Diurnal changes in hypothalamic neuropeptide and SOCS-3 expression: effects of lactation and relationship with serum leptin and food intake.

Rats normally eat about 85% of their food at night. Lactation increases food intake 3- to 4-fold, but the diurnal pattern of food intake persists. The mechanisms responsible for the diurnal and lactation-induced changes in food intake are still unresolved, hence we have further investigated the possible roles of serum leptin and hypothalamic expression of neuropeptide Y (NPY), agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in rats. Suppressor of cytokine signalling-3 (SOCS-3) acts as a feedback inhibitor of leptin signalling in the hypothalamus, hence changes in expression of SOCS-3 were also investigated. Changes in expression of NPY, AgRP or POMC alone could not account for the diurnal changes in intake and their alteration by lactation. However, there were increased AgRP mRNA:POMC mRNA ratios at night and also during lactation, which were very similar to estimated changes in food intake. Such changes in expression may result in dominance of the orexigenic AgRP peptide over the appetite-suppressing POMC-derived peptides, and so could contribute to the hyperphagia in these states. Diurnal and lactation-related changes in the AgRP mRNA:POMC mRNA ratio and food intake are not due to changes in leptin alone. However, hypoleptinaemia, possibly through increased expression of NPY, may contribute to the hyperphagia of lactation. In the dark, expression of SOCS-3 was decreased in non-lactating rats; lactation decreased SOCS-3 expression in both light and dark phases. However, such changes are likely to enhance the ability of leptin-responsive neurones to transmit the leptin signal, and so are unlikely to contribute to either the nocturnal increase in appetite or the hyperphagia of lactation.

Lactation modulates diurnal expression profiles of specific leptin receptor isoforms in the rat hypothalamus.

We investigated the effects of lactation on diurnal changes in serum leptin and hypothalamic expression of the leptin receptor isoforms, Ob-Ra, -Rb, -Rc, -Re and -Rf in rats. In non-lactating rats, serum leptin concentration was increased at night while hypothalamic mRNA levels of Ob-Rb, -Rc and -Re decreased; by contrast, expression of Ob-Ra and Ob-Rf was unchanged at night. There were significant negative correlations between serum leptin and mRNA expression of Ob-Rb (P<0.001) and Ob-Re (P<0.05), which were independent of time of day. In lactating rats, the nocturnal rise in serum leptin was attenuated. Daytime hypothalamic Ob-Rb mRNA levels were significantly lower than in non-lactating controls, and the normal nocturnal decreases in expression of Ob-Rb, -Rc and -Re were lost. The relationship between serum leptin and Ob-Re expression was not changed by lactation. Lactation had no effect on the expression of Ob-Ra mRNA in the hypothalamus. Decreased daytime Ob-Rb expression could lead to reduced hypothalamic sensitivity to leptin, and thus contribute to increased daytime appetite in lactating rats. Moreover, maintaining high levels of Ob-Re expression could, by increasing hypothalamic leptin-binding protein concentration and reducing local leptin bioavailability, further accentuate hyperphagia. Thus, selective changes in expression of specific isoforms of the leptin receptor may contribute to the hyperphagia of lactation in rats.

Lipogenic enzyme activities in different adipose depots of Pirenaican and Holstein bulls and heifers taking into account adipocyte size.

The effects of sex, genotype, and adipose depot on lipogenic enzyme activity have been investigated in Holstein and Pirenaican bulls and heifers, taking into account differences in adipocyte size. Fifteen Pirenaican bulls and 15 heifers and 15 Holstein bulls and 13 heifers were fattened until slaughter (12 to 13 mo old and 450 to 500 kg of body weight). During the fattening period, animals had ad libitum access to commercial concentrates and straw. The 10th rib was dissected to determine the fat content. Adipocyte size and activities of the following lipogenic enzymes were determined: glycerol 3-phosphate dehydrogenase, fatty acid synthase, nicotinamide adenine dinucleotide phosphate (NADP)-malate dehydrogenase, glucose 6-phosphate dehydrogenase, and NADP-isocitrate dehydrogenase, in the omental, perirenal, subcutaneous, and intermuscular adipose depots, respectively. Because adipocyte mean cell volume varied with sex, breed, and depot, regression analyses of log(e) activity per cell and log(e) cell volume were used to compare activities per unit volume. Sex, breed and depot had no effect (P > 0.05) on the gradients of regressions, which did not differ significantly from 1. Thus, activity per unit volume did not vary with cell size. Consequently, sex, breed, and depot effects on the regression analyses were equivalent to effects on activity per unit volume. Females had greater amounts of fat in the 10th rib (P < 0.001), larger adipocytes (P < 0.001) and, in general, greater (P < 0.05) lipogenic activity per cell, even when adjusted for cell size, than males. These findings suggest that differences in adiposity between sexes are mainly due to females having a greater capacity for lipid synthesis, and hence, hypertrophy, than males. When adjusted for differences in carcass weight, Holsteins had larger adipocytes than Pirenaicans. The abdominal depots, omental and perirenal, had a greater adipocyte size (P < 0.001) and, in general, greater lipogenic enzyme activities per cell (P < 0.05) than the subcutaneous and intermuscular carcass depots. However, when activity per cell was adjusted for cell size, subcutaneous depots had greater fatty acid synthae, glucose 6-phosphate dehydrogenase, and NADP-malate dehydrogenase activities than omental and perirenal, indicating that other factors such as nutrient supply may restrict hypertrophy of carcass adipocytes.

Regulation of serum leptin and its role in the hyperphagia of lactation in the rat.

The factors regulating serum leptin concentration and its relationship to the hyperphagia of lactation have been investigated in rats. Lactation results in hypoleptinaemia and loss, or at least marked attenuation, of the nocturnal rise in serum leptin. Litter removal resulted in a fall in food intake and restoration of the nocturnal rise in serum leptin. Returning the litter to the mother after a 48-h absence increased food intake and began to reinitiate milk production, but the nocturnal serum leptin levels were still increased at 48 h after litter restoration. Adjusting litter size to four, eight, ten or fourteen pups at parturition resulted in different rates of litter growth and food intake during the subsequent lactation, but had no effect on the degree of hypoleptinaemia. Reducing litter size from ten to four pups at mid-lactation resulted in a transient increase in both serum leptin and pup growth rate, while food intake fell to a level found in rats suckling four pups throughout lactation. Reducing milk production by injection of bromocriptine increased serum leptin, but did not restore the nocturnal rise in serum leptin; food intake decreased, but remained much higher than in non-lactating rats. Feeding a varied, high-energy diet resulted in a decrease in the weight of food ingested, but no change in calorie intake, and had no effect on the hypoleptinaemia. These studies suggested that the hypoleptinaemia of lactating rats is due to negative energy balance, but the loss of the nocturnal rise in serum leptin is due to the suckling stimulus. The negative energy balance of lactation does not appear to be caused by a physical constraint on food intake. While the hypoleptinaemia should facilitate the hyperphagia of lactation, other orexigenic signals must also be involved.

Leptin and the adaptations of lactation in rodents and ruminants.

Lactation markedly increases nutrient requirements in both rodents and ruminants. This is met mostly by increased food intake, but there are also adaptations to increase metabolic efficiency. Despite such changes, lactating animals usually experience periods of negative energy balance. This is not due to a physical constraint on food intake, at least in the rat. Leptin, a hormone secreted by adipocytes, plays an important role in the regulation of appetite and energy balance. During lactation, serum leptin concentration is decreased in both rodents and ruminants, and the nocturnal rise in concentration is lost in rats. Hypoleptinaemia in lactation is primarily a result of negative energy balance. There is also increased clearance of serum leptin, and the attenuation of the nocturnal rise in leptin in rats is at least partly due to the suckling stimulus. Hypoleptinaemia is not the major factor driving hyperphagia in lactating rats, but it probably facilitates the increased food intake. Leptin may play a more important role in this respect in lactating ruminants. Leptin is probably involved in other adaptations that increase metabolic efficiency during lactation. The ability of hypothalamic neuropeptides to respond to leptin does not appear to be altered by lactation in either rodents or ruminants. The reason why lactating animals do not respond to hypoleptinaemia with a further increase in appetite, thereby achieving energy balance, appears to be due to a failure to respond to changes in neuropeptides which mediate the effects of leptin.

Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation during the lactation cycle of the rat.

The removal of the litter from lactating rats results in a decrease in the lipolytic response to catecholamines in maternal adipocytes; this effect can be prevented by concomitant treatment of the rats with growth hormone. The decrease in response to catecholamines following litter removal was not due to a change in the amount of either hormone-sensitive lipase (HSL) or perilipin per adipocyte or in the proportion of either of these proteins associated with the fat droplet. Incubation in vitro with isoproterenol did not cause any apparent net translocation of HSL to the fat droplet in adipocytes from the mature female rats in any state used in this study, but isoproterenol did cause a movement of perlipin away from the fat droplet. This translocation of perilipin was not altered by litter removal. Thus, the decrease in response to catecholamines found on litter removal from lactating rats appears to be due to a diminished ability to activate HSL associated with fat droplet.

Effects of recombinant ovine leptin on in vitro lipolysis and lipogenesis in subcutaneous adipose tissue from lactating and nonlactating sheep.

Direct effects of recombinant ovine leptin on adipose metabolism in sheep were investigated. Lipolytic and lipogenic rates were assessed following preincubation of subcutaneous adipose tissue explants with recombinant ovine leptin. Leptin had no consistent effect on the basal (unstimulated) lipolytic rate in adipose tissue from wethers. Lipolytic rate measured in the presence of combinations of adenosine deaminase, isoprenaline, and N6-phenylisopropyl adenosine was unaffected by pretreatment with leptin. In lactating ewes, there was no relationship between increasing concentrations of leptin and basal lipolytic rate. Leptin had no effect on basal (unstimulated) lipogenesis, or on insulin-stimulation or growth hormone inhibition of lipogenesis in adipose tissue from wethers. Lipogenesis in adipose tissue from lactating ewes was also unaffected by preincubation with leptin; however, at supraphysiological concentrations of leptin, there was a small reduction in the rate of insulin-stimulated lipogenesis. Leptin failed to induce phosphorylation of the signal transducers and activators of transcription, STAT 3 and STAT 5, in sheep adipocytes. These results suggest that leptin does not have a direct physiological effect on subcutaneous adipose tissue metabolism in sheep.

Food restriction selectively increases hypothalamic orexin-B levels in lactating rats.

Orexins are hypothalamic peptides implicated in the regulation of ingestive and other behaviours. Here we investigated prepro-orexin expression and hypothalamic orexin-A and -B levels in lactating rats, which display marked hyperphagia, with or without food restriction for 2 days or treatment with bromocriptine, which inhibits milk production and thus reduces the energy losses of lactation. Neither prepro-orexin gene expression nor hypothalamic orexin-A peptide levels were changed in any of these lactating groups compared with age-matched virgin controls. However, hypothalamic orexin-B levels were significantly higher in lactating rats that were food-restricted for 2 days (P<0.05) compared with non-lactating controls and with lactating rats that were either freely-fed or bromocriptine-treated. Thus, food restriction superimposed on lactation selectively increases hypothalamic orexin-B levels, suggesting that orexin-A and -B may be differentially released or cleared. Changes in orexin-B availability may influence physiological activities other than energy homeostasis, perhaps inducing arousal.

Signals of adiposity.

Adipose tissue, a reserve of energy, has played an essential role in mammalian evolution. Adipose tissue differs from other tissues in that its mass has considerable capacity to expand, which while beneficial in decreasing the risk of starvation, increases the risk of predation. Adipose tissue mass is thus under tight control in nondomestic species. Adipose tissue secretes a variety of factors, some of which (leptin, tumor necrosis factor (TNF) alpha, resistin) are thought to be involved in modulation of adipose mass. Leptin has a variety of functions, primarily targetting the hypothalamus where it acts to decrease appetite and increase energy expenditure. Leptin is also involved in the adaptations to fasting, and leptin is also required for normal reproductive and immune function. TNF alpha and resistin appear to have key paracrine roles, attenuating the anabolic effects of insulin on adipose tissue metabolism.

Adipocyte studies: systems for investigating effects of growth hormone and other chronically acting hormones.

Adipose tissue is very amenable to study in vitro. Collagenase digestion yields free adipocytes which usually respond well to acute stimulation/inhibition by hormones and other factors. Chronic effects of hormones are best studied using explants of adipose tissue which, from some species (e.g. sheep), can be maintained in culture for up to a week without loss of function. Alternatively, preadipocytes can be readily isolated from adipose tissue and induced to proliferate and differentiate in culture, while various adipocyte-like cell-lines have been established, which can be used for chronic studies. Use of these various systems for investigating the mechanisms of action of growth hormone are described.

Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes.

Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resided predominantly in the cytosol and unphosphorylated perilipin upon the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the beta-adrenergic agonist, isoproterenol, HSL translocated from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats had markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age.

Ovine adipose tissue monounsaturated fat content is correlated to depot-specific expression of the stearoyl-CoA desaturase gene.

The basis for the variation in fatty acid composition in different ovine adipose tissue depots was investigated. The proportion of stearic (C18:0) and oleic (C18:1) acids vary in a site-specific fashion; abdominal depots (omental and perirenal) contain relatively more C18:0 than C18:1, and carcass depots, especially sternum, have a markedly higher proportion of C18:1. Additionally, expression of a number of lipogenic enzyme genes (stearoyl-CoA desaturase [SCD], acetyl-CoA carboxylase-alpha [ACC-alpha], lipoprotein lipase [LPL]) and the cytoskeletal protein gene alpha-tubulin vary among depots, although the pattern of variation differs for each mRNA. When these expression data were related to the mean cell volume of adipocytes pooled from all depots, a significant pattern emerged: expression of the ACC-alpha, LPL, and alpha-tubulin genes was highly correlated with the size of adipocytes. In contrast, when the expression of SCD mRNA was assessed as a function of mean cell volume, two populations of adipocytes emerged: no significant correlation was found between the expression of SCD mRNA per adipocyte and mean cell volume for the abdominal depots, although a highly significant correlation was observed between SCD gene expression and mean cell volume for the carcass and epicardial depots. Similarly, a highly significant correlation was found for the amount of C18:1 per adipocyte and the abundance of SCD mRNA per adipocyte for the carcass and epicardial depots, whereas no significant correlation was observed for these traits for the omental and perirenal depots. Thus, the SCD gene seems to be regulated in a depot-specific fashion and in a manner distinct from that of the ACC and LPL genes.

Present and future studies on lipogenesis in animals and human subjects.

Lipogenesis occurs in all vertebrate species and has a critical role in energy balance, providing a means whereby excess energy can be stored as a fat. The metabolic pathways involved and their tissue distribution in different species, including man, are well known. The responses of lipogenesis to diet and to physiological and pathological states have been the subject of many studies. At a molecular level the major rate-controlling enzymes have been identified and their acute, and to a lesser extent chronic, control by hormones have been investigated extensively. However, there is no reason to suppose that all factors regarding lipogenesis have been identified (e.g. the recent discovery of acylation-stimulating protein). Little is known about the movement of newly-synthesized triacylglycerols in cells, either for secretion or storage. The production of leptin and tumour necrosis factor alpha by adipocytes provides a novel means of feedback control of triacylglycerol production, leptin by decreasing appetite and tumour necrosis factor alpha by inducing insulin resistance. The synthesis of these peptides appears to vary with the amount of triacylglycerol in adipocytes, but the molecular basis of this process is unknown. Elucidation of the signalling systems involved in the acute and chronic regulation of lipogenesis is also important, both with respect to some homeorhetic adaptations and also in some pathological conditions (e.g. non-insulin-dependent diabetes). Finally, molecular biology is revealing unexpected complexities, such as multiple promoters and different isoforms of enzymes (e.g. acetyl-CoA carboxylase; EC 6.4.1.2) exhibiting tissue specificity. Molecular biology, through transgenesis, also offers novel and powerful means of manipulating lipogenesis.

Neuropeptide Y receptor alterations in the hypothalamus of lactating rats.

Hypothalamic neuropeptide Y (NPY) neurons are influenced by circulating levels of insulin and leptin and are thought to be involved in mediating hunger following underfeeding. We have investigated hypothalamic NPY receptor subtypes in lactating rats, which are markedly hyperphagic throughout the day and night. NPY receptors were measured by using [125I] peptide YY, a high-affinity ligand, and Y1 receptors were masked by using the highly specific antagonist BIBP 3226. Freely fed lactating rats showed no changes in the densities of Y1, or non-Y1, NPY binding sites in whole hypothalamic homogenates or in individual hypothalamic regions (measured by quantitative autoradiography) examined during the day or night (P > 0.05; n = 10/group, and n = 6/group, respectively). However, reducing food intake by 35% had a more profound effect on NPY receptor density in lactating than in control rats, producing down-regulation of non-Y1 receptors in the ventromedial, dorsomedial, and perifornical lateral areas (all P < 0.05; n = 7/group) and reduction of plasma insulin and leptin levels (both P < 0.01). Thus, although the NPY system may not have a major role in the hyperphagia of freely fed lactating rats, it appears to have an important function in the response to undernutrition in such animals.

Regulation of differentiation of sheep subcutaneous and abdominal preadipocytes in culture.

Factors regulating the differentiation of sheep subcutaneous and abdominal (omental or perirenal) preadipocytes from foetal lambs, suckling lambs and fattening sheep have been investigated using a serum-free cell culture system. Differentiation was assessed by changes in the activity of the enzyme glycerol 3-phosphate dehydrogenase. Insulin or IGF-I was essential for differentiation. Dexamethasone, a lipid supplement (Excyte) and the thiazolidinedione, rosiglitazone (BRL 49653) (a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist) all enhanced preadipocyte differentiation, whereas fibroblast growth factor and GH were inhibitory. The most effective combination was insulin, triiodothyronine, dexamethasone and rosiglitazone. Under suboptimal conditions, preadipocytes from fattening sheep differentiated less well than cells from suckling and foetal lambs. Also, under suboptimal conditions, abdominal preadipocytes did not differentiate as well as subcutaneous preadipocytes. However, age and depot differences were minimised when cells were cultured with insulin, triiodothyronine, rosiglitazone and either dexamethasone or lipid. The results suggest that variation in the ability to produce the natural ligand for PPAR-gamma contributes to depot- and age-specific differences in the ability of preadipocytes to differentiate.

Regulation of the GTP-binding protein-based antilipolytic system of sheep adipocytes by growth hormone.

Chronic exposure of sheep adipose tissue to growth hormone (GH) in vitro decreases the ability of the adenosine analogue, N6-phenylisopropyladenosine (PIA), to inhibit isoprenaline-stimulated lipolysis by a mechanism which is dependent on both gene transcription and protein serine/threonine phosphorylation. The inhibition is not due to a change in ligand binding to the adenosine receptor, the amounts of the three isoforms of the inhibitory GTP-binding protein, Gi, or the maximum (forskolin-stimulated) adenylate cyclase activity. The ability of GH to modulate the PIA-activated adenosine receptor to stimulate dissociation of heterotrimeric Gi was assessed by measurement of pertussis toxin-catalysed ADP-ribosylation of Gi; GH does not appear to alter the interaction between the activated receptor and Gi. The ability of GH to alter the ability of activated Gi to inhibit adenylate cyclase activity was assessed by measuring the ability of a GTP analogue, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), to inhibit forskolin-stimulated adenylate cyclase activity; chronic exposure to GH prevented this effect of p[NH]ppG. Thus the attenuation of the inhibition of lipolysis by PIA by chronic exposure of adipocytes to GH appears to be due to an impairment in the interaction between adenylate cyclase and the alpha subunit of one or more isoforms of Gi.

Dephosphorylation of perilipin by protein phosphatases present in rat adipocytes.

By incubating 32P-labelled adipocytes, and extracts from these cells, in the presence or absence of specific inhibitors, we evaluated the contribution of protein phosphatases PP1, PP2A and PP2C, to the dephosphorylation of perilipin, an acutely hormone-regulated adipocyte phosphoprotein. Under conditions to completely inhibit PP2A activity, perilipin phosphatase activity in extracts remain unaffected, but PP1 inhibition results in abolition of perilipin phosphatase activity. Inhibition of PP1 (and 2A) in intact adipocytes stimulated lipolysis and increased phosphorylation of perilipin. No involvement of PP2C was found. Hence, PP1 constitutes the predominant if not sole perilipin phosphatase in adipocytes.

Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation.

In this study we first established, by immunoblotting with specific antibodies, the temporal changes in cellular levels of protein kinase C (PKC) isoforms during differentiation of 3T3-F442A pre-adipocytes. Both pre-adipocyte and adipocyte 3T3-F442A cells were found to express PKC-alpha, -gamma, -delta, -epsilon, -zeta and -mu. However we were unable to detect PKC-beta, -eta or -theta. The same PKC isoform expression profile was found in rat adipocytes. The alpha, delta and gamma isoforms displayed similar temporal patterns of expression during differentiation of 3T3-F442A cells; all increased rapidly, peaking at day 2 of differentiation. Subsequently, the expression of these isoforms decreased, resulting in lower levels in fully differentiated adipocytes than in pre-adipocytes. The expression of PKC-epsilon increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKC-mu increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process itself. No change was observed in PKC-zeta levels during adipocyte development. Anti-sense oligodeoxynucleotides (ODNs) were used to deplete selectively the individual PKC subtypes. Each of the ODNs used effectively depleted the specific isoforms to undetectable levels and did not affect expression of the other PKC subtypes. This approach indicated that pre-adipocyte differentiation is not dependent upon PKC-zeta but that PKC-alpha,-delta and -mu each exert an inhibitory influence upon differentiation. Use of anti-sense ODNs to deplete PKC-epsilon and -gamma revealed that pre-adipocyte differentiation is dependent upon each of these isoforms. However, PKC-gamma, but not PKC-epsilon, appeared to be necessary for the clonal expansion of differentiating cells, suggesting that PKC-epsilon is required at a later phase in the differentiation process, when its expression is elevated, for the attainment and maintenance of the adipocyte phenotype.

Lactation suppresses diurnal rhythm of serum leptin.

Rats consume most of their daily food intake at night; serum leptin levels and adipose tissue leptin mRNA content are elevated at night in non-lactating rats fed ad libitum. Lactation induces massive hyperphagia with most food still consumed at night, but the nocturnal increase in leptin secretion was not observed in lactating rats. Thus the link between nocturnal food intake and increased serum leptin is broken during lactation and the hypoleptinaemia may be an important factor promoting the hyperphagia of lactation.

Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic AMP-specific phosphodiesterase in 3T3-F442A preadipocytes.

The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.