Blood Glucagon Levels Predict the Hemoglobin A1c Response to Saxagliptin in Patients with Type 2 Diabetes Inadequately Controlled with Metformin

Background Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used as second-option medications when metformin fails. Variance of the glycated hemoglobin (HbA1c) response to DPP-4 inhibitions in patients with type 2 diabetes mellitus (T2DM) has been observed, but the characteristics which predict the response to DPP-4 inhibitor therapy are unclear. The aim of this study was to investigate the characteristics of α- and β-cell functions which might predict the efficacy of saxagliptin and facilitate personalization of treatment. Methods We studied 60 patients with T2DM who had inadequate glycemic control [HbA1c7.0–13.0% (53–119 mmol/mol)) with metformin alone. The patients were treated with saxagliptin (5 mg, daily) and metformin (1000–2000 mg as former) for 12 weeks. Oral glucose tolerance tests were carried out at baseline and endpoint to evaluate α- and β-cell functions, and blood C-peptide, insulin, glucagon levels were tested. Blood glucose, HbA1c and weight were also observed. Results Significant reduction of weight, HbA1c and glucagon was observed after 12-week treatment, while C-peptide, insulin and homeostasis model assessment-β increased (P < 0.05). Linear regression and receiver operating characteristic analysis showed that baseline HbA1c and 30 min-glucagon were correlated with the HbA1c response to saxagliptin, while the weight loss was correlated with gender, age and fasting-insulin level. Further analysis showed the 30 min-glucagon of 49.1 pmol/L was the optimal cutoff value to predict the efficacy of saxagliptin. Conclusions Saxagliptin added to metformin significantly improved glycemic control and α- and β-cell function. Blood glucagon level was a good predicting factor for the HbA1c response to saxagliptin, and it will help appropriate patient selection. Trial registration Chinese Clinical Trial Register identifier, ChiCTR-PPR-15007045.


INTRODUCTION
Dipeptidyl peptidase-4 (DPP-4) inhibitors are a kind of oral anti-diabetic agents that increase circulating concentrations of the glucagon-like peptide-1 (GLP-1) in non-insulin-dependent diabetes mellitus (T2DM) [1]. DPP-4 inhibitors may improve b-cell function in patients with T2DM [2] and inhibit a-cell function [3]. HbA1c and fasting glucose contributed to the reduction of HbA1c [4]. Previous studies also found that about 30% of patients with T2DM had resistance to GLP-1 which may be caused by gene variants, such as GLP1R, transcription factor 7-like 2 (TCF7L2) and wolframin ER transmembrane glycoprotein (WSF-1) gene variation [5][6][7]. All of these genes affect b-cell functions, including insulin production and secretion and b-cell proliferation [8][9][10]; GLP1R and TCF7L2 were also identified in a-cells and were found to affect glucagon secretion [11,12]. Therefore, we inferred that a-and b-cell functions, influenced by both genetic factors, and acquired disposition like blood glucose and duration of diabetes, may predict the efficacy of DPP-4 inhibitors. However, the influence of baseline a-and b-cell function on the efficacy of DPP-4 inhibitors in patients with T2DM has not been observed.
Saxagliptin is demonstrated to be effective and safe comparing with placebo in Asian patients with T2DM who had inadequate glycemic control with or without metformin and diet and lifestyle modification [13,14].
There was no difference in fasting insulin or glucagon between saxagliptin group and the placebo group in previous study, but postprandial C-peptide and b-cell function based on the homeostasis model assessment (HOMA)-b were improved by saxagliptin treatment [14]. Another study in patients with T2DM poorly controlled with metformin monotherapy showed that saxagliptin suppressed glucagon response significantly [15]. However, the relationship between postprandial glucagon and efficacy of saxagliptin was unobserved. In the present study, oral glucose tolerance tests (OGTTs) were used for evaluating a-and b-cell functions in patients with T2DM inadequately controlled with metformin treated with saxagliptin as add-on therapy, thereby trying to find predictive factors on the efficacy of saxagliptin.

Patients
The patients with T2DM who had inadequate glycemic control with metformin were consecutively recruited from the Nanjing First

Patients and Saxagliptin Efficacy
Among the 64 enrolled patients, 60 patients

Saxagliptin Efficacy Comparison
The patients were divided into high/ low-glucagon and high/low HbA1c groups according to the results of ROC analysis. There was no difference in baseline HbA1c, weight, BMI and HOMA-b between low-glucagon (n = 23, baseline 30 min-glucagon \49.1 pmol/ L) and high-glucagon (n = 37, baseline 30 min-glucagon C49.1 pmol/L) groups. However, the changes in HbA1c, weight, BMI and HOMA-b after 12-weeks of saxagliptin treatment were significantly greater in the high-glucagon group than in the low-glucagon group (Fig. 3a). The HbA1c levels at baseline and endpoint were lower in low-HbA1c group (n = 23, baseline HbA1c\8.7% [72 mmol/mol]) than in high-HbA1c group [n = 37, baseline HbA1c C8.7% (72 mmol/mol)]; however, no difference was observed in weight, BMI or HOMA-b between the two groups (Fig. 3b).

DISCUSSION
The prevalence of T2DM increased rapidly in recent years, and the number of patients with T2DM is expected to rise to 552 million by 2030 [17]. Due to the large amount of patients and the availability of at least 10 drug classes for the treatment of T2DM, the ideal drug sequence after metformin failure is an area of increasing uncertainty [18]. Thus, precision medicine, taking account of human individuality in genes, environment, and lifestyle for early disease diagnosis, and individualized therapy, have been required in the treatment of T2DM at this time.

DPP-4 inhibitors, including saxagliptin, are
accepted by more and more patients due to their effectiveness in glycemic control and less side-effect in hypoglycemia and weight gain [19]. Our study confirms that saxagliptin in combination with metformin is effective in improving glycemic control and losing weight. The effects of restoring a-and b-cell functions in patients with T2DM were also observed, which were consistent with previous studies [20,21].
In our study, blood glucagon level, especially the 30 min-glucagon in OGTT, was significantly associated with HbA1c response to saxagliptin according to the linear regression and ROC analysis, which may be considered as a good predictive factor of saxagliptin efficacy.
However, b-cell function, such as insulin, C-peptide and AUC (INS), had been slightly  The mechanism of glucagon level decreased by GLP-1 remains unclear. Gromada et al. found that GLP-1 has minimal activity with glucagon receptor [27]. However, most studies have not localized the GLP-1 receptor to a-cells [28]. Blood glucagon level may correlate with the capacity of GLP-1 production in vivo, because GLP-1 is a product of proglucagon that has nearly 50% homology to glucagon [29]. Recently, Kushiyama et al. found that blood glucagon level had no correlation with active GLP-1 level, though active GLP-1 level in plasma was associated with the efficacy of DPP-4 inhibitors in patients with T2DM [30].
However, GLP-1 has a short half-life (less than 2 min) due to rapid degradation by the enzyme DPP-4, and is difficult to be detected routinely; by contrast, glucagon is stable and detectable as a predictor used in clinical medicine.
According to our study, blood glucagon level was more outstanding than baseline HbA1c, which was the predictor recommended in previous studies [4,31]. There were more significant improvements not only in HbA1c but also in weight and b-cell function in high-glucagon group than in low-glucagon group, while baseline HbA1c could only predict the HbA1c response to saxagliptin combined with metformin.
Blood glucagon levels may also be related to efficacy of other drugs which affect glucagon secretion, such as insulin and sodium-glucose cotransporter-2 (SGLT2) inhibitors. SGLT2 inhibitors have an opposite effect on glucagon compared with DPP-4 inhibitors [32,33], and the relationship between blood glucagon levels and the drug efficacy needs further investigation.
Considering the effects of metformin and saxagliptin on losing weight, we chose overweight patients with BMI[24 kg/m 2 .
Previous study showed that the suppression of glucagon secretion during hyperglycemia was impaired in obese mice [34]. Fasting hyperglucagonemia was observed in obese people who, despite their insulin-resistant state, have normal glucose tolerance and may constitute very early steps in the pathophysiology of T2DM as well as a reduced incretin effect [35]. Therefore, our study showed significant higher blood glucagon level compared with previous study  T2DM patients were divided into high-and low-HbA1c groups according to baseline HbA1c = 8.65%. There was no difference in weight, BMI and HOMA-b between the two groups, while baseline HbA1c and DHbA1c were significantly higher in high-HbA1c group than in low-HbA1c group. *P\0.001; **P\0.05.
BL baseline, BMI body mass index, HbA1c glycated hemoglobin, HOMA-b homeostasis model assessment, T2DM type 2 diabetes mellitus (baseline mean glucagon: 57.6 vs. 19.0 pmol/L) [14]. This may also explain the significant decrease of HbA1c after saxagliptin treatment. There was no significant weight loss after saxagliptin treatment in Chinese patients in previous studies [13,14]. However, in our study, weight and BMI decreased after saxagliptin treatment. Younger and high 120 min-GLA/ INS may be correlated with increased weight loss. The patients recruited in our study had lower insulin and C-peptide levels than in previous studies [14,19], which may have been caused by a higher glucotoxicity.
Limitations of this study include the lack of a placebo control which precludes effects of metformin, dietary and lifestyle modifications.
In the previous study with a duration of 24 weeks, the decline of HbA1c was only 0.4% (4 mmol/mol) in metformin ? placebo group with reinforced diet and lifestyle [14], while other studies showed that metformin had no effect on glucagon suppression [15,36]. However, the effects of metformin, dietary and lifestyle modifications may not be ignored.