D. Vernon Rayner

Effects of fasting and refeeding on ob gene expression in white adipose tissue of lean and obese (ob/ob) mice

A 33‐mer antisense oligonucleotide has been utilized as a probe for the rapid chemiluminescence‐based detection of ob (obese) mRNA. Expression of the ob gene was evident in several white adipose tissue depots of mice (epididymal, highest; subcutaneous and omental, lowest), but not in other organs. Fasting (24 h) induced a substantial fall in ob mRNA in the epididymal fat of lean mice, which was rapidly reversed on refeeding, responses consistent with the putative role of ob in energy balance. Fasting had no effect, however, on ob mRNA levels in obese (ob/ob) mice.

Regulation of leptin production: sympathetic nervous system interactions

Abstract. Leptin is secreted primarily from white adipose tissue and stimulates long-form OB-Rb receptors in the hypothalamus to decrease food intake and increase energy expenditure. A variety of neuropeptides are involved in these responses, including neuropeptide Y, agouti-related protein, the prepro-melanocortin system and cocaine- and amphetamine-regulated transcript. OB-Rb receptors (and other receptor isoforms) are also found in peripheral tissues. Leptin is now known to have a wide range of peripheral actions and is involved in activating the immune system, haematopoiesis, angiogenesis and as a growth factor, as well as being a regulator of many cellular functions. The identification of leptin has led to reappraisal of the role of white adipose tissue from being an organ concerned primarily with energy storage as fat to an understanding that it is also a major endocrine and secretory organ. While the importance of the sympathetic nervous system in mobilising fatty acids from adipose tissue has long been known, it has become apparent that the sympathetic system is a key regulator of leptin production in white adipose tissue as well. Sympathomimetic amines and cold exposure or fasting (which lead to sympathetic stimulation of white fat), decrease leptin gene expression in the tissue and leptin production. On the other hand, sympathetic blockade often increases circulating leptin and leptin gene expression, and it is possible that the sympathetic system has a tonic inhibitory action on leptin synthesis. Apart from the few instances where leptin is absent, leptin levels are increased in obesity, while the sympathetic sensitivity of adipose tissue is reduced, consistent with the high leptin levels that are seen. The dysregulation of energy balance leading to obesity may partly involve a decrease in leptin sensitivity, or the leptin system may be set to have maximal effects at low leptin levels.

Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice

Leptin receptor gene expression has been measured in arcuate and ventromedial hypothalamic nuclei. Receptor mRNA in both hypothalamic areas was higher in obese mice than in lean littermates. Twice daily leptin administration for 7 days profoundly affected food intake, reduced leptin receptor mRNA in the arcuate nucleus, and had a similar effect on neuropeptide Y gene expression. A single leptin injection was ineffective. Exposure of lean mice to cold for 24 h caused an induction of leptin receptor and NPY mRNA which was normalized when animals were returned to the warm. Regulation of receptor gene expression may be an important component in the reading of the leptin signal.

The sympathetic nervous system in white adipose tissue regulation

Sympathetic stimulation has long been recognized to mobilise fatty acids from white adipose tissue. However, it is now apparent that adipose tissue is not only concerned with energy storage as fat, but is a major endocrine and secretory organ. This change has resulted from the identification of leptin as a hormone of energy balance secreted by white adipose tissue. The sympathetic system is a key regulator of leptin production in white fat. Sympathomimetic amines, cold exposure or fasting (which lead to sympathetic stimulation of white fat), decrease ob gene expression in the tissue and leptin production. On the other hand, sympathetic blockade often increases circulating leptin and ob gene expression, and it is postulated that the sympathetic system has a tonic inhibitory action on leptin synthesis. In rodents this action is through stimulation of, beta3-adrenoceptors. The adrenal medulla (as opposed to the direct sympathetic innervation) has been thought to play only a minor role in the catecholaminergic regulation of white adipose tissue. However, in rodents responses of the leptin system to adrenergic blockade vary with the method used. Changes in leptin and ob gene expression are considerably less using methods of blockade that only effect the terminal adrenergic innervation, rather than medullary secretions as well. Stimulation of the leptin system increases sympathetic activity and hence metabolic activity in many tissues. As well as leptin, other (but not all) secretions from white adipose tissue are subject to sympathetic regulation. In obesity the sympathetic sensitivity of adipose tissue is reduced and this factor may underlie the dysregulation of leptin production and other adipose tissue secretions.

Hyperleptinaemia in mice induced by administration of the tyrosine hydroxylase inhibitor α-methyl-p-tyrosine

α‐methyl‐p‐tyrosine (αMPT), an inhibitor of tyrosine hydroxylase, was administered to mice to block noradrenaline synthesis. Ten hours after injection of αMPT there was a 6‐fold increase in plasma leptin. The level of ob mRNA in epididymal white adipose tissue was also increased, but UCP1 mRNA in brown fat fell. In contrast to lean mice, ob mRNA in white fat of ob/ob mice was not increased by αMPT. αMPT raised plasma leptin in fasted as well as fed mice. Hyperleptinaemia was attenuated by treatment with a β3‐adrenoceptor agonist. Inhibition of noradrenaline synthesis leads to the rapid induction of hyperleptinaemia; it is suggested that sympathetic tone plays a pivotal role in regulating leptin production.

Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status

Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high‐fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative 125I‐labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob‐R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high‐fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. 125I‐labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. 125I‐Labelled leptin was reduced by leptin challenge and increased by high‐fat feeding. Ob‐Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob‐Rb expression in the ARC, but not after high‐fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob‐Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high‐fat diet. Thus, early loss of leptin sensitivity after high‐fat feeding is unrelated to down‐regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease 125I‐labelled leptin binding and the loss of ability of leptin to up‐regulate Ob‐Rb expression in the ARC after high‐fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high‐fat diet.

Effect of flavour of liquid Ensure diet supplement on energy intake in male SD rats.

Outbred male Sprague-Dawley rats were provided with one of the four flavours of the liquid diet, Ensure, in addition to chow pellets, to examine whether differences in flavour lead to differences in energy intake i.e. degree of over-consumption. For half the rats, the Ensure supplement was provided for 14 days and then withdrawn for the final 8 days of the study, whereas the remaining animals were allowed to consume Ensure for 22 days. All four flavours of Ensure, chocolate, vanilla, coffee and asparagus, induced a sustained increase in daily energy intake of approximately 15%. There was an effect of flavour on initial consumption of the Ensure diet, with coffee and asparagus flavours being consumed less avidly than vanilla or chocolate. However, this effect was short-lived. Overall, there was no effect of flavour on body weight gain, energy intake from Ensure, total energy intake, body composition, or measured blood hormones and metabolites. Withdrawal of Ensure resulted in reductions in body weight gain, total energy intake, fat but not lean tissue mass, and concentrations of blood leptin, non-esterified fatty acids and triglycerides, but there was no effect of the flavour of Ensure previously supplied on any of these parameters. The ability of the liquid diet, Ensure, to stimulate long-term caloric over-consumption is not due to its flavouring. Rather, other attributes of Ensure must be more important, such as its intrinsic flavour, liquid formulation, macronutrient composition, and ease of ingestion, digestion and absorption.

Dietary supplementation with conjugated linoleic acid plus n-3 polyunsaturated fatty acid increases food intake and brown adipose tissue in rats.

The effect of supplementation with 1% conjugated linoleic acid and 1% n-3 long chain polyunsaturated fatty acids (CLA/n-3) was assessed in rats. Food intake increased with no difference in body weights. White adipose tissue weights were reduced whereas brown adipose tissue and uncoupling protein-1 expression were increased. Plasma adiponectin, triglyceride and cholesterol levels were reduced while leptin, ghrelin and liver weight and lipid content were unchanged. Hypothalamic gene expression measurements revealed increased expression of orexigenic and decreased expression of anorexigenic signals. Thus, CLA/n-3 increases food intake without affecting body weight potentially through increasing BAT size and up-regulating UCP-1 in rats.

Leptin Signals and Secretions from White Adipose Tissue

The classical view of white adipose tissue is of an organ concerned with the storage of lipid, responding passively to changes in energy balance. It is now evident that white fat is an important secretory and endocrine organ which plays a wide role in metabolic and physiological regulation. It secretes, in particular, a critical signal in the control of energy balance, i.e. leptin. This hormone is also implicated in a range of other processes, from reproduction to immunity and angiogenesis. A wide variety of protein secretions from white fat have now been identified, including angiotensinogen, plasminogen activator inhibitor- l, retinol binding protein adiponectin and interleukin-6, and these are associated with processes as diverse as haemostasis and the control of blood pressure. It is clear that white adipose tissue has come ‘in from the cold’.