR) has been shown to increase the number and activity of osteoblasts [5,70]. In a human osteoblastic cell line (Saos-2), GIP has been shown to induce a cyclic increase in the intracellular concentration of cAMP in osteoblasts, which induces bone formation. In addition, mice lacking the GIPR gene had lower bone formation parameters and a greater number of osteoclasts than wild-type mice, indicating the knockout mice had high-turnover osteoporosis [71]. In vitro, GIP has been shown to inhibit the resorptive activity of osteoclasts (e.g., bone resorption in organ culture) [72] and to act as an anabolic hormone in a mouse model that overexpresses GIP [73].

GLP-1R also plays a role in controlling bone resorption. In a mouse model, GLP-1R-knockout mice had increased numbers of osteoclasts and bone resorption compared with wild-type mice [74]. The GLP-1R knockout mice also had more fragile bones and developed cortical osteopenia. GLP-1 itself had no direct effect on either osteoclasts or osteoblasts. Therefore, GLP-1R signaling may play an essential role in controlling bone resorption indirectly, possibly via a calcitonin-dependent pathway [69,74].

A second glucagon-like peptide, GLP-2, has also been shown to act as an antiresorptive hormone. GLP-2 was first recognized as a growth factor in 1996. Intestinal GLP-2 is produced in L cells, which are abundant in the distal jejunum, ileum and colon [75]. GLP-2 has numerous biological effects, including stimulation of intestinal mucosal growth and blood flow, upregulation of nutrient absorption, inhibition of gastric emptying and acid secretion and reduction of intestinal permeability and nutrient-dependent bone resorption. GLP-2 acts through the GLP-2 receptor (GLP-2R), which has been shown to be located in the proximal small intestine, brain and lung tissue, and osteoclasts [76].

Secretion of GLP-2 is biphasic in response to food. The first rise is a rapid increase in GLP-2 following food intake that is thought to be caused by early, indirect L-cell stimulation via neurohumoral signaling pathways [77]. The second rise is through direct L-cell stimulation due to nutrients reaching the distal intestine. Of interest, postprandial GLP-2 secretion depends on both caloric load and nutrient composition, where carbohydrates and fats, but not proteins, have been established as potent GLP-2 secretagogues [78].

Bone remodeling occurs according to a circadian rhythm, with bone resorption occurring primarily at night. Healthy, postmenopausal women treated for 14 days with GLP-2 at bedtime showed a decrease in the nocturnal rise in bone resorption without suppressing bone formation [79]. In a randomized, placebo-controlled clinical trial of postmenopausal women with low bone mineral density (BMD), 4 months of treatment with GLP-2 caused a significant increase in hip BMD [80].

Taken together, the roles of GIP, GLP-1, and GLP-2 have a positive impact on bone mass and quality. Therefore, incretin therapies that increase levels or bioactivity of GIP and the GLPs may positively impact the quantity and quality of human bone [69].

Effects of GLP-1R agonists & DPP4 inhibitors on bone in preclinical investigations

GLP-1R agonists have shown a positive effect on BMD in animal models [81]. In rat models of Type 2 diabetes or insulin resistance, exenatide promoted bone formation and increased BMD through the Wnt (wingless-type) signaling pathway [82].

Similar to GLP-1R agonists, DPP4 inhibitors have shown a positive effect on BMD in animal studies [81]. In mice fed a high-fat diet, treatment with sitagliptin significantly improved vertebral volumetric BMD and trabecular architecture [83].

Clinical trials of GLP-1R agonists & DPP4 inhibitors with bone outcomes

In patients with Type 2 diabetes, 44 weeks of exenatide treatment caused a 6% decrease in body weight but did not affect BMD or have an effect on levels of bone turnover markers in the serum [84]. To date, GLP-1R agonists have shown no skeletal effects in clinical trials [69].

Unlike GLP-1R agonists, DPP4 inhibitors have been associated with a reduced risk of fractures in clinical trials [69]. A meta-analysis was done of all randomized clinical trials lasting at least 24 weeks where patients with Type 2 diabetes were treated with DPP4 inhibitors compared with placebo or active drugs. In 28 trials that enrolled 11,800 patients on DPP4 inhibitors and 9175 patients on comparators, DPP4 inhibitors were found to be associated with a reduced risk of bone fractures [85]. There were a number of limitations to this meta-analysis: the trials that were included were of short duration; fractures were captured as an adverse event and were not a primary outcome in any of the trials; only fractures that were considered as serious AEs were included in the analysis; the number of events was too small to analyze the fracture site; and there was no information on sex or menopausal status.

The potential mechanism of action for reduced bone fractures could be the increase in circulating levels of GLP-1 and GIP, both involved in the regulation of bone metabolism, due to the presence of a DPP4 inhibitor [85]. However, another plausible mechanism of action revolves around DPP4 inhibitors extending the half-life of GLP-2 [76], which essentially increases the circulating levels of GLP-2. Results from a randomized, placebo-controlled clinical trial of postmenopausal women treated with GLP-2 at bedtime showed a dose-dependent increase in BMD, particularly at sites enriched in cortical bone, such as the hip, and a decrease in bone resorption but no decrease in biomarkers associated with bone formation [80]. Nocturnal GLP-2 treatment shifted the bone turnover balance in favor of bone formation. These observations suggest that GLP-2 could be a potential treatment for osteoporosis. However, larger clinical trials with primary fracture outcomes will shed more light on the role of incretin therapies in bone.

One clinical trial of a DPP4 inhibitor with cardiovascular end points, SAVOR-TIMI 53, also looked at fracture outcomes. As discussed in the cardiovascular section of this paper, patients in the SAVOR-TIMI 53 trial were given either saxagliptin or a placebo. The number of patients with bone fractures was similar between the two groups (n = 241 [2.9%] vs. n = 240 [2.9%], for saxagliptin and placebo, respectively) [45]. Clinical trials with bone outcomes that are ongoing or recruiting are outlined in Table 6 for DPP4 inhibitors and Table 7 for GLP-1R agonists.

Table 6. Clinical trials with Dipeptidyl peptidase-4 inhibitors and bone outcomes.

Trial	Drug	Design	Outcomes	Sample size	Date reporting	Ref.
Placebo control
DRTC NCT01374568	Sitagliptin	Phase IV postmenopausal women with T2DM	Primary: bone turnover measured by OC, urine NTX, BALP	40	2014	[111]
Active control
BoneGlyc NCT01679899	Vildagliptin vs gliclazide MR	Phase IV menopausal women with T2DM	Primary: markers of bone remodeling measured by blood levels of OC, BALP, CTX, NTX	38	2015	[112]

BALP: Bone-specific alkaline phosphatase; CTX: Carboxy-terminal telopeptide of type I collagen; MR: Modified release; NTX: Amino-terminal telopeptide of type I collagen; OC: Osteocalcin; T2DM: Type 2 diabetes mellitus.

Table 7. Placebo-controlled clinical trials with Glucagon-like peptide-1 receptor agonists and bone outcomes.

Trial	Drug	Design	Outcomes	Sample size	Date reporting	Ref.
NCT01381926	Exenatide	Phase IV postmenopausal women with Type 2 diabetes mellitus	Primary: changes in bone turnover and bone resorption markers	30	2014	[113]

While results from the SAVOR-TIMI 53 trial did not show any benefit in terms of bone fracture, the results of upcoming clinical trials will help determine if incretin-based therapies may be beneficial for bone health.

Summary

Incretin therapies, originally developed to lower blood glucose levels in patients with Type 2 diabetes, have shown effects independent of glycemic control. Preclinical data from studies of GLP-1R agonists have shown beneficial effects on memory, motor activity and spatial learning, demonstrating significant neuroprotective effects, as well as an increase in bone formation and increased BMD. Preclinical and early clinical data from investigations using DPP4 inhibitors suggest these agents may have cardioprotective effects and may be associated with a reduced risk of bone fractures and increased BMD. While these preclinical and early clinical data look promising, large-scale randomized clinical trials with end points specific to cardiovascular, cognitive and bone outcomes will provide important data on the potential of incretin therapies in preventing cardiovascular events, and in treating mood and bone disorders.

Expert commentary

Despite a wealth of data from preclinical and early phase studies, and now, large-scale Phase III clinical trials, many questions remain unanswered as they pertain to incretin therapy for diabetes. For example, it is unclear as to whether DPP4 inhibitors or GLP-1R agonists will provide superior or equivalent outcomes compared with other antihyperglycemic agents. Similarly, it is unclear as to how metabolites of GLP-1 may influence diabetes-related complications. While many of these questions will be addressed over the next 5 years with the reporting of large clinical trials, further extensive preclinical and targeted clinical research is necessary to improve our understanding of incretin biology.

Many of the diabetes-related complications with greatest morbidity, such as heart failure with preserved ejection fraction, remain poorly studied in terms of the role incretin therapy plays in either disease progression or disease protection. Furthermore, the effects of ‘off-target’ substrates for DPP4, while being potentially beneficial in one area (e.g., SDF-1α in heart failure), may result in negative effects in other areas (e.g., endothelial function) [86].

Further studies are also needed to determine the role of incretin therapy in CNS function and in bone health. Peripheral GLP-1 and GLP-1R agonists are able to cross the BBB, suggesting a therapeutic role in the treatment of cognitive deficits [2]. In animal models, GLP-1 has reduced levels of beta-amyloid peptides, protected against oxidative stress, and enhanced cognitive function, including memory impairments [2,56,59]. At present, human Phase III and IV clinical trials are looking at the role of GLP-1R agonists in cognitive function as well as improvement in psychiatric symptoms. GLP-1 is a well-characterized and validated target for drug discovery in applications for the treatment of domain-specific outcomes in brain disorders, as defined by the National Institutes of Health research domain criteria.

The GLP-1R has also been shown to have effects on bone: GLP-1R knockout mice had increased numbers of osteoclasts and bone resorption leading to fragile bones and osteopenia [74]. In clinical trials, to date, GLP-1R agonists have shown no skeletal effects [69]. In contrast, clinical trials and meta-analyses of DPP4 inhibitors have suggested a decrease in fracture rate, potentially by increasing the circulating half-life of GLP-2 [69,85]. However, most recently, the SAVOR-TIMI 53 study did not show a difference in fracture rate between saxagliptin and placebo [45]. The results of ongoing, large-scale, randomized clinical trials will shed more light on the role of the nonglycemic effects of incretin therapies in cardiovascular disease, bone and cognitive function and their potential to impact disease management.

Five-year view

Incretin therapy represents a significant advance in the treatment of diabetes and its complications. However, many questions remain unanswered about the nonglycemic effects of these therapies on cardiovascular disease, cognitive function, and bone health. For example, the DPP4 inhibitors used in the SAVOR-TIMI 53 and EXAMINE trials did not demonstrate any increase in the incidence of MACE within the short time frame studied [45,46]. However, the effect of these interventions on long-term outcomes remains unclear. Similarly, studies have shown the benefit of incretin therapy in prevention or amelioration of disorders associated with brain amyloid precursor protein [2]. While the role of incretin agents in brain function is a new field, these agents have the potential to change the course, and possibly the frequency, of degenerative neurological disorders such as Alzheimer’s disease. However, large, well-designed clinical trials are needed to provide conclusive evidence. Finally, if incretin therapy, particularly GLP-2, can be shown to protect bone and improve fragility fractures in long-term studies, then there may be benefits for two chronic and costly diseases (i.e., improvement in glycemic control and a decrease in diabetes mellitus-related complications, including fractures that are now regarded as a complication of diabetes mellitus).

The next 5 years represent an exciting time with the reporting of multiple major clinical trials likely to determine the effects of incretin therapy independent of glycemic control, including their role in preventing macrovascular and microvascular complications. In addition, ongoing preclinical studies should clarify the role of GLP-1 metabolites and may also lead to the development of novel therapeutic strategies.

Key issues

• The incretins, glucose-dependent insulinotropic peptide and glucagon-like peptide-1 (GLP-1), are gastrointestinal hormones that play a major role in glucose homeostasis. GLP-1 has both receptor-dependent and receptor-independent effects.

• Incretin therapies, glucagon-like peptide-1 receptor (GLP-1R) agonists and dipeptidyl-peptidase-4 (DPP4) inhibitors, were developed to lower blood glucose levels in patients with Type 2 diabetes but have also been shown to have nonglycemic effects on cardiac events, brain function and bone health.

• To date, large clinical trials with DPP4 inhibitors have demonstrated no adverse cardiovascular effects. Preclinical and early clinical data suggest DPP4 inhibitors may be associated with cardioprotective effects.

• In animal models, GLP-1 has reduced levels of beta-amyloid peptides, protected against oxidative stress, and enhanced cognitive function, including memory impairments. Clinical trials involving patients with Parkinson’s or Alzheimer’s disease have started, and GLP-1R agonists show promise in treating a range of neurodegenerative diseases.

• In animal models, both GLP-1R agonists and DPP4 inhibitors have shown a positive effect on bone mineral density. While GLP-1R agonists have shown no skeletal effects in clinical trials to date, DPP4 inhibitors may be associated with reduced risk of bone fracture and increased bone mineral density. The potential mechanism of action for reduced bone fractures could be the increase in circulating levels of GLP-1 and glucose-dependent insulinotropic peptide due to the presence of a DPP4 inhibitor or the extension of the half-life of GLP-2.

• Large-scale, randomized clinical trials with specific cardiovascular, cognitive and bone outcomes will provide important data on the potential role of incretin therapies, independent of glycemic control.

Financial & competing interests disclosure

This work was supported by an unrestricted grant from the Boehringer Ingelheim/Eli Lilly alliance. Writing assistance was received from Cathie Bellingham of New Evidence. A Hanna has received speaking honoraria from Merck, AstraZeneca, Boehringer Ingelheim, Eli Lilly, and Novo Nordisk, manufacturers of DPP4 inhibitors and GLP-1R agonists. KA Connelly is funded by a CIHR New Investigator Award and has received research support and honoraria from Merck, AstraZeneca, Bristol-Myers Squibb, and Boehringer Ingelheim, all of which manufacture DPP4 inhibitors. RG Josse has received speaking honoraria from Merck, AstraZeneca, Eli Lilly, and Novo Nordisk, manufacturers of DPP4 inhibitors and GLP-1R agonists. RS McIntyre has received research funding and speaking honoraria from AstraZeneca, Bristol-Myers Squibb, France Foundation, GlaxoSmithKline, Janssen-Ortho, Eli Lilly, Organon, Lundbeck, Pfizer, Shire, Merck, I3CME, Physicians’ Postgraduate Press, CME Outfitters, and Optum Health. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.