Efficacy of canagliflozin against nonalcoholic fatty liver disease: a prospective cohort study.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease worldwide and is characterized by insulin resistance, hepatic steatosis and often prediabetes or diabetes. Canagliflozin, a selective sodium glucose cotransporter 2 inhibitor, is a new oral anti-diabetic drug that reduces hyperglycaemia by promoting urinary glucose excretion. Glycosuria produced by canagliflozin is associated with weight loss, mainly due to reduced fat volume and improve insulin resistance. Reduced body weight and improvement of insulin resistance by canagliflozin may be an effective treatment for NAFLD. METHODS: Thirty-five patients with NAFLD (17 men and 18 women) were enrolled and administered canagliflozin (100 mg). Body weight and serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GTP), low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides (TG), blood sugar (BS), glycated haemoglobin (HbA1C), uric acid (UA) and ferritin, and fibrosis-4 (FIB-4) index values were measured at baseline and at 3-month and 6-month follow-up visits. RESULTS: Body weight and serum levels of AST, ALT, γ-GTP, TG, UA, HbA1C, BS and ferritin decreased significantly after 3 and 6 months of canagliflozin treatment. Serum BS levels and FIB-4 index values decreased slightly following 3 months of treatment; these results reached significance after 6 months. Reduced serum ALT levels at 6 months were significantly correlated with baseline HbA1C and ferritin levels. Moreover, a significant correlation between reduced body weight and serum ALT levels was observed at 6 months. Decreased serum ALT levels were significantly correlated with decreased serum ferritin at 6 months. CONCLUSIONS: Canagliflozin significantly reduced the serum levels of BS, HbA1C, TG, UA and ferritin, as well as FIB-4 index values and body weight, with improved liver function. Sodium glucose cotransporter 2 inhibitors may be an important therapeutic modality for improving liver injury in NAFLD patients.

Hepatic arterial infusion of 5-fluorouracil in combination with subcutaneous interferon-alpha for advanced hepatocellular carcinoma.

BACKGROUND/AIMS: Transcatheter arterial chemoinfusion (TACI) is the main therapeutic modality for advanced hepatocellular carcinoma (HCC) with portal thrombus. However, TACI is not sufficient to improve prognosis. In this study, we evaluated the response to hepatic arterial infusion of 5-fluorouracil (5-FU) in combination with subcutaneous interferon (IFN)-alpha in patients with advanced HCC. METHODOLOGY: Ten patients (men, 8; women, 2; mean age, 55-77) with advanced HCC were enrolled in this study. Hepatic arterial infusion of 5-FU (500 mg/24 hrs) was performed for 5 days on the first and second week. IFN-alpha (5 x 10(6) International Units) was subcutaneously administered three times a week for 4 weeks (1 therapeutic course). Response to therapy was evaluated by abdominal computed tomography at the end of two courses of therapy. RESULTS: Seven patients received more than two courses of therapy. One patient (14%) showed complete response (CR). Four patients had stable disease (SD) (57%) and the remaining 2 patients had progressive disease (PD) (29%). Tumor markers decreased in all patients except 1 with PD. The 6-month survival rate was 40%. Therapy was discontinued in 3 patients due to severe adverse effects; all of these patients were over 70 years old, and had moderate liver dysfunction (Child-Pugh score of Grade B) before initiation of therapy. CONCLUSIONS: The goal of the therapy with hepatic arterial infusion of 5-FU in combination with subcutaneous IFN-alpha was attained in only 14% among our advanced HCC patients. The tumor completely disappeared in 1 patient, suggesting that this therapeutic modality may be of potential benefit in advanced HCC patients. However, this therapy should be performed with caution in patients with poor hepatic function (grade B or C of Child-Pugh score) and in those more than 70 years old.

Transfecting the multidrug resistance protein 2 gene improves transcellular organic anion transport.

Eisai hyperbilirubinuria rats (EHBRs) lack functionally active multidrug resistance protein 2 (Mrp2), which causes impaired biliary excretion of numerous organic anions. We previously reported that Mrp3 expression is enhanced while organic anion transporting polypeptide 1 (Oatp1) or Oatp2 expression is reduced in the liver of EHBRs. Mrp3 mediates basolateral efflux of organic anions but not canalicular export. In this study, we transfected the human MRP2 gene into the liver of EHBRs and evaluated whether its transfection improves transcellular transport of organic anions in hepatocytes of EHBRs. The protein expression vector (pDEST26) including the full-length human MRP2 cDNA was developed. This vector was mixed with the hemagglutinating virus of Japan (HJV) envelope protein and transfected into the liver of EHBRs via the portal vein. Expression of Mrp3, Oatp1 and Oatp2 was evaluated by reverse transcription-polymerase chain reaction and Western blot analysis. mRNA and protein expression of MRP2 were detected in hepatocytes from transfected EHBRs. The serum-conjugated bilirubin level in EHBRs decreased to a normal level (35.7 to 6.4 micromol/l) with the expression of human MRP2. The change in expression of Mrp3, Oatp1 and Oatp2 in the liver of EHBRs was normalized by transfecting MRP2. Transfection of human MRP2 was performed using the HJV envelope protein. Transfection of MRP2 is useful for improving the transcellular transport of organic anions in the livers of EHBRs.

Dubin-Johnson-like black liver with normal bilirubin level.

Black liver is a common finding in Dubin-Johnson syndrome (DJS), which is caused by the lack of multidrug resistance-associated protein 2 (MRP2). Impaired excretion of epinephrine metabolites is believed to be a cause of black liver in DJS. Recently, we experienced a patient with black liver whose serum bilirubin level was normal. Coarse brown granules were observed in the hepatocytes, and this finding closely resembled that observed in DJS. However, the granules were negative for Schmorl staining. The MRP2 gene did not show any mutation. Immunostaining study demonstrated MRP2 protein expression in the liver, and it was localized in the canalicular membranes of hepatocytes. This case illustrates for the first time that DJS is not the only cause of black liver.

Genetic polymorphisms of bilirubin uridine diphosphate-glucuronosyltransferase gene in Japanese patients with Crigler-Najjar syndrome or Gilbert's syndrome as well as in healthy Japanese subjects.

BACKGROUND AND AIM: Numerous mutations of bilirubin uridine diphosphate-glucuronosyltransferase gene (UGT1A1) have been reported in patients with familial unconjugated hyperbilirubinemia. The UGT1A1 mutation appears to be considerably different among ethnic groups. To clarify the incidence of this gene mutation in the Japanese population, the presence of UGT1A1 mutation was investigated in a group of Japanese patients with Crigler-Najjar syndrome type 2 (CNS2) and Gilbert's syndrome (GS), as well as in healthy anicteric subjects. METHODS: Four patients with CNS2, 63 patients with GS, and 71 healthy subjects were enrolled in the study. The promoter and coding regions of UGT1A1 were amplified by polymerase chain reaction (PCR) from genomic DNA isolated from leukocytes. The PCR products were directly sequenced by a dye terminating method. The UGT1A1 enzyme activity was determined in COS7 cells transfected with wild or P364L (1091 C > T) mutant DNA. RESULTS: Homozygous Y486D was observed in all four patients with CNS2. The GS patients had UGT1A1 mutations with 13 different genotypes in the promoter and coding region. Homozygous TA insertion in the TATA box (TA7) of the promoter region (TA7/7; 33%), homozygous G71R (9%), and combination of TA7/6 and heterozygous G71R (17%) were the most frequent findings in GS patients. Homozygous or heterozygous Y486D (8%) and P229Q (8%) were also observed in GS. A novel mutation, heterozygous P364L, was also identified in a GS patient. In addition to GS patients, homozygous or heterozygous TA7, G71R, and heterozygous Y486D were also observed in healthy subjects. The allele frequency of G71R and TA7 was 0.183 and 0.113 in healthy subjects, respectively. The P364L UGT1A1 enzyme activity was 64.4% lower than the wild-type enzyme activity. CONCLUSIONS: Polymorphisms in the coding region of UGT1A1 were commonly observed in Japanese patients with GS and in healthy subjects. The genetic basis of hyperbilirubinemia appears to be different between the Japanese and Caucasian populations.

Modulation of organic anion transporting polypeptide 1 and multidrug resistance protein 3 expression in the liver and kidney of Gunn rats.

Background: Gunn rat is an animal model of Crigler-Najjar syndrome (CNS) type I that develops jaundice due to defect of bilirubin conjugation. Bilirubin UDP-glucuronosyltransferase (UGT1A1), which plays a critical role in bilirubin glucuronidation, has been reported to be deficient in CNS type 1. On the other hand, little is known about the expression of organic anion transporters in Gunn rats. In the present study, we evaluated expressions of organic anion transporting polypeptide (oatp) 1 and 2, multidrug resistance-associated protein (mrp) 2 and mrp3 in the liver and kidney of Gunn rats. Methods: Serum samples, liver and kidney tissues were obtained from Gunn rats and normal SD rats ( [Formula: see text], in each group). Semi-quantitative mRNA expression of oatp1, oatp2, mrp2, and mrp3 mRNA was evaluated by constructed reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions were determined by Western blotting and by immunohistochemistry. Results: Marked elevation of serum unconjugated bilirubin concentration ( [Formula: see text] micromol/l) was observed in Gunn rats. Hepatic expression of oatp1 and oatp2 mRNA was 44% ( [Formula: see text] ) and 35% ( [Formula: see text] ) lower in Gunn rats than in SD rats, respectively. Hepatic oatp1 protein expression was 37% ( [Formula: see text] ) lower in Gunn rats than in SD rats. In contrast to oatp1, hepatic expression of mrp3 mRNA and protein was 76% ( [Formula: see text] ) and 557% ( [Formula: see text] ) higher in Gunn rats than in SD rats, respectively. Hepatic expression of oatp2 and mrp2 protein was not significantly different between Gunn rats and SD rats. Like the Western blot analysis, immunohistochemical staining disclosed decrease of oatp1 and increase of mrp3 protein expressions in the liver of Gunn rats. Decrease of oatp1 and increase of mrp3 expressions were also observed in the kidney of Gunn rats. Conclusion: Decreased expression of oatp1 and increased expression of mrp3 were observed in the liver and kidney of Gunn rats. Deficient UGT1A1 activity-associated retention of unconjugated bilirubin in the hepatocytes may modulate the expressions of these transporters in Gunn rats.

Increased hepatic and renal expressions of multidrug resistance-associated protein 3 in Eisai hyperbilirubinuria rats.

BACKGROUND AND AIM: Eisai hyperbilirubinuria rats (EHBR) are animal models of Dubin-Johnson syndrome, which suffer from jaundice due to impaired biliary excretion of bilirubin glucuronides. In EHBR, deficiency of multidrug resistance-associated protein 2 (mrp2) causes defective biliary excretion of numerous organic anions. However, little is known about the expression of other organic anion transporters in this mrp2-deficient model. The aim of the present study was to investigate adaptive expressions of mrp1, mrp3, mrp6, organic anion transporting polypeptide 1 (oatp1) and oatp2 in liver and kidney of EHBR. METHODS: For the present study, EHBR (n = 5) were used. Hepatic and renal mRNA expression of the aforementioned transporters was determined by constructed semiquantitative reverse transcription polymerase chain reaction assay. Their protein expression was determined by western blotting. Localization of hepatic and renal mrp3 was confirmed by immunohistochemistry. Sprague-Dawley (SD) rats (n = 5) were used as normal controls. RESULTS: Deficiency of mrp2 protein was confirmed in EHBR. Hepatic and renal expression of mrp3 mRNA was 53.6% (P < 0.001) and 82.9% (P < 0.001), and its protein expression was 298.9% (P < 0.001) and 245.0% (P = 0.001) higher in EHBR than in SD rats, respectively. Hepatic and renal expression of mrp1 and mrp6 mRNA was not significantly different between EHBR and SD rats. The mrp1 and mrp6 proteins were expressed in very low amounts in the liver and kidney of both EHBR and SD rats. In contrast to mrp3, hepatic expression of oatp1 and oatp2 mRNA was 33.9% (P = 0. 001) and 38.6% (P < 0.001), and their protein expression was 57.4% (P < 0.05) and 51.0% (P < 0.01) lower in EHBR than in SD rats, respectively. Hepatic and renal mrp3 protein was localized at the basolateral membrane. CONCLUSIONS: Mrp3 plays an important role in the compensation of mrp2 deficiency in liver and kidney of EHBR. Hepatic expressions of mrp3, oatp1 and oatp2 changed adaptively in this animal model. This is a compensatory mechanism for reducing injury to hepatocytes from cytotoxic materials that increase in mrp2 deficiency.