Rapid chemiluminescent detection of mRNAs on Northern blots with digoxigenin end‐labelled oligonucleotides

Trayhurn, Paul; Duncan, J.L.; Nestor, Antoinette; Thomas, Moira E. A.; Eastmond, Nigel C.; Rayner, D V

doi:10.1002/elps.1150160157

Cited by 26 publications

(13 citation statements)

References 10 publications

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“…This size of the transcript is smaller than that originally described by Zhang et al [1] (about 4.5 kb) but similar to that more recently reported by Trayhurn et al [11]. A much fainter signal was also detected at a size of 2.3 kb.…”

Section: Resultssupporting

confidence: 88%

“…The same explanation holds true for the effect of acute fasting and chronic food restriction in WAT. The finding of a substantial fall in the ob gene mRNA level in this tissue during fasting is in line with the recent report of Trayhurn et al [11]. Under condition of acute cold exposure the transient fall in the ob gene mRNA in BAT can be associated with a transient lipid depletion in this tissue during the first day, i.e.…”

Section: Discussionsupporting

confidence: 92%

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Modulation of obese gene expression in rat brown and white adipose tissues

et al. 1995

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The ob gene mRNA expression in rat brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) was measured on Northern blots hybridized with a rat ob gene probe. The level of ob gene mRNA in BAT was about 40% of that in WAT. Fasting (36 h) or semi-starvation (10 days) decreased the ob gene mRNA level in both tissues by 62-68%, and cold exposure at 6°C (24 h) decreased it in BAT (-84%) but not in WAT. Acute administration of the /3a-adrenergic agonist Ro 16-8714 decreased the ob gene mRNA level in BAT (-51%) and WAT (-28%) of lean Zucker rats and only in BAT (-74%) of obese fa/fa rats. This study demonstrates that, in the rat, the ob gene is not only expressed in WAT but also in BAT, and suggests that in these two tissues, the modulation of the ob gene expression might be more closely associated with known alterations in cell lipid content than with changes in sympathetic activity.Key words." ob Gene; Fast; Cold; Rat brown and white adipose tissue db/db mouse and that would account for the up-regulation of the ob gene expression in the WAT of these animals.To date, the ob gene expression has been demonstrated in WAT [1,2] and also detected in brown adipose tissue (BAT) [3]. Since BAT is known to play an important role in the regulation of energy balance in rodents [4], the expression of the ob gene was examined in this tissue. The aims of this study therefore were: (i) to quantify the ob gene expression in rat interscapular BAT and to compare its level to that measured in epididymal WAT; and (ii) to investigate if the ob gene level is modulated by conditions which are known to reduce or increase BAT sympathetic and thermogenic activity, namely caloric restriction [5,6], cold exposure [6,7], or the administration of a fl3-adrenoceptor agonist [8]. Under each of these conditions, possible changes in the ob gene expression in epididymal WAT were also studied in parallel. Materials and methods

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Section: Resultssupporting

confidence: 88%

Section: Discussionsupporting

confidence: 92%

Modulation of obese gene expression in rat brown and white adipose tissues

et al. 1995

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“…RNA fractionation, blotting, hybridization and chemiluminescent detection were carried out according to previously published procedures (Trayhurn et al 1994). In brief, RNAs were fractionated by agarose gel electrophoresis, transferred to a positively charged nylon membrane (Roche Molecular Biochemicals, Mannheim, Germany) by capillary blotting and crosslinked with UV light using a Foto/UV 21 (Fotodyne, WI, USA).…”

Section: Methodsmentioning

confidence: 99%

The anorectic effect of oestradiol does not involve changes in plasma and cerebrospinal fluid leptin concentrations in the rat

Rocha

Grueso²,

Puerta

2001

Journal of Endocrinology

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Oestradiol is a potent anorectic agent that reduces both food intake and body weight. Since leptin is known to reduce food intake, we first analysed if the anorectic effect of oestradiol is driven by an increased leptin concentration in either cerebrospinal fluid or plasma. Oestradiol also reduces body weight and fat mass. Accordingly, a decrease in plasma leptin concentration can also be expected after an oestradiol-driven reduction in fat mass. To test this hypothesis was the second aim of this study. Female Wistar rats received oestradiol chronically during 14 days. During the first week of treatment there was a reduction in food intake, body weight and fat mass that returned to initial values during the second week, but no changes in ob mRNA levels were found in white adipose tissue depots. There was no effect of treatment or time on plasma and cerebrospinal fluid leptin concentrations. Therefore, the anorectic effect of oestradiol is not driven by an increase in leptin concentration either in plasma or in cerebrospinal fluid, and the reduction in fat mass that oestradiol produces is not followed by a reduction leptin concentration.

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“…Fresh blocking buffer (5 ml) containing 1:10,000 diluted anti-DIG Fab fragment (Boehringer Mannheim) was added, and the incubation continued for 30 min at room temperature. Posthybridization washes and immunological detection with the DIG-labeled probe were conducted according to Trayhurn et al, 23 …”

Section: Analytic Methodsmentioning

confidence: 99%

Hormonal regulation of human leptin in vivo: effects of hydrocortisone and insulin

2000

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OBJECTIVE: To investigate the effects of continuous i.v. infusion of hydrocortisone or insulin on leptin secretion in humans. SUBJECTS: Six, nonfasting healthy adults (four women, two men), aged (mean AE s.e.m.) 36.6 AE 1.7 y; body mass index (BMI) 27.6 AE 0.9 kgam 2 . DESIGN: Randomized, placebo-controlled, cross-over study, with a 2-week`wash-out' period. INTERVENTIONS: Intravenous infusion of hydrocortisone (3.3 m mga(kg min)), insulin (1 mUa(kg min)) or normal saline (placebo) for 24 h. MEASUREMENTS: Blood sampling every 1 ± 2 h for measurement of glucose, insulin, cortisol and leptin; subcutaneous abdominal fat biopsy for determination of leptin mRNA expression. RESULTS: Plasma cortisol increased to 50.0 AE 0.4 m mgadl during hydrocortisone infusion, but was unaltered during saline or insulin infusion. The plasma insulin levels were: 28.5 AE 4.7 m mUaml (placebo), 40.8 AE 9.2 m mUaml (hydrocortisone, P 0.214), and 243 AE 23.0 m mUaml (insulin, P 0.0002). Peak hyperleptinemia occurred after 16 h of insulin and 20 h of hydrocortisone infusion; peakabaseline plasma leptin levels (ngaml) were 18.2 AE 4.2a15.1 AE 3.3 (placebo, P 0.056), 42.1 AE 7.0a16.0 AE 3.8 (hydrocortisone, 163%, P 0.008) and 30.2 AE 4.3a16.6 AE 2.7 (insulin, 83%, P 0.024). Adipocyte leptin mRNA increased by 350% after the hydrocortisone infusion. CONCLUSION: Hydrocortisone, a natural glucocorticoid, induces hyperleptinemia in vivo, with a potency greater than that of insulin. The interaction between glucocorticoids and leptin may be of metabolic signi®cance in humans.

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Rapid chemiluminescent detection of mRNAs on Northern blots with digoxigenin end‐labelled oligonucleotides

Cited by 26 publications

References 10 publications

Modulation of obese gene expression in rat brown and white adipose tissues

Modulation of obese gene expression in rat brown and white adipose tissues

The anorectic effect of oestradiol does not involve changes in plasma and cerebrospinal fluid leptin concentrations in the rat

Hormonal regulation of human leptin in vivo: effects of hydrocortisone and insulin

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