Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

To evaluate hypoxic cells in live mouse liver tissues, immunohistochemistry for protein adducts of reductively activated pimonidazole (PARaPi) was performed using the “in vivo cryotechnique (IVCT)” followed by freeze-substitution fixation. This method was used because cryotechniques have some merits for examining biological events in living animal organs with improved time-resolution compared to conventional perfusion and/or immersion chemical fixation. Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were quickly frozen by IVCT. The frozen liver tissues were freeze-substituted in acetone containing 2% paraformaldehyde, and routinely embedded in paraffin wax. De-paraffinized sections were immunostained for PARaPi. In liver tissues of mice without hypoxia, almost no immunostained cells were detected. However, in liver tissues with 30 s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. After 60 s of hypoxia, many hepatocytes were immunostained with various degrees of staining intensity in all lobular zones, indicating different reactivities of pimonidazole in the hepatocytes to hypoxia. At this time, the general immunoreactivity also appeared to be stronger around the central veins than other portal areas. Although many hepatocytes were immunostained for PARaPi in the liver tissues with perfusion fixation via heart, those with perfusion via portal vein were not immunostained. Thus, IVCT is useful to detect time-dependent hypoxic states with pimonidazole treatment in living animal organs.     

Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice

Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation.     

Dynamics of glycogen content in the normal and cirrhotic liver following glucose administration to starving rats

Using biochemical, cytofluorimetric and television cytophotometric methods, glycogen contents were studied in normal and cirrhotic rat liver at various intervals after glucose administration to fasting animals. The obtained data indicate that after a 48 h fasting glycogen contents in normal and cirrhotic liver are equally poor. A marked rise of glycogen content in cirrhotic liver was observed only 20-30 min after glucose administration to rats. It has been established that at all intervals after glucose administration to rats hepatocytes of the portal lobule zone, both in normal and in cirrhotic liver, accumulate more glycogen than those of the central zone. Again, the intensity of glycogen accumulation in cirrhotically altered liver is significantly lower than in normal liver, due, presumably, to a lower rate of glycogen synthesis in pathologically changed liver.     

Histochemical analyses of living mouse liver under different hemodynamic conditions by “in vivo cryotechnique”

Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using “in vivo cryotechnique” (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid–Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5 min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.     

Metabolic heterogeneity of glycogen in hepatocytes of patients with liver cirrhosis

Concentrations of the total glycogen (TG) and of its labile and stable fractions (LF and SF, respectively) were determined in hepatocytes of portal and central zones of the normal human liver and in the liver of patients with cirrhosis of viral and alcohol etiology. Using the PAS reaction, TG and its LF and SF were revealed in histological sections of the material obtained by liver punction biopsies. Concentrations of TG and its fractions were measured by television cytophotometry. In liver cirrhosis, concentrations of TG, LF, and SF in both zones of the hepatic lobule were much higher than in the normal liver. The ratio between hepatocyte TG concentration in the portal zone and that in the central zone (P/C ratio), both in norm and in viral cirrhosis, exceeds 1.0 to reach, respectively, 1.26 +/- 0.02 and 1.03 +/- 0.01. The glycogen fraction composition in cells of both liver lobule zones in viral cirrhosis does not significantly differ from that in norm. On the contrary, in the liver of patients with alcoholic cirrhosis, the P/C ratio falls to 0.82 +/- 0.02 to be accompanied by qualitative changes in glycogen composition.     

Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.     

胚胎肝细胞用于肝组织工程的体外培养研究

目的:观察三维受控组装系统下,胚胎肝细胞在三维立体结构的体外生长状态,探讨胚胎肝细胞在肝组织工程中应用的可行性。方法:用清华大学机械工程系研制的"三维受控组装系统",将第15 d小鼠胚胎肝细胞作为肝组织工程的种子细胞,与以明胶为主的复合材料混合,构建成复杂三维立体结构,观察其体外生长发育状态。对体外培养1周及4周的三维类肝组织标本进行苏木精-伊红(HE)染色,免疫组织化学方法检测甲胎蛋白(AFP)及白蛋白(ALB)的表达,并对体外培养4周的三维类肝组织用PAS显色法检测肝糖原表达。结果:HE染色结果显示体外培养的胚胎肝细胞在三维支架材料中,可形成含有类血管和肝组织样结构;体外培养1周的类肝组织AFP表达呈阳性,体外培养4周的三维类肝组织ALB表达呈阳性,PAS显色亦呈阳性。结论:在三维受控组装系统的构建下,呈立体状生长的胚胎肝细胞,可逐渐形成肝组织样结构,并显示一定的肝脏功能。     

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD.     

Complementary expression of glutamine synthetase and carbamoylphosphate synthetase I in ornithine carbamoyltransferase-deficient mouse liver (spf–ash mouse)

 Glutamine synthetase and carbamoylphosphate synthetase I expression was examined immunohistochemically in livers of spf–ash homozygous and hemizygous mice, in which one of the urea cycle enzymes (ornithine carbamoyltransferase) is deficient and hyperammonemic disorders are obvious. In the mutant adult mouse liver, only hepatocytes lining central veins expressed glutamine synthetase. In contrast, other hepatocytes expressed carbamoylphosphate synthetase I but not glutamine synthetase. This complementary expression pattern is similar to that seen in wild-type mouse liver. In the liver of mutant young mice, which showed severe retarded growth and abnormal hair and skin development, the developmental expression pattern of both enzymes was also similar to that of the corresponding wild-type liver. However, suppression of carbamoylphosphate synthetase I expression in the pericentral hepatocytes occurred later in the mutant than in wild-type liver. These results show that high plasma concentrations of ammonium ions, which are one of the substrates for both the enzymes, do not change their complementary expression. Instead they support the idea that factor(s) associated with central veins rather than humoral factors direct pericentral hepatocytes to express glutamine synthetase and to suppress carbamoylphosphate synthetase I expression. Accepted: 24 April 1997     

Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.     

The metabolic zonation of glycogen synthesis in rat liver after fasting and refeeding.

The quantitative analysis of total glycogen and two fractions of the glycogen content was made by means of cytophotometry in hepatocytes with respect to the portal and central zones of the liver lobule after 48 hr starvation and 15, 30, 60, 120 min after refeeding using the Magiscan image analyzer. It was shown that glycogen content was minimal after 48 hr starvation, although a few cells of the central zone contained a noticeable glycogen quantity. Glycogen synthesis initiation was observed after 15 min refeeding. Glycogen synthesis has been characterized by an increasing glycogen content in the portal zone of the liver lobule compared to the pericentral zone, and this difference increased with time. The distinctive morphological changes were observed in the total glycogen content as well as fractions with different optical density in the process of glycogen synthesis after starvation of rats.     

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

Ischemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age‐mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23–26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near‐complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co‐overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C‐terminus of MFN2, leading to autophagy activation. The SIRT1‐MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.     

Dynamic glycogen synthesis in the hepatocytes of different areas of the hepatic lobes in rats

Using image analyser Magiscan, a quantitative analysis of the total glycogen and of its two fractions was made in hepatocytes of portal and central zones of the liver lobule of rats after a 48 hour starvation and 15, 30, 60, 120 minutes after refeeding. Glycogen content was the lowest after a 48 hour starvation and only a few cells of the central zone contained a noticeable glycogen quantity. Glycogen synthesis initiation began 15 minutes after refeeding. Glycogen synthesis is characterized by a higher glycogen content in the portal zone of liver lobule, and further this difference was even more increased. Different changes were observed in the content of glycogen fractions in the process of glycogen resynthesis after starvation of rats.     

Cellular organization of normal mouse liver: a histological,quantitative immunocytochemical,and fine structural analysis

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 μm) and small (0.03 μm) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.     

Dantrolene prevents hepatic steatosis by reducing cytoplasmic Ca2+ level and ER stress

IntroductionOur previous studies demonstrated that dantrolene, a ryanodine receptor stabilizer, prevents endoplasmic reticulum (ER) stress in the heart. ER stress is a strong mediator of impaired lipid metabolism in the liver, thereby contributing to fatty liver disease. In this study, we investigated the effects of dantrolene on fatty liver disease in mice and ER stress in hepatocytes.Methods and resultsEight weeks old C57BL/6 mice were fed high-fat diet (HFD) for 8 weeks with or without the oral administration of dantrolene (100 mg/kg/day). The livers of mice without dantrolene (HFD group) showed severe fatty liver, whereas the livers of the mice treated with dantrolene (HFD + DAN group) only showed slightly fatty liver. To address the preventive effects of dantrolene, primary hepatocytes were cultured with palmitate in the presence or absence of dantrolene. Dantrolene reduced lipid load and prevents palmitate-induced increase in cytoplasmic Ca²⁺ and ER stress. Based on these findings, we propose that dantrolene is a potential new therapeutic agent against fatty liver disease.     

Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Summary Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.Supported by grants of the Forschungsförderung des Landes Nordrhein-Westfalen, No. 40002585 and the Verein der Freunde und Förderer der Universität KölnDedicated to Professor Dr. Dietrich Eichner on the occasion of his 65th birthday     

青少年食蟹猴糖尿病模型的肝脏病理生理学研究

目的通过对胰岛素用量不足条件下链脲佐菌素诱导的青少年食蟹猴1型糖尿病模型肝脏病理生理学的研究,探讨长期高血糖所致青少年食蟹猴肝损伤特点及机制。方法通过静脉注射68 mg/kg的链脲佐菌素,诱导4只3岁的食蟹猴成为1型糖尿病模型,然后经长期的血糖监测和静脉糖耐量实验来评价该模型的可靠性及稳定性,造模4年后,对模型猴进行血生化、PAS染色、苏丹III染色及普通病理和超微病理等指标的检测,另外选取4只健康与模型猴年龄匹配的猴作为正常对照组,同时进行相应的检测。结果与正常对照组比较,糖尿病猴血清学检测指标中总胆汁酸、尿素氮、谷丙转氨酶、谷草转氨酶、胆碱酯酶、乳酸脱氢酶、总胆固醇、甘油三酯和低密度脂蛋白胆固醇明显升高。组织化学染色结果显示,与正常猴比较,糖尿病猴中央静脉区肝实质细胞肿胀,肝细胞PAS染色（糖原染色）加深,苏丹Ⅲ染色（脂肪染色）阳性细胞增多;电镜结果显示糖尿病猴肝细胞内胞质糖原颗粒增多;线粒体电子密度显著增高,结构不清;窦周隙内含有大量脂滴的肝星状细胞明显增多。结论在长期胰岛素用量不足血糖控制不理想的条件下,青少年食蟹猴1型糖尿病模型肝脏特异性的病理改变是肝糖原贮积和含有大量脂滴的肝星状细胞增生,这些病理改变与非酒精性脂肪肝病的病变特点存在显著不同,但其机制目前尚不清楚。     

Microphotometric determination of glycogen in single fibres of human quadriceps muscle

Synopsis A technique for the quantitation of glycogen in single fibres of human skeletal muscle is described. By using microphotometry the loss of glycogen from cryostat sections during a PAS-staining procedure was shown to be negligible. Further, it was found that nearly all the PAS-positive material (98.5%) inside a muscle fibre is glycogen. A significantly higher mean glycogen concentration (P<0.001) was found in type II fibres than in type I fibres in the resting quadriceps muscle of sedentary young males. The coefficient of variation for the glycogen concentration within each fibre type was found to be 17% and 15% for type I and type II respectively. The specificity of the PAS-staining technique for glycogen was confirmed by a statistically significant correlation (r=0.78,P<0.001) between the glycogen concentration measured biochemically and that calculated from microphotometry and area and thickness measurements. With the technique described, it seems possible to measure the glycogen concentration of single muscle fibres in serial sections and to calculate this in standard biochemical terms.     

Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis

Extrahepatic cholestasis induced by ligation and transsection of the common bile duct caused a change in the parenchyma/stroma relationship in rat liver. Two weeks after ligation, the periportal zones of the parenchyma were progressively invaded by expanding bile ductules with surrounding connective tissue diverging from the portal areas. Parenchymal disarray developed and small clumps of hepatocytes or isolated hepatocytes were scattered within the expanded portal areas. These cells showed normal activity of lactate, succinate and glutamate dehydrogenase and may, therefore, be considered to be functionally active. After cholestasis the remainder of the liver parenchyma showed adaptational changes with respect to glucose homeostasis, as demonstrated by histochemical means. Glycogen stores disappeared completely whereas glycogen phosphorylase activity increased about ten fold. The increased glycogen phosphorylase activity and glycogen depletion indicate a greater glycogenolytic capacity in liver parenchyma after bile duct ligation to maintain as far as possible a normal plasma glucose concentration. The parenchymal distribution pattern of glucose-6-phosphatase activity did not change significantly after bile duct ligation. The isolated hepatocytes within the expanded portal tracts showed a high activity of this enzyme whereas the pericentral parenchyma was only moderately active. The distribution patterns of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in the liver parenchyma were also largely unchanged after bile duct ligation, but the histochemical reaction for glucose-6-phosphate dehydrogenase activity demonstrated infiltration of the remainder of the parenchyma by non-parenchymal cells, possibly Küpffer cells and leucocytes as part of an inflammatory reaction. Under normal conditions the mitochondrial enzymes succinate and glutamate dehydrogenase show an opposite heterogenous distribution pattern in liver parenchyma. Following cholestasis both enzymes became uniformly distributed. The underlying regulatory mechanism for these different changes in distribution patterns of enzyme activities is not yet understood.     

Altered liver glycogen metabolism in fed genetically obese mice.

The cyclic AMP and glycogen concentrations and the activities of phosphorylase kinase, phosphorylase a and glycogen synthase a were not different in livers from lean or ob/ob mice despite increased plasma glucose and insulin in the obese group. The liver water content was decreased by 10% in the obese mice. In hepatocytes isolated from lean mice and incubated with increasing glucose concentrations (14-112 mM), a sequential inactivation of phosphorylase and activation of glycogen synthase was observed. In hepatocytes from obese mice the inactivation of phosphorylase was not followed by an activation of synthase. The inactivation of phosphorylase occurred more rapidly and was followed by an activation of synthase in hepatocytes isolated from both groups of mice when in the incubation medium Na+ was replaced by K+ or when Ca2+ was omitted and 2.5 mM-EGTA included. The inactivation of phosphorylase and activation of synthase were not different in broken-liver-cell preparations from lean and obese animals. The re-activation of phosphorylase in liver filtrates in the presence of 0.1 microM-cyclic AMP and MgATP was inhibited by about 70% by EGTA and stimulated by Ca2+ and was always greater in preparations from ob/ob mice. The apparent paradox between the impairment of glycogen metabolism in isolated liver preparations and the situation in vivo in obese mice is discussed.