Inhibition by insulin of cellular autophagy in proximal tubular cells of rat kidney

Adult male Sprague-Dawley rats were injected intraperitoneally with 5 U insulin/kg body wt (45 animals). As determined by quantitative electron microscopy, the volume fraction and the numerical density of autophagic vacuoles (AV) in proximal tubular cells decreased within 10 min by 46 and 26%, respectively. A partial recovery of the AV volume fraction was observed 20 and 30 min after the injection contrary to our previous findings with liver (J. Cell Biol. 78: 152-167, 1978). In an additional experiment (12 animals) it was shown that an insulin dose of 0.5 U but not of 0.05 U/kg body wt reduced the AV volume fraction to an extent similar to that of 5 U. To eliminate possible secondary effects, Ringer solution containing 0.8 microM insulin was dropped intravitally for 15 min to one pole of the decapsulated kidney and Ringer solution without additions to the other pole (8 animals). After intravital fixation, the AV volume fraction and numerical density in proximal tubular cells was found to be reduced under the influence of insulin by 22 and 36%, respectively. This data shows that insulin inhibits the process of cellular autophagy in proximal tubular cells of the kidney.     

Heterogeneity of clara cell glutathione. A possible basis for differences in cellular responses to pulmonary cytotoxicants

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.     

匹诺塞林缓解大鼠肝细胞系BRL-3A低氧/复氧损伤

目的探讨匹诺塞林(PIN)对低氧/复氧(H/R)诱导的大鼠肝细胞损伤的保护作用及其可能机制。方法将大鼠肝细胞(BRL-3A细胞系)分为正常对照组、PIN实验组、低氧复氧损伤模型组和PIN预处理组。CCK-8检测细胞存活率;Annexin V-FITC/PI双染法流式细胞计量术检测细胞凋亡;检测细胞培养液中谷丙转氨酶(ALT)活性;ELISA检测TNF-α和IL-1β含量;Western blot检测细胞中TLR4、IκB-α和NF-κB P65蛋白水平;RT-q PCR检测细胞中TLR4、IκB-α和NF-κB P65mRNA表达。结果在H/R条件下细胞存活率明显降低(P0.01),细胞凋亡率增高(P0.001),ALT活性升高(P0.01),IL-1β和TNF-α含量增多(P0.01),TLR4和NF-κB P65蛋白与mRNA表达水平显著提高(P0.01)而IκB-α降低(P0.05);经PIN预处理后,细胞存活率显著提高(P0.01),细胞凋亡率显著减小(P0.001),ALT活性降低(P0.01),IL-1β和TNF-α含量降低(P0.01),TLR4和NF-κB P65蛋白与mRNA表达水平明显降低(P0.01)而IκB-α升高(P0.05)。结论 PIN对H/R诱导的BRL-3A肝细胞的损伤具有保护作用,且该作用可能是通过TLR4/NF-κB信号通路实现的。     

In vitro responses to a B-lymphoblastoid cell line in immunodeficiency diseases

The use of a B-lymphoblastoid cell line (B-LCL) as a stimulator cell in the mixed leukocyte reaction (MLR) was investigated in patients with immunodeficiency disorders. The kinetics of the MLR stimulated by B-LCL are similar to those stimulated by normal allogeneic leukocytes, however, B-LCL stimulate a greater quantitative response. Comparison of LCL-stimulated and normal allogeneic lymphocyte-stimulated MLR in 32 patients and normal controls demonstrated that variation in the MLR was reduced when B-LCL were used as stimulator cells. The decrease in variability allowed for more sensitivity in the determination of abnormal responses; 9 of 32 patients had abnormal B-LCL-stimulated MLC responses, compared with 5 of 32 patients with abnormal responses to normal allogeneic leukocytes. Dose-response studies showed that vigorous responses could be obtained with low doses of B-LCL stimulator cells which served to better define deficient patient responses. Several patients demonstrated dissociation between the B-LCL-stimulated MLR and mitogen responses. The use of a B-LCL as a stimulator cell in the MLR is a valuable tool for the assessment of the immune status of patients with a variety of immunodeficiency disorders.     

A spleen cell derived factor imparts resistance to NK cell mediated lysis in a mouse lymphoma cell line

The effect of interleukin-2 (IL-2) preparations on the susceptibility of YAC cells to mouse natural killer (NK) cells was examined. Crude IL-2 preparations induced a significant decrease in the susceptibility of YAC cells. Highly purified IL-2 preparations, however, did not have a similar effect. Since the IL-2 preparations used in this study were purified from the culture supernatant of Con A activated spleen cells (Con A Sup.), our results indicated the presence in Con A Sup. of a lysis resistance inducing factor (LRIF), distinct from IL-2. We were able to purify LRIF from Con A Sup. by preparative isoelectric focusing and determined its isoelectric pH to be 4.8.     

Induction of antigen expression of follicular dendritic cells in a monoblastic cell line. A contribution to its cellular origin

Experiments were carried out to elucidate the cellular origin of the dendritic reticulum cell (DRC). The monoblastic cell line THP-1, the histiocytic cell line U-937, and the mononuclear cell fraction from peripheral blood (PMC) were stimulated with supernatants from lectin-stimulated peripheral blood lymphocytes and from stimulated T- and B-cell lines. Differentiation towards DRC was assessed by immunocytochemical demonstration of the DRC-specific antigen Ki-M4. Supernatants from isolated peripheral T lymphocytes and from T- and B-cell lines were capable of stimulating THP-1 to Ki-M4 antigen expression, whereas U-937 and the PMC fraction remained negative for this antigen throughout the experiments. These results provide further evidence for a relationship of DRCs with the mononuclear-phagocytic cell system and hence for their bone marrow origin. Furthermore, the data suggest that soluble factors of T and/or B cells are involved in mediating the differentiation process of precursor cells to DRC.     

Cytogenetic responses of human uroepithelial cell lines and a malignant bladder carcinoma cell line to X-rays.

The responses to X-rays of three simian virus 40 (SV40) immortalized but non-tumourigenic human bladder epithelial cell lines have been compared with a malignant bladder epithelial line using the micronucleus assay. A linear increase in induced micronuclei (MN) was observed for all four cell lines with increasing X-ray dose. The three SV40 immortalized lines were found to be significantly less sensitive than the malignant cell line. Spontaneous levels of MN indicate that certain cell lines within the SV40 immortalized lines have a higher genetic instability. These cell lines may have a predisposition towards the generation of a fully transformed phenotype when treated with carcinogenic agents.     

Cardiac cellular responses to altered nutrition in the neonatal rat

Rats reared in litters of 18, 12, and 6 to determine whether preweanling nutritional state would alter rates of cardiac cell division, weighed 31.7, 39.1, 48.2 g, respectively, at 21 days of age. Weights of left ventricles also increased (93.2, 123.5, and 167.2 mg) as did those of right ventricles (29.9, 43.2, and 54.3 mg). Total DNA content rose in both ventricles in the pups reared 6 per litter vs. those reared 18 per litter (6/litter vs. 18/litter), but more so in the left ventricle (79%) than in the right (24%). Autoradiography confirmed that this increase in ventricular DNA resulted from increased proliferation of cardiac muscle cells, fibroblasts, and vascular endothelial cells. When 3H-labeled thymidine was injected on day 1, autoradiographs prepared on day 21 reflected an increased dilution of label in the 6/litter rats, consistent with enhanced proliferation. The labeling index and grains per nucleus of left ventricular muscle cells of the 6/litter rats were 29% (P less than 0.005) and 20% (P less than 0.001) less than those of the 18/litter rats. Less vigorous but definite hyperplasia occurred in the right ventricle, which appeared to respond with an increase more in cell size than in cell number.     

Distribution and cellular origin of undulin in rat liver.

BACKGROUND: Undulin is a novel large glycoprotein of the interstitial extracellular matrix belonging to the fibronectin-tenascin glycoprotein gene family. The distribution in diseased liver and the cellular origin of this protein are unknown. EXPERIMENTAL DESIGN: Immunohistochemistry studies were performed on cryostat sections of normal and damaged rat livers (CCl4 model). Hepatocytes, Kupffer cells, fat-storing cells (FSC), and sinusoidal endothelial cells (EC) were isolated by standard methods and kept in culture. Undulin biosynthesis in vitro was studied by indirect immunofluorescence and by immunoprecipitation of endogenously labeled protein followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. RESULTS: Undulin was demonstrated in portal stroma, in vascular adventitia, and inside the space of Disse of normal liver. Acutely and chronically damaged livers revealed strong staining reactions in damaged areas, scars, and sinusoids. The overall distribution of undulin resembled the pattern noted for fibronectin. In contrast to undulin, tenascin was not detectable within the adventitia of vascular and ductular structures of normal and damaged livers, and tenascin accumulated preferentially at scar-parenchyma interfaces in fibrotic livers. In vivo, desmin and smooth muscle alpha-actin positive cells were in part codistributed with undulin fibers as shown by double staining techniques. In vitro, undulin was detected in granules of freshly isolated FSCs and ECs and was localized as fibers in the extracellular matrix of cultivated FSCs and ECs. Synthesis of undulin was demonstrated by immunoprecipitation of the protein from cultured FSCs and ECs. No experimental evidence was found for undulin synthesis in vitro by hepatocytes and Kupffer cells. CONCLUSIONS: The novel glycoprotein undulin is present in the normal rat liver and accumulates during acute and chronic liver injury. Our results suggest that among the resident cells of the liver, FSCs and ECs are the major sources of undulin.     

环氧化酶对大鼠胃黏膜细胞系的保护作用

应用MTT法、DNA条带分析及流式细胞仪分析法比较环氧化酶(cyclooxygenase)(COX-1和COX-2)非选择性抑制剂消炎痛和COX-2选择性抑制剂NS-398对大鼠胃黏膜上皮细胞株(RGM-1)的作用,并用Northern blot印迹法分析脂多糖(LPS)作用于RGM-1后,COX-1和COX-2的不同变化。结果发现消炎痛在一定的浓度范围内可诱导RGM-1细胞的凋亡、抑制其增殖,并有明显的量效关系,而NS-398对RGM-1则无明显作用。若预先用LPS作用于细胞后,消炎痛对细胞的作用明显减轻,而NS-398的作用无明显变化。Northern blot分析表明,LPS作用于细胞后,RGM-1细胞COX-2mRNA的水平明显增加。放射免疫分析(RIA)证实LPS的作用可提高细胞PG的水平。提示,COX-1和COX-2在维持胃黏膜细胞的完整性中可能起不同的作用。     

Evidence against a direct role of klotho in insulin resistance

The klotho gene may be involved in the aging process. Klotho is a coactivator of FGF23, a regulator of phosphate and vitamin D metabolism. It has also been reported to be downregulated in insulin resistance syndromes and paradoxically to directly inhibit IGF-1 and insulin signaling. Our aim was to study klotho’s regulation and effects on insulin and IGF-1 signaling to unravel this paradox. We studied klotho tissue distribution and expression by quantitative real-time polymerase chain reaction and Western blotting in obese Zucker rats and high-fat fed Wistar rats, two models of insulin resistance. Klotho was expressed in kidneys but at much lower levels (<1.5%) in liver, muscle, brain, and adipose tissue. There were no significant differences between insulin resistant and control animals. We next produced human recombinant soluble klotho protein (KLEC) and studied its effects on insulin and IGF-1 signaling in cultured cells. In HEK293 cells, FGF23 signaling (judged by FRS2-α and ERK1/2 phosphorylation) was activated by conditioned media from KLEC-producing cells (CM-KLEC); however, IGF-1 signaling was unaffected. CM-KLEC did not inhibit IGF-1 and insulin signaling in L6 and Hep G2 cells, as judged by Akt and ERK1/2 phosphorylation. We conclude that decreased klotho expression is not a general feature of rodent models of insulin resistance. Further, the soluble klotho protein does not inhibit IGF-1 and/or insulin signaling in HEK293, L6, and HepG2 cells, arguing against a direct role of klotho in insulin signaling. However, the hypothesis that klotho indirectly regulates insulin sensitivity via FGF23 activation remains to be investigated.     

Hypotonic cell swelling stimulates permeability to cAMP in a rat colonic cell line

This study characterized the membrane permeability to cAMP in a cell line derived from the rat colon (CC531^mdr+) by comparison of fluxes of ³H-cAMP, ³H-8-bromo-cAMP, ³H-taurine, ³H-adenosine and ³H-5AMP under various experimental conditions including cell membrane depolarization and hypotonic cell swelling. Cell volume was modified by changing the osmolality and composition of the extracellular medium. Incubation in iso- and hypotonic KCl media induced graded increases in cell volume and stable activation of volume-sensitive channels that was reflected in an increased efflux of ³H-taurine. Incubation in hypotonic KCl solution also enhanced the efflux of ³H-8-Br-cAMP (a non-hydrolysable analogue of cAMP). Both the efflux of ³H-taurine and of ³H-8-Br-cAMP were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 100 µM) suggesting the involvement of volume-sensitive anion channels. To gain further insight into the route mediating cAMP permeability, the uptakes of ³H-cAMP, ³H-8-Br-cAMP and ³H-taurine were determined over short (5-min) periods. Uptakes of these substrates demonstrated close similarities: comparable increases were observed that correlated with the increases in cell volume in iso- and hypoosmotic KCl media; they were inhibited strongly by NPPB (100 µM) and metabolic inhibitors (deoxyglucose, 20 mM together with the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 10 µM) while barely reduced by dipyridamole (100 µM) and they were not affected by adenosine (1 mM). In contrast, the uptakes of ³H-adenosine and ³H-5AMP had strikingly different properties; they were insensitive to cell swelling; barely inhibited by NPPB (100 µM) and metabolic inhibitors (deoxyglucose and FCCP) while strongly reduced by dipyridamole (100 µM). Unlike the uptakes of ³H-cAMP, ³H-8-Br-cAMP and ³H-taurine, the uptakes of ³H-adenosine and ³H-5AMP were reduced in Na⁺-free media, suggesting the presence in this cell line of two different adenosine carriers, one sodium-dependent and one sodium-independent. Taken together the present data show that in this rat colonic cell line, cAMP permeability is increased by cell swelling in hypotonic KCl medium and inhibited by NPPB and metabolic inhibitors. The similarity of these characteristics to those of taurine permeability suggests the involvement of a volume-sensitive anion pathway.     

The role of pioglitazone in antioxidant,anti-inflammatory,and insulin sensitivity in a high fat-carbohydrate diet-induced rat model of insulin resistance

We explored the cascade effects of a high fat-carbohydrate diet (HFCD) and pioglitazone (an anti-diabetic therapy used to treat type 2 diabetes mellitus (T2DM)) on lipid profiles, oxidative stress/antioxidant, insulin, and inflammatory biomarkers in a rat model of insulin resistance. Sixty albino rats (80-90 g) were randomly divided into three dietary groups; 1) standard diet; 2) HFCD diet for 12 weeks to induce an in vivo model of insulin resistance; and 3) HFCD diet plus pioglitazone. Blood and tissue samples were taken to assess hepatic function, lipid profiles, oxidative biomarkers, malondialdehyde (MDA) levels, antioxidant defense biomarkers, including reduced glutathione (GSH), superoxide dismutase (SOD), and the inflammatory markers interleukin-6 (IL-6) and tumor necrotic factor (TNF-α). HFCD-fed rats had significantly (P≤0.05) increased serum triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein (LDL), alanine transaminase (ALT), and bilirubin levels, but decreased high-density lipoprotein (HDL) levels compared with the normal group. Moreover, serum leptin, resistin, TNF-α, and IL-6 levels were increased significantly in HFCD animals compared with controls. Similarly, HFCD-induced insulin resistance caused antioxidant and cytokine disturbances, which are important therapy targets for pioglitazone. Importantly, administration of this drug ameliorated these changes, normalized leptin and resistin and inflammatory markers by reducing TNF-α levels. Metabolic cascades of elevated lipid profiles, oxidative stress, insulin, and inflammatory biomarkers are implicated in insulin resistance progression. HFCD induced metabolic cascades comprising hypertriglyceridemia, hyperglycemia, insulin resistance, obesity-associated hormones, and inflammatory biomarkers may be alleviated using pioglitazone.     

Antibody-secreting cell responses in the mouse liver.

To elucidate the origins of biliary IgA antibodies, we investigated the isotype and specificity of antibody-secreting cells (ASC) in the liver in comparison with the spleen and intestinal lamina propria of mice immunized by peroral or parenteral routes. The profile of specific IgM, IgG1, IgG2a, and IgA ASC in the liver resembled that of the spleen rather than the lamina propria, regardless of the route of immunization. Peroral immunization increased the proportion of specific IgA ASC in all three organs. However, liver mononuclear cells (MNC) contained a higher proportion of total IgA-secreting cells than spleen cells. After immunization, the number and proportion of B220+ B cells were increased in the liver but not in the spleen. Although the predominant isotype of Ig and specific antibody in bile in response to immunization by either route was IgA, IgM and IgG were clearly detectable. However, specific activities of biliary antibodies relative to total Ig isotype were generally higher than in serum. The predominance of IgA-secreting cells in the liver and the large amount of IgA secreted in the bile resemble the situation at other secretory sites of the mucosal immune system. However, specific antibody-secreting cells appear to accumulate in the liver promptly after immunization, regardless of isotype, and contribute locally produced antibodies to the bile.     

Induction of selective biological responses to chemoattractants in a human monocyte-like cell line

The availability of monocyte cell lines that can be induced to differentiate in a predictable fashion can provide important tools for the study of the biochemical mechanisms of specific cellular responses. The U937 human monocyte cell line was previously shown to differentiate into chemotactically responsive cells when incubated with supernatants of lectin-stimulated lymphocytes (conditioned medium). Considering the heterogeneous nature of stimulated lymphocyte supernatants, attempts were made to identify well-defined agents that could reproducibly induce U937 cell differentiation. Both dimethyl sulfoxide and dibutyryl cAMP induced expression of receptors for the N-formylated oligopeptide chemoattractants in U937 cells. Unstimulated U937 cells contained no detectable receptors. After cells were exposed to 1 mM dibutyryl cAMP, 1.3% dimethyl sulfoxide, or 5% conditioned medium for 72 h, the average number of oligopeptide chemoattractant receptors per U937 cell was 33,000, 4,000, and 3,400, respectively. Specific binding proteins for the chemoattractants were identified by covalent affinity labeling on the differentiated U937 cells as well as on normal human monocytes. Cells exposed to conditioned medium responded chemotactically, secreted lysosomal enzymes, and formed superoxide anion when incubated with the chemoattractant. Treatment of U937 cells with dibutyryl cAMP resulted in the most reproducible and rapid increase in the number of chemoattractant receptors as well as in chemotactic responsiveness. The receptors on dibutyryl cAMP-treated cells and on dimethyl sulfoxide-treated cells initiated chemotaxis and lysosomal enzyme secretion in response to chemoattractants, but not the formation of superoxide anion. These findings demonstrate that development of the chemotactic and respiratory burst functions during the differentiation of a monocyte-like cell line can occur independently.