Possible Alterations in Appetite-related Molecules Αfter the Elimination of Hepatitis C Virus

Abstract

Background/Aim: Recent advances in antiviral treatment have achieved a sustained viral response (SVR) in over 95% of hepatitis C virus (HCV) infections. HCV elimination is suggested to improve several lifestyle-related factors; however, few studies have focused on dietary habit-/appetite-related factors.

Patients and Methods: HCV-infected patients who received Daclatasvir/Asnaprevir (DCV/ASV) therapy were enrolled, and the changes in appetite-related molecules after antiviral therapy were assessed with a multiple cytokine-measuring system.

Results: Among 119 HCV-infected patients who received DCV/ASV treatment, 104 (87.3%) achieved an SVR. In the SVR group, DCV/ASV treatment improved several liver-related variables at 24 weeks after the completion of therapy. In patients with an SVR, the values of glucagon-like peptide 1 (GLP-1) and leptin were significantly increased at 24 weeks after completing direct-acting antiviral therapy. However, no significant change was observed in non-SVR patients, regardless of the receipt of direct-acting antiviral treatment.

Conclusion: Gastrointestinal hormones related to the dietary habit and/or appetite may be influenced by HCV elimination.

Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease (CLD). Recent advances in antiviral treatment with direct-acting antivirals (DAAs) have achieved a sustained viral response (SVR) in over 95% (around 98%-99%) of HCV-infected patients (1-3). Based on this highly successful ratio of HCV elimination, various effects after SVR achievement have been reported, as HCV elimination is known to affect not only the liver function but also its related metabolism (4,5). In addition, HCV elimination is suggested to improve several lifestyle-related factors, including the activities of daily living (ADL) and quality of life (QOL) (6,7). Regarding lifestyle-related factors, the dietary habit is particularly important, as is exercise. However, few studies have focused on dietary habit-/appetite-related factors. Such data may provide new information regarding the effects of an SVR on HCV-infected patients.

Interferon (IFN) therapy has been suggested to influence the patient condition, in not only those with an SVR but even those without an SVR (8-10). Thus, the effects of HCV elimination after DAA therapy should be precisely clarified by a comparison with a control group that did not achieve an SVR. However, because of the extremely low ratio of non-SVR patients with current DAA therapies, such a comparison has been difficult to conduct.

In Japan, Daclatasvir/Asnaprevir (DCV/ASV) combination therapy was first approved as IFN-free therapy for hepatitis C in 2014. The SVR ratio with DCV/ASV therapy is around 85-89% (11,12), so clinical data from a moderate number of patients without an SVR (non-SVR patients after DAA therapy) are available.

In the current study, we preliminarily evaluated the changes in metabolism- and appetite-related molecules in patients with and without an SVR after the same DAA therapy.

Patients and Methods

Patients and DAA therapy. Among the HCV-infected patients who received DCV/ASV treatment in our Department, we assessed those whose preserved sera could be used for the current study. An SVR was defined when serum HCV RNA was undetectable at 24 weeks after the completion of the treatment. Non-SVR was defined when serum HCV RNA was still detected within 24 weeks after the termination of the DCV/ASV treatment.

Measurement of metabolic molecules. To measure multiple hormones, we used the BioPlex^® protein assay system (Bio-Rad, Hercules, CA, USA) (13,14). We selected eight appetite-related gastrointestinal hormones, including total amylin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1), ghrelin, insulin, leptin, pancreatic polypeptide (PP) and peptide YY (PYY) from the MILLIPLEX™ MAP Human Metabolic Hormone Panel (Millipore, Billerica, MA, USA) and measured these hormones according to the manufacturer’s instructions (15-18). We used 200 μl of preserved sera per sample and directly applied it to the system for measuring multiple hormones simultaneously. Among those hormones, the results of amylin, ghrelin and PYY were excluded from the analyses, as more than half of the samples were shown to be below the quantification limits (amylin, 13 pg/ml; ghrelin, 13 pg/ml; PYY, 28 pg/ml), and median values could not be determined. Thus, we used the data of the remaining five hormones.

We compared the values of the hormones at the initiation of the DAA therapy and at 24 weeks after the completion of the therapy. The hormones were measured both in 15 non-SVR (control) patients and in 15 patients with an SVR. We screened for SVR patients whose preserved sera were available, and selected 15 SVR patients who have similar clinical characteristics to the non-SVR patients regarding their age and the degree of liver fibrosis (histological fibrosis stage or platelet count). The FIB-4 index, which is a highly used biomarker for liver fibrosis, was determined based on the following formula: FIB-4=Age [years]×AST [U/l]/(platelets [10⁹/l]×(ALT [U/l])^1/2) (19).

The current study was conducted with the approval by the ethics committee of the institutional review board (Nos. 1831 and 3321).

Statistical analyses. Numerical variables were shown as the median (interquartile range), and the differences between two groups were evaluated using the Mann-Whitney U-test. Differences in the frequency of categorized variables between two groups were evaluated using Fisher’s exact test. The changes in the values of appetite-related hormones between the pre- and post-treatment phases were assessed using Wilcoxon’s signed-rank test. A p-value less than 0.05 was defined as statistically significant.

Results

Antiviral effects of DCV/ASV therapy. Among a total of 119 HCV-infected patients who received DCV/ASV treatment, 104 (87.3%) achieved an SVR, and the remaining 15 were classified as non-SVR patients. The basic characteristics of patients were not significantly different between the SVR and non-SVR groups (Table I). In the SVR group, DCV/ASV treatment improved several liver-related variables at 24 weeks after the completion of therapy (Figure 1).

Table I. Basic clinical data of patients who received daclatasvir and asunaprevir combination therapy.

Numerical variables were shown as the median (interquartile range). SVR: Patients with a sustained viral response; Non-SVR: patients without an SVR; AST: aspartate aminotransferase; ALT: alanine aminotransferase; γ-GTP: γ-glutamyl transpeptidase; ALP: alkaline phosphatase; ND: not determined; NS: not significant.

Figure 1. Changes in the liver-related variables after HCV elimination. Several liver-related variables at the initiation of the DAA therapy and at 24 weeks after the completion of the therapy in patients with a sustained viral response (N=104) were compared. Box plots: 10^th percentile, 25^th percentile; median, 75^th percentile and 90^th percentile. AST: Aspartate aminotransferase; ALT: alanine aminotransferase; γ-GTP: γ-glutamyl transpeptidase; ALP: alkaline phosphatase.

Changes in metabolic hormones after DCV/ASV therapy. To assess the changes in metabolism- and appetite-related hormones after HCV elimination, we selected 15 SVR patients who have similar clinical characteristics to the 15 non-SVR patients in relation to their age and degree of liver fibrosis. No significant difference was found in the variables, including the FIB-4 index, a major biomarker for liver fibrosis (19) (Table II). As described in the “Patients and Methods” section, we analyzed the data of the five appetite-related gastrointestinal hormones, including GIP, GLP-1, insulin, leptin and PP. In patients with an SVR, the values of GLP-1 and leptin were significantly increased at 24 weeks after the completion of DAA therapy (Table III). However, no significant changes were observed in non-SVR patients, regardless of the receipt of DAA treatment (Table IV).

Table II. Basic clinical data of patients who received daclatasvir and asunaprevir combination therapy.

Numerical variables were shown as the median (interquartile range). SVR: Patients with a sustained viral response; Non-SVR: patients without an SVR; ND: not determined; NS: not significant.

Table III. Changes in the appetite-related hormones in patients with an SVR.

Numerical variables were shown as the median (interquartile range). SVR, Sustained viral response; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; PP, pancreatic polypeptide; NS, not significant.

Table IV. Changes in the appetite-related hormones in patients without an SVR.

Numerical variables were shown as the median (interquartile range). SVR, Sustained viral response; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; PP, pancreatic polypeptide; NS, not significant.

Discussion

Because of advances in the treatment of viral hepatitis, a nationwide survey showed that the rate of hepatitis virus-associated liver cirrhosis, particularly HCV-related cirrhosis decreased in Japan (20). HCV elimination is known to improve various physical and mental statuses, including changes in the metabolism of patients (4-7). Using the multiple cytokine assay system, we simultaneously measured several molecules with a small volume of sera (200 μl per sample) and explored whether or not appetite-related hormones were affected by HCV elimination.

‘Lifestyle’ covers various aspects, including the physical exercise and dietary habits. Recently, sarcopenia (muscle volume loss) has been recognized as a clinically important factor, and the importance of exercise is being increasingly recognized (21-24). However, factors associated with the dietary habit are less frequently assessed, and in particular, changes in appetite-related hormones in response to HCV elimination have barely been examined. Unlike non-SVR patients, GLP-1 and leptin levels were increased in those who achieved an SVR in our study. Previous studies have suggested that various metabolic functions, including the glucose metabolism, are altered in HCV-eliminated patients (4,5). Our results suggest that changes in appetite-related hormones may be involved in the changes to the metabolism in HCV-eliminated patients.

The current study is unique in that we investigated the changes in appetite-related hormones in response to HCV elimination. In addition, data from patients who did not achieve an SVR after DAA therapy were used as the control set. Unlike IFN therapy (8-10), the failure of antiviral therapy was suggested to have relatively little influence on patients who received DAA therapy (Table Ⅳ). Recently, a study that analyzed patients with non-SVR was reported (25); however, the results were obtained from patients who received various types of DAA therapies. Given the recently reported high SVR rate (98-99%), comparing data between SVR and non-SVR patients who receive the same treatment protocol is not easy and is specific to this study. To our knowledge, the changes in appetite-related hormones in response to HCV elimination have not been previously reported, though ‘eating habits’ are quite important for human health (26). Thus, our data would provide new information regarding the effects of an SVR on HCV-infected patients.

Several limitations associated with the present study warrant mention. First, this was a retrospective study using preserved sera. Second, based on the fact that 10-15% of patients failed to achieve an SVR following DCV/ASV therapy, we used these patients as non-SVR cases; however, this number was still relatively small. Third, we did not clarify the clinical impact of the changes in appetite-related hormones, such as the influence on the appetite, dietary habit and body weight, although a recent report suggested that an SVR cause a gain in body weight (27). Thus, the present study is a preliminary one, and our results should be evaluated in another study with a larger number of patients.

In summary, this is the first report regarding the changes in gastrointestinal hormones in response to HCV elimination. Our results suggest that some hormones that are related to the dietary habit and/or appetite may be influenced by HCV elimination following DAA treatment.

Conflicts of Interest

The Authors declare no conflicts of interest in association with the present study.

Authors’ Contributions

YS proposed the study concept, collected the data, and analyzed the data; HE designed the study, analyzed the data, and wrote the manuscript. NA, TT, NI, YY, AF, KY, RY, SK, SO, RN, HS, and TN collected and interpreted the data; HI collected the data and supervised the study. All of the Authors reviewed and edited the manuscript and approved the final version of the manuscript.

Acknowledgements

We are grateful to Kanazawa N, Kasuya Y, Inui S, Fujii S, Kido H, Shimoji Y and Minemoto K (Hyogo College of Medicine) for their technical and secretarial assistance.