Maternal high-fat diets cause insulin resistance through inflammatory changes in fetal adipose tissue.

OBJECTIVES: Epidemiological and animal studies have shown that maternal obesity predisposes the offspring to obesity and the metabolic syndrome, possibly via late-onset metabolic programming of the fetus. Little is known, however, about the metabolic effect of maternal obesity on the fetus. This study investigated the effect of a maternal high-fat diet (HFD) on fetal growth and glucose metabolism using a diet-induced obesity mouse model. STUDY DESIGN: Female mice (6 weeks old; C57BL/6N) were fed either a normal chow diet (NCD, 10 kcal% fat) or an HFD (60 kcal% fat) for 4 weeks before mating and throughout pregnancy. At 17 days of gestation, gene expression of inflammatory markers and adipokines in fetal subcutaneous adipose tissue was analyzed by quantitative real-time polymerase chain reaction. RESULTS: HFD mice were overweight, glucose intolerant and insulin resistant compared with NCD mice of the same gestational age. Although fetal body weight was not significantly different, fetal plasma glucose and insulin levels were higher in the HFD group than the NCD group. Furthermore, examination of fetal subcutaneous adipose tissue in the HFD group revealed hypertrophy with an increase in the levels of cluster of differentiation-68, chemokine receptor-2 and tumor necrosis factor-α mRNA, but a decrease in the level of glucose transporter-4 mRNA. CONCLUSION: Maternal HFD causes inflammatory changes in the adipose tissue of offspring.

Transgenic mice overproducing human thioredoxin-1, an antioxidative and anti-apoptotic protein, prevents diabetic embryopathy.

AIMS/HYPOTHESIS: Experimental studies have suggested that apoptosis is involved in diabetic embryopathy through oxidative stress. However, the precise mechanism of diabetic embryopathy is not yet clear. Thioredoxin (TRX) is a small, ubiquitous, multifunctional protein, which has recently been shown to protect cells from oxidative stress and apoptosis. Using transgenic mice that overproduce human TRX-1 (TRX-Tg mice), we examined whether oxidative stress is involved in fetal dysmorphogenesis in diabetic pregnancies. METHODS: Non-diabetic and streptozotocin-induced diabetic (DM) female mice were mated with male TRX-Tg mice. Pregnant mice were killed either at day 10 or day 17 of gestation, and viable fetuses and their placentas were recovered, weighed and assessed for gross and histological morphology, biochemical markers and gene expression. RESULTS: In both wild-type (WT) and transgenic (Tg) groups, fetal and placental weights in the diabetic group were significantly decreased compared with the non-diabetic group. The incidence of malformation was higher in the diabetic group, and was significantly decreased in the TRX-Tg group (DM-WT vs DM-Tg; 28.6% vs 10.4%). Oxidative stress markers such as thiobarbituric acid reactive substances and 8-hydroxy-2'-deoxyguanosine were increased in DM-WT group fetuses but were decreased in fetuses from the DM-Tg group. Furthermore, immunohistochemically assayed apoptosis and cleaved caspase-3 production in embryonic neuroepithelial cells was significantly increased in the DM-WT group, and was significantly decreased in the DM-Tg group. CONCLUSIONS/INTERPRETATION: These results indicate that oxidative stress is involved in diabetic embryopathy, and that the antioxidative protein TRX at least partially prevents diabetic embryopathy via suppression of apoptosis.

Effects of biliary obstruction on the endocytic activity in hepatocyte and liver sinusoidal endothelial cells in rats.

Obstructive jaundice impairs function in liver parenchymal and sinusoidal cells. In this study, the endocytic activity in sinusoidal endothelial cells in the rats with biliary obstruction was measured by plasma clearance of radiolabeled formaldehyde-treated bovine serum albumin. The endocytic activity in hepatocytes was also measured with asialofetuin. The clearance of asialofetuin significantly decreased after 1 week of biliary obstruction and the clearance was reduced to 42% of the controls at 4 weeks. In contrast, the clearance of formaldehyde-treated bovine serum albumin was essentially unchanged until 4 weeks of biliary obstruction. The maximal removal rate which was assessed by kinetic analysis of injected protein for formaldehyde-treated bovine serum albumin showed no significant decrease at 2 weeks compared with the controls, while that for asialofetuin was significantly decreased to 50% of the controls. These results suggest that the endocytosis of formaldehyde-treated bovine serum albumin in endothelial cells is maintained until the advanced stage of biliary obstruction, whereas the endocytic activity for asialofetuin in hepatocytes is impaired earlier.

The effect of perioperative portal venous injection of donor spleen cells on renal allograft survival in dogs.

Preoperative portal venous injection of donor lymphocytes has been shown to extend allograft survival in inbred animals. However, the effect of perioperative injection in a large animal model has not been carefully studied. We examined the effect of perioperative (day 0) donor spleen cell administration in a canine kidney transplant model using both a double-donor transplantation and a single-donor survival study. In the double-donor model, two kidneys from two separate donors were transplanted into a single recipient. Spleen cells obtained from only one of the donors were injected through the portal vein immediately after reperfusion of the allografts. Both allografted kidneys were resected 14 days after transplantation and studied. There was only a slight cellular infiltrate in the kidney derived from the donor from whom spleen cells had been obtained, whereas a much more extensive cellular infiltrate as well as edema and hemorrhage were present in the other kidney. In a survival study of single allografts, the injection of 2 x 10(9) spleen cells significantly prolonged the mean survival time (15.0 +/- 3.4 days) compared with untreated recipients (6.6 +/- 2.2 days). Addition of cyclosporine did not significantly prolong survival in the spleen cell inoculated animals. These findings suggest that perioperative protal venous injection of donor cells may be a specific immunosuppressive method useful in clinical organ transplantation.

Bile acid inhibition of P-glycoprotein-mediated transport in multidrug-resistant cells and rat liver canalicular membrane vesicles.

To study the effect of bile acids on P-glycoprotein-mediated drug transport, we performed experiments using multidrug resistant cells and rat canalicular membrane vesicles. Cellular accumulation and efflux of rhodamine 123 were measured in drug-resistant cells by means of computerized quantitative image analysis and fluorescence microscopy. ATP-dependent [3H]daunomycin transport was studied by means of rapid filtration in canalicular membrane vesicles prepared from normal rats. Doxorubicin-sensitive (PSI-2) and -resistant (PN1A) 3T3 cells and human-derived hepatocellular carcinoma doxorubicin-sensitive and -resistant cells were used. Taurochenodeoxycholate and glycochenodeoxycholate, taurolithocholate and ursodeoxycholate (50 to 200 mumol/L) inhibited rhodamine 123 and [3H]daunomycin transport in multidrug-resistant cells and canalicular membrane vesicles, respectively, whereas taurocholate, taurodeoxycholate and tauroursodeoxycholate did not. Primary and secondary unconjugated bile acids had no effect. These results reveal that taurolithocholate, taurochenodeoxycholate and glycochenodeoxycholate and ursodeoxycholate inhibit P-glycoprotein-mediated drug transport function in multidrug resistant cell lines and in canalicular membrane vesicles. These results suggest possible interaction between P-glycoprotein function and bile acids in cholestasis and after treatment of patients with ursodeoxycholic or chenodeoxycholic acid.

Changes in receptor-mediated endocytosis in liver sinusoidal cells after partial hepatectomy in the rat.

Liver sinusoidal cells play an important role in host defense by clearing particulate matter and macromolecules from the circulation. In this study, receptor-mediated endocytosis in sinusoidal cells was examined in two-thirds hepatectomized rats using 125I-labeled formaldehyde-treated bovine serum albumin (fBSA) as an endocytable macromolecule. The liver-weight to body-weight ratio in hepatectomized rats returned to the control value 10 days after hepatectomy. The endocytotic index for fBSA in sinusoidal cells decreased significantly to 0.0210 +/- 0.0017 (controls, 0.0598 +/- 0.0019) on the first day, then returned to the control level at 5 days (0.0554 +/- 0.0030). The changes in hepatic uptake for fBSA showed a similar time course of the endocytotic index. A transient increase in the uptake of fBSA per unit weight of liver of 22-39% above control occurred 2 to 3 days after hepatectomy. In contrast to fBSA, the endocytotic index in hepatocytes evaluated with 125I-labeled asialofetuin reached the minimum level on the second day, and then recovered to the control level 10 days after hepatectomy. These results suggest that endocytosis of fBSA by sinusoidal cells decreases after hepatectomy and rapidly recovers to normal before the completion of liver regeneration, whereas endocytosis of asialofetuin by hepatocytes decreases following hepatic resection and returns to normal when regeneration is substantially complete.

Prolongation of survival for carcinoma at the hepatic duct confluence.

BACKGROUND: Because of poor long-term results in resection for carcinoma at the hepatic duct confluence, we have adopted a more aggressive approach. METHODS: The records of 46 patients with carcinoma of the hepatic duct confluence were reviewed. Twenty-four patients underwent hepatic resection and 10 underwent local resection. The remaining 12 patients had unresectable lesions and received palliative treatment. Out of 24 patients who underwent hepatic resection, 17 underwent combined resection of the caudate lobe. Five patients who underwent hepatic resection and three patients who underwent local resection received intraoperative radiotherapy in a dose of 30 Gy of electron beam. Postoperative radiotherapy with a total dose of 50 Gy of external electron beam was performed for five patients who underwent nonradical hepatic resection and one patient who underwent nonradical local resection. RESULTS: None of the 10 patients who underwent local resection and only 1 (4.2%) of 24 patients who underwent hepatic resection died in the hospital. Among patients who underwent hepatic resection, the 1-, 3-, and 5-year survival rates were 62%, 37%, and 25%, respectively, whereas the respective survival rates in the group that underwent local resection were 50%, 20%, and 20%. The 1-, 3-, and 5-year survival rates for patients who underwent combined caudate lobectomy were 69.7%, 53.4%, and 23%, respectively, compared with 57.1%, 26%, and 14.3% for those who did not. The median survival time for patients who received radiotherapy after nonradical resection was 17 months, compared with 5 months for those who did not. CONCLUSIONS: These results suggest that major hepatic resection combined with caudate lobectomy should be performed for carcinoma of the heptic duct confluence. Postoperative radiotherapy after nonradical resection may be effective in prolonging survival.

Defective ATP-dependent bile canalicular transport of organic anions in mutant (TR-) rats with conjugated hyperbilirubinemia.

TR- mutant Wistar rats secrete markedly fewer organic anions other than bile acids from the liver into the bile than do control rats. Fluorescence-image analysis of isolated normal and TR- hepatocyte "doublets", which retain a bile canaliculus between them, revealed that normal hepatocytes readily transport a fluorescent bile acid (fluorescein isothiocyanate glycocholate) and a nonbile acid organic anion (carboxydichlorofluorescein diacetate) into the canaliculus. Hepatocyte doublets from TR- rats also transported fluorescein isothiocyanate glycocholate normally, but transport of carboxydichlorofluorescein diacetate into the canaliculus was negligible. Vesicles derived from the canicular domain of the plasma membrane of hepatocytes (CMV) from control and TR- rats were used to characterize the transport process for 35S-labeled bromosulphthalein and 35S-labeled bromosulphthalein glutathione, which represent nonbile acid organic anions. CMV from normal rat hepatocytes had an ATP- and temperature-dependent, saturable transport process for these 35S-labeled compounds that was absent in CMV from TR- rats. CMV from TR- rats retained normal ATP-dependent transport of daunomycin, and immunologic blots with a monoclonal antibody against the multidrug resistance gene product, P-glycoprotein, revealed no difference between normal and TR-CMV. These studies reveal that the bile canaliculus in normal rats contains an ATP-dependent organic anion transport system that is functionally absent in TR- mutant rats. The defect in TR- mutant rats is phenotypically similar to that seen in mutant Corriedale sheep and in the Dubin-Johnson syndrome in man.

The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles.

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.

Plasma clearance of intravenously injected aspartate aminotransferase isozymes: evidence for preferential uptake by sinusoidal liver cells.

Both cytosolic (c-AAT) and mitochondrial (m-AAT) isozymes of aspartate aminotransferase (EC 2.6.1.1) appear in serum in some diseases including hepatobiliary dysfunction. The present study aimed at elucidation of the mechanism by which AAT isozymes are cleared from blood. Intravenous injection into rats of m-AAT and c-AAT purified from rat liver exhibited a biphasic clearance curve with an overall half-life of 42 min and 4.7 hr, respectively. The tissue distribution of the radioactivity following intravenous administration of 125I-labeled isozymes revealed that the liver is a major organ involved in plasma clearance of these isozymes. This conclusion was also supported by the significant retardation in plasma clearance of m-AAT in hepatectomized as well as CCl4-intoxicated rats. Furthermore, clearance rate of each AAT isozyme in an isolated perfused liver exhibited a single exponential process with the uptake rate for m-AAT being much faster than that for c-AAT. Separation of hepatocytes and sinusoidal liver cells from the rat intravenously injected with 125I-labeled AAT isozymes revealed that sinusoidal cells were responsible for the plasma clearances. In vitro uptake study showed that both isozymes were exclusively taken up by sinusoidal liver cells. The uptake rate for m-AAT was considerably greater than that for c-AAT. Endocytotic index for uptake by sinusoidal cells was 16 times with c-AAT and 34 times with m-AAT as compared with that for inulin or dextran which are taken up by fluid-phase endocytosis, suggesting involvement of adsorptive endocytosis in the uptake of the isozymes.

Hepatic clearance of rat liver aspartate aminotransferase isozymes: evidence for endocytotic uptake via different binding sites on sinusoidal liver cells.

Rat liver aspartate aminotransferase (AAT) (EC 2.6.1.1) exists in two isozymic forms, cytosolic (c-AAT) and mitochondrial (m-AAT). The previous study (Kamimoto, Y. et al., Hepatology an accompanying paper in this issue) demonstrated that these isozymes were cleared from blood at different half-lives via adsorptive endocytosis by sinusoidal liver cells. To understand the cellular mechanism for the differential uptake of the isozymes, we have further studied in vivo uptake of 125I-labeled AAT isozymes by sinusoidal cells. The results indicated that the uptake of the isozymes occurred via a typical endocytotic pathway: the initial binding, internalization and subsequent degradation in the lysosomes. Quantitation of the isozymes bound to the cell surface prior to internalization either by binding at 4 degrees C or by a combined use of anti-AAT antibody and 125I-protein A at 37 degrees C revealed that the differential plasma clearance of AAT isozymes could be attributable to the isozymic difference in capacity of surface membranes to bind the isozymes. The uptake of 125I-c-AAT was inhibited by unlabeled c-AAT more significantly than by m-AAT, but not by other ligands known to be taken up by sinusoidal cells via receptor-mediated pathways. Similarly, the uptake of 125I-m-AAT was inhibited predominantly by unlabeled m-AAT. Similar ligand specificity was also demonstrated in the binding study at 4 degrees C. The binding of 125I-m-AAT and c-AAT followed saturation kinetics with an apparent Kd of 1.3 X 10(-6) M and 1.7 X 10(-6) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)