Effects of neprilysin and neprilysin inhibitors on glucose homeostasis: Controversial points and a promising arena

1 INTRODUCTION

Neprilysin (NEP) is also known as cluster of differentiation 10, enkephalinase, neutral endopeptidase, membrane metalloendopeptidase, and common acute lymphoblastic leukemia antigen (CALLA).¹ NEP is a transmembrane zinc-dependent metalloproteinase that inactivates various peptide hormones including natriuretic peptides, bradykinin, oxytocin, neurotensin, substance P, enkephalins, angiotensin II (AngII), endothelin 1 (ET-1), and glucagon-like peptide 1 (GLP-1). NEP is also involved in the degradation of amyloid beta (Aβ) and could be effective in the prevention development of Alzheimer's disease (AD)^2-4 (Figure 1).

NEP is widely expressed on the surface of endothelial cells, neutrophils, and fibroblasts and various tissues including the brain, kidneys, lungs, testes, gastrointestinal tract, and heart.⁵ Soluble NEP is found in the blood, which reflects the concentration and activity of membrane-bound NEP.⁶ Indeed, NEP is mainly involved in neurohumoral activation and regulation of the sympathetic nervous system and renin-angiotensin system (RAS) in different diseases including heart failure.^{2, 4, 7} Higher expression of NEP is associated with poor prognostic outcomes in patients with heart failure.⁸ However, in the cohort and mixed cohort studies, NEP level was of no clinical value in the prognosis of heart failure.^{9, 10}

Different types of NEP inhibitors are developed in the management of heart failure and hypertension and as analgesic agents.¹¹ NEP inhibitors including sacubitril, RB-101, omapatrilat, ecadotril, and candoxatril are rarely used alone but used in combination with AngII receptor blockers (ARBs) in the management of heart failure through modulation of RAS and expression of natriuretic peptides (Figure 2).

Sacubitril was the first NEP inhibitor approved in 2015 for the management of heart failure.¹¹ Sacubitril in combination with valsartan was initially named LCZ696, which is an ARB and NEP inhibitor (ARNI). LCZ696 decreases blood pressure more than valsartan alone in hypertensive patients.¹² In the PARADIGM-HF study, LCZ696 was more effective than angiotensin-converting enzyme inhibitor (ACEI) enalapril in the management of heart failure.¹³ The combo of NEP inhibitor/Valsartan (approved by the Food and Drug Administration in 2015) has been a significant addition to heart failure therapy in diabetes and no major complications have been seen.¹³

NEP inhibitors are also effective in the management of type 2 diabetes mellitus (T2DM) by increasing the circulating level of GLP-1, which is degraded by NEP.¹⁴ However, the acute effect of NEP inhibitors leads to detrimental effects by increasing blood glucose independent of GLP-1 increment.¹⁵ These findings suggest a controversial point regarding the potential role of NEP inhibitors in T2DM patients. Thus, this perspective aimed to elucidate the controversial points concerning the role of NEP inhibitors in T2DM.

2 ROLE OF PANCREATIC NEP IN T2DM

T2DM is a metabolic disorder characterized by hyperglycemia and insulin resistance (IR) due to dysfunction of pancreatic β cells and insulin insensitivity.^{16, 17} T2DM represents 90% of all diabetes and commonly occurs in adults aged >40 years.¹⁸ T2DM is associated with cardiometabolic disorders including obesity, hypertension, and dyslipidemia.^{19, 20} Accumulation of amyloids in the pancreatic β cells induces progressive pancreatic β cell loss with the development of pancreatic β cell dysfunction and overt T2DM.²¹ The chief constituent of pancreatic β cell amyloids is islet amyloid polypeptide (IAPP) also known as amylin, which is mainly cleaved by pancreatic NEP. Progressive deposition of IAPP triggers the development of pancreatic β cell toxicity.²¹ NEP blocks the deposition of extracellular pancreatic IAPP and prevents pancreatic β cell dysfunction. Therefore, NEP seems to be protective against the development of T2DM through modulation of pancreatic IAPP and accumulation of amyloids.²¹

Aggregation of IAPP in the pancreas leads to the development of T2DM whereas accumulation of Aβ in the brain promotes the development of AD.^{22, 23} Therefore, both T2DM and AD share a similarity in the pathogenesis of brain IR and cognitive dysfunction. In this state, AD is classified as type 3 diabetes mellitus.^{22, 24} IAPP is derived from pre-pro-IAPP to form 37 amino acid peptide.²⁵ Notably, IAPP is co-secreted with insulin from pancreatic β cells into circulation. Progressive accumulation of IAPP induces the formation of oligomers and fibrils with subsequent amyloid deposits observed in T2DM.²⁵ Of interest, human IAPP lacks proline residues that prevent its deposition as seen in rat IAPP.²⁶ Notoriously, impairment of human IAPP processing due to defects in the activity of autophagy and proteasome may contribute toward IAPP-induced pancreatic β cytotoxicity.²⁵ Therefore, restoration of IAPP proteostasis may be a promising option in the prevention and treatment of T2DM.²⁵

Degradation of IAPP is done by peptidase enzymes like an insulin-degrading enzyme (IDE) and NEP to prevent the accumulation of IAPP in the pancreatic β cells and the development of T2DM.²¹ Downregulation or upregulation of these enzymes affects the functional capacity of IAPP. For example, inhibition of IDE by bacitracin accelerates amyloid deposition in insulinoma cell lines by inhibiting the degradation of pancreatic IAPP.²⁷ NEP is highly expressed in the pancreatic β cells and is involved in the clearance of both pancreatic amyloids and brain Aβ (Figure 3).^{3, 28} Moreover, an experimental study with transgenic mice illustrated that inhibition of NEP promotes pancreatic β cell amyloidogenesis.²⁸ However, NEP activators or upregulation of pancreatic β cell NEP reduce pancreatic β cell amyloidogenesis and apoptosis in cell lines.²⁸ NEP prevents amyloid fibrilization in the pancreatic β cells by about 98% compared to neuronal Aβ fibrilization.²¹ NEP blocks human IAPP through noncatalytic and catalytic interactions.²¹ Intracellular accumulation of soluble IAPP in the pancreatic β cell is started initially.²⁹ However, NEP inactivates extracellular IAPP, which is more intricate in the propagation of pancreatic β cytotoxicity.³⁰ In vitro study demonstrated that IAPP was associated with β cell apoptosis through caspase-3 activation. In addition, pancreatic β cells are more susceptible to IAPP toxicity than α cells cultured under the same conditions.³⁰ As well, in vitro study conducted by Zraika et al³¹ observed that NEP inhibitors increase amyloid deposition in the pancreatic β cells by 54% and induce pancreatic β apoptosis by 75%. However, NEP activators or NEP upregulation led to a decrease in apoptosis and amyloid deposition in the pancreatic β cells by 79%. NEP reduces apoptosis and amyloid deposition in the pancreatic β cells through inhibition of IAPP fibril generation rather than degrading of IAPP.³¹ Recently, an experimental study conducted by Parilla et al³² revealed that NEP deficiency is linked with the expansion of pancreatic β cell mass in mice.

These findings suggest that NEP expression in the pancreatic β cells has a protective role against pancreatic β cell dysfunction and apoptosis through inhibition of pancreatic amyloidogenesis. Thus, NEP activators could be an effective therapeutic strategy in the prevention and treatment of T2DM.

3 NEP AND BLOOD GLUCOSE HOMEOSTASIS

It has been reported that NEP contributes to the impairment of glucose homeostasis through modulation of IR, pancreatic β cell mass, and development of pancreatic β cell dysfunction as observed in T2DM.³³ Notably, the expression of NEP is increased in both T2DM and nutrient excess.^{34, 35} Different experiment studies revealed that NEP activity was augmented in mice fed on a high-fat diet and correlated with the reduction of pancreatic β cell mass and development of IR.^{34, 36} NEP-deficient mice experience good glycemic control with augmentation of GLP-1 activity.³⁶ Genetic ablation of NEP improves glucose homeostasis through the GLP-1-dependent pathway. As well, pharmacological inhibition of NEP in humans increases GLP-1 activity.³⁷ Therefore, inhibition of NEP may improve insulin sensitivity and glucose tolerance and protect pancreatic β cells from glucotoxicity.

Overexpression of NEP had been associated with the development of IR and metabolic syndrome in a study that involved 318 subjects with metabolic syndrome.³⁴ NEP activity correlated with body mass index (BMI) and IR with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma. Thus, NEP is associated with cardiometabolic risk in the presence of IR and obesity.³⁴ Therefore, NEP activity is positively correlated with IR and BMI in obese patients with cardiometabolic disorders.^{23, 34}

Reduction of pancreatic β cell mass by the effect of NEP inhibitors is not fully elucidated; however, increasing NEP activity is associated with a compensatory increase in pancreatic β cell mass to overcome glucose dyshomeostasis.³⁸ When overt T2DM is developed, the pancreatic β cell mass is reduced due to apoptosis.³⁸ However, the experimental study confirmed that NEP deficiency or use of NEP inhibitors improves pancreatic β cell mass in mice through modulation expression of GLP-1 receptors, which promotes survival, differentiation, and proliferation of pancreatic β cells.³² Therefore, NEP deficiency may increase pancreatic β cell mass by increasing the GLP-1signaling pathway, which may explain the increase of pancreatic β cell mass in NEP deficient mice treated with a high-fat diet.

Furthermore, NEP increases dipeptidyl peptidase-4 (DPP4) activity and contributes to increasing active GLP-1 proteolysis.³⁶ Therefore, the activity of plasma DPP4 is reduced in NEP-deficient mice on a high-fat diet.³⁶ Thus, the acute effect of NEP inhibitors reduces DPP4 activity when NEP activity is exaggerated as in a high-fat diet; however, under the normal physiological condition; there is no interaction between NEP and DPP4.³⁶ Simonsen et al³⁹ showed that NEP inhibitors did not improve glycemic control in diabetic rats on DPP4 inhibitors. Remarkably, 50% of GLP-1 activity is reduced by the effect of NEP; thus the combination of NEP inhibitors with DPP4 inhibitors is more effective in maintaining GLP-1 activity.⁴⁰ An experimental study observed that NEP inhibitor candoxatril alone or in the combination with DPP4 inhibitor valine pyrrolidide increases GLP-1. However, the combination was superior to the DPP4 inhibitor when used alone in the elevation of GLP-1.⁴⁰ These findings suggest that NEP inhibitors may improve glycemic control through increasing endogenous GLP-1 activity and reduction of DPP4 activity (Figure 4).

In addition, NEP inhibitors increase other substrates cleaved by NEP-like bradykinin, which improves insulin sensitivity in mice.⁴¹ Similarly, bradykinin through activation of the bradykinin 2 receptor enhances glucose uptake and oxidation.⁴² Wu et al⁴³ demonstrated that bradykinin could protect endothelial cells from the effect of high blood glucose through modulation of the Akt pathway. Therefore, NEP inhibitors produce a protective effect against the development of diabetic complications via GLP-1-dependent and independent mechanisms.

However, different studies confirmed that NEP deficiency did not affect glycemic control and did not produce any benefit against high-fat diet-induced dysglycemia.^{34, 44} NEP-deficient mice did not experience any changes in IR, glucose tolerance, and body/epidermal fat weight compared to the controls.³⁴ Likewise, treatment with NEP inhibitors produces insignificant effects on glucose control in rats.³⁹ Of interest, follow-up of NEP-deficient mice for 1 year showed they develop obesity and impairment of glucose tolerance.⁴⁵

These findings indicated that NEP plays a minor role in the regulation of glucose homeostasis and insulin sensitivity. However, these experimental studies may not take into consideration the following points: pharmacological inhibition of NEP does not completely reduce NEP activity as in genetic ablation, and the duration of experimental studies regarding the use of NEP inhibitors and high diet period may affect the final data. These findings proposed that NEP inhibitors might not be effective in controlling blood glucose in T2DM patients.

4 NEP INHIBITORS AND GLUCOSE HOMEOSTASIS

5 PROS AND CONS OF NEP INHIBITORS' EFFECTS ON GLUCOSE HOMEOSTASIS

Regarding the safety profile of NEP inhibitors, it has been shown that long-term use of NEP inhibitors may increase IAPP aggregation with the development of pancreatic β cell dysfunction and apoptosis.³¹ However, NEP inhibitor-induced GLP-1 elevation may reduce pancreatic β cell apoptosis.⁸¹ These findings from experimental studies are not confirmed in human studies. Furthermore, NEP inhibitors affect the clearance of brain Aβ,⁶⁹ which raises a concern about the risk of AD in relation to long-term therapy with NEP inhibitors. Therefore, long-term follow-up for the effects of NEP inhibitors on cognitive function and AD risk is recommended. Long-term and short-term effects of NEP inhibitors may lead to detrimental effect on insulin sensitivity and glucose homeostasis through different mechanisms including augmentation of substrates that adversely affect insulin signaling and glucose homeostasis. Furthermore, long-term use of NEP inhibitors may increase the risk for the development of T2DM by increasing the deposition of pancreatic IAPP. To reduce the detrimental effects of NEP inhibitors and increase their efficacy, NEP inhibitors might be combined with ARBs. This combination reduces the harmful adverse effects mediated by increasing AngII levels.⁵³ In addition, ARNIs are more effective than NEP inhibitors on insulin sensitivity and glucose homeostasis.⁸²

Taken together, NEP inhibitors can affect insulin sensitivity and glucose homeostasis in bidirectional ways that could be beneficial or harmful depending on the time, duration, dose, and route of administration. Therefore, a large-scale prospective study to elucidate the effects of NEP inhibitors or ARNIs on glucose homeostasis in both diabetic patients and healthy subjects is recommended. As well, long-term follow-up of patients receiving NEP inhibitors or ARNIs by measuring IAPP serum level is advisable to outweigh the risk/benefit ratio in relation to AD risk.

6 CONCLUSIONS

NEP enzyme inactivates various peptide hormones including NPs, bradykinin, oxytocin, neurotensin, substance P, enkephalins, AngII, ET-1, and GLP-1. NEP expression in the pancreatic β cells has a protective role against pancreatic β cell dysfunction and apoptosis through inhibition of pancreatic amyloidogenesis. Overexpression of NEP enzyme is associated with development of IR, poor glucose homeostasis, and cardiometabolic disorders by the increase DPP4 activity and reduction of circulating GLP-1. Therefore, NEP inhibitors improve glucose homeostasis by inhibition of DPP4 activity and increasing of circulating GLP-1. NEP inhibitors are rarely used alone but are used in combination with ARBs in the management of heart failure through modulation of RAS and expression of natriuretic peptides. Taken together, NEP inhibitors produce beneficial rather than detrimental effects on glucose homeostasis. Therefore, large prospective studies are suggested in this regard.

AUTHOR CONTRIBUTIONS

Hayder M. Al-kuraishy and Ali I. Al-Gareeb: conceptualization and writing of the original draft. Athanasios Alexiou, Marios Papadakis, Hebatallah M. Saad, and Gaber El-Saber Batiha: preparation of figures; writing, correcting, and amending of the article. Faisal Holil AlAnazi, Hanan A. Ogaly, and Yousef Abud Alanazi: editing and polishing of the manuscript and responding to reviewers' comments. All authors contributed significantly to the manuscript and approved the submitted version.

FUNDING INFORMATION

Open Access funding enabled and organized by Projekt DEAL. This work was supported by the University of Witten-Herdecke Germany.