Integrative analysis of multiple genomic data from intrahepatic cholangiocarcinoma organoids enables tumor subtyping.

As genomic analysis technology has advanced, it has become possible to sub-classify intrahepatic cholangiocarcinoma (ICC) at the histological or molecular level. Here, we verify the recently suggested two subgroups of ICC in the organoids model, compare the characteristics between types. ICC patients are subclassified into small-duct (SD) and large-duct (LD) subtype according to histological characteristics. ICC organoids are established, and unsupervised principal component analysis clustering separates each type of ICC. Differential gene expression reveals enrichment on KRAS, TGFß and ERBB2 signaling pathways in LD-type compared with SD-type (P < 0.05). Gene set enrichment analysis demonstrates that the cholangiocarcinoma class 2 signature, defined by Andersen et al., is enriched in the LD-type (enrichment Score = 2.19, P < 0.001). A protein-protein interaction network analysis identifies ZNF217 as a significant hub protein (odds ratio = 4.96, P = 0.0105). We perform prospective modeling of histological subtype using patient-derived organoids. Moreover, gene expression profiling of ICC organoids enables identification of type-specific targetable pathways.

Anti-Cancer Effects of YAP Inhibitor (CA3) in Combination with Sorafenib against Hepatocellular Carcinoma (HCC) in Patient-Derived Multicellular Tumor Spheroid Models (MCTS).

PURPOSE: To assess the expression levels of YAP and TAZ in patient-derived HCC tissue and identify the effects of YAP/TAZ inhibition depending on the baseline YAP/TAZ expression when combined with sorafenib using a patient-derived multicellular tumor spheroid (MCTS) model. METHODS: Primary HCC cell lines were established from patient-derived tissue. Six patient-derived HCC cell lines were selected according to YAP/TAZ expression on Western blot: high, medium, low. Then, MCTS was generated by mixing patient-derived HCC cells and stroma cells (LX2, WI38, and HUVECs) and YAP/TAZ expression was assessed using Western blot. Cell viability of MCTS upon 48 h of drug treatment (sorafenib, sorafenib with CA3 0.1 µM, and CA3 (novel YAP1 inhibitor)) was analyzed. RESULTS: Out of six patient-derived HCC cell lines, cell lines with high YAP/TAZ expression at the MCTS level responded more sensitively to the combination therapy (Sorafenib + CA3 0.1 µM) despite the potent cytotoxic effect of CA3 exhibited in all of the patient-derived HCCs. CONCLUSION: Targeting YAP/TAZ inhibition using the novel YAP1 inhibitor CA3 could be a promising therapeutic strategy to enhance sensitivity to sorafenib especially in HCCs with high YAP/TAZ expression in MCTS.

A micellized bone morphogenetic protein-7 prodrug ameliorates liver fibrosis by suppressing transforming growth factor-ß signaling.

Bone morphogenetic protein-7 (BMP-7) antagonizes transforming growth factor-ß (TGF-ß), which is critically involved in liver fibrogenesis. Here, we designed a micelle formulation consisting of a protein transduction domain (PTD) fused BMP-7 polypeptide (mPTD-BMP-7) to enhance endocytic delivery, and investigated its ability to ameliorate liver fibrosis. The mPTD-BMP-7 formulation was efficiently delivered into cells via endocytosis, where it inhibited TGF-ß mediated epithelial-mesenchymal transition. After successfully demonstrating delivery of fluorescently labeled mPTD-BMP-7 into the murine liver in vivo, we tested the mPTD-BMP-7 formulation in a murine liver fibrosis model, developed by repeated intraperitoneal injection of hepatotoxic carbon tetrachloride, twice weekly from 4 to 16 weeks. mPTD-BMP-7 effects were tested by injecting the mPTD-BMP-7 formulation (or vehicle control) into the lateral tail at a dose of 50 (n=8) or 500 µg/kg (n=10), also twice per week from 4 to 16 weeks. Vehicle-treated control mice developed fibrous septa surrounding the liver parenchyma and marked portal-to-portal bridging with occasional nodules, whereas mice treated with mPTD-BMP-7 showed only fibrous expansion of some portal areas, with or without short fibrous septa. Using the Ishak scoring system, we found that the fibrotic burden was significantly lower in mPTD-BMP-7 treated mice than in control mice (all P<0.001). Treatment with mPTD-BMP-7 protected tight junctions between hepatocytes and reduced extracellular matrix protein levels. It also significantly decreased mRNA levels of collagen 1A, smooth muscle α-actin, and connective tissue growth factor compared with that in control mice (all P<0.001). Collectively, out results indicate that mPTD-BMP-7, a prodrug formulation of BMP-7, ameliorates liver fibrosis by suppressing the TGF-ß signaling pathway in a murine liver fibrosis model.

Impaired antibacterial response of liver sinusoidal Vγ9+Vδ2+ T cells in patients with chronic liver disease.

OBJECTIVE: The liver acts as a frontline barrier against diverse gut-derived pathogens, and the sinusoid is the primary site of liver immune surveillance. However, little is known about liver sinusoidal immune cells in the context of chronic liver disease (CLD). Here, we investigated the antibacterial capacity of liver sinusoidal γδ T cells in patients with various CLDs. DESIGN: We analysed the frequency, phenotype and functions of human liver sinusoidal γδ T cells from healthy donors and recipients with CLD, including HBV-related CLD (liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC)), alcoholic LC and LC or HCC of other aetiologies, by flow cytometry and RNA-sequencing using liver perfusates obtained during living donor liver transplantation. We also measured the plasma levels of D-lactate and bacterial endotoxin to evaluate bacterial translocation. RESULTS: The frequency of liver sinusoidal Vγ9+Vδ2+ T cells was reduced in patients with CLD. Immunophenotypic and transcriptomic analyses revealed that liver sinusoidal Vγ9+Vδ2+ T cells from patients with CLD were persistently activated and pro-apoptotic. In addition, liver sinusoidal Vγ9+Vδ2+ T cells from patients with CLD showed significantly decreased interferon (IFN)-γ production following stimulation with bacterial metabolites and Escherichia coli. The antibacterial IFN-γ response of liver sinusoidal Vγ9+Vδ2+ T cells significantly correlated with liver function, and inversely correlated with the plasma level of D-lactate in patients with CLD. Repetitive in vitro stimulation with E. coli induced activation, apoptosis and functional impairment of liver sinusoidal Vγ9+Vδ2+ T cells. CONCLUSION: Liver sinusoidal Vγ9+Vδ2+ T cells are functionally impaired in patients with CLD. Bacterial translocation and decreasing liver functions are associated with functional impairment of liver sinusoidal Vγ9+Vδ2+ T cells.

Pharmacological Inhibition of Sonic Hedgehog Signaling Suppresses Tumor Development in a Murine Model of Intrahepatic Cholangiocarcinoma.

Cholangiocarcinoma (CCC) is the second most primary liver cancer with an aggressive biological behavior, and its incidence increases steadily. An aberrant up-regulation of the sonic hedgehog signaling pathway has been reported in a variety of hepatic diseases including hepatic inflammation, fibrosis, as well as cancer. In this study, we determined the effect of a sonic hedgehog inhibitor, vismodegib, on the development of CCC. Through database analyses, we found sonic hedgehog signaling was up-regulated in human CCC, based on overexpression of its target genes, GLI1 and GLI2. Further, human CCC cells were highly sensitive to the treatment with vismodegib in vitro. Based on the data, we investigated the in vivo anti-cancer efficacy of vismodegib in CCC employing a murine model of CCC developed by hydrodynamic tail vein injection method. In the murine model, CCC induced by constitutively active forms of TAZ and PI3K exhibited up-regulated sonic hedgehog signaling. Treatment of vismodegib significantly suppressed tumor development in the murine CCC model, based on comparison of gross morphologies and liver weight/body weight. It is expected that pharmacological inhibition of sonic hedgehog signaling would be an effective molecular target therapy for CCC.

YAP/TAZ Suppress Drug Penetration Into Hepatocellular Carcinoma Through Stromal Activation.

BACKGROUND AND AIMS: HCC is the most predominant type of liver cancer affecting 800,000 people globally each year. Various small-molecule compounds targeting diverse oncogenic signaling pathways have been tested for patients with HCC, and clinical outcomes were not satisfactory. In this study, we investigated molecular signaling that determines the efficiency of drug delivery into HCC. APPROACH AND RESULTS: Hydrodynamics-based transfection (HT) was performed to develop mouse models for HCC induced by various oncogenes. Mice bearing liver cancer were treated with verteporfin at 5 weeks after HT. Multicellular HCC organoid (MCHO) models were established that contained various types of stromal cells, such as hepatic stellate cells, fibroblasts, and endothelial cells together with HCC cells. Tumor organoids were treated with verteporfin, and distributions of the drug in the organoids were assessed using fluorescence microscopy. Murine HCC models developed by HT methods showed that a high Yes-associated protein/Transcriptional co-activator with PDZ-binding motif (YAP/TAZ) activity in HCC cells impaired verteporfin penetration into the cancer. Activation of tumor stroma was observed in HCC with a high YAP/TAZ activity. Consistent with the findings in the in vivo models of HCC, MCHOs with activated YAP/TAZ signaling showed stromal activation and impaired penetration of verteporfin into the tumor organoids. Inhibition of YAP/TAZ transcriptional activity in HCC cells significantly increased drug penetration into the MCHO. CONCLUSIONS: Drug delivery into liver cancer is impaired by YAP/TAZ signaling in tumor cells and subsequent activation of stroma by the signaling. Disrupting or targeting activated tumor stroma might improve drug delivery into HCC with an elevated YAP/TAZ activity.

Genetically Engineered Mouse Models for Liver Cancer.

Liver cancer is the fourth leading cause of cancer-related death globally, accounting for approximately 800,000 deaths annually. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, comprising approximately 80% of cases. Murine models of HCC, such as chemically-induced models, xenograft models, and genetically engineered mouse (GEM) models, are valuable tools to reproduce human HCC biopathology and biochemistry. These models can be used to identify potential biomarkers, evaluate potential novel therapeutic drugs in pre-clinical trials, and develop molecular target therapies. Considering molecular target therapies, a novel approach has been developed to create genetically engineered murine models for HCC, employing hydrodynamics-based transfection (HT). The HT method, coupled with the Sleeping Beauty transposon system or the CRISPR/Cas9 genome editing tool, has been used to rapidly and cost-effectively produce a variety of HCC models containing diverse oncogenes or inactivated tumor suppressor genes. The versatility of these models is expected to broaden our knowledge of the genetic mechanisms underlying human hepatocarcinogenesis, allowing the study of premalignant and malignant liver lesions and the evaluation of new therapeutic strategies. Here, we review recent advances in GEM models of HCC with an emphasis on new technologies.

High Risk of Hepatocellular Carcinoma Development in Fibrotic Liver: Role of the Hippo-YAP/TAZ Signaling Pathway.

Liver cancer is the fourth leading cause of cancer-related death globally, accounting for approximately 800,000 deaths annually. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, making up about 80% of cases. Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for HCC. A fibrotic liver typically shows persistent hepatocyte death and compensatory regeneration, chronic inflammation, and an increase in reactive oxygen species, which collaboratively create a tumor-promoting microenvironment via inducing genetic alterations and chromosomal instability, and activating various oncogenic molecular signaling pathways. In this article, we review recent advances in fields of liver fibrosis and carcinogenesis, and consider several molecular signaling pathways that promote hepato-carcinogenesis under the microenvironment of liver fibrosis. In particular, we pay attention to emerging roles of the Hippo-YAP/TAZ signaling pathway in stromal activation, hepatic fibrosis, and liver cancer.

Effect of tibolone on the survival of early stage cervical adenocarcinoma patients.

OBJECTIVE: Gynecologic oncologists are uncertain about the safety of tibolone application in cervical adenocarcinoma (AC) patients. This study examined the possible adverse effects of tibolone on the survival of cervical AC patients. METHODS: Medical records of 70 cervical AC patients with International Federation of Gynecology and Obstetrics stages IA to IB were reviewed. A bilateral salpingo-oophorectomy was performed in all patients, and survival outcomes between tibolone users (n=38) and non-users (n=32) were compared. RESULTS: A comparison of the tibolone users with non-users revealed similar clinicopathological variables. Progression-free survival (P=0.34) and overall survival (P=0.22) were similar in the users and non-users. The risks of progression (hazard ratio [HR], 1.71; 95% confidence interval [CI], 0.46-6.37; P=0.43) and death (HR, 1.59; 95% CI, 0.06-45.66; P=0.79) were also similar in both groups. CONCLUSION: Tibolone has no adverse effect on the survival of cervical AC patients and can be administered safely to this population. These findings may be helpful in improving the quality of life of cervical AC patients.

Deubiquitinase YOD1 potentiates YAP/TAZ activities through enhancing ITCH stability.

Hippo signaling controls the expression of genes regulating cell proliferation and survival and organ size. The regulation of core components in the Hippo pathway by phosphorylation has been extensively investigated, but the roles of ubiquitination-deubiquitination processes are largely unknown. To identify deubiquitinase(s) that regulates Hippo signaling, we performed unbiased siRNA screening and found that YOD1 controls biological responses mediated by YAP/TAZ. Mechanistically, YOD1 deubiquitinates ITCH, an E3 ligase of LATS, and enhances the stability of ITCH, which leads to reduced levels of LATS and a subsequent increase in the YAP/TAZ level. Furthermore, we show that the miR-21-mediated regulation of YOD1 is responsible for the cell-density-dependent changes in YAP/TAZ levels. Using a transgenic mouse model, we demonstrate that the inducible expression of YOD1 enhances the proliferation of hepatocytes and leads to hepatomegaly in a YAP/TAZ-activity-dependent manner. Moreover, we find a strong correlation between YOD1 and YAP expression in liver cancer patients. Overall, our data strongly suggest that YOD1 is a regulator of the Hippo pathway and would be a therapeutic target to treat liver cancer.

Effect of number of retrieved lymph nodes on prognosis in FIGO stage IB-IIA cervical cancer patients treated with primary radical surgery.

AIM: In the treatment of cervical cancer, the extent of lymphadenectomy is a matter of debate. The goal of the current study was to examine the question of whether the number of retrieved lymph nodes (RLN) can influence survival of patients with early stage cervical cancer. METHODS: The medical records of 180 FIGO stage IB-IIA cervical cancer patients treated with primary radical surgery were reviewed. Patients were divided into two groups: those with ≤ 40 RLN and those with > 40 RLN. Patients were also assigned to either the bulky (tumor size > 4 cm) cervical cancer group or the non-bulky (tumor size ≤ 4 cm) cervical cancer group. RESULTS: The number of RLN had a statistically significant effect on both disease-free survival (P = 0.04) and overall survival (P = 0.02) of all patients. Patients with > 40 RLN had better prognoses than those with ≤ 40 RLN. In the bulky cervical cancer group, the number of RLN was an independent prognostic factor. In multivariate analysis for the bulky cervical cancer group, > 40 RLN had a significant positive effect on disease-free survival (adjusted hazard ratio, 0.36; 95% confidence interval, 0.13-0.97) and overall survival (adjusted hazard ratio, 0.23; 95% confidence interval, 0.06-0.90). However, number of RLN was not an independent prognostic factor in the non-bulky cervical cancer group. CONCLUSIONS: A more extensive lymphadenectomy increased the survival of bulky cervical cancer patients. This finding may be helpful in determining surgical extent before surgery for cervical cancer.

Signaling pathways implicated in alpha-melanocyte stimulating hormone-induced lipolysis in 3T3-L1 adipocytes.

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes.

IL-18 E42A mutant is resistant to the inhibitory effects of HPV-16 E6 and E7 oncogenes on the IL-18-mediated immune response.

Our previous studies showed that the down-modulation of IL-18-induced immune response caused by oncoproteins E6 and E7 as one of the mechanisms underlying immune escape in HPV-induced cervical cancer cells. E42 residue of IL-18 also appears to be critical in the activity of IL-18. Single point mutation E42 in IL-18 show promise in the study of IL-18 binding motifs for HPV oncoproteins. We attempted to ascertain whether site-specific IL-18 mutant E42A would modulate the inhibitory effects of IL-18-induced immune responses via the HPV 16 E6 and E7 oncoproteins. Compared to wild type IL-18, E42A-induced IFN-gamma production was not inhibited by HPV 16 E6 and E7. In vitro and in vivo binding assays have also revealed that E6 and E7 do not result in the inhibition of the binding of E42A to its IL-18 receptor alpha chain. There were no effects on the E42A-induced phosphorylations of p38 and JNK observed in the presence of E6 or E7. The degradation of IkappaB by E42A was not affected by E6 or E7 in NK0 cells. Moreover, E42A-induced NF-kappaB activation was also not inhibited by these oncoproteins. These results suggest that E42A is a stronger activator than wild type IL-18, and is not susceptible to inhibition by the HPV oncoproteins E6 and E7. Thus, it is suggested that E42A could be used in immunotherapy for patients with cervical cancer.

E7-expressing HaCaT keratinocyte cells are resistant to oxidative stress-induced cell death via the induction of catalase.

Cervical carcinoma is one of the most prevalent cancers in women worldwide, and human papillomavirus (HPV) type 16 is the most common agent linked to human cervical carcinoma. In order to identify various relevant factors affected by the E7 oncogene, we established a stable cell line, which constitutively expressed E7 using the HaCaT human keratinocyte cell line. The increased expression and activity of catalase in the E7-expressing HaCaT cells (HaCaT/E7) were verified via matrix-assisted laser desorption/ionization-time of flight, Western blot, and reverse transcription-polymerase chain reaction analyses. The regulation of catalase by E7 was investigated by the detection of catalase promoter activity. E7 enhanced the activities of both the catalase promoter and nuclear factor-kappaB, one of the major transcription factors regulating the expression of the catalase gene. HaCaT/E7 cells produced lower quantities of intracellular reactive oxygen species (ROS), and appeared to be more resistant to H(2)O(2)-induced cell death. Moreover, in order to test the specific effects of E7 on catalase induction, the HaCaT/E7 cells were transiently transfected with E7 antisense vector, resulting in reductions in both the expression and activity of catalase, and a recovery of intracellular ROS levels, thus resulting in recovered sensitivity to H(2)O(2)-induced cell death. These results suggest that the HPV 16 E7 oncogene induces higher resistance to ROS-induced cell injury in the E7-infected cells, probably via the modulation of several anti-oxidant enzymes, including catalase.

Survivin reduces activation-induced T cell death in G1 phase.

A process termed activation-induced cell death (AICD) is responsible for peripheral T cell tolerance after negative selection of self-reactive T cells, and deletion of hyperactivated T cells following the immune response. Cells in G1 phase of the cell cycle are most susceptible to AICD. We have investigated the relationship between the induction of AICD by phorbol 12-myristate 13-acetate plus ionomycin during the cell cycle and the expression of survivin, an inhibitor of the apoptosis protein (LAP) family. AICD was highly induced in cells of the human T cell line Jurkat E6.1 arrested in G1 phase, whereas survivin was hardly expressed in G1 and instead it was highly expressed in G2/M. Moreover, transient over-expression of survivin in G1 partially blocked the induction of AICD. These results suggest that survivin inhibits the induction of AICD, especially in G1 phase.

Alpha-lipoic acid decreases thiol reactivity of the insulin receptor and protein tyrosine phosphatase 1B in 3T3-L1 adipocytes.

Alpha-lipoic acid is known to increase insulin sensitivity in vivo and to stimulate glucose uptake into adipose and muscle cells in vitro. In this study, alpha-lipoic acid was demonstrated to stimulate the autophosphorylation of insulin receptor and glucose uptake into 3T3-L1 adipocytes by reducing the thiol reactivity of intracellular proteins. To elucidate mechanism of this effect, role of protein thiol groups and H(2)O(2) in insulin receptor autophosphorylation and glucose uptake was investigated in 3T3-L1 adipocytes following stimulation with alpha-lipoic acid. Alpha-lipoic acid or insulin treatment of adipocytes increased intracellular level of oxidants, decreased thiol reactivity of the insulin receptor beta-subunit, increased tyrosine phosphorylation of the insulin receptor, and enhanced glucose uptake. Alpha-lipoic acid or insulin-stimulated glucose uptake was inhibited (i) by alkylation of intracellular, but not extracellular, thiol groups downstream of insulin receptor activation, and (ii) by diphenylene iodonium at the level of the insulin receptor autophosphorylation. alpha-Lipoic acid also inhibited protein tyrosine phosphatase activity and decreased thiol reactivity of protein tyrosine phosphatase 1B. These findings indicate that oxidants produced by alpha-lipoic acid or insulin are involved in activation of insulin receptor and in inactivation of protein tyrosine phosphatases, which eventually result in elevated glucose uptake into 3T3-L1 adipocytes.

Alpha-lipoic acid inhibits adipocyte differentiation by regulating pro-adipogenic transcription factors via mitogen-activated protein kinase pathways.

Obesity is associated with a number of pathological disorders such as non-insulin-dependent diabetes, hypertension, hyperlipidemia, and cardiovascular diseases. alpha-Lipoic acid (LA) has been demonstrated to activate the insulin signaling pathway and to exert insulin-like actions in adipose and muscle cells. Based on this similarity LA is expected to promote adipogenesis in pre-adipocytes. Here, however, we report that LA inhibited differentiation of 3T3-L1 pre-adipocytes induced by a hormonal mixture or troglitazone. Northern blot analysis of cells demonstrated that this inhibition was accompanied with attenuated expression of adipocyte-specific fatty acid-binding protein and lipoprotein lipase. Electrophoretic mobility shift assay and Western blot analysis of cells demonstrated that LA modulates transcriptional activity and/or expression of a set of anti- or pro-adipogenic transcription factors. LA treatment of 3T3-L1 pre-adipocytes also resulted in prolonged activation of major mitogen-activated protein kinase signaling pathways but showed little or no effect on the activity of the insulin receptor/Akt signaling pathway. These findings suggest that LA inhibits insulin or the hormonal mixture-induced differentiation of 3T3-L1 pre-adipocytes by modulating activity and/or expression of pro- or anti-adipogenic transcription factors mainly through activating the MAPK pathways.

R-alpha-lipoic acid action on cell redox status, the insulin receptor, and glucose uptake in 3T3-L1 adipocytes.

The insulin signaling pathway has been reported to mediate R-alpha-lipoic acid- (R-LA-)-stimulated glucose uptake into 3T3-L1 adipocytes and L6 myotubes. We investigated the role of the thiol antioxidant dihydrolipoic acid (DHLA) and intracellular glutathione (GSH) in R-LA-stimulated glucose transport and explored the hypothesis that R-LA could increase glucose uptake into 3T3-L1 adipocytes in an oxidant-mimetic manner. R-LA pretreatment of 3T3-L1 cells stimulated glucose transport at early time points (30 min - 6 h), whereas it inhibited glucose uptake at later time points. Analysis of the oxidized and reduced content of LA in cells and medium showed that >90% of lipoic acid present was in its oxidized form. Furthermore, all oxidized forms of LA (S-, R-, and racemic LA) stimulated glucose uptake, whereas the reduced form, dihydrolipoic acid, was ineffective. Intracellular GSH levels were not changed at the early time points (before 12 h), while longer preincubation (24 - 48 h) of cells with R-LA significantly increased intracellular GSH. Pretreatment of adipocytes with R-LA increased intracellular peroxide levels at early time points (30 min - 6 h), after which it was decreased (12 - 48 h). R-LA also increased tyrosine phosphorylation of immunoprecipitated insulin receptors from 3T3-L1 adipocytes. These results indicate that (i) 3T3-L1 adipocytes have a low capacity to reduce R-LA and the oxidized form of lipoic acid is responsible for stimulating glucose uptake, (ii) R-LA modulates glucose uptake by changing the intracellular redox status, and (iii) the insulin receptor is a potential cellular target for R-LA action.