Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1 +/ − mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1 +/ − mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.
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- Abstract: Diabetes x
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- Abstract: Pancreas x
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- Abstract: Hypoglycemia x
- Abstract: Insulinoma x
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K E Lines, P J Newey, C J Yates, M Stevenson, R Dyar, G V Walls, M R Bowl, and R V Thakker
Hong Liu, Jian Guo, Lin Wang, Ning Chen, Andrew Karaplis, David Goltzman, and Dengshun Miao
To assess the roles of 1,25-dihydroxyvitamin D (1,25(OH)2D) and parathyroid hormone (PTH) in hard tissue formation in oro-facial tissues, we examined the effect of either 1,25(OH)2D or PTH deficiency on dentin and dental alveolar bone formation and mineralization in the mandibles, and osteoblastic bone formation in long bones of 1α-hydroxylase knockout (1α(OH)ase−/−) mice. Compared with wild-type mice, the mineral density was decreased in the teeth and mandibles, and unmineralized dentin (predentin and biglycan immunopositive dentin) and unmineralized bone matrix in the dental alveolar bone were increased in 1α(OH)ase−/− mice. The dental volume, reparative dentin volume, and dentin sialoprotein immunopositive areas were reduced in 1α(OH)ase−/− mice. The cortical thickness, dental alveolar bone volume, and osteoblast number were all decreased significantly in the mandibles; in contrast, the osteoblast number and surface were increased in the trabecular bone of the tibiae in 1α(OH)ase−/− mice consistent with their secondary hyperparathyroidism. The expression of PTH receptor and IGF1 was reduced slightly in mandibles, but enhanced significantly in the long bones in the 1α(OH)ase−/− mice. To control for the role of secondary hyperparathyroidism, we also examined teeth and mandibles in 6-week-old PTH−/− mice. In these animals, dental and bone volumes in mandibles were not altered when compared with their wild-type littermates. These results suggest that 1,25(OH)2D3 plays an anabolic role in both dentin and dental alveolar bone as it does in long bones, whereas PTH acts predominantly in long bones rather than mandibular bone.
Almas R Juma, Pauliina E Damdimopoulou, Sylvia V H Grommen, Wim J M Van de Ven, and Bert De Groef
Pleomorphic adenoma gene 1 (PLAG1) belongs to the PLAG family of zinc finger transcription factors along with PLAG-like 1 and PLAG-like 2. The PLAG1 gene is best known as an oncogene associated with certain types of cancer, most notably pleomorphic adenomas of the salivary gland. While the mechanisms of PLAG1-induced tumorigenesis are reasonably well understood, the role of PLAG1 in normal physiology is less clear. It is known that PLAG1 is involved in cell proliferation by directly regulating a wide array of target genes, including a number of growth factors such as insulin-like growth factor 2. This is likely to be a central mode of action for PLAG1 both in embryonic development and in cancer. The phenotype of Plag1 knockout mice suggests an important role for PLAG1 also in postnatal growth and reproduction, as PLAG1 deficiency causes growth retardation and reduced fertility. A role for PLAG1 in growth and reproduction is further corroborated by genome-wide association studies in humans and domestic animals in which polymorphisms in the PLAG1 genomic region are associated with body growth and reproductive traits. Here we review the current evidence for PLAG1 as a regulator of growth and fertility and discuss possible endocrine mechanisms involved.
A Alidibbiat, C E Marriott, K T Scougall, S C Campbell, G C Huang, W M Macfarlane, and J A M Shaw
Generation of new β-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true β-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. β-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the β-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted β-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new β-cells.
JA Shaw, MI Delday, AW Hart, HM Docherty, CA Maltin, and K Docherty
The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.
Galya Vassileva, Weiwen Hu, Lizbeth Hoos, Glen Tetzloff, Shijun Yang, Li Liu, Ling Kang, Harry R Davis, Joseph A Hedrick, Hong Lan, Timothy Kowalski, and Eric L Gustafson
G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1 −/− mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1 −/− mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1 −/− mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1 −/− mice on HFD displayed increased hepatic steatosis when compared with Gpbar1 + / + males and Gpbar1 −/− females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.
Junhong Chen, Jing Sun, Michelle E Doscas, Jin Ye, Ashley J Williamson, Yanchun Li, Yi Li, Richard A Prinz, and Xiulong Xu
p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKTS473/T308 and S6K1T389 phosphorylation but decreased S6S235/236 and IRS-1S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKTS473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.
H Del Zotto, L Massa, R Rafaeloff, GL Pittenger, A Vinik, G Gold, A Reifel-Miller, and JJ Gagliardino
The possible relationship between changes in islet cell mass and in islet neogenesis-associated protein (INGAP)-cell mass induced by sucrose administration to normal hamsters was investigated. Normal hamsters were given sucrose (10% in drinking water) for 5 (S8) or 21 (S24) weeks and compared with control (C) fed hamsters. Serum glucose and insulin levels were measured and quantitative immunocytochemistry of the endocrine pancreas was performed. Serum glucose levels were comparable among the groups, while insulin levels were higher in S hamsters. There was a significant increase in beta-cell mass (P<0.02) and in beta-cell 5-bromo-2'-deoxyuridine index (P<0.01), and a significant decrease in islet volume (P<0.01) only in S8 vs C8 hamsters. Cytokeratin (CK)-labelled cells were detected only in S8 hamsters. INGAP-positive cell mass was significantly larger only in S8 vs C8 hamsters. Endocrine INGAP-positive cells were located at the islet periphery ( approximately 96%), spread within the exocrine pancreas ( approximately 3%), and in ductal cells (<1%) in all groups. INGAP positivity and glucagon co-localization varied according to topographic location and type of treatment. In C8 hamsters, 49.1+/-6. 9% cells were INGAP- and glucagon-positive in the islets, while this percentage decreased by almost half in endocrine extra-insular and ductal cells. In S8 animals, co-expression increased in endocrine extra-insular cells to 36.3+/-9.5%, with similar figures in the islets, decreasing to 19.7+/-6.9% in ductal cells. INGAP-positive cells located at the islet periphery also co-expressed CK. In conclusion, a significant increase of INGAP-positive cell mass was only observed at 8 weeks when neogenesis was present, suggesting that this peptide might participate in the control of islet neogenesis. Thus, INGAP could be a potentially useful tool to treat conditions in which there is a decrease in beta-cell mass.
Monisha Rajasekaran, Ok-Joo Sul, Eun-Kyung Choi, Ji-Eun Kim, Jae-Hee Suh, and Hye-Seon Choi
Obesity is strongly associated with chronic inflammation for which adipose tissue macrophages play a critical role. The objective of this study is to identify monocyte chemoattractant protein-1 (MCP-1, CCL2) as a key player governing M1–M2 macrophage polarization and energy balance. We evaluated body weight, fat mass, adipocyte size and energy expenditure as well as core body temperature of Ccl2 knockout mice compared with wild-type mice. Adipose tissues, differentiated adipocyte and bone marrow-derived macrophages were assessed by qPCR, Western blot analysis and histochemistry. MCP-1 deficiency augmented energy expenditure by promoting browning in white adipose tissue and brown adipose tissue activity via increasing the expressions of Ucp1, Prdm16, Tnfrsf9, Ppargc1a, Nrf1 and Th and mitochondrial DNA copy number. MCP-1 abrogation promoted M2 polarization which is characterized by increased expression of Arg1, Chil3, Il10 and Klf4 whereas it decreased M1 polarization by decreased p65 nuclear translocation and attenuated expression of Itgax, Tnf and Nos2, leading to increased browning of adipocytes. Enhanced M2 polarization and attenuated M1 polarization in the absence of MCP-1 are independent. Collectively, our results suggest that the action of MCP-1 in macrophages modulates energy expenditure by impairing browning in adipose tissue.
V Csernus, AV Schally, and K Groot
Antagonistic analogs of GHRH inhibit growth of various human cancers both in vivo and in vitro. To elucidate the mechanism of direct action of the antagonistic analogs of GHRH on tumor cells, cultured human cancer cells were exposed to GHRH, vasoactive intestinal peptide (VIP), secretin, glucagon, neuropeptide-Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), and VIP analogs in a superfusion system, and changes in cAMP and IGF-II release from the cells were measured. Various human cancer cell lines, such as mammary (MDAMB-468 and ZR-75-1), prostatic (PC-3), pancreatic (SW-1990 and Capan-2), ovarian (OV-1063), and colorectal (LoVo) responded to pulsatile stimuli with GHRH (0.5-20 nM), VIP (0.02-10 nM), and PACAP-38 (0.05-5 nM) with a rapid, transient increase in cAMP release from the cells. The VIP antagonist, PG-97-269, and the adenylate cyclase inhibitor, MDL-12330A, but not SQ-22536 or pertussis toxin, blocked the cAMP responses to these peptides. Stimulation of the cells with 100 nM secretin, glucagon or NPY did not alter the cAMP release. Our results suggest that GHRH receptors different from the type expressed in the pituitary are involved in mediating these effects. As cAMP is a potent second messenger controlling a wide variety of intracellular functions, including those required for cell growth, our results indicate that GHRH might have a direct stimulatory effect on growth of human cancers. Blockade of the autocrine/paracrine action of GHRH with its antagonistic analogs may provide a new approach to tumor control.