Growth Hormone Stops Excessive Inflammation After Partial Hepatectomy, Allowing Liver Regeneration and Survival Through Induction of H2-Bl/HLA-G.

BACKGROUND AND AIMS: Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. APPROACH AND RESULTS: PHx was performed on C57BL/6 mice lacking GHR (Ghr-/- ), disabled for all GH-dependent Janus kinase 2 signaling (Box1-/- ), or lacking only GH-dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391-/- ), and wild-type littermates. C57BL/6 Ghr-/- mice showed striking mortality within 48 hours after PHx, whereas Box1-/- or Ghr391-/- mice survived with normal liver regeneration. Ghr-/- mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2-Bl, a key immunotolerance protein, which is up-regulated by PHx through a GH-mediated, Janus kinase 2-independent, SRC family kinase-dependent pathway. GH treatment was confirmed to up-regulate expression of the human homolog of H2-Bl (human leukocyte antigen G [HLA-G]) in primary human hepatocytes and in the serum of GH-deficient patients. We find that injury-associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr-/- mice by adenoviral delivery of H2-Bl or by infusion of HLA-G protein. Further, H2-Bl knockdown in wild-type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr-/- backcrossed on a strain with high endogenous H2-Bl expression showed a high rate of survival following PHx. CONCLUSIONS: GH induction of H2-Bl expression is crucial for reducing innate immune-mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA-G may lead to improved clinical outcomes following liver surgery or transplantation.

The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects.

The growth hormone receptor (GHR), although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK-STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling.

Role of the glucose-sensing receptor in insulin secretion.

Glucose is a primary stimulator of insulin secretion. It has been thought that glucose exerts its effect by a mechanism solely dependent on glucose metabolism. We show here that glucose induces rapid Ca2+ and cyclic AMP signals in ß-cells. These rapid signals are independent of glucose-metabolism and are reproduced by non-metabolizable glucose analogues. These results led us to postulate that glucose activates a cell-surface receptor, namely the glucose-sensing receptor. Rapid signals induced by glucose are blocked by inhibition of a sweet taste receptor subunit T1R3 and a calcium-sensing receptor subunit CaSR. In accordance with these observations, T1R3 and CaSR form a heterodimer. In addition, a heterodimer of T1R3 and CaSR is activated by glucose. These results suggest that a heterodimer of T1R3 and CaSR is a major component of the glucose-sensing receptor. When the glucose-sensing receptor is blocked, glucose-induced insulin secretion is inhibited. Also, ATP production is significantly attenuated by the inhibition of the receptor. Conversely, stimulation of the glucose-sensing receptor by either artificial sweeteners or non-metabolizable glucose analogue increases ATP. Hence, the glucose-sensing receptor signals promote glucose metabolism. Collectively, glucose activates the cell-surface glucose-sensing receptor and promotes its own metabolism. Glucose then enters the cells and is metabolized through already activated metabolic pathways. The glucose-sensing receptor is a key molecule regulating the action of glucose in ß-cells.

Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic ß-Cells.

Glucose is a primary stimulator of insulin secretion in pancreatic ß-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse ß-cells, glucose induced triphasic changes in cytoplasmic Ca(2+) concentration ([Ca(2+)]c); glucose evoked an immediate elevation of [Ca(2+)]c, which was followed by a decrease in [Ca(2+)]c, and after a certain lag period it induced large oscillatory elevations of [Ca(2+)]c. Initial rapid peak and subsequent reduction of [Ca(2+)]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca(2+), glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca(2+)]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.

Glucose-Sensing Receptor T1R3: A New Signaling Receptor Activated by Glucose in Pancreatic ß-Cells.

Subunits of the sweet taste receptors T1R2 and T1R3 are expressed in pancreatic ß-cells. Compared with T1R3, mRNA expression of T1R2 is considerably lower. At the protein level, expression of T1R2 is undetectable in ß-cells. Accordingly, a major component of the sweet taste-sensing receptor in ß-cells may be a homodimer of T1R3 rather than a heterodimer of T1R2/T1R3. Inhibition of this receptor by gurmarin or deletion of the T1R3 gene attenuates glucose-induced insulin secretion from ß-cells. Hence the T1R3 homodimer functions as a glucose-sensing receptor (GSR) in pancreatic ß-cells. When GSR is activated by the T1R3 agonist sucralose, elevation of intracellular ATP concentration ([ATP]i) is observed. Sucralose increases [ATP]i even in the absence of ambient glucose, indicating that sucralose increases [ATP]i not simply by activating glucokinase, a rate-limiting enzyme in the glycolytic pathway. In addition, sucralose augments elevation of [ATP]i induced by methylsuccinate, suggesting that sucralose activates mitochondrial metabolism. Nonmetabolizable 3-O-methylglucose also increases [ATP]i and knockdown of T1R3 attenuates elevation of [ATP]i induced by high concentration of glucose. Collectively, these results indicate that the T1R3 homodimer functions as a GSR; this receptor is involved in glucose-induced insulin secretion by activating glucose metabolism probably in mitochondria.

Lactisole inhibits the glucose-sensing receptor T1R3 expressed in mouse pancreatic ß-cells.

Glucose activates the glucose-sensing receptor T1R3 and facilitates its own metabolism in pancreatic ß-cells. An inhibitor of this receptor would be helpful in elucidating the physiological function of the glucose-sensing receptor. The present study was conducted to examine whether or not lactisole can be used as an inhibitor of the glucose-sensing receptor. In MIN6 cells, in a dose-dependent manner, lactisole inhibited insulin secretion induced by sweeteners, acesulfame-K, sucralose and glycyrrhizin. The IC50 was ∼4 mmol/l. Lactisole attenuated the elevation of cytoplasmic Ca2+ concentration ([Ca2+]c) evoked by sucralose and acesulfame-K but did not affect the elevation of intracellular cAMP concentration ([cAMP]c) induced by these sweeteners. Lactisole also inhibited the action of glucose in MIN6 cells. Thus, lactisole significantly reduced elevations of intracellular [NADH] and intracellular [ATP] induced by glucose, and also inhibited glucose-induced insulin secretion. To further examine the effect of lactisole on T1R3, we prepared HEK293 cells stably expressing mouse T1R3. In these cells, sucralose elevated both [Ca2+]c and [cAMP]c. Lactisole attenuated the sucralose-induced increase in [Ca2+]c but did not affect the elevation of [cAMP]c. Finally, lactisole inhibited insulin secretion induced by a high concentration of glucose in mouse islets. These results indicate that the mouse glucose-sensing receptor was inhibited by lactisole. Lactisole may be useful in assessing the role of the glucose-sensing receptor in mouse pancreatic ß-cells.

Return of the glucoreceptor: Glucose activates the glucose-sensing receptor T1R3 and facilitates metabolism in pancreatic ß-cells.

Subunits of the sweet taste receptor, namely T1R2 and T1R3, are expressed in mouse pancreatic islets. Quantitatively, the expression of messenger ribonucleic acid for T1R2 is much lower than that of T1R3, and immunoreactive T1R2 is in fact undetectable. Presumably, a homodimer of T1R3 could function as a signaling receptor. Activation of this receptor by adding an artificial sweetener, sucralose, leads to an increase in intracellular adenosine triphosphate ([ATP]c). This increase in [ATP]c is observed in the absence of ambient glucose. Sucralose also augments elevation of [ATP]c induced by methylsuccinate, a substrate for mitochondria. Consequently, activation of T1R3 promotes metabolism in mitochondria and increases [ATP]c. 3-O-Methylglucose, a non-metabolizable analog of glucose, also increases [ATP]c. Conversely, knockdown of T1R3 attenuates elevation of [ATP]c induced by glucose. Hence, glucose promotes its own metabolism by activating T1R3 and augmenting ATP production. Collectively, a homodimer of T1R3 functions as a cell surface glucose-sensing receptor and participates in the action of glucose on insulin secretion. The glucose-sensing receptor T1R3 might be the putative glucoreceptor proposed decades ago by Niki et al. The glucose-sensing receptor is involved in the action of glucose and modulates glucose metabolism in pancreatic ß-cells.

Identification of differentially expressed genes during proliferative response of the liver induced by follistatin.

The liver mass is controlled strictly and maintained constant in normal and pathological situations. An exception is observed after an administration of follistatin, which induces proliferation in intact liver. In the present study, we identified genes differentially expressed in proliferating liver caused by overexpression of follistatin-288. Adenovirus vector encoding follistatin-288 (Ad-FS) or green fluorescent protein was injected intraperitoneally in rats. Changes in the liver weight, expression of follistatin and nuclear bromodeoxyuridine labeling were measured. Samples taken on day 5 and day 7 were used to prepare RNA for microarray analysis. The expression of the genes was confirmed by quantitative reverse transcriptase PCR. After the injection of Ad-FS follistatin mRNA peaked on day 3, which was followed by progressive increase in the protein expression. A peak in bromodeoxyuridine labeling was observed on day 7. Microarray data from day 5 and day 7 samples showed that follistatin modified the expression of 907 genes, of which 575 were overexpressed and 332 were downregulated taking into consideration a two fold change reference compared to control rats. In particular, significant increases and time related changes in gene expression after the Ad-FS injection were found in nine genes including growth differentiation factor 15 and fibroblast growth factor 21. This study confirmed that follistatin induced proliferation in intact liver, and identified candidate genes involved in follistatin-induced liver cell growth.

Adenovirus-mediated overexpression of the activin betaC subunit accelerates liver regeneration in partially hepatectomized rats.

BACKGROUND/AIMS: The expression level of the activin betaC subunit is high in normal liver and reduces after partial hepatectomy, but its function is controversial. METHODS: To determine the role of the betaC subunit during liver regeneration, we overexpressed the betaC subunit gene in the liver by infusing adenovirus vector encoding the flag-tagged betaC subunit into the portal vein. Adenovirus vector encoding the beta-galactosidase was also infused as a control. Seventy percent hepatectomy was performed 4 days after the infection. RESULTS: Approximately 20% of hepatocytes expressed the flag-tagged betaC subunit at the time of hepatectomy and approximately 50% of hepatocytes expressed the betaC subunit 3 days after hepatectomy. In betaC-infected liver, bromodeoxyuridine labeling was significantly greater at 24 and 48 h after partial hepatectomy compared with the control liver. Consistent with this observation, the liver regeneration rate was significantly greater in betaC-transfected liver at 72 and 96 h after hepatectomy. Many of the bromodeoxyuridine-positive nuclei were observed in or by the betaC-transfected hepatocytes. CONCLUSIONS: These results indicate that liver regeneration is accelerated in betaC-overexpressing liver. The betaC subunit may function to promote replication of hepatocytes during liver regeneration.

Comparison of the function of the beta(C) and beta(E) subunits of activin in AML12 hepatocytes.

To investigate the function of the beta(C) and beta(E) subunits of activin, we overexpressed these subunits in AML12 cells, a normal hepatocyte cell line, using adenovirus vector. Overexpression of the beta(C) subunit increased [3H]thymidine incorporation and the cell number. In contrast, both [3H]thymidine incorporation and the cell number were reduced in the beta(E) overexpressing cells. When AML cells overexpressing the beta(E) subunit were cultured in medium containing 1% serum for 48 h, many of the cells died by apoptosis, whereas cells overexpressing the beta(C) subunit or beta-galactosidase survived in the same condition. To examine dimer formation, the beta(C) and beta(E) subunits were expressed in AML12 cells. In these cells, the beta(C) homodimer, the beta(E) homodimer and the beta(C)-beta(E) heterodimer were detected. When the expression level of the beta(E) subunit was increased, formation of the beta(E) homodimer was increased, while formation of the beta(C)-beta(E) heterodimer was slightly reduced. Overexpression of the beta(E) subunit did not significantly affect the formation of the beta(C) homodimer. These results indicate that the beta(C) and beta(E) subunits form homo- and heterodimers, and that the functions of the two subunits are quite different.