Abstract

Background

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance resulting in glucose intolerance and hyperglycaemia [1].

Since insulin effectiveness is reduced in diabetes, research into other signalling pathways that support insulin actions or that reduce blood glucose is ongoing. One of these strategies focus on the use of the incretins, a class of peptide hormones that helps to normalise insulin signaling and also improves blood sugar levels. Incretins increase the release of insulin during high blood sugar levels, the so-called 'incretin effect'. Drugs that mimic incretin hormones can maintain glucose homeostasis and improve multiple symptoms of type 2 diabetes like the risk of hypoglycaemia, inadequate post-prandial blood glucose control, glucose fluctuations, β-cell failure, and weight gain [1,2].

GLP-1 is an endogenous 30-amino acid peptide hormone. Numerous novel long-lasting GLP-1 receptor agonists have been developed by several companies. Exendin-4 (Byetta) has been on the market as a T2DM treatment for several years [3]. Liraglutide (Victoza) also has been released onto the market several years ago [4]. A third drug is lixisenatide (Lyxumia), which will be released onto the market soon [5].

T2DM has been identified as a risk factor for AD, indicating that insulin signaling failure may be a factor in initiating or accelerating the development of AD. Epidemiological studies found a clear correlation between T2DM and the risk of developing AD [6-8]. It was also shown that insulin receptors in the brain are desensitised in AD patients [9,10]. Therefore, a promising strategy to treat AD is the use of such GLP-1 analogues [11]. GLP-1 receptors are found on neurons in the brains of rodents and humans [12,13]. The GLP-1 receptor agonists exendin-4, liraglutide, lixisenatide, and (Val8)GLP-1 have neuroprotective properties. The protease resistant and long-lasting GLP-1 analogue Val(8)GLP-1 enhanced synaptic plasticity in acute and chronic application and preserved synaptic functionality in the brains of a mouse model of AD [14,15]. The novel GLP-1 analogue liraglutide lowered plaque formation, protected memory and synaptic plasticity, and reduced inflammation in the brains of a mouse model of Alzheimer disease [16]. All of these effects in the brain were observed after peripheral injection. Therefore, it is likely that these peptides have to be able to cross the blood brain barrier (BBB).

The focus of this study was to measure the kinetics of incretin drugs of crossing the blood brain barrier, activation of incretin receptors by measuring cAMP levels, and physiological effects in the brain on cell proliferation and neurogenesis.

Results

Experiment 1

There were no significant levels of Liraglutide found 5 min post i.p. injection, in the brains of mice administered with a 2.5 nmol/kg bw, 25 nmol/kg bw or 250 nmol/kg bw dose of the peptide (see Figure ​Figure1a).1a). However, significant levels of the GLP-1R agonist were found at both 30 min and 3 h post injection with the 250 nmol/kg dose (p < 0.01 and p < 0.05 respectively). The medium dose of 25 nmol/kg bw was also sufficient to raise peptide levels in the brain 30 min post injection (p < 0.05) but not after 3 h (Figure ​(Figure1b1b and ​and1c).1c). To determine whether Liraglutide activates the GLP-1R in the brain we measured total brain cAMP levels 30 min post i.p. injection with 25 nmol/kg bw of the peptide. cAMP is the second messenger of the GLP-1R. Significant levels of cAMP (p < 0.05) were detected 30 min post administration indicating GLP-1R activation (Figure ​(Figure1d1d).

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Figure 1

At 5 min post- ip. injection, there was no significant increase of liraglutide in the brains of mice (A). There was an increase in liraglutide levels in the brains of mice injected with 25 or 250 nmol/kg bw at 30 min (B) and only those injected with the highest dose at 3 h post-injection (C). Brain levels of cAMP were increased 30 min post injection with 25 nmol/kg bw ip.. * = p < 0.05. Values are the mean ± S.E.M.

Experiment 2

Lixisenatide was found in significant concentrations following administration with 2.5, 25 or 250 nmol/kg bw of the peptide at 30 min post injection (p < 0.01, p < 0.05 and p < 0.05) (Figure ​(Figure2a).2a). Similarly at 3 h post administration Lixisenatide was still present in a significant amount in the brain 3 h post injection in the mice given the 2 lower doses (p < 0.05), however, not with the highest dose due to huge variation within this particular group (Figure ​(Figure2b).2b). Interestingly, for this peptide there is as much detected in the brain when administered the lowest dose of 2.5 nmol/kg bw as with the highest dose of 250 nmol/kg bw. Furthermore, there is little or no decrease in the concentration of Lixisenatide from 30 min to 3 h post injection.

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Figure 2

(A) Lixisenatide levels in the brains at 30 min following i.p. (2.5, 25 or 250 nmol/kg body weight) injection were increased. (B) Lixisenatide levels in the brains at 3 h following i.p. injection were only increased for the lower doses (2.5, 25 nmol/kg body weight). (C) The number of neuronal progenitor cells in the dentate gyrus was increased after 3 weeks of ip injection 25 nmol/kg bw once-daily. (D) The number of young neurons in the dentate gyrus was also increased. (E) The level of cAMP was enhanced in the brains after injection with 25 nmol/kg bw ip. (F) When directly comparing the effects of liraglutide with lixisenatide, a significant difference between drugs is found.* = p < 0.05, ** = p < 0.01. Micrographs: BrdU stain: A Saline control B Lixisenatide treated, 3 weeks once daily i.p. injection. Blue arrows point to BrdU positively stained cells. DCX stain: Doublecortin stained immature neurons in dentate gyrus. A Saline control B Lixisenatide treated, 3 weeks once daily i.p. administration.

There was a 1.8-fold increase in cell proliferation as measured by BrdU analysis (p < 0.01, Figure ​Figure2c).2c). Correspondingly, there was a 1.7-fold increase (p < 0.05) in young neurons as determined by Double Cortin analysis (Figure ​(Figure2d).2d). The level of cAMP was inhanced in the brains after injection with 25 nmol/kg bw ip. (p < 0.01), see Figure ​Figure2e.2e. When directly comparing the effects of liraglutide with lixisenatide, a significant difference between drugs is found (p < 0.05, Figure ​Figure2f2f).

Discussion

We have investigated the GLP-1 receptor agonists Liraglutide and Lixisenatide. We have provided clear evidence that both peptides have the ability to cross the BBB, as we found significantly elevated levels of these peptides in the brain 30 min following i.p. injection. Liraglutide levels in the brain were higher with increasing doses injected ip. Also, there was a time-dependence as only the highest dose injected showed a significant increase in the brain after 3 h. The brain levels of cAMP were increased, demonstrating the activation of the GLP-1 receptor which is linked to an adenylate cyclase [1]. Previous studies already had demonstrated that liraglutide also enhances neuronal progenitor proliferation and increased neurogenesis in the brain of a mouse model of Alzheimer disease [16].

Lixisenatide also entered the brain in a sufficiently detectable amount, even at the lowest dose tested of 2.5 nmol/kg bw. 30 min post-injection. At 3 h post-injection, the highest dose did not show a significant increase in the brain. This observation suggests that the transport across the BBB is highly regulated, and that this transport system may shut down at doses that are abnormally high. Similar observations have been made previously when testing exendin-4 [17]. The brain levels of cAMP were increased, demonstrating the activation of the GLP-1 receptor. We were also able to demonstrate that lixisenatide, when injected once daily for 3 weeks, promotes neuronal progenitor proliferation in the dentate gyrus, and neurogenesis in the brain, demonstrating that this drug not only crosses the BBB, but also shows clear physiological effects in the brain. Interestingly, lixisenatide is transported across the BBB at a lower dose than liraglutide, and shows enhanced cAMP at equal dose compared with liraglutide.

Our results show that incretin analogues can be injected peripherally and are taken up into the brain where they show physiological activity. Previous studies have shown similar effects in the brain, in particular on cell proliferation and neurogenesis [14,18-21], and our results add considerably to those findings.

Exendin-4 is currently on the market as a T2DM treatment and is injected peripherally twice-daily. Liraglutide is also on the market and is injected peripherally once daily [1]. Lixisenatide is under development to be used as a once-daily treatment for T2DM [5]. Such drugs can be taken by non-diabetic people as they only affect blood sugar levels in hyperglycaemia [22].

Conclusion

If the improvements in memory and synaptic plasticity, reduction in neurotoxic amyloid oligomer levels, numbers of amyloid plaques and of the inflammation response, and increased neurogenesis observed in previous studies [11,23] translate to humans, GLP-1 analogues such as Liraglutide or GLP-1 receptor agonists such as exendin-4 and lixisenatide are promising new treatment strategies for Alzheimer's disease. Importantly, clinical trials of the effects of exendin-4 in patients with Parkinson's disease (UCL, London, UK) have been started, and clinical trials in patients with MCI or early-phase AD are on their way (NIH/NIA, USA) (see an update on http://www.clinicaltrials.gov). A third clinical trial that aims to test liraglutide in AD patients is currently being prepared.

Methods

Competing interests

Prof. Holscher is a named inventor on a patent application of liraglutide as a treatment of neurodegenerative disease, and on a second patent application of lixisenatide as a treatment of neurodegenerative disease.

Authors' contributions

KH conducted the experiments, CH wrote the paper and raised the research funds. All authors read and approved the final manuscript.

Authors' information

Dr. Kerry Hunter is a post-doc in the lab of Prof. Christian Holscher. Prof. Holscher is the group leader of the Neuroscience research group at the University of Ulster. He has a long track record in investigating the effects of incretin hormones and analogues on neurodegeneration.

References