Masahito Nagaki

Is there a delayed gastric emptying of patients with early-stage, untreated Parkinson’s disease? An analysis using the 13C-acetate breath test

During the pre-symptomatic stage of Parkinson’s disease (PD), the idiopathic PD related abnormal synuclein immunostaining is confined to the medulla oblongata and olfactory bulb, according to Braak. In the study of the enteric nervous system of PD, it has reported that Lewy bodies were found in the Auerbach’s and Meissner’s plexuses. These lesions may cause dysfunction of the gastrointestinal tract (GI) as pre-clinical symptoms of PD. However, because l-dopa therapy itself may worsen the symptoms of the digestive tract function, it is needed to evaluate the gastrointestinal tract function in patients with early-stage, untreated (de novo) PD. In the present study, using the 13C-acetate breath test (13C-ABT), we investigated gastric emptying in 20 untreated, early-stage PD patients and 40 treated, advanced-stage PD patients, and 20 healthy volunteers. Gastric emptying was examined by the 13C-ABT [the half emptying time (HET), the peak time of the 13C% dose-excess curve (Tmax)]. The Tmax and HET of gastric emptying as assessed using the 13C-ABT was significantly delayed in untreated, early-stage PD patients as compared to the controls (Pxa0<xa00.001). The Tmax and HET of gastric emptying were not significantly delayed in untreated, early-stage PD patients as compared to treated, advanced-stage PD patients. The results demonstrated that delay in gastric emptying did not differ between untreated, early-stage and treated, advanced-stage PD patients. Gastric emptying of untreated, early-stage PD is already delayed. Delayed gastric emptying may be one of markers of the pre-clinical stage of PD.

Transcription factor HNF and hepatocyte differentiation

To know the precise mechanisms underlying the life or death and the regeneration or differentiation of cells would be relevant and useful for the development of a regenerative therapy for organ failure. Liver‐specific gene expression is controlled primarily at a transcriptional level. Studies on the transcriptional regulatory elements of genes expressed in hepatocytes have identified several liver‐enriched transcriptional factors, including hepatocyte nuclear factor (HNF)‐1, HNF‐3, HNF‐4, HNF‐6 and CCAAT/enhancer binding protein families, which are key components of the differentiation process for the fully functional liver. The transcriptional regulation by these HNFs, which form a hierarchical and cooperative network, is both essential for hepatocyte differentiation during mammalian liver development and also crucial for metabolic regulation and liver function. Among these liver‐enriched transcription factors, HNF‐4 is likely to act the furthest upstream as a master gene in transcriptional cascade and interacts with other liver‐enriched transcriptional factors to stimulate hepatocyte‐specific gene transcription. A link between the extracellular matrix, changes in cytoskeletal filament assembly and hepatocyte differentiation via HNF‐4 has been shown to be involved in the transcriptional regulation of liver‐specific gene expression. This review provides an overview of the roles of liver‐enriched transcription factors in liver function.

Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression

Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine‐1‐phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild‐type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase‐3β. In addition, ASM induced up‐regulation of glucose transporter 2 accompanied by suppression of AMP‐activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase‐1 (SphK1) diminished ASM‐mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM‐mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high‐dose glucose increased glycogen deposition and lipid accumulation in wild‐type hepatocytes but not in ASM_/_ cells. This result is consistent with glucose intolerance in ASM_/_ mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.—Osawa, Y., Seki, E., Kodama, Y., Suetsugu, A., Miura, K., Adachi, M., Ito, H., Shiratori, Y., Banno, Y., Olefsky, J. M., Nagaki, M., Moriwaki, H., Brenner, D. A., Seishima, M. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP‐activated protein kinase suppression. FASEBJ. 25, 1133‐1144 (2011). www.fasebj.org

Imaging the recruitment of cancer-associated fibroblasts by liver-metastatic colon cancer.

The tumor microenvironment (TME) is critical for tumor growth and progression. However, the formation of the TME is largely unknown. This report demonstrates a color‐coded imaging model in which the development of the TME can be visualized. In order to image the TME, a green fluorescent protein (GFP)‐expressing mouse was used as the host which expresses GFP in all organs but not the parenchymal cells of the liver. Non‐colored HCT‐116 human colon cancer cells were injected in the spleen of GFP nude mice which led to the formation of experimental liver metastasis. TME formation resulting from the liver metastasis was observed using the Olympus OV100 small animal fluorescence imaging system. HCT‐116 cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. GFP‐expressing host cells were recruited by the metastatic tumors as visualized by fluorescence imaging. A desmin positive area increased around and within the liver metastasis over time, suggesting cancer‐associated fibroblasts (CAFs) were recruited by the liver metastasis which have a role in tumor progression. The color‐coded model of the TME enables its formation to be visualized at the cellular level in vivo, in real‐time. This imaging model of the TME should lead to new visual targets in the TME. J. Cell. Biochem. 112: 949–953, 2011.

Role of acid sphingomyelinase of Kupffer cells in cholestatic liver injury in mice

Kupffer cells, resident tissue macrophages of the liver, play a key role in the regulation of hepatic inflammation, hepatocyte death, and fibrosis that characterize liver diseases. However, it is controversial whether Kupffer cells promote or protect from liver injury. To explore this issue we examined the role of Kupffer cells in liver injury, cell death, regeneration, and fibrosis on cholestatic liver injury in C57BL/6 mice using a model of partial bile duct ligation (BDL), in which animals do not die and the effects of BDL can be compared between injured ligated lobes and nonligated lobes. In cholestatic liver injury, the remaining viable cells represented tolerance for tumor necrosis factor alpha (TNF‐α)‐induced hepatocyte apoptosis and regenerative features along with AKT activation. Inhibition of AKT by adenovirus expressing dominant‐negative AKT abolished the survival and regenerative properties in hepatocytes. Moreover, Kupffer cell depletion by alendronate liposomes increased hepatocyte damage and the sensitivity of TNF‐α‐induced hepatocyte apoptosis in ligated lobes. Kupffer cell depletion decreased hepatocyte regeneration and liver fibrosis with reduced AKT activation. To investigate the impact of acid sphingomyelinase (ASMase) in Kupffer cells, we generated chimeric mice that contained ASMase‐deficient Kupffer cells and ‐sufficient hepatocytes using a combination of Kupffer cell depletion, irradiation, and the transplantation of ASMase‐deficient bone marrow cells. In these mice, AKT activation, the tolerance for TNF‐α‐induced apoptosis, and the regenerative responses were attenuated in hepatocytes after BDL. Conclusion: Kupffer cells have a protective role for hepatocyte damage and promote cell survival, liver regeneration, and fibrosis in cholestatic liver disease. Kupffer cell‐derived ASMase is crucial for AKT activation of hepatocytes that is required for the survival and regenerative responses. (HEPATOLOGY 2009.)

Critical role of CD44 in hepatotoxin-mediated liver injury

BACKGROUND/AIMSnBlocking of adhesion molecules is considered to be one of the therapeutic strategies inflammatory diseases, although it remains unclear whether this strategy is beneficial.nnnMETHODSnWe used CD44-deficient mice to assess whether inhibition of CD44 could control liver injury caused by carbon tetrachloride (CCl(4)).nnnRESULTSnCD44-deficient mice exhibited suppressed liver inflammation during the early phase (within 6h) after CCl(4) injection due to reduced inflammatory cell infiltration and cytokine production, but showed severe liver inflammation with increased numbers of apoptotic hepatocytes at the late phase (after 12h). The induction of hepatocyte apoptosis was triggered by reduced NF-kappaB activity, which was induced by the low inflammatory cytokine concentrations. Furthermore, macrophages contributed to the induction of hepatocyte apoptosis, since neutralization by an anti-CD11b antibody significantly protected against hepatocyte apoptosis. Finally, we found that blocking of MIP-2 and TNF-alpha reduced hepatocyte apoptosis with decreased numbers of intrahepatic leukocytes and reduced inflammatory cytokine production.nnnCONCLUSIONSnThese findings suggest that targeting of CD44 as a therapeutic approach for inflammatory liver diseases may require caution for particular immune systems in the liver.

Simultaneous color-coded imaging to distinguish cancer "stem-like" and non-stem cells in the same tumor.

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133+ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133− Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133+ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133+ cells were labeled with GFP and CD133− cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010.

Differentiation of mouse hepatic progenitor cells induced by hepatocyte nuclear factor-4 and cell transplantation in mice with liver fibrosis.

Background. Hepatocyte nuclear factor-4 (HNF-4) plays a central role in the differentiation process of hepatic cells. We investigated the effects of an overexpression of HNF-4 on hepatic progenitor cells isolated from a fetal mouse liver and transplantation of the cells in a mouse model of liver fibrosis. Methods. Hepatic progenitor cells were isolated from the embryonic day 14.0 fetal mouse livers and were purified by magnetic cell sorting to remove the hematopoietic cells. We transfected adenovirus-mediated HNF-4 into the cells, and analyzed the expressions of the liver-specific functions using reverse-transcription polymerase chain reaction and Northern blotting. HNF-4-overexpressing hepatic progenitor cells were then injected into recipient mice, which were treated with dimethylnitrosamine and 30% partial hepatectomy. Results. After 5 days of culture, the cells located in the center of the aggregates were stained positive for albumin, but the peripheral cells for cytokeratin 19. Adenovirus-mediated HNF-4 gene transfer resulted in increases in the expressions of HNF-4, apolipoprotein (Apo)A1, ApoC3, and pregnane X receptor messenger RNA. The mice treated with HNF-4-transfected progenitor cells survived significantly longer than the control mice (P=0.004). The plasma levels of albumin, total cholesterol, and glucose were higher in the mice treated with cells transfected by HNF-4 than in the control mice. Conclusions. These findings demonstrate that adenovirus-mediated HNF-4 transfection induces the differentiation from hepatic progenitor cells to hepatic parenchymal cells in vitro. These cells may be useful as a source for cell transplantation in liver diseases.

Role of interleukin-18 in intrahepatic inflammatory cell recruitment in acute liver injury.

Although the innate immune system has been demonstrated to play important roles as the first line of defense against various infections, little is known about the interactions between intrahepatic inflammatory cells and the cytokine network in the liver. Here, we examined the role of IL‐18 in IHL recruitment in acute liver injury. C57BL/6 mice were injected with an αCD40 mAb, and their serum IL‐18 levels were observed to increase, with subsequent recruitment of IHLs into the liver. NKT cells were involved in this liver injury, as the serum ALT levels were reduced in NKT KO mice through the suppression of macrophage and monocyte migration and cytokine production. In contrast, depletion of neutrophils exacerbated the liver injury associated with high levels of TNF‐α and IL‐18 and increased numbers of macrophages and monocytes. Treatment with a neutralizing antibody against IL‐18 reduced the serum ALT levels and inflammatory cell accumulation in the liver. Finally, additional administration of rIL‐18 with αCD40 injection caused severe liver injury with increased IFN‐γ production by NK cells. In conclusion, these findings demonstrate that IL‐18 modulates liver inflammation by the recruitment of inflammatory cells, including NKT cells, macrophages, monocytes, and neutrophils.

Pathological role of CD44 on NKT cells in carbon tetrachloride-mediated liver injury.

Aim:u2002 CD44 has a variety of functions in immune regulation and signal transduction. Although CD44 is involved in the induction of several inflammatory diseases, it remains unknown whether CD44‐targeting therapies are useful for liver diseases. Here, we examined whether CD44 blockade is effective in a chemical‐induced liver injury model.