Erika Harno

Effects of methyl β-cyclodextrin on EDHF responses in pig and rat arteries; association between SKCa channels and caveolin-rich domains

The small and intermediate conductance, Ca2+‐sensitive K+ channels (SKCa and IKCa, respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IKCa and SKCa channels was investigated.

Impairment of endothelial SKCa channels and of downstream hyperpolarizing pathways in mesenteric arteries from spontaneously hypertensive rats

Background and purpose:  Previous studies have shown that endothelium‐dependent hyperpolarization of myocytes is reduced in resistance arteries from spontaneously hypertensive rats (SHRs). The aim of the present study was to determine whether this reflects down‐regulation of endothelial K+ channels or their associated pathways.

The expression and function of Ca2+‐sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes

The extracellular calcium‐sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate‐conductance, calcium‐sensitive K+ channels (IKCa) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes.

Evidence for the presence of GPRC6A receptors in rat mesenteric arteries

In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.

Developmental programming of hypothalamic neuronal circuits: impact on energy balance control

The prevalence of obesity in adults and children has increased globally at an alarming rate. Mounting evidence from both epidemiological studies and animal models indicates that adult obesity and associated metabolic disorders can be programmed by intrauterine and early postnatal environment- a phenomenon known as “fetal programming of adult disease.” Data from nutritional intervention studies in animals including maternal under- and over-nutrition support the developmental origins of obesity and metabolic syndrome. The hypothalamic neuronal circuits located in the arcuate nucleus controlling appetite and energy expenditure are set early in life and are perturbed by maternal nutritional insults. In this review, we focus on the effects of maternal nutrition in programming permanent changes in these hypothalamic circuits, with experimental evidence from animal models of maternal under- and over-nutrition. We discuss the epigenetic modifications which regulate hypothalamic gene expression as potential molecular mechanisms linking maternal diet during pregnancy to the offsprings risk of obesity at a later age. Understanding these mechanisms in key metabolic genes may provide insights into the development of preventative intervention strategies.

Will treating diabetes with 11β-HSD1 inhibitors affect the HPA axis?

Inhibitors of 11β-HSD1 are in clinical trials for the treatment of type 2 diabetes. These compounds act by decreasing the cortisol generated in liver and adipose tissue, and therefore reducing tissue-specific gluconeogenesis and fatty acid metabolism. However, there is concern that reduction in tissue-regenerated cortisol might decrease feedback to the hypothalamic-pituitary-adrenal (HPA) axis, resulting in upregulation of cortisol from the adrenal gland. This review considers evidence from 11β-HSD1 knockout and transgenic mice, inhibitor studies and results from clinical trials evaluating HPA axis biomarkers. It is clear that analysis of the HPA axis is not sufficiently detailed, and there is a need to understand the subtle changes in the axis associated with pulsatility, diurnal rhythm and stress.

11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitors Still Improve Metabolic Phenotype in Male 11β-HSD1 Knockout Mice Suggesting Off-Target Mechanisms

The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11β-HSD1 inhibitor (compound C) inhibited liver 11β-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)–fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11β-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11β-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting “off-target” mechanisms. Based on the patent literature, we synthesized another 11β-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11β-HSD1 inhibitors occurs via 11β-HSD1–independent pathways, and only limited efficacy is achievable from selective 11β-HSD1 inhibition. These data challenge the concept that inhibition of 11β-HSD1 is likely to produce a “step-change” treatment for diabetes and/or obesity.

11-Dehydrocorticosterone Causes Metabolic Syndrome, Which Is Prevented when 11β-HSD1 Is Knocked Out in Livers of Male Mice

Metabolic syndrome is growing in importance with the rising levels of obesity, type 2 diabetes, and insulin resistance. Metabolic syndrome shares many characteristics with Cushings syndrome, which has led to investigation of the link between excess glucocorticoids and metabolic syndrome. Indeed, increased glucocorticoids from intracellular regeneration by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) drives insulin resistance and increases adiposity, but these metabolic changes are assumed to be due to increased circulating glucocorticoids. We hypothesized that increasing the substrate for 11β-HSD1 (11-dehydrocorticosterone, 11-DHC) would adversely affect metabolic parameters. We found that chronic administration of 11-DHC to male C57BL/6J mice resulted in increased circulating glucocorticoids, and down-regulation of the hypothalamic-pituitary-adrenal axis. This elevated 11β-HSD1-derived corticosterone led to increased body weight gain and adiposity and produced marked insulin resistance. Surprisingly liver-specific 11β-HSD1 knockout (LKO) mice given 11-DHC did not show any of the adverse metabolic effects seen in wild-type mice. This occurred despite the 11-DHC administration resulting in elevated circulating corticosterone, presumably from adipose tissue. Mice with global deletion of 11β-HSD1 (global knockout) were unaffected by treatment with 11-DHC, having no increase in circulating corticosterone and exhibiting no signs of metabolic impairment. Taken together, these data show that in the absence of 11β-HSD1 in the liver, mice are protected from the metabolic effects of 11-DHC administration, even though circulating glucocorticoids are increased. This implies that liver-derived intratissue glucocorticoids, rather than circulating glucocorticoids, contribute significantly to the development of metabolic syndrome and suggest that local action within hepatic tissue mediates these effects.

Reply by authors to letter from Sandow and Grayson.

Dear Professor McGrath, Thank you for giving us the opportunity to respond to the letter addressed to yourself from Drs Sandow and Grayson concerning our recent peer-reviewed paper (Weston et al., 2010), which was itself the subject of a complimentary Commentary article (Garland, 2010). We are confident that Weston et al. (2010) is a paper of significant scientific merit that contains novel findings that will stimulate discussion and further experiments. PREAMBLE ‘Many of our concerns about the Western blot data arise because full length blots including molecular weight markers are not presented, and so it is not possible to determine the precise nature and relevance of the excised bands in the Western blot figures. This is a common problem with the presentation of Western blot images and journal space is often cited as an excuse.’ ANSWER In the same BJP issue (Issue 4, June 2010) as Weston et al. (2010), there were several other research papers that contained Western blot data (Chung et al., 2010; GomezMonterrey et al., 2010; Ikeda-Matsuo et al., 2010; Iqbal et al., 2010; Iwanski et al. (2010); Müller et al., 2010). In all these instances, only the bands of interest were shown (and the paper by Ikeda-Matsuo et al. (2010) was also the subject of a Commentary article; Andreasson, 2010). In other words, the sole depiction of bands of interest has become the norm amongst peer-reviewed journals for the presentation of data of this type. We do not regard this as a ‘problem’, and we have never been constrained by Editors because of restrictions on journal space. In Weston et al. (2010), we therefore used the normal BJP convention, and this format was not an issue raised by our peer reviewers. For the benefit of our research colleagues, we now present our ‘raw data full-length blots’ (Figure 1) in the hope that they will appreciate the quality of our data. In any case, we would have been happy to have provided any necessary clarification about molecular markers, etc. CONCERN 1 ‘The apparent Kir2.1 protein band that is recognised by the Alomone antibody is marked at 52 kDa. However, the antibody data sheet provided by Alomone (http://www.alomone.com/System/UpLoadFiles/DGallery/ Docs/APC-026.pdf) shows the Kir2.1 band at 62 kDa; whereas in fact, the Kir2.1 protein is 48 200 Da and is not glycosylated.’ ANSWER Prior to the publication of Weston et al. (2010), we were aware (in 2008) of the discrepancy between the anticipated band size for Kir2.1 (48.2 kDa) and the band size given by Alomone, the suppliers of the antibody, on their data sheet and we therefore contacted Alomone Laboratories Technical support. They replied that the expected MW for the rat Kir 2.1 channel (48.2 kDa) is based solely on the amino acid sequence and does not include glycosylations, phosphorylations or other transcriptional and post-translational alterations which may affect the apparent MW visualized in a Western blot. In addition, they commented that the above parameters can vary between tissues and/or species and different sample preparation protocols may yield different results. Our published Western Blot data indeed show a band below the 52 kDa marker. Please note that the reference for 52 kDa for the Kir2.1 blot was provided by a molecular weight marker ladder, over which computer bars were placed for clarity in our figure presentation, as is the worldwide convention. We do not believe that the resolution of a Western Blot is fine enough to single out relatively tiny weight differences. Where bands fell between two markers, our interpretation of the molecular weight of the protein from the standard ladder was indicated. CONCERN 2 ‘The paper states that protein concentrations were determined using the Bradford assay, and the consistency of protein loading in each sample lane of the blot images presented in figure 4 was visually assessed using both Ponceau S and b actin staining. However, the quantity of protein that was loaded in each sample lane was not stated.’ ANSWER The protein loading was 10 mg per lane for Caveolin-1 and for Kir2.1, 30 mg per lane for SK3. Ponceau S stain was used simply to visualize protein transfer from the gel onto the membrane. Nowhere in Weston et al. (2010) do we state that b-actin was used as a visual reference for equal protein loading, and indeed, it was not. Densitometry analysis was used (see Weston et al., 2010; ‘samples were adjusted for loading errors, using the b-actin blot density to standardize, prior to normalization relative to WKY values’) to normalize all results, and thus, any variation in the protein loading of the original gel would have been accounted for. CONCERN 2 CONTINUED ‘Furthermore, the amount of tissue from which the protein was extracted, and the total yield of protein that was obtained from the tissue in each sample, was not mentioned.’ ANSWER Each sample was taken from mesenteric artery branches 1 to 4/5 (or small as we could dissect) from a whole BJP British Journal of Pharmacology DOI:10.1111/j.1476-5381.2011.01284.x www.brjpharmacol.org

POMC: The Physiological Power of Hormone Processing

Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon.