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Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. The majority of FGR cases are idiopathic and are associated with placental insufficiency, which can result from placental thrombosis. Evidence suggests that Dermatan Sulfate (DS) is an important anti-coagulant in placentae of uncomplicated pregnancies. This study hypothesised that the expression of biglycan proteoglycan, a source of DS, is decreased in idiopathic FGR placentae compared with placentae from uncomplicated pregnancies. This study aimed to determine biglycan mRNA, protein expression and spatial distribution in idiopathic FGR placentae compared with the placentae from gestation-matched controls. Biglycan mRNA expression, protein expression and spatial distribution was determined in 26 idiopathic FGR-affected placentae and 27 placentae from gestation-matched controls (27-40 weeks gestation) using real-time PCR, immunoblotting and immunohistochemistry, respectively. Mean biglycan mRNA expression was significantly decreased in FGR placentae compared with control placentae (2.87 +/- 0.55, (n = 26) vs. 4.48 +/- 0.85, (n = 27); t-test p = 0.01). FGR placentae demonstrated a trend towards decrease in mean biglycan protein expression compared with control placentae (0.86 +/- 0.22 (n = 9, FGR) vs, 1.9 +/- 0.56 (n = 7, control) p = 0.07). Biglycan immunoreactivity was detected in endothelial cells and sub-endothelial cells of the perivascular region of fetal capillaries. Semi-quantitative analyses demonstrated a significant decrease in immunoreactive biglycan in FGR placentae compared with control placentae (51.1 +/- 19.3 vs, 500.7 +/- 223, n = 6, p < 0.001). This is the first study to demonstrate decreased biglycan expression in idiopathic FGR placentae compared to gestation-matched controls. Reduced biglycan expression may contribute to placental thrombosis within the fetal vasculature, and may contribute to the pathogenesis of idiopathic FGR.

The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell "niche" within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, alpha-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Fetal growth restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. Most cases of FGR are idiopathic and are associated with placental thrombosis. Previous studies suggest that proteoglycans, such as decorin, that contain the glycosaminoglycan dermatan sulfate are the principal anticoagulants in the normal placenta. The present study investigated decorin expression in placentas from pregnancies complicated by idiopathic FGR (n = 26) and gestation-matched controls (n = 27). Real-time polymerase chain reaction demonstrated significantly reduced decorin mRNA expression in FGR compared with control (1.52 +/- 0.14 v. 2.21 +/- 0.22, respectively; P < 0.01). Immunoblotting revealed decreased decorin protein (40 kDa) expression in FGR compared with controls (420.8 +/- 39.0 v. 690.1 +/- 42.2, respectively; n = 12 in each group; P = 0.0007). Immunohistochemistry demonstrated the presence of immunoreactive decorin protein in the placental villous stroma surrounding the fetal capillaries and a significant decrease in decorin protein presence in FGR compared with control (1.75 +/- 0.66 v. 2.98 +/- 1.12, respectively; n = 6 in each group; P < 0.01, t-test). This is the first study to demonstrate reduced decorin in idiopathic FGR, indicating a potentially significant role for decorin in the aetiology of placental thrombosis in idiopathic FGR.

Fetal growth restriction (FGR), a clinically significant pregnancy disorder, is poorly understood at the molecular level. This study investigates idiopathic FGR associated with placental insufficiency. Previously, we showed that the homeobox gene HLX is expressed in placental trophoblast cells and that HLX expression is significantly decreased in human idiopathic FGR. Here, we used the novel approach of identifying downstream targets of HLX in cell culture to detect potentially important genes involved in idiopathic FGR. Downstream targets were revealed by decreasing HLX expression in cultured trophoblast cells with HLX-specific small interfering RNAs to model human idiopathic FGR and comparing these levels with controls using a real-time PCR-based gene profiling system. Changes in candidate HLX target mRNA levels were verified in an independent trophoblast cell line, and candidate target gene expression was assessed in human idiopathic FGR-affected placentae (n = 25) compared with gestation-matched controls (n = 25). The downstream targets RB1 and MYC, cell cycle regulatory genes, showed significantly increased mRNA levels in FGR-affected tissues compared with gestation-matched controls, whereas CCNB1, ELK1, JUN, and CDKN1 showed significantly decreased mRNA levels (n = 25, P < 0.001, t-test). The changes for RB1 and CDKN1C were verified by Western blot analysis in FGR-affected placentae compared with gestation-matched controls (n = 6). We conclude that cell cycle regulatory genes RB1, MYC, CCNB1, ELK1, JUN, and CDKN1C, which control important trophoblast cell functions, are targets of HLX.

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.

Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the microvasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. We have recently shown that placental homeobox genes HLX, DLX3, DLX4, MSX2 and GAX are expressed in placental endothelial cells. Hence, the novel homeobox genes TLX1, TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were detected that have not been reported in endothelial cells previously. Importantly, these homeobox genes have not been previously reported in placental endothelial cells and, with the exception of HEX, PHOX1 and HOXB7, have not been described in any other endothelial cell type. Reverse transcriptase PCR was performed on cDNA from freshly isolated placental microvascular endothelial cells (PLEC), and the human placental microvascular endothelial cell line HPEC. cDNAs prepared from control term placentae, human microvascular endothelial cells (HMVEC) and human umbilical vein macrovascular endothelial cells (HUVEC) were used as controls. PCR analyses showed that all novel homeobox genes tested were expressed by all endothelial cells types. Furthermore, real-time PCR analyses revealed that homeobox genes TLX1, TLX2 and PHOX1 relative mRNA expression levels were significantly decreased in HUVEC compared with microvascular endothelial cells, while the relative mRNA expression levels of MEIS2 and TGIF were significantly increased in macrovascular cells compared with microvascular endothelial cells. Thus we have identified novel homeobox genes in microvascular endothelial cells and have shown that homeobox genes are differentially expressed between micro- and macrovascular endothelial cells.

Homeobox genes are a large family of transcription factors. Of these, the HLX homeobox gene (previously known as HLX1 and HB24) is important for normal placentation. We have previously shown that HLX mRNA expression is significantly reduced in fetal growth-restricted human placentae compared with control placentae. In this study, a rabbit polyclonal antibody to the homeodomain protein HLX was raised and characterised. Western analysis revealed a protein of 50 kDa. HLX protein was detected in cellular nuclei in the cytotrophoblast-derived cell lines HTR8/SVneo, SGHPL-4, JEG-3, JAR and BeWo. Dual labelling with cytokeratin 7 was used to determine the spatial distribution of HLX in the early placenta and fetal membranes, showing both a perinuclear and punctate nuclear distribution for HLX. In the early pregnancy placenta HLX was localised to villous cytotrophoblast, and extravillous cytotrophoblast nuclei in the proximal regions of the cytotrophoblast cell columns, but was not detected at significant levels in the syncytiotrophoblast. In first trimester placental bed biopsies, HLX expression was not localised to the nucleus but instead was found in the cytoplasm. We conclude that HLX is primarily expressed in cytotrophoblast cell types in the human placenta and propose that HLX is involved in cytotrophoblast proliferation and downregulation of cell differentiation.

The efflux pump ATP binding cassette superfamily member G2 (ABCG2)/breast cancer resistance protein (BCRP) is highly expressed in human placenta. We have investigated the role of BCRP in the protection of the human placental trophoblasts from apoptosis and its expression in idiopathic fetal growth restriction, a condition associated with abnormal placental apoptosis. Inhibition of BCRP activity with the selective inhibitor Ko143 augmented cytokine (tumor necrosis factor-alpha/interferon-gamma)-induced apoptosis and phosphatidylserine externalization in primary trophoblast and trophoblast-like BeWo cells. Silencing of BCRP expression in BeWo cells significantly increased their sensitivity to apoptotic injury in response to cytokines and exogenous C6 and C8 ceramides. BCRP silencing also increased intracellular ceramide levels after cytokine exposure but did not affect cellular protoporphyrin IX concentrations or sensitivity to activators of the intrinsic apoptotic pathway. BCRP expression in placentas from pregnancies complicated by idiopathic fetal growth restriction was decreased compared with controls, suggesting reduced transport of its substrates from the placenta. We conclude that BCRP may play a hitherto unrecognized survival role in the placenta, protecting the trophoblast against cytokine-induced apoptosis and possibly other extrinsic activators via modulation of ceramide signaling. Decreased placental BCRP expression may result in reduced viability and hence functional deficit, contributing to the fetal growth restriction phenotype.

The cytokine colony stimulating factor-1 (CSF-1) is a key regulator of the proliferation, differentiation and activation of mononuclear phagocytes. CSF-1 also plays an important role in reproduction. CSF-1 is produced in the placenta and activates signal transduction pathways that significantly increase the proliferation of placental trophoblast cells in culture. The target genes activated by CSF-1 mediated signal transduction in the nucleus are not well understood. Here, we use placental trophoblast cells to investigate potential downstream effector genes of CSF-1. HLX1 is a homeobox gene that controls proliferation in embryonic cell types and haematopoietic cell lineages. We have shown HLX1 is expressed in placental trophoblast cells but its functional role in the placenta is unknown. Following CSF-1 stimulation, HLX1 mRNA expression was significantly increased in SGHPL-4 and HTR-8/SVNeo cultured trophoblast cells (p<0.001, n=3). siRNA-mediated reduction of HLX1 mRNA levels with four independent oligonucleotides (siRNAs) resulted in significantly decreased cell proliferation in both cell lines (p<0.001, n=4). When HLX1 mRNA levels were reduced in the presence of CSF-1 stimulation, proliferation remained significantly decreased (p<0.001, n=4) in both the cell lines. We have shown for the first time that a homeobox gene, HLX1, is a downstream effector gene of CSF-1, that HLX1 regulates placental cell proliferation and that CSF-1 acts, at least in part, through HLX1 to control cell proliferation.

Angiogenesis is fundamental to normal placental development. Aberrant angiogenesis within the placental terminal villi is a characteristic of significant placental pathologies and includes structural and vascular abnormalities as well as altered endothelial cell function, which substantially impacts on maternal-fetal exchange. Homeobox gene transcription factors regulate vascular development in embryonic and adult tissues, but their role in the placental microvasculature is not well known. In this study, we isolated and enriched human placental microvascular endothelial cells (PLEC) by a perfusion-based method and compared homeobox gene expression between PLEC and macrovascular human umbilical vein endothelial cells (HUVEC). Reverse transcriptase PCR detected mRNA expression of homeobox genes DLX3, DLX4, MSX2, GAX and HLX1 in both PLEC and HUVEC. DLX4 and HLX1 have not been previously detected in PLEC and with the exception of GAX, none of these homeobox genes have been previously identified in HUVEC. There was lower expression of HLX1 mRNA in HUVEC compared with PLEC. Using real-time PCR analysis PLEC HLX1 mRNA expression relative to housekeeping gene GAPDH was 0.9+/-0.06 fold of the calibrator (n=6) versus 0.2+/-0.06 (n=6) for HUVEC, p<0.001. These data provided evidence of heterogeneity in homeobox gene expression between microvascular PLEC and macrovascular HUVEC that most likely reflects significant differences in endothelial cell function in the two different cellular environments.

Idiopathic fetal growth restriction (FGR) is often associated with placental insufficiency. Previously, we isolated and characterized homeobox gene DLX4 from the placenta and provided evidence that DLX4 may regulate placental development. Here, we have investigated whether DLX4 expression levels were altered in idiopathic FGR. FGR-affected placentae were collected based on strict clinical criteria. DLX4 mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Initial RT-PCR results showed a qualitative increase in DLX4 mRNA in both FGR-affected placentae and gestation-matched controls. Real-time PCR showed a 3-fold increase in DLX4 mRNA levels in FGR-affected placentae compared with gestation-matched controls (P < 0.005). Western immunoblotting using a rabbit DLX4 polyclonal antibody revealed significantly increased levels of DLX4 protein in term FGR-affected placentae compared with term controls [5500.1 +/- 21.8 (n = 10) versus 3533.2 +/- 22.4 (n = 10); P < 0.001]. Qualitative immunohistochemical analyses of term placentae showed moderately increased immunoreactivity for DLX4 antigen in the FGR-affected placentae in syncytiotrophoblasts, residual cytotrophoblast cells and endothelial cells of the fetal capillaries compared with gestation-matched control term placentae. We conclude that the increased expression of homeobox gene DLX4 may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae.

Fetal growth restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. This study involved idiopathic FGR, which is frequently associated with placental dysfunction. Here, we investigated mRNA levels of the human placental homeobox gene ESX1L in pre-term and term idiopathic FGR pregnancies compared with gestation-matched controls. Real-time PCR quantitation showed ESX1L levels in control placentae decreased between pre-term and term [0.7 +/- 0.20 (27-35 weeks, n = 13) versus 0.2 +/- 0.06 (36-41 weeks, n = 12), t-test, P < 0.005]. ESX1L levels in FGR-affected placentae were significantly lower than in gestation-matched controls, and there was no significant change between pre-term FGR and term FGR [0.32 +/- 0.04 (27-36 weeks, n = 11) versus 0.31 +/- 0.02 (36-41 weeks, n = 14), t-test, P = 0.82]. Multiple linear regression analysis revealed a rapid decline in ESX1L expression in control placentae [0.075-fold of the calibrator for each week of gestation (95% CI = -0.105 to -0.045, P < 0.0005)]. In FGR-affected placentae, ESX1L levels were lower than in gestation-matched controls, and the decline in ESX1L levels with gestation was not significant [0.001-fold of the calibrator for each week of gestation (95% CI = -0.030 to 0.010, P < 0.3]. The linear relationship between ESX1L mRNA levels in FGR-affected placentae and gestation-matched controls during gestation was significantly different (likelihood ratio test for interaction, P = 0.0005). Our findings were consistent with a potential role for the ESX1L gene within the growth control mechanism of the fetus, through its effect on placental function.

Fetal growth restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. Identifiable causes of FGR account for approximately 30% of cases, but the remainder are idiopathic and are frequently associated with placental malfunction. Previously, we isolated the homeobox gene HLX1 and provided evidence for a regulatory role in normal placental development. Here, we investigated whether placental HLX1 expression levels are changed in placentas from idiopathic FGR pregnancies. Real-time polymerase chain reaction quantitation showed reduced HLX1 mRNA levels with advancing gestation age (preterm control placentas, 27 to 35 weeks, 1.1 +/- 0.3, n = 13, versus term placentas 36 to 41 weeks, 0.74 +/- 0.02, n = 12, P < 0.005). FGR-affected placentas had significantly lower levels of HLX1 expression compared with gestation age-matched controls (0.36 +/- 0.07 versus 1.05 +/- 0.2, n = 25, P < 0.001). Immunoblotting with a rabbit polyclonal HLX1 antibody revealed reduced levels of HLX1 in FGR-affected placentas compared with controls (481.07 +/- 12.3 versus 2766.7 +/- 30.3, n = 10, P < 0.001). Immunohistochemistry showed a qualitative decrease in HLX1 immunoreactivity in FGR-affected term placentas compared with controls. This is the first demonstration that a homeobox transcriptional regulator shows altered expression in an important human placental disorder, suggesting that decreased HLX1 levels contribute to the abnormalities in placental developmental seen in idiopathic FGR.

Background and Aim: Previous studies using isolated perfused rat liver in vivo have suggested that during the erythrocytic phase of malaria infection, overall phagocytosis by Kupffer cells is enhanced. The aim of the present study was to further investigate the individual phagocytic capacity and prostaglandin E(2) (PGE(2)) secretion of isolated Kupffer cells in vitro, and the immunohistochemical characteristics of Kupffer cells in vivo., Methods: Malaria was induced in male Sprague-Dawley rats (n = 12) by inoculation with parasitized red cells containing Plasmodium berghei. Kupffer cells were isolated by centrifugal elutriation., Results: A significantly increased yield of Kupffer cells was obtained from malaria-infected livers compared to controls (36.7 +/- 4.5 vs 11.8 +/- 1.1 x10(6) cells, P < 0.0001, n = 12). There was an increased internalization by phagocytosis of [(3)H]-BSA latex microspheres after 60 min in malaria-infected Kupffer cells compared to controls (65.05 +/- 1.5 vs 48.6 +/- 0.7, P < 0.001, n = 12). PGE(2) secretion into the cell culture medium was significantly suppressed in malaria-infected Kupffer cells compared to controls (1167 +/- 88 vs 4537 +/- 383 pg per 10(6) cells, P < 0.001, n = 5). Staining of ED1, ED2 and PCNA was greater in malaria-infected livers compared to control., Conclusion: The results indicate that the number of Kupffer cells is significantly increased and their phagocytic activity on a cell-by-cell basis is enhanced during the erythrocytic stage of malaria.

The aim of the study was to determine the incidence and spatial distribution of apoptotic cell in fetal membranes obtained from human pregnancies complicated with fetal growth restriction (FGR) for which there was no established cause. Fetal membrane samples from normal (n=10) and FGR-affected (n=10) pregnancies were collected and stored following delivery. The incidence of apoptosis and the number of apoptotic cells in normal and FGR-affected fetal membranes were determined using immunohistochemistry and a monoclonal antibody for neo-epitope of cytokeratin-18, M30. The level of apoptotic proteins in FGR-fetal membranes compared to the normal tissue was determined using Western immunoblotting analysis. Multiple labeling of trophoblast cells using immunofluorescence markers was used to investigate regional differences in localization of apoptotic cells between normal and FGR-affected fetal membranes. Apoptosis was detected in both normal and FGR-affected fetal membranes. However, quantitative analysis of apoptotic cells by immunohistochemistry showed a significant increase in FGR-affected fetal membranes compared to normal (p<0.005). Furthermore, it was observed that apoptotic cells were predominantly localized to chorio-decidual layer of the fetal membrane. By using semi-quantitative analysis of Western immunoblotting, a significant increase in the levels of the apoptotic marker proteins poly-ribo (ADP) polymerase (PARP) and the neo-epitope of cytokeratin-18 were observed in FGR-affected fetal membranes compared to normal (p<0.005). Immunofluorescence studies further confirmed the restriction of the apoptotic cells to the chorionic trophoblast cells in FGR-affected fetal membranes. Our results document for the first time an increased incidence of apoptosis in FGR-affected fetal membranes, with the apoptotic cells restricted primarily to the chorionic trophoblast layer of the fetal membranes. Increased apoptosis in FGR-affected fetal membranes may impair functions of the fetal membranes that are important for normal fetal development and growth. Elucidation of the molecular mechanisms involved in the control of apoptosis in the chorionic trophoblast layer in the FGR-affected fetal membranes may provide further insights into the etiology of FGR.

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.

Objective: Elevated levels of proteases are linked to the malignant phenotype in a wide variety of solid tumors. Therefore, the expression of plasminogen, matrix metalloproteinases (MMP-2 and MMP-9), and of the serine protease urokinase-type plasminogen activator (uPA) in serous epithelial carcinoma of the ovary were investigated., Methods: Plasminogen antigen was analyzed in tissue extracts and in the urine of patients with normal (n = 12), benign (n = 6), borderline (n = 9), and invasive serous tumors (n = 22) by Western immunoblotting using rabbit polyclonal plasminogen and murine monoclonal angiostatin antibodies. In the same tissue extracts, semiquantitative estimates of MMP-2, MMP-9, total MMP activity, and uPA activity were determined using semiquantitative gelatin zymography in the presence or absence of human plasminogen., Results: Bands corresponding to Glu-plasminogen (approximately 92 kDa) and Lys-plasminogen (approximately 86 kDa) were detected in all ovarian tissues and in corresponding urine samples. Densitometric analysis of combined Glu- or Lys-plasminogen levels showed significantly decreased levels in malignant compared to normal tissue. In Grade 3 cancers, there was no evidence of Glu-plasminogen or angiostatin. MMP activity was significantly elevated in both borderline and in Grade 3 ovarian cancer tissues. Increased tissue uPA activity on zymograms was detected only in Grade 3 ovarian cancer tissue., Conclusion: These data suggest that proteolytic activity of the plasminogen activation cascade increases in serous epithelial ovarian carcinoma.

The effect of centpiperalone [2-piperazino-4(3H)-quinazolinone monoacetate], a potent hypoglycemic compound on glucose and oxygen uptake, and lactic acid formation by rat hemidiaphragms suspended in glucose-phosphate buffer in Warburg bath at 37 degrees C and 80 shakes/min was studied in vitro. Although centpiperalone enhanced glucose and oxygen uptake by itself, no increment in the above parameters was seen when insulin was added simultaneously along with centpiperalone. Preincubation of hemidiaphragms with centpiperalone and their subsequent incubation with insulin did not indicate any potentiation of insulin action as indicated by uptake of oxygen and glucose. However when 16 times diluted serum from centpiperalone-treated rats was added to hemidiaphragms suspended in glucose-phosphate buffer, significant increase in glycogen content was observed compared to that seen in hemidiaphragms after addition of serum from normal untreated and alloxanized centpiperalone-fed rats. Definite degranulation of B-cells was seen in centpiperalone-fed rats using the aldehyde fuchsin staining technique. These data strongly suggest that centpiperalone induces hypoglycemia through liberation of insulin from B-cells.

Administration of alloxan (40 mg/kg i.v.) caused a marked decrease in the lipolytic and esterolytic activities of rat aorta. This effect was completely reversed when the diabetic rats were treated with low doses of insulin (2-3 U/die) for 3 days. The insulin-dependence of esterolysis and lipolysis in rat aorta and the role of hydrolases in atherogenesis in experimental diabetes have been discussed.