Abstract The histological spectrum of nonalcoholic fatty liver diseases (NAFLD) ranges from hepatic steatosis to steatohepatitis and fibrosis. Berberine (BBR) is known for its therapeutic effect on obesity, hyperglycaemia and dyslipidaemia; however, its effect on NAFLD has yet to be thoroughly explored. Db/db mice and methionine-choline-deficient diet-fed mice were administered BBR via gavage. We found that BBR-treated mice were more resistant to steatosis in the liver than vehicle-treated mice and that BBR significantly reduced hepatic inflammation, fibrosis and lipid peroxides. The beneficial effect of BBR was associated with suppressing endoplasmic reticulum (ER) stress. Additionally, BBR decreased the free fatty acid-induced lipid accumulation and tunicamycin-induced ER stress in primary hepatocytes and hepatocyte cell lines. We demonstrated that BBR exhibited chaperone activity, reduced protein aggregation in vitro and alleviated tunicamycin-induced triglyceride and collagen deposition in vivo . Finally, we showed that BBR could reverse ER stress-activated lipogenesis through the ATF6/SREBP-1c pathway in vitro. These results indicated that BBR may be a new therapeutic strategy against hepatic steatosis and non-alcoholic steatohepatitis.
A non-targeted metabolomics method was employed to study metabolic characteristics in subjects with different glucose tolerance. Plasma samples of 120 participants with normal glucose tolerance (NGT), impaired glucose regulation (IGR), and type 2 diabetes (T2D) were collected. Gas chromatography/mass spectrometry (GC/MS) was used to profile and compare the plasma metabolome among the three groups. Through the use of multivariate statistical analysis, we found distinct metabolome change from NGT to IGR and to T2D. ANOVA found that the IGR and T2D groups had perturbations of monosaccharide and lipid metabolism, disorders of glucogenic amino acids, and branched-chain amino acid catabolism. Furthermore, we also found that the levels of 2-hydroxybutyrate and 2-ketoisocaproate were progressively increased with glucose tolerance severity. The results from this study help us better understand the relationship between plasma metabolism and glucose tolerance states and also suggest that 2-hydroxybutyrate and 2-ketoisocaproate may be closely associated with the development of T2D.
The phosphatases of regenerating liver (PRLs) are highly over-expressed in metastatic cancers yet their mechanism of action is poorly understood.The PRL family belongs to the PTP (protein tyrosine phosphatase) superfamily and is comprised of three closely related proteins: PRL1, PRL2, and PRL3.They all contain a C-terminal prenylation site and a single catalytic domain of roughly 170 amino acids.Like other members of the family of protein tyrosine phosphatases, their phosphatase activity occurs through a two-step catalytic cycle involving the transient phosphorylation of a catalytic cysteine residue.In PRL phosphatases, this intermediate is extremely long-lived leading to the accumulation of a cysteine-phosphorylated form of the enzyme both in vitro and in cells [1,2].While a number of different cellular substrates have been proposed, there is no consensus about their physiological substrate due, in part, to their slow rate of overall catalysis.The catalytic cysteine of PRLs readily forms a disulfide with the adjacent cysteine residue, which further decreases their effectiveness in dephosphorylating physiological substrates [1].Recently, CNNM proteins, a family of membrane proteins involved in magnesium homeostasis, were identified as PRL-binding partners [3].Disruption of the PRL-CNNM interaction promotes tumor formation and invasiveness in animal and cellular models, strongly suggesting that the physiological function of PRLs is to regulate CNNM magnesium transport.Here, we determined five crystal structures of PRL3 or PRL2 bound to the CBS-pair domain of CNNM3.In the structures, the CBS-pair domain is present as a dimer in the head-to-head arrangement that is typical for other CBS-pair domains.The CNNM3 CBS-pair domain contains a long loop that extends away from the dimerization interface and contacts the PRL catalytic site.The side chain of aspartic acid 426 sits in the pocket formed by the phosphatase P-loop and WPFDD motif and likely mimics the negatively charged phosphate of a bound substrate.Mutagenesis showed that Asp426 of CNNM3 is required for high affinity binding, suggesting that the CNNM CBS-pair domain might act as a pseudo-substrate.Addition of the CBS-pair domain inhibited phosphatase activity and CNNM3 binding was blocked by phosphorylation of the PRL active site cysteine.In addition to the many polar contacts, Pro427 and Tyr429 provide a hydrophobic surface that contacts Leu105 of PRL2 (Leu108 of PRL3).The structures reveal why disulfide formation dramatically decreases binding affinity and confirm that all three PRLs bind to CNNMs.We used isothermal titration calorimetry (ITC) experiments and extensive mutagenesis to probe the importance of PRL residues for CNNM binding.Comparison of binding activity and in vitro phosphatase activity shows that they are strongly correlated with the notable exception of the PRL3 R138E mutant which showed weak CNNM3 binding but normal phosphatase activity.These results support the hypothesis that PRLs function as pseudophosphatases in regulating the action of CNNM proteins in cancer.
Urothelial carcinoma (UC) comprises a heterogeneous group of epithelial neoplasms with diverse biological behaviors and variable clinical outcomes. Distinguishing UC histological subtypes has become increasingly important because prognoses and therapy can dramatically differ among subtypes. In clinical work, overlapping morphological findings between low-grade noninvasive UC (LGNUC), which exhibits an inverted growth pattern, and inverted urothelial papilloma (IUP) can make subclassification difficult. We propose a combination of immunohistochemistry (IHC) and molecular cytogenetics for subtyping these clinical entities. In our study, tissue microarray immunohistochemical profiles of Ki-67, p53, cytokeratin 20 (CK20) and cyclinD1 were assessed. Molecular genetic alterations such as the gain of chromosomes 3, 7 or 17 or the homozygous loss of 9p21 were also assessed for their usefulness in differentiating these conditions. Based on our analysis, Ki-67 and CK20 may be useful for the differential diagnosis of these two tumor types. Fluorescence in situ hybridization (FISH) can also provide important data in cases in which the malignant nature of an inverted urothelial neoplasm is unclear. LGNUC with an inverted growth pattern that is negative for both Ki-67 and CK20 can be positively detected using FISH.
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