Short-term hyperglycaemia causes non-reversible changes in arterial gene expression in a fully 'switchable' in vivo mouse model of diabetes.

AIMS/HYPOTHESIS: Irreversible arterial damage due to early effects of hypo- or hyperglycaemia could account for the limited success of glucose-lowering treatments in preventing cardiovascular disease (CVD) events. We hypothesised that even brief hypo- or hyperglycaemia could adversely affect arterial gene expression and that these changes, moreover, might not be fully reversible. METHODS: By controlled activation of a 'switchable' c-Myc transgene in beta cells, adult pIns-c-MycER(TAM) mice were rendered transiently hypo- and then hyperglycaemic, after which they were allowed to recover for up to 3 months. Immediate and sequential changes in aortic global gene expression from normal glycaemia through hypo- and hyperglycaemia to recovery were assessed. RESULTS: Gene expression was compared with that of normoglycaemic transgenic and tamoxifen-treated wild-type controls. Overall, expression of 95 genes was significantly affected by moderate hypoglycaemia (glucose down to 2.5 mmol/l), whereas over 769 genes were affected by hyperglycaemia. Genes and pathways activated included several involved in atherogenic processes, such as inflammation and arterial calcification. Although expression of many genes recovered to initial pre-exposure levels when hyperglycaemia was corrected (74.9%), in one in four genes this did not occur. Quantitative reverse transcriptase PCR and immunohistochemistry verified the gene expression patterns of key molecules, as shown by global gene arrays. CONCLUSIONS/INTERPRETATION: Short-term exposure to hyperglycaemia can cause deleterious and persistent changes in arterial gene expression in vivo. Brief hypoglycaemia also adversely affects gene expression, although less substantially. Together, these results suggest that early correction of hyperglycaemia and avoidance of hypoglycaemia may both be necessary to avoid excess CVD risk in diabetes.

Secretion of adiponectin by human placenta: differential modulation of adiponectin and its receptors by cytokines.

AIMS/HYPOTHESIS: Pregnancy, a state of insulin resistance, is associated with elevated levels of cytokines and profound alterations in metabolism. Serum adiponectin, an adipokine with anti-inflammatory and insulin-sensitising properties, has been shown to be lower in patients with gestational diabetes mellitus, a state of greater insulin resistance than normal pregnancies. Hypothesising that the human placenta is a source of adiponectin, we investigated its expression and secretion, and the regulation by cytokines of adiponectin and its receptors. METHODS: Real-time RT-PCR, radioimmunoassay, Western blotting, radioligand binding and immunofluorescent analyses were applied to demonstrate the expression, secretion and functionality of placental adiponectin. RESULTS: Adiponectin gene expression and protein were found in the human term placenta, with expression primarily in the syncytiotrophoblast. RIA of conditioned media from explant experiments revealed that the placenta can secrete adiponectin in vitro. Addition of conditioned media to HEK-293 cells transfected with the gene for adiponectin receptor-1 (ADIPOR1) altered the phosphorylation status of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase, an effect abolished after preabsorption with adiponectin antibody. Cytokines, including TNF-alpha, IFN-gamma, IL-6 and leptin, differentially modulated placental adiponectin receptors as well as adiponectin gene expression and secretion. Interestingly, in placentae from women with gestational diabetes mellitus, we observed significant downregulation of adiponectin mRNA, significant upregulation of ADIPOR1 expression, and a non-significant increase in ADIPOR2 expression. CONCLUSIONS/INTERPRETATION: Our results indicate that the human placenta produces and secretes adiponectin, and that adiponectin and its receptors are differentially regulated by cytokines and their expression altered in women with gestational diabetes mellitus. Collectively, our novel data suggest that adiponectin may play a role in adapting energy metabolism at the materno-fetal interface.

Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells.

It is well known that the smooth muscle of the human myometrium is a target for the steroid hormones progesterone (P4) and estrogen. Progesterone is believed to participate in the maintenance of pregnancy, while estrogen is possibly involved in the process of parturition by promoting cervical dilatation. We examined the combined effects of P4 and 17beta-estradiol (E2) on components of signalling pathways in human myometrial cells in vitro by immunoblotting. Long-term treatment of myometrial cells with a series of concentrations of P4 and E2 in combination caused a change in the phosphorylation status of p42/44 mitogen-activated protein kinase and of c-Jun N-terminal kinase (SAPK/JNK). P4 and E2 caused a decrease in protein expression of Gqalpha, Gzalpha, Gi1/2alpha and, to a lesser extent, G0alpha. The two steroids caused a decrease in the expression of the two small GSalpha isoforms. Cyclo-oxygenase-2 expression was increased by 2.5-fold after steroid treatment, while proliferating cell nuclear antigen expression levels remained unchanged. These observations show that the combination of P4 and E2 influences intracellular and membrane-bound components of signal transduction pathways in human myometrial cells. The implications of the two steroid hormones on intracellular signalling pathways in the human myometrium merits further investigation.

Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia.

Menorrhagia (excessive menstrual bleeding) is a common clinical problem of unknown aetiology. The free-radical and vasodilator nitric oxide (NO) relaxes the myometrial smooth muscle and is a strong candidate for the cause of excessive blood loss in menorrhagic patients. The aim of this study was to measure NO production in women with and without menorrhagia to detect nitric oxide synthase (NOS) isoforms in uterine cells and to investigate any steroid effects on myometrial NOS expression. We showed for the first time that menorrhagic endometrium produces significantly higher amounts of NOx (the sum of NO(2-) and NO(3-)) than control endometrium (P < 0.01). Inducible NOS (iNOS) protein was detected by immunoblotting in endometrial and myometrial tissue extracts. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments revealed an induction of myometrial smooth muscle endothelial NOS (eNOS) expression by progesterone and 17beta-oestradiol, while myometrial iNOS expression was unaffected by steroid hormones. These results are consistent with the hypothesis that NO plays a role in excessive menstrual bleeding and provide the first evidence on steroid regulation of eNOS in the human non-pregnant uterus.