Margareta Halin Lejonklou

Co‐expression of ghrelin and its receptor in pancreatic endocrine tumours

Objective  Expression of ghrelin has been reported in pancreatic endocrine tumours, but data on ghrelin receptor protein expression are lacking. The aim of this study was to examine the ghrelin receptor, as well as ghrelin, in a selected series of these tumours, including multiple endocrine neoplasia 1 (MEN1) associated tumours, and to correlate data with clinical features including body mass index.

Uppsala Consensus Statement on Environmental Contaminants and the Global Obesity Epidemic

Summary: From the lectures presented at the 2nd International Workshop on Obesity and Environmental Contaminants, which was held in Uppsala, Sweden, on 8–9 October 2015, it became evident that the findings from numerous animal and epidemiological studies are consistent with the hypothesis that environmental contaminants could contribute to the global obesity epidemic. To increase awareness of this important issue among scientists, regulatory agencies, politicians, chemical industry management, and the general public, the authors summarize compelling scientific evidence that supports the hypothesis and discuss actions that could restrict the possible harmful effects of environmental contaminants on obesity.

Lack of Nuclear Expression of Hairy and Enhancer of Split-1 (HES1) in Pancreatic Endocrine Tumors

The Notch signaling cascade plays a vital role in the proliferation and differentiation of cells during pancreatic development. Cell line experiments have suggested the involvement of Notch signaling in pancreatic endocrine tumorigenesis. We investigated the expression of NOTCH1, HES1, HEY1 and ASCL1 in pancreatic endocrine tumors and compared the data to tumor phenotype including hormone production, heredity, and WHO classification. Real-time quantitative PCR and immunohistochemistry were performed on samples of 26 pancreatic endocrine tumors. For comparison, 10 specimens of macroscopically normal pancreas were analyzed using immunohistochemistry. The subcellular localization of proteins was determined. Neither hormone production, nor heredity, or WHO classification was found to be associated with the expression of these proteins. There were discrepancies between mRNA and protein expression levels. All tumors displayed ASCL1 immunoreactivity. HES1 immunoreactivity was lacking altogether in 46% of the tumors, and in the remaining lesions its expression was weak and confined to the cytoplasm. In the nontumorous pancreatic endocrine cells, weak nuclear expression of HES1 as well as of HEY1 and NOTCH1 was observed. There was a significant positive correlation between NOTCH1 and HES1 mRNA levels, but no indication that HES1 was inhibiting ASCL1 transcription was found. No nuclear expression of HES1 was found in the tumors. This lack of nuclear expression of HES1 may contribute to the abundance of ASCL1 and to tumorigenesis in the endocrine pancreas.

Neurogenin 3 and neurogenic differentiation 1 are retained in the cytoplasm of multiple endocrine neoplasia type 1 islet and pancreatic endocrine tumor cells

Objectives: To investigate if transcription factors involved in pancreatic differentiation and regeneration are present in pancreatic endocrine tumors and if they are differentially expressed in normal pancreas compared with multiple endocrine neoplasia type 1 (MEN1) nontumorous pancreas. Methods: The expression of neurogenin 3 (NEUROG3), neurogenic differentiation 1 (NEUROD1), POU class 3 homeobox 4 (POU3F4), pancreatic duodenal homeobox factor 1 (PDX1), ribosomal protein L10 (RPL10), &dgr;-like 1 homolog (Drosophila; DLK1), and menin was analyzed by immunohistochemistry in normal pancreas and pancreatic endocrine tumors from 6 patients with MEN1 and 16 patients with sporadic tumors, as well as pancreatic specimens from Men1 heterozygous and wild type mice. Quantitative polymerase chain reaction was performed in a subset of human tumors. Results: Tumors and MEN1 nontumorous endocrine cells showed a prominent cytoplasmatic NEUROG3 and NEUROD1 expression. These factors were significantly more expressed in the cytoplasm of Men1 heterozygous mouse islet cells compared with wild type islets; the latter showed an exclusively nuclear reactivity. The degree of Pou3f4, Rpl10, and Dlk1 immunoreactivities differed significantly between islets of heterozygous and wild type mice. The expressions of RPL10 and NEUROD1 were prominent in the MEN1 human and heterozygous mouse exocrine pancreas. Insulinomas had significantly higher PDX1 and DLK1 messenger RNA levels compared with other tumor types. Conclusions: Transcription factors involved in pancreatic development show altered expression and subcellular localization in MEN1 nontumorous pancreas and pancreatic endocrine tumors.

Low-dose developmental exposure to bisphenol A induces sex-specific effects in bone of Fischer 344 rat offspring

Background: Bisphenol A (BPA) is a component of polycarbonate plastics to which humans are regularly exposed at low levels, and an endocrine disruptor with effects on several hormonal systems. Bone is a sensitive hormone target tissue, and we have recently shown that in utero and lactational exposure to 25 &mgr;g BPA/kg BW/day alters femoral geometry in rat offspring. Objective: To investigate bone effects in rat offspring after developmental exposure to a BPA dose in the range of human daily exposure (0.1–1.5 &mgr;g/kg BW/day) as well as a dose to corroborate previous findings. Methods: Pregnant Fischer 344 rats were exposed to BPA via drinking water corresponding to 0.5 &mgr;g/kg BW/day: [0.5], (n=21) or 50 &mgr;g/kg BW/day: [50], (n = 16) from gestational day 3.5 until postnatal day 22, while controls were given only vehicle (n = 25). The offspring was sacrificed at 5 weeks of age. Bone effects were analyzed using peripheral quantitative computed tomography (pQCT), the 3‐point bending test, plasma markers of bone turnover, and gene expression in cortical bone and bone marrow. Results: Compared to controls, male offspring developmentally exposed to BPA had shorter femurs. pQCT analysis revealed effects in the [0.5] group, but not in the [50] group; BPA reduced both trabecular area (−3.9%, p < 0.01) and total cross sectional area (−4.1%, p < 0.01) of femurs in the [0.5] group, whereas no effects were seen on bone density. Conversely, bone length and size were not affected in female offspring. However, the procollagen type I N‐terminal propeptide (P1NP), a peptide formed during type 1 collagen synthesis, was increased in plasma (42%: p < 0.01) in female offspring exposed to [0.5] of BPA, although collagen gene expression was not increased in bone. The biomechanical properties of the bones were not altered in either sex. Bone marrow mRNA expression was only affected in male offspring. Conclusions: Developmental low‐dose exposure to BPA resulted in sex‐specific bone effects in rat offspring. A dose approximately eight times lower than the current temporary EFSA human tolerable daily intake of 4 &mgr;g/kg BW/day, reduced bone length and size in male rat offspring. Long‐term studies are needed to clarify whether the increased plasma levels of P1NP in female offspring reflect development of fibrosis. HighlightsLow‐dose developmental exposure to bisphenol A has sex‐specific effects in rats.Effects were seen at a dose considerably lower than the current temporary human TDI.Reduced bone size was found in male offspring.Increased plasma propeptide of type I collagen (P1NP) was found in female offspring.Increased P1NP may reflect increased bone formation and/or fibrogenesis.

Accelerated proliferation and differential global gene expression in pancreatic islets of five-week-old heterozygous Men1 mice: Men1 is a haploinsufficient suppressor.

Individuals carrying heterozygous (hz) MEN1 (Multiple Endocrine Neoplasia Syndrome Type 1) germ line mutations develop endocrine tumors as a result of somatic loss of the wild-type (wt) allele. However, endocrine cell proliferation has been observed despite wt allele retention, indicating haploinsufficiency. To study downstream molecular effects of the hz haplotype, a germ line Men1 hz mouse model was used to explore differences in global endocrine pancreatic gene expression. Because islet cells of 5-wk-old hz mice express Menin from the retained wt Men1 allele, these were isolated after collagenase digestion of the pancreas, and used for global gene expression array. Wild-type littermates were used for comparison. Array findings were corroborated by quantitative PCR, Western blotting, in situ proximity ligation assay, and immunohistochemistry. The hz islets show increased proliferation: the Ki-67 index was twice as high as in wt islets (3.48 vs. 1.74%; P = 0.024). The microarray results demonstrated that several genes were differentially expressed. Some selected genes were studied on the protein level, e.g. the cytoskeletal regulator myristoylated alanine-rich protein kinase C substrate (Marcks) was significantly less expressed in hz islets, using in situ proximity ligation assay and Western blotting (P < 0.001 and P < 0.01, respectively). Further, gene ontology analysis showed that genes with higher mRNA expression in the hz endocrine pancreas were associated with e.g. chromatin maintenance and apoptosis. Lower mRNA was observed for genes involved in growth factor binding. In conclusion, despite retained Menin expression, proliferation was accelerated, and numerous genes were differentially expressed in the endocrine pancreas of 5-wk-old hz Men1 mice, corroborating the hypothesis that MEN1 is a haploinsufficient suppressor.

Low-dose developmental bisphenol A exposure alters fatty acid metabolism in Fischer 344 rat offspring

Background: Bisphenol A (BPA) is an endocrine disruptor and also a suggested obesogen and metabolism‐disrupting chemical. Accumulating data indicates that the fatty acid (FA) profile and their ratios in plasma and other metabolic tissues are associated with metabolic disorders. Stearoyl‐CoA desaturase 1 (SCD‐1) is a key regulator of lipid metabolism and its activity can be estimated by dividing the FA product by its precursor measured in blood or other tissues. Objective: The primary aim of this study was to investigate the effect of low‐dose developmental BPA exposure on tissue‐specific FA composition including estimated SCD‐1 activity, studied in 5‐ and 52‐week (wk)‐old Fischer 344 (F344) rat offspring. Methods: Pregnant F344 rats were exposed to BPA via their drinking water corresponding to 0: [CTRL], 0.5: [BPA0.5], or 50 &mgr;g/kg BW/day: [BPA50], from gestational day 3.5 until postnatal day 22. Results: BPA0.5 increased SCD‐16 (estimated as the 16:1n‐7/16:0 ratio) and SCD‐18 (estimated as the 18:1n‐9/18:0 ratio) indices in inguinal white adipose tissue triglycerides (iWAT‐TG) and in plasma cholesterol esters (PL‐CE), respectively, in 5‐wk‐old male offspring. In addition, BPA0.5 altered the FA composition in male offspring, e.g. by decreasing levels of the essential polyunsaturated FA linoleic acid (18:2n‐6) in iWAT‐and liver‐TG. No differences were observed regarding the studied FAs in 52‐wk‐old offspring, although a slightly increased BW was observed in 52‐wk‐old female offspring. Conclusions: Low‐dose developmental BPA exposure increased SCD‐16 in iWAT‐TG and SCD‐18 in PL‐CE of male offspring, which may reflect higher SCD‐1 activity in these tissues. Altered desaturation activity and signs of altered FA composition are novel findings that may indicate insulin resistance in the rat offspring. These aforementioned results, together with the observed increased BW, adds to previously published data demonstrating that BPA can act as a metabolism disrupting chemical. HighlightsLow‐dose developmental exposure to BPA alters fatty acid composition in rats.Increased fatty acid markers of SCD‐1 activity in male offspring.Decreased levels of linoleic acid, an essential fatty acid, in male offspring.Increased body weight was found in female offspring.The observed altered fatty acid composition may reflect insulin resistance.

Low-dose exposure to bisphenol A in combination with fructose increases expression of genes regulating angiogenesis and vascular tone in juvenile Fischer 344 rat cardiac tissue.

Abstract Objectives: Epidemiological studies report associations between exposure to the high-volume chemical and endocrine disruptor bisphenol A (BPA) and cardiovascular disorders, but there is a lack of experimental studies addressing the mechanisms of action of BPA on the cardiovascular system. In the present study, effects on markers for cardiovascular function of exposure to BPA and fructose in vivo in rat cardiac tissues, and of BPA exposure in human cardiomyocytes in vitro, were investigated. Materials: Juvenile female Fischer 344 rats were exposed to 5, 50, and 500 μg BPA/kg bodyweight/day in their drinking water from 5 to 15 weeks of age, in combination with 5% fructose. Further, cultured human cardiomyocytes were exposed to 10 nM BPA to 1 × 104 nM BPA for six hours. Expression of markers for cardiovascular function and BPA target receptors was investigated using qRT-PCR. Results: Exposure to 5 μg BPA/kg bodyweight/day plus fructose increased mRNA expression of Vegf, Vegfr2, eNos, and Ace1 in rat heart. Exposure of human cardiomyocytes to 1 × 104 nM BPA increased mRNA expression of eNOS and ACE1, as well as IL-8 and NFκβ known to regulate inflammatory response. Conclusions:. Low-dose exposure of juvenile rats to BPA and fructose induced up-regulation of expression of genes controlling angiogenesis and vascular tone in cardiac tissues. The observed effects of BPA in rat heart were in line with our present and previous studies of BPA in human endothelial cells and cardiomyocytes. These findings may aid in understanding the mechanisms of the association between BPA exposure and cardiovascular disorders reported in epidemiological studies.

Prenatal Bisphenol A Exposure is Linked to Epigenetic Changes in Glutamate Receptor Subunit Gene Grin2b in Female Rats and Humans

Bisphenol A (BPA) exposure has been linked to neurodevelopmental disorders and to effects on epigenetic regulation, such as DNA methylation, at genes involved in brain function. High doses of BPA have been shown to change expression and regulation of one such gene, Grin2b, in mice. Yet, if such changes occur at relevant doses in animals and humans has not been addressed. We investigated if low-dose developmental BPA exposure affects DNA methylation and expression of Grin2b in brains of adult rats. Furthermore, we assessed associations between prenatal BPA exposure and Grin2b methylation in 7-year old children. We found that Grin2b mRNA expression was increased and DNA methylation decreased in female, but not in male rats. In humans, prenatal BPA exposure was associated with increased methylation levels in girls. Additionally, low APGAR scores, a predictor for increased risk for neurodevelopmental diseases, were associated with higher Grin2b methylation levels in girls. Thus, we could link developmental BPA exposure and low APGAR scores to changes in the epigenetic regulation of Grin2b, a gene important for neuronal function, in a sexual dimorphic fashion. Discrepancies in exact locations and directions of the DNA methylation change might reflect differences between species, analysed tissues, exposure level and/or timing.

Low-dose exposure to Bisphenol A during development has limited effects on male reproduction in midpubertal and aging Fischer 344 rats

Low doses of Bisphenol A (BPA) during development may affect reproduction. In this study, Fischer 344 rats were exposed to 0.5 or 50 μg BPA/kg bw/day via drinking water from gestational day 3.5 to postnatal day 22. Anogenital distance, organ weight, histopathology of reproductive organs, hormone analysis and sperm morphology were evaluated in male offspring. In this study no major effects of BPA on male reproduction in midpubertal (postnatal day 35) or adult (12-month-old) rats were revealed, apart from a higher prevalence of mild inflammatory cell infiltrate in cauda epididymis in adult rats exposed to 50 μg BPA/kg bw/day. No BPA-related effects on sexual development were seen but care should be taken when evaluating histopathology in midpuberty testis due to large morphological variation. Results from the present study show no major signs of altered male reproduction in rats exposed to low doses of BPA during gestation and lactation.