The authors have declared that no competing interests exist.
Conceived and designed the experiments: JA MRG. Performed the experiments: IR MdMDH JAdC AR IC JDB. Analyzed the data: JA MRG. Contributed reagents/materials/analysis tools: DN MM BF. Wrote the paper: JA MRG.
To investigate the influence of metformin use on liver dysfunction and hepatic encephalopathy in a retrospective cohort of diabetic cirrhotic patients. To analyze the impact of metformin on glutaminase activity and ammonia production
Eighty-two cirrhotic patients with type 2 diabetes were included. Forty-one patients were classified as insulin sensitizers experienced (metformin) and 41 as controls (cirrhotic patients with type 2 diabetes mellitus without metformin treatment). Baseline analysis included: insulin, glucose, glucagon, leptin, adiponectin, TNFr2, AST, ALT. HOMA-IR was calculated. Baseline HE risk was calculated according to minimal hepatic encephalopathy, oral glutamine challenge and mutations in glutaminase gene. We performed an experimental study
Hepatic encephalopathy was diagnosed during follow-up in 23.2% (19/82): 4.9% (2/41) in patients receiving metformin and 41.5% (17/41) in patients without metformin treatment (logRank 9.81; p = 0.002). In multivariate analysis, metformin use [H.R.11.4 (95% CI: 1.2–108.8); p = 0.034], age at diagnosis [H.R.1.12 (95% CI: 1.04–1.2); p = 0.002], female sex [H.R.10.4 (95% CI: 1.5–71.6); p = 0.017] and HE risk [H.R.21.3 (95% CI: 2.8–163.4); p = 0.003] were found independently associated with hepatic encephalopathy. In the enzymatic assay, glutaminase activity inhibition reached 68% with metformin 100 mM. In Caco2 cells, metformin (20 mM) decreased glutaminase activity up to 24% at 72 hours post-treatment (p<0.05).
Metformin was found independently related to overt hepatic encephalopathy in patients with type 2 diabetes mellitus and high risk of hepatic encephalopathy. Metformin inhibits glutaminase activity
Hepatic encephalopathy (HE) is one of the major complications of liver cirrhosis affecting one third of cirrhotic patients
MET group (n = 41) | Non-MET group (n = 41) | Significance | |
|
60.2±9 | 60.4±10 | 0.908 |
|
34 (82.9%) | 28 (68.3%) | 0.123 |
|
5.9±1.0 | 6.3±1.6 | 0.194 |
|
9.0±2.4 | 9.9±4.2 | 0.285 |
|
0.476 | ||
|
26 (63.4%) | 20 (48.8%) | |
|
9 (22%) | 13 (31.7%) | |
|
1 (2.4%) | 1 (2.4%) | |
|
0 (0%) | 2 (4.9%) | |
|
5 (12.2%) | 5 (12.2%) | |
|
8.3±5.2 | 6.7±4.3 | 0.203 |
|
24.2±16.7 | 19.5±12.9 | 0.231 |
|
8.5±5.4 | 9.3±3.4 | 0.619 |
|
101.2±45.5 | 111.9±66 | 0.547 |
|
14.3±8.9 | 18.2±7.7 | 0.203 |
|
20.2±16.6 | 20±16.9 | 0.977 |
|
12.7±3.4 | 17.6±11.7 | 0.159 |
|
55.1±63.8 | 44.3±25.8 | 0.553 |
|
43.6±43.9 | 45.3±45.3 | 0.905 |
|
7 (17.1%) | 18 (43.9%) | 0.008 |
|
5 (12.2%) | 6 (14.6%) | 0.746 |
|
39.6±28.3 | 45.4±26.5 | 0.344 |
Hepatic encephalopathy | Significance | |
|
0.002 (Log Rank 9.81) | |
|
4.9% (2/41) | |
|
41.5% (17/41) | |
|
0.018 (Log Rank 5.57) | |
|
21.4% (6/28) | |
|
45.4% (10/22) | |
|
0.006 (Log Rank 7.57) | |
|
45.8% (11/24) | |
|
19.2% (5/26) |
GLS: glutaminase. OGC: oral glutamine challenge. MHE: minimal hepatic encephalopathy.
The aim of this study was to determine whether the metformin use was associated with decreased risk of hepatic encephalopathy in diabetic cirrhotic patients and to analyze the ability of metformin to inhibit glutaminase activity
Eighty-two consecutive diabetic cirrhotic patients from the Unit for Clinical Management of Digestive Diseases, University Hospital of Valme, were included. The study started either with the first visit to Hepatology office or with the first hospital admission and outcomes to finish were survival and liver transplantation. Exclusion criteria were: age≤18 years; non-diabetic patients; patients with type 1 diabetes mellitus; and patients with treatment ongoing for cirrhosis. The protocol was approved by the CEIC of University Hospital of Valme (Sevilla, Spain) and all patients provided written informed consent to participate in this study. The study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice. A total of 41 cases and 41 controls were included. They were classified according to insulin sensitizers experienced. Cases were defined as patients who underwent metformin treatment, while controls were defined as cirrhotic patients with type 2 diabetes mellitus without metformin treatment. Metformin-experienced average time was 33.4±26.7 months. Type 2 diabetes mellitus was diagnosed according to the
Hepatic encephalopathy | Multivariate |
|
[H.R. 11.4 (95% CI: 1.2–108.8); p = 0.034] |
|
[H.R. 1.12 (95% CI: 1.04–1.2); p = 0.002] |
|
[H.R. 10.4 (95% CI: 1.5–71.6); p = 0.017] |
|
[H.R. 21.3 (95% CI: 2.8–163.4); p = 0.003] |
Baseline analysis, using commercial tests, included: insulin, glucose, glucagon, TNFr2, leptin, adiponectin, AST and ALT. HOMA-IR was calculated [glucose (mmol/L) * Insulin (IU/ml)/22,5]. Cirrhosis was defined and based on liver biopsy, ultrasound, endoscopic analysis and biochemical parameters.
Each bar represents the mean±SD (all experiments were conducted by triplicate).
3A) Glutaminase activity inhibition in cells assay (%), according to metformin concentration; 3B) Ammonia concentration in cells assay, according to metformin concentration. Each bar represents the mean ± SD (all experiments were conducted by triplicate). *p≤0.05 vs. the corresponding control sample. #p≤0.05 vs. the same group collected at the previous time point.
Minimal hepatic encephalopathy (MHE) was diagnosed based on psychometric hepatic encephalopathy score (PHES) and critical flicker frequency (CFF) (Hepatonorm™ Analyzer (R&R Medi-Business Freiburg GmbH, Freiburg, Germany)). This battery comprises the digit symbol test (DST), the number connection test A (NCT-A), the number connection test B (NCT-B), the serial dotting test (SDT), and the line drawing test (LDT). Patients were classified as having MHE when the PHES score was less than −4 points or the CFF value was below the cut-off (38 Hz)
We performed an experimental study
First, in an enzymatic assay, we tested different metformin doses (0, 10, 25, 50 and 100 µM) with a constant glutamine concentration (100 mM). Ammonia production was measured to determine the glutaminase activity.
On the other hand, human colonic epithelial mammalian cell line of Caco2 (American Type Culture Collection, ATCC) was maintained in DMEM medium pH 7.4 supplemented with 10% fetal bovine serum, 2.2 g/L HCO3Na, 100 mM sodium pyruvate, 0.292 gr/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin in 5% CO2 at 37°C. Cell assay was initiated 24 hr after seeding. Caco2 cells (10000 cells/cm2) were cultured in presence of different metformin doses (0, 20, 50, 100 and 200 mM) and samples (cell pellet and cultured medium) were collected after 0, 24, 48 and 72 hr post-treatment. Glutaminase activity was determined by the measurement of ammonia production.
Results are expressed as mean±SD of 3 independent experiments. Data were compared using ANOVA with the Least Significant Difference (LSD) test as posthoc multiple comparison analysis. We used the Kaplan-Meier method (log rank test to compare curves), Chi-square and T-student. Cox’s regression was used for univariate analysis and those variables with p<0.150 were entered into the multivariate analysis. The statistical differences were placed at p≤0.05.
Baseline epidemiological, biochemical and liver function test from both groups of patients are shown in
Patients with MHE (PHES<−4 or CFF<38 Hz) and altered OGC (NH3>128 mg/dl at 60 minutes) or showing genetic profile (Large/large microsatellite or non-TACC haplotype) were classified as high risk of HE development. Metformin use, in these cohorts, was associated with lower HE bouts, both in high-risk and low risk patients (log Rank 7.57; p = 0.006).
In chemical assay, 17.5% of glutaminase activity inhibition was obtained with a metformin concentration of 10 mM and up to 68% inhibition was reached using 100 mM. Therefore, a dose-dependent glutaminase activity was observed with metformin use (
The major findings from this work are: first, in the experimental study, we obtained a partial inhibition of glutaminase activity (about 20%), both in the chemical and cells assays when compared with control experiments. Glutaminase converts glutamine in glutamic acid, which is indispensable for cell function, together with ammonia and free radicals. Therefore, this partial inhibition is probably enough to prevent complications in cirrhotic patients (in particular hepatic encephalopathy), preserving the beneficial effects of glutaminase. Second, we observed an eight-fold lower risk of hepatic encephalopathy in metformin-experienced patients (4.9% vs 41.5%; p = 0.002), despite both cohort were similar in liver function and HE risk score. Metformin effects on glutaminase activity and inflammatory state (modulated by glycemic control) could explain, at least in part, this result. HOMA-IR correlates with protein-C-reactive activity and patients receiving metformin showed a trend to lowering TNFr2 levels than non-metformin treated patients (data not shown). Interestingly, in Child-Pugh A patients, HOMA index was independently associated with higher rate of overt HE, supporting the hypothesis that insulin resistance syndrome could promote inflammation and increased risk of overt HE. Indeed, fecal calprotectin correlated with critical flicker frequency and HE grading
Type 2 diabetes mellitus has been found associated with hepatic encephalopathy in patients with Hepatitis C-related cirrhosis. The mechanisms by which diabetes could promote hepatic encephalopathy includes: a) inflammation states in cirrhotic patients has been associated with bacterial translocation, hepatic encephalopathy and risk of spontaneous bacterial peritonitis. Insulin resistance syndrome and type 2 diabetes mellitus are considered as an inflammatory state due to increased production of pro-inflammatory cytokines, such as TNFα and IL-6
The effect of metformin on hepatic encephalopathy was stronger than expected. In spite of all these data support an active effect of metformin on cirrhotics, a selection bias could not be excluded in a retrospective analysis. Moreover, although metformin seems to be safer than exogenous insulin preventing cirrhosis complications, it may be difficult to maintain adequate blood glucose levels with insulin sensitizers alone. Thus, a balance between glucose control to avoid diabetes progression and insulin sensitivity to avoid cirrhosis complications is required. Other cirrhosis outcomes, particularly ascites, were also modified by metformin use (probably due to decrease inflammation
In conclusion, our results indicated that metformin use reduced the risk of hepatic encephalopathy in diabetic cirrhotic patients, probably by two mechanisms: inhibiting partially glutaminase activity and improving insulin sensitivity. A randomized control trial is warranted to confirm or not these data defining the usefulness of metformin in the management of liver cirrhosis.