Impact of Sitagliptin on Neprilysin and Glycemic Control in Newly Diagnosed Type 2 Diabetes Patients

Introduction

Insulin resistance and progressive loss of pancreatic beta cell functions lead to hyperglycemia over time in patients with type 2 diabetes, which has been estimated by the WHO to be the seventh leading cause of death worldwide despite breakthrough advances in diagnosis and treatment [1].

Also known as enkephalinase, neutral endopeptidase, or EC 3.4.24.11, neprilysin (NEP) is ubiquitously found in the brain, vascular endothelial cells, cardiac myocytes, neutrophils, smooth muscle cells, and renal tubular cells [2]. Neprilysin (NEP) is involved in the degradation of various peptides, including GLP-1, and plays a significant role in glucose metabolism and cardiovascular function. NEP inhibition has been associated with improved insulin sensitivity and reduced systemic inflammation in diabetes [3]. NEP has been shown to play a role in glucose homeostasis [4]. Neprilysin substrates, including incretin glucagon-like peptide-1 (GLP-1) and bradykinin, are known to mediate glucose metabolism [5,6]. Neprilysin exerts its glycemic effect mainly by inactivation and cleavage of GLP-1. Increased NEP activity in metabolic tissues of mice with induced obesity has been associated with decreased insulin sensitivity and reduced beta cell functioning [7]. NEP deficiency has been found to be correlated with the expansion of islet β-cell mass in mice fed a high-fat diet [8]. Some studies have shown a glucose-lowering effect of NEP inhibition via increasing plasma levels of its substrates [9]. In addition to the above-mentioned substrates, NEP also degrades many other peptides that have the potential to modulate glucose metabolism, such as incretin glucose-dependent insulinotropic peptide (GIP) and pancreatic polypeptide.

Dipeptidyl peptidase-4 (DPP-4) is an enzyme responsible for the degradation of incretin hormones, such as GLP-1 and GIP. Inhibition of DPP-4, as seen with sitagliptin, prolongs incretin activity, increasing insulin secretion and suppressing glucagon release, thereby improving glucose control in type 2 diabetes patients [10]. Studies have demonstrated that sitagliptin plays a significant role in regulating glucose metabolism and improving metabolic parameters, which is aligned with recent research on its effectiveness in diabetes management.

Neprilysin activity has been linked to increased oxidative stress and inflammatory responses, potentially contributing to metabolic dysfunction [12]. Experimental models have demonstrated that neprilysin inhibition improves insulin sensitivity and reduces systemic inflammation, further supporting its role in metabolic regulation [3]. The very important role of NEP in inactivating natriuretic peptides and its involvement in the renin-angiotensin system (RAS) has led researchers to explore the utility of NEP inhibitors in the treatment of certain forms of cardiovascular and renal diseases that are among the most important complications of type 2 diabetes [13,14]. NEP inhibitors have been approved for use in humans with heart failure, of which approximately 35% also had diabetes mellitus [15].

Despite the well-established role of NEP inhibitors in cardiovascular disease, data regarding their potential use in diabetes management remain limited. However, recent studies have provided evidence supporting the metabolic benefits of NEP inhibition. Jordan et al reported that sacubitril/valsartan therapy leads to improved lipid and glucose metabolism, reinforcing the importance of NEP in metabolic regulation [16]. Similarly, Seferovic et al demonstrated that NEP inhibition can enhance glycemic control in diabetic patients [17]. These findings raise important questions regarding the interplay between NEP inhibition and glucose homeostasis, yet the direct effects of DPP-4 inhibition on neprilysin levels remain unclear. Given that both NEP and DPP-4 contribute to GLP-1 degradation, further investigation is needed to clarify whether DPP-4 inhibition, such as with sitagliptin, influences NEP levels and its metabolic effects in type 2 diabetes patients. This study assessed the effects of sitagliptin monotherapy on serum neprilysin levels and their relationship with metabolic parameters in patients newly diagnosed with type 2 diabetes.

Material and Methods

STUDY DESIGN AND PARTICIPANTS:

A prospective observational study was conducted involving 35 patients over the age of 18 who were newly diagnosed with type 2 diabetes mellitus and treated at the outpatient endocrinology clinic of our hospital. The diagnosis of type 2 diabetes mellitus was established according to the criteria of the American Diabetes Association (ADA), based on fasting plasma glucose ≥126 mg/dL, postprandial plasma glucose ≥200 mg/dL, or HbA1c ≥6.5% [18]. Patients who were initiated on sitagliptin monotherapy were consecutively enrolled. Patients with acute illness, chronic heart failure, renal, hepatic or pulmonary diseases, malignancies, alcohol abuse, or active smoking were excluded from the study.

DATA COLLECTION:

Baseline demographic characteristics (age, sex) and anthropometric measurements, including weight, height, body mass index (BMI), waist circumference, hip circumference, waist-to-hip ratio, and fat percentage were recorded. Glycemic parameters (fasting blood glucose, postprandial glucose, glycosylated hemoglobin [HbA1c], homeostasis model assessment-insulin resistance [HOMA-IR] index, albumin creatinine ratio [ACR]) and lipid profile (total cholesterol, HDL-C, LDL-C, triglycerides), were measured at baseline and at 3 months after sitagliptin therapy. Glycemic control was assessed by measuring fasting blood glucose (FBG), postprandial blood glucose (PPG), and glycosylated hemoglobin (HbA1c) levels at baseline and after 3 months of sitagliptin therapy. Body weight (kg) and body mass index (BMI, kg/m²) were measured at baseline and after 3 months of treatment. Weight loss was defined as a reduction in either body weight or BMI during the study period.

TREATMENT PROTOCOL:

All patients received sitagliptin monotherapy at a daily dose of 100 mg. No other antidiabetic agents were prescribed during the study period. Dietary counseling and lifestyle modification advice were provided to all participants.

BLOOD SAMPLING AND LABORATORY MEASUREMENTS:

Venous blood samples were collected after an overnight fast. Routine biochemical parameters were analyzed using standardized methods. Biochemical parameters were measured using an automated analyzer (Cobas 6000 series, Roche Diagnostics, Basel, Switzerland) following standardized laboratory protocols. Glycemic parameters, including fasting plasma glucose (FBG), postprandial plasma glucose (PPG), and glycosylated hemoglobin (HbA1c), were measured using standardized enzymatic methods. Metabolic parameters – serum lipid profile (total cholesterol, LDL-C, HDL-C, triglycerides), liver enzymes (AST, ALT), and HOMA-IR index – were also assessed following standard laboratory protocols. Serum samples for neprilysin measurement were immediately separated and stored at −80°C until analysis. Serum neprilysin levels were determined by enzyme-linked immunosorbent assay (ELISA) using the Human Neprilysin (NEP/CD10) ELISA Kit (Cat. No. ELK2196, ELK Biotech, Wuhan, China). The intra-assay and inter-assay coefficients of variation were <8% and <10%, respectively.

SAMPLING METHOD:

Patients were recruited from among those who were newly prescribed sitagliptin monotherapy at the outpatient endocrinology clinic. Thus, a convenience sampling method was used. The non-random nature of patient selection may have introduced selection bias, which is acknowledged as a limitation of the study.

ETHICS STATEMENT:

This study was approved by the Ethics Committee of Uludag University (Approval No: 2023-27/30). All participants provided written informed consent.

STATISTICAL ANALYSIS:

All statistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). The normality of continuous variables was assessed using the Kolmogorov-Smirnov test. Since the data were not normally distributed, changes in dependent variables were compared using the Wilcoxon signed-rank test. Multivariable regression analysis was conducted to evaluate the effect of changes in neprilysin levels on BMI, waist circumference, and HOMA-IR. Neprilysin levels were log-transformed to stabilize variance. A p-value <0.05 was considered statistically significant. A post hoc power analysis was performed for the primary outcome (BMI change). Assuming an effect size of 0.3, with a sample size of 35 patients and a significance level of 0.05, the achieved statistical power was approximately 55%, which indicates a moderate ability to detect significant changes in BMI within the study cohort.

Results

DEMOGRAPHIC AND ANTHROPOMETRIC DATA:

Thirty-five newly diagnosed diabetic patients were included in the study, of which 20 (57.14%) were women. The mean age of the patients was 53.31±8.09 years. The mean body weight was 86.10±15.26 kg at the beginning of the therapy. The mean baseline BMI value was calculated as 32.30±5.80 kg/m2. The mean weight and BMI values were significantly lower at the 3rd month compared to the beginning of the treatment (for both, p=0.002). Anthropometric measurements of the patients at baseline and the 3rd month of the treatment are presented in Table 1.

BIOCHEMICAL AND GLYCEMIC PARAMETERS:

Biochemical, glycemic parameters, and lipid profiles of the patients included in the study were measured and compared between the beginning and the 3rd month of the treatment. Accordingly, postprandial blood glucose, HbA1c, AST, and ALT values of the patients were significantly lower at the end of the 3-month study period compared to the baseline levels (for all, p<0.05). There was no significant change in the other studied parameters. Biochemical parameters at the beginning and 3rd month of the treatment are given in Table 2.

NEPRILYSIN LEVELS:

The mean baseline NEP value was 1420.53±1658.64, while this value was 1260.82±1456.57 at the end of the 3-month study period. The difference was not statistically significant (p=0.789) (Table 2).

MULTIVARIABLE REGRESSION ANALYSIS:

In the multivariable regression analysis, changes in neprilysin levels were found to have a significant negative association with BMI change (β=−0.0003, p=0.001), suggesting that an increase in neprilysin levels is linked to a reduction in BMI (Table 3). However, no significant association was observed between changes in neprilysin levels and waist circumference change (β=−0.0003, p=0.464) or HOMA change (β=0.0004, p=0.383) (Table 3). Age showed a borderline negative effect on BMI change (β=−0.0384, p=0.067), while sex was not significantly associated with changes in any dependent variable. Pearson’s correlation analysis showed a weak negative correlation between baseline neprilysin levels and BMI (r=−0.104). In contrast, a weak positive correlation was observed between neprilysin levels at the third month and BMI (r=0.042). However, neither correlation was statistically significant (p>0.05) (Table 3).

Discussion

In our study, sitagliptin monotherapy led to a significant reduction in BMI after 3 months of treatment. Glycemic control improved as expected, with significant reductions in postprandial blood glucose and HbA1c. However, neprilysin levels decreased slightly, but this change was not statistically significant. These findings suggest that sitagliptin has a positive effect on glycemic control and weight loss, but does not significantly affect serum neprilysin levels in newly diagnosed type 2 diabetes patients.

In recent years, research has focused on achieving glycemic control through inhibition of certain peptidases, such as DPP-4 and NEP, that are known to exert their effects through cleavage and inactivation of GLP-1 [4]. Both NEP and DPP-4 are produced by islet cells, and their upregulation mediates insulin secretory dysfunction [19]. Increasing evidence suggests that these peptidases are associated with impaired glucose homeostasis and that systemic inhibition of NEP and DPP-4 enhances glycemic control in part through improved beta cell functioning [7,17,20]. Esser et al proposed that islet NEP is at least as important as islet DPP-4 [9].

Inhibition of these peptidases has been associated with a glucose-lowering effect, resulting in increased circulatory GLP-1 [21]. After being approved by the FDA in 2006, DPP-4 inhibitors have been developed for clinical use and are now widely used for the treatment of type 2 diabetes [22]. The inhibition of DPP-4 has been extensively studied, with several studies documenting its glucose-lowering effects [23–26]. In contrast, NEP inhibition is a relatively new area of investigation, represented mostly by animal studies [4,7,20]. NEP is known to degrade multiple bioactive peptides, including GLP-1 and glucose-dependent insulinotropic peptide (GIP), both of which are crucial for insulin secretion and glucose metabolism. Therefore, NEP inhibition could theoretically enhance glycemic control by prolonging the activity of these incretins. However, human studies investigating this effect remain limited [16,17]. In a study by Albrechtsen et al, a combination of DPP-4 and neprilysin was examined in healthy men, showing higher GLP4 levels compared to inhibition of DPP-4 alone. However, the study was conducted in normoglycemic subjects, and the results were obtained without any change in glucose or insulin levels [27]. This suggests that while NEP inhibition can contribute to incretin regulation, its direct impact on glucose metabolism in diabetic patients is still uncertain. Further research is needed to determine whether NEP inhibitors alone or in combination with DPP-4 inhibitors can significantly enhance glycemic control in type 2 diabetes. Moreover, while NEP inhibitors such as sacubitril/valsartan have been approved for heart failure treatment [17], their potential metabolic benefits remain under investigation. Given the concerns regarding increased heart failure risk with DPP-4 inhibitors [28], NEP inhibitors could present an alternative therapeutic strategy for diabetic patients with cardiovascular comorbidities. These findings highlight the need for further studies to clarify the role of NEP in glucose metabolism and its clinical relevance in diabetes management.

To bring a new perspective to research on this issue, the present study investigated the potential relationship between DPP-4 inhibition and serum NEP levels in patients with newly diagnosed type 2 diabetes. In our multivariable regression analysis, changes in neprilysin levels showed a significant negative association with BMI change (p=0.001), suggesting that an increase in neprilysin levels contributes to a reduction in BMI. However, no significant association was observed between changes in neprilysin levels and waist circumference or HOMA levels. This discrepancy may be due to the complex and multifactorial nature of glucose metabolism and body composition regulation. Log transformation was applied to neprilysin levels to normalize the data distribution and reduce the effect of outliers, which improved the interpretability and stability of the regression model. We could not find a significant difference between NEP levels at the beginning and after three-month therapy with sitagliptin (p=0.789). Our findings indicate that serum neprilysin levels remain unchanged despite improvements in glycemic control with sitagliptin monotherapy, reinforcing that neprilysin is not a predictive marker of glycemic regulation in newly diagnosed type 2 diabetes patients. Although no significant correlation was found between neprilysin levels and glycemic parameters, the significant association with BMI suggests that neprilysin may play a more specific role in body composition regulation rather than glycemic control. This finding aligns with previous studies showing that neprilysin modulates glucose homeostasis and lipid metabolism through complex peptide cleavage pathways. This finding suggests that NEP and DPP-4 may be involved in glycemic homeostasis via different and independent underlying mechanisms that should be further studied. Our measurements were made at the beginning and third month of the study period, and shorter or longer measurements may have produced different results.

Sitagliptin is known to reduce HbA1c, fasting, and postprandial glucose by glucose-dependent stimulation of insulin secretion and inhibition of glucagon secretion [29]. Although it differs among studies, the standard recommended dose of sitagliptin is 100 mg once daily, as in our study [30]. As expected, in the present study, Hb1Ac and postprandial glucose levels were significantly reduced as a result of 3-month sitagliptin therapy. Although fasting blood glucose was also decreased, the difference was not statistically significant. This finding indicates that 3-month sitagliptin therapy improved glycemic control without affecting NEP levels.

Our study has several limitations. First, it had a relatively small sample size of 35 patients and was conducted at a single center, limiting the generalizability of our findings. The achieved statistical power in our study was 55%, indicating a moderate ability to detect significant changes in BMI and other outcomes, and the relatively short follow-up period of 3 months may not be sufficient to capture long-term changes in NEP levels or glycemic parameters. Second, our study did not directly measure incretin hormones such as GLP-1 and GIP, which are crucial in mediating the effects of DPP-4 inhibitors like sitagliptin. Direct assessment of these hormones would have helped clarify the mechanistic role of NEP in glucose regulation. Additionally, the absence of a control group receiving an alternative treatment or a placebo limits our ability to draw direct comparisons, particularly with other commonly used diabetes therapies. Moreover, our convenience sampling method introduced a potential selection bias, as patients were not randomly selected. Despite these limitations, our findings provide valuable insights and could inform future, larger multi-center studies with more diverse populations and longer follow-up periods.

Conclusions

Sitagliptin monotherapy significantly improved glycemic control and reduced BMI in patients with newly diagnosed type 2 diabetes. However, neprilysin levels were not significantly affected by sitagliptin therapy. These findings suggest that while sitagliptin effectively manages blood glucose and weight, it does not affect neprilysin levels in this patient population.