Introduction
Diabetes treatment has substantially improved in recent years. Yet significant numbers of individuals with diabetes still do not meet their treatment goals or experience undesired side effects. In addition, none of the major diabetes drugs currently in use specifically addresses the inadequate muscular glucose uptake in individuals with insulin-resistant disease. As about 80% of blood glucose in the postprandial state is disposed in skeletal muscle in healthy individuals [
1], a lack of this function results in hyperglycaemia and increased risk of muscle fatigue. Individuals with diabetes are also at significantly higher risk of developing chronic complications, including microvascular nephropathy, neuropathy and retinopathy, and macrovascular diabetic foot, as well as cardiovascular disease (CVD) [
2,
3]. Novel treatment alternatives that adequately address several aspects of this complex disease are required.
HDL and its major protein constituent apolipoprotein A-I (ApoA-I) have important and well-established functions in the transport and metabolism of cholesterol and other lipids in the circulation, and are considered to prevent atherosclerosis and CVD [
4]. ApoA-I has also been implicated in the regulation of glucose control [
5,
6], suggesting that ApoA-I may be an important link between diabetes and CVD. Indeed, ApoA-I/HDL stimulates glucose uptake to murine and human cultured myotubes [
7‐
10]. This translates into mouse models of chronic [
10] and acute [
11] upregulation of human ApoA-I protein that leads to increased glucose-stimulated insulin secretion (GSIS) during GTT, as well as direct stimulation of muscle tissue [
12]. Moreover, radiolabelled glucose analogue distribution and positron emission tomography/computed tomography analyses have confirmed muscle as target for increased glucose uptake [
12,
13], and also identified the heart as a significant in vivo target [
12,
14].
Collectively, ApoA-I/HDL show great promise in novel approaches to treat cardiometabolic diseases. However, several clinical HDL formulations are based on a complex mixture of phospholipids and two ApoA-I proteins per macromolecule. Drugs based on shorter peptides are thus desired. Indeed, in addition to their cholesterol efflux capacity, administration of ApoA-I mimetic peptides to obese mice have been shown to increase insulin sensitivity and improve glucose tolerance [
15,
16]. We have previously identified the C-terminal region (54 amino acids; RG54) as the bioactive domain for induction of glucose uptake in vitro and ex vivo [
7] and as sufficient for HDL formation [
17]. Importantly, the RG54 peptide retains sequence homology with ApoA-I while also adopting a reconstituted (r)HDL-resembling structure in solution [
7], thus potentially retaining the many biological functions of the ApoA-I protein in glucose and lipid metabolism. A drug based on the RG54 peptide sequence would therefore hold promise for the treatment of individuals with diabetes and severe insulin resistance. To explore this, we here investigate the RG54 peptide in relevant in vitro and in vivo model systems for its function in insulin secretion, glucose uptake, cholesterol efflux and atherosclerotic plaque formation.
Discussion
The current study investigates the potential of the short RG54 peptide derived from the ApoA-I protein to be used as a treatment in diabetes and cardiometabolic disease via multiple functions.
The RG54 peptide efficiently catalyses cholesterol efflux from macrophages and reduces the formation of atherosclerotic plaques in a rodent atherosclerosis model. Notably, while treatment with the RG54 peptide significantly reduced both aortic plaque area and the number of plaques in atherosclerotic
Apoe−/− mice, the two comparators, i.e. liraglutide (a glucagon-like peptide 1 receptor agonist) and the ApoA-I protein, failed to prevent atherosclerotic plaque formation in the current study. This finding was unexpected, since earlier studies using experimental animal models have demonstrated that both have anti-atherosclerotic effects. The reason for this may be due to comparably less frequent injections in our study. For example, Gaspari et al described that
Apoe−/− mice treated twice daily with liraglutide had decreased plaque area and increased plaque stability compared with littermate control animals after 4 weeks of treatment [
21], indicating that the dosage of liraglutide in our study was below the cut-off level to achieve significant protection. Similarly, Shah et al convincingly showed that ApoA-I treatment prevented the progression of aortic atherosclerosis in
Apoe−/− mice treated more frequently and with higher doses of ApoA-I than in the current study [
22]. Importantly, since
Apoe−/− mice treated with the RG54 peptide clearly exhibited reduced levels of plaques despite the relatively few administrations per week, this indicates that additionally improved efficacy can likely be reached by further optimisation of the pharmacokinetics of the RG54 peptide.
Our data also conclude that the RG54 peptide acts on glucose control via dual mechanisms, i.e. by directly stimulating glucose uptake in cultured myotubes and by priming beta cells for improved secretion of insulin following glucose stimulation, which mimics the postprandial state. The in vitro studies are verified by treatments with the RG54 peptide in two rodent diabetes models (DIO and
db/db mice), which both show improved capabilities to clear blood glucose in the GTTs. Importantly, while the improved glucose tolerance in the DIO mice was accompanied by increased insulin secretion, the RG54 peptide treatment of the
db/db mice at 5.5 weeks of age led to a significantly improved capability to clear glucose in the GTT without increases in plasma insulin. This finding, which was also true for the animal groups treated in parallel with the ApoA-I and liraglutide comparators, validates the direct and insulin-independent effects of the RG54 peptide on glucose disposal in peripheral tissues. The data also show that s.c. administration is a viable route for the RG54 peptide. This is of relevance, since current diabetes drugs are limited to increasing secretion of endogenous insulin, reducing the reabsorption of glucose in the kidneys, preventing absorption of monosaccharides in the intestine, by lowering liver glucose production and increasing gut energy utilisation, or by directly replacing the endogenous insulin. Drugs based on mechanisms of action that directly stimulate uptake of glucose by skeletal muscle tissues, independent of endogenous and exogenous insulin, are thus needed for individuals who have developed insulin resistance or are experiencing undesired side effects with current treatments. Additional experimental exploration at the cell and tissue level focused on understanding the mechanism of the RG54 peptide would significantly support such ambitions and the design of clinical trials. In order to capture the molecular and cellular impacts, such studies should preferably be performed using metabolically challenged animals, as indicated by our signalling analyses (ESM Fig.
6) and our previous studies on lean animals treated with ApoA-I protein [
12].
The increased risk for CVD in diabetes is another challenging problem. Data from large clinical trials focused on CVD outcomes following treatment with glucose-lowering drugs, sodium–glucose co-transporter-2 inhibitors and glucagon-like peptide-1 analogues, have shown significant cardioprotective benefits of these drugs [
23,
24]. The biological mechanism that leads to this improved situation for the patient population is not clear. Since the two classes of diabetes drugs both lower blood glucose but through completely different mechanisms, it is plausible that establishing glycaemic control is a strongly contributing factor to the reduced CVD risk, which may involve a reduction in AGEs ([
25] and refs therein), potentially including the ApoA-I protein [
20,
26,
27]. While only speculative, the finding that the RG54 peptide contributes to glucose control and also prevents atherosclerosis in rodent models suggests that diabetes treatments based on the RG54 peptide may show even greater effects on CVD risk.
The demonstrated biological effects of the RG54 peptide hold promise for the development of a novel diabetes drug with a special focus on treating individuals with moderate to severe insulin resistance. However, the described studies have several limitations, including that the translatability of the biological effects of the RG54 peptide is unclear and has to be tested. A small-scale clinical study performed by Drew et al [
8] showed that the reduction of blood glucose in individuals with type 2 diabetes after acute HDL infusion was significantly larger that in placebo control participants, indicating that similar findings on glucose control by HDL as seen cellular and animal model systems are also observed in humans. Another limitation is the administration route. Although many current diabetes drugs are injectables, non-invasive administration is preferred. This is a challenge for a peptide-based treatment, as the peptide will be efficiently hydrolysed in the gut before absorption and entering circulation. However, novel delivery strategies such as inhaled biodegradable calcium-phosphate nanoparticles charged with therapeutic peptides hold promise and may revolutionise the field of peptide-based drugs [
28]. In parallel with explorations in oral delivery systems, technological advances in drug pumps already now provide better possibilities for individuals that could also potentially be utilised as a means of administering an optimised RG54 peptide.
This study demonstrates that RG54 peptide improves glucose control and reduces atherosclerotic burden in clinically relevant models, providing a rationale for RG54 peptide as a potential treatment for individuals with type 2 diabetes with poor glucose control and/or at increased risk of CVD, justifying further preclinical evaluation and subsequent clinical testing of this peptide.
Acknowledgements
A. Knutsson and S. Hsiung (Lund University, Sweden) for training and advice regarding the Apoe−/− model and aortic preparation; E. Krupinska and W. Knecht (Lund University Protein Production Platform [LP3], Sweden) for providing protein; R. Del Giudice (Lund University, Sweden) for peptide purification and technical advice; Red Glead Discovery (Lund, Sweden) for peptide synthesis and solubility studies; and the SciLifeLabs Drug Discovery and Development platform (Stockholm, Sweden) for the stability studies. Part of this work was previously published as an abstract at the 23rd Annual Scandinavian Atherosclerosis Conference, Humlebæk, Denmark, 2017; and the 24th edition IDF Congress, Abu Dhabi, United Arab Emirates, 2017.
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