Pharmacokinetics of leptin in female mice.

Pharmacokinetics of leptin in mammals has received limited attention and only one study has examined more than two time points and this was in ob/ob mice. This study is the first to observe the distribution of leptin over a time course in female mice. A physiologic dose (12 ng) of radiolabelled leptin was injected in adult female mice via the lateral tail vein and tissues were dissected out and measured for radioactivity over a time course up to two hours. Major targets for administered leptin included the liver, kidneys, gastrointestinal tract and the skin while the lungs had high concentrations of administered leptin per gram of tissue. Leptin was also found to enter the lumen of the digestive tract intact from the plasma. Very little of the dose (<1 %) was recovered from the brain at any time. Consequently we confirm that the brain is not a major target for leptin from the periphery, although it may be very sensitive to leptin that does get to the hypothalamus. Several of the major targets (GI tract, skin and lungs) for leptin form the interface for the body with the environment, and given the ability of leptin to modulate immune function, this may represent a priming effect for tissues to respond to damage and infection.

New insights into polar overdominance in callipyge sheep.

The callipyge phenotype in sheep involves substantial postnatal muscle hypertrophy and other changes to carcass composition. A single nucleotide polymorphism in the DLK1-DIO3 imprinted gene cluster alters gene expression of the paternal allele-specific protein-coding genes and several maternal allele-specific long noncoding RNA and microRNA when the mutation is inherited in cis. The inheritance pattern of the callipyge phenotype is polar overdominant because muscle hypertrophy only occurs in heterozygous animals that inherit a normal maternal allele and the callipyge SNP on the paternal allele (+/C). We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms, indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the downregulated genes are putative targets of the maternal allele-specific microRNA with gene ontology, indicating regulatory and cell signaling functions. These results suggest that the trans-effect of the maternal noncoding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development.

The ruminant developmental transition from late foetus to lamb is associated with marked changes in skeletal muscle structure and function that reflect programming for new physiological demands following birth. To determine whether epigenetic changes are involved in this transition, we investigated the genomic architecture of the chromatin modification, histone 3 lysine 27 trimethylation (H3K27me3), which typically regulates early life developmental processes; however, its role in later life processes is unclear. Chromatin immunoprecipitation coupled with next-generation sequencing was used to map H3K27me3 nucleosomes in ovine longissimus lumborum skeletal muscle at 100 days of gestation and 12 weeks post-partum. In both states, H3K27me3 modification was associated with genes, transcription start sites and CpG islands and with transcriptional silencing. The H3K27me3 peaks consisted of two major categories, promoter specific and regional, with the latter the dominant feature. Genes encoding homeobox transcription factors regulating early life development and genes involved in neural functions, particularly gated ion channels, were strongly modified by H3K27me3. Gene promoters differentially modified by H3K27me3 in the foetus and lamb were enriched for gated ion channels, which may reflect changes in neuromuscular function. However, most modified genes showed no changes, indicating that H3K27me3 does not have a large role in late muscle maturation. Notably, promyogenic transcription factors were strongly modified with H3K27me3 but showed no differences between the late gestation foetus and lamb, likely reflecting their lack of involvement in the myofibre fusion process occurring in this transition. H3K27me3 is a major architectural feature of the epigenetic landscape of ruminant skeletal muscle, and it comments on gene transcription and gene function in the context of late skeletal muscle development.

Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria.

The bovine Muc1 protein is synthesized by mammary epithelial cells and shed into milk as an integral component of the milk fat globule membrane; however, the structure and functions of this mucin, particularly in relation to lactation, are poorly defined. The objectives of this investigation were to investigate the Muc1 gene and protein structures in the context of lactation and to test the hypothesis that Muc1 has a role in innate immune defense. Polymerase chain reaction analysis of genomic DNA from 630 cattle revealed extensive polymorphism in the variable number of tandem repeats (VNTR) in the bovine Muc1 gene. Nine allelic variants spanning 7 to 23 VNTR units, each encoding 20 AA, were identified. Three alleles, containing 11, 14, and 16 VNTR units, respectively, were predominant. In addition, a polymorphism in one of the VNTR units has the potential to introduce a unique site for N-linked glycosylation. Statistical analysis indicated weak associations between the VNTR alleles and milk protein and fat percentages in a progeny-tested population of Holstein-Friesian dairy cattle. No association with somatic cell count could be demonstrated. Bovine Muc1 was purified from milk fat globule membranes and characterized. The protein was highly glycosylated, primarily with O-linked sialylated T-antigen [Neu5Ac(alpha2-3)-Gal(beta1-3)-GalNAcalpha1] and, to a lesser extent, with N-linked oligosaccharides, which together accounted for approximately 60% of the apparent mass of Muc1. Purified bovine Muc1 directly bound fluorescently labeled Escherichia coli BioParticles (Invitrogen, Mount Waverley, Australia) and inhibited their binding to bovine mammary epithelial cells grown in vitro. It was also demonstrated that the expression of Muc1 mRNA in bovine mammary epithelial cells was markedly upregulated by lipopolysaccharide. Muc1 may be a pattern recognition protein that has the capacity to sequester bacteria and prevent their attachment to epithelial surfaces by immobilizing and subsequently shedding Muc1-bound bacteria from the cell surface. It was concluded that bovine Muc1 is probably an inducible innate immune effector and an important component of the first line of defense against bacterial invasion of epithelial surfaces, particularly mammary epithelial surfaces and the neonatal gut.

Adipogenesis in mouse 3T3L1 cells: the effects of Rtl1 over-expression.

Rtl1 and Dlk1 are two genes in an imprinted gene cluster on mouse chromosome 12. Rtl1 is a paternally expressed gene encoding a retrotransposon-like protein, but as yet, there is no evidence that a protein is produced by this gene although a transcript is produced. Dlk1 is also paternally expressed and it encodes a plasma membrane-bound protein known to act as a negative regulator of adipogenesis. Genes at other imprinted loci often show coordinate expression and may interact at a functional level. We tested the hypothesis that Dlk1 and Rtl1 are functionally related. Rtl1 and Dlk1 mRNA expression patterns show similar time courses of down-regulation during induced adipogenesis of 3T3L1 cells. Despite the coordinate down-regulation of Dlk1 and Rtl1 mRNA levels, Rtl1 over-expression did not affect the time course of adipogenesis or Dlk1 expression. It is concluded that these two genes are neither directly nor indirectly functionally related.

Analysis of gene expression during the onset of muscle hypertrophy in callipyge lambs.

The callipyge mutation causes postnatal muscle hypertrophy in heterozygous lambs that inherit a paternal callipyge allele (+/CLPG). Our hypothesis was that the up-regulation of one or both of the affected paternally expressed genes (DLK1 or PEG11) initiates changes in biochemical and physiological pathways in skeletal muscle to induce hypertrophy. The goal of this study was to identify changes in gene expression during the onset of muscle hypertrophy to identify the pathways that are involved in the expression of the callipyge phenotype. Gene expression was analysed in longissimus dorsi total RNA from lambs at 10, 20, and 30 days of age using the Affymetrix Bovine Expression Array. An average of 40.6% of probe sets on the array was detected in sheep muscle. Data were normalized and analysed using a two-way anova for genotype and age effects with a false discovery rate of 0.10. From the anova, 13 genes were significant for the effect of genotype and 13 were significant for effect of age (P < 0.10). No significant age-by-genotype interactions were detected (P > 0.10). Of the 13 genes indicating an effect of genotype, quantitative PCR assays were developed for all of them and tested on a larger group of animals from 10 to 200 days of age. Nine genes had significantly elevated transcript levels in callipyge lambs. These genes included phosphofructokinase, a putative methyltransferase protein, a cAMP phosphodiesterase, and the transcription factor DNTTIP1.

Differential expression of Dlk-1 in bovine adipose tissue depots.

Dlk-1, a type 1 membrane glycoprotein, is a member of the Epidermal Growth Factor-like family of homeotic proteins that are typically involved in cell fate decisions and in mice it has been implicated in the control of differentiation of adipocytes. The aim of this study was to determine whether there were tissue-specific expression patterns of Dlk-1 splice variants in bovine tissues. Only the Dlk-1-C2 variant was expressed in adult bovine tissues while both Dlk-1-C2 and Dlk-1-A variants were expressed in foetal tissues. Quantitative real-time PCR revealed large differences in the relative levels of expression of the Dlk-1-C2 variant in adult adipose tissue depots with no expression in subcutaneous and brisket adipose tissues. Expression was also demonstrated in three adult skeletal muscle samples. The large variation in the level of expression of Dlk-1-C2 in different adult tissues may reflect the relative preadipocyte content of those tissues and consequently their potential for generating new adipocytes. A low abundance soluble glycoprotein (bFA1) was purified from bovine amniotic fluid. Analyses of its amino acid sequence revealed that it corresponded to most of the extracellular domain of bovine Dlk-1 and was derived by proteolytic processing from the full-length Dlk-1 protein encoded by the Dlk-1-A variant. The tissues expressing the Dlk-1-A variant have not been identified but are likely to be foetal in origin. Splice variants of Dlk-1 may have varied functional roles with the foetal Dlk-1-A form capable of generating a protein that undergoes proteolytic processing to release a soluble ecto-domain of Dlk-1. In contrast the Dlk-1-C2 splice variant codes for a protein lacking this processing site and therefore it probably remains bound to the cell membrane.

Identification of an immuno-protective mucin-like protein, peritrophin-55, from the peritrophic matrix of Lucilia cuprina larvae.

A mucin-like glycoprotein, peritrophin-55 was isolated and purified from the peritrophic matrix of Lucilia cuprina larvae. When injected into sheep, peritrophin-55 induced an immune response that inhibited larval growth by 51-66% when larvae subsequently fed on sera from the vaccinated sheep. The protein may have potential use as an immunogen probably accompanying other antigens to protect sheep from the cutaneous myiasis caused by these larvae. Peritrophin-55 was uniformly distributed throughout the peritrophic matrix where it probably lubricates the surface of the peritrophic matrix and protects the midgut from invasion by bacteria. The protein consists of an 8-cysteine amino-terminal domain (peritrophin-B domain) and a carboxy-terminal proline and threonine-rich domain with high probability for extensive O-linked glycosylation. The gene consists of two exons separated by a small intron. Peritrophin-55 mRNA was only detected in the larval cardia and midgut and to a minor extent in the hindgut. Sequence upstream of the transcriptional start site contained a putative promoter region, sequence similar to an ecdysone response element, sequence related to the Drosophila transposon S element and a tetranucleotide repeat region. A putative Drosophila melanogaster ortholog or paralog of peritrophin-55 (CG7714) was located within a 3458 bp intron of the Cha gene (choline-O-acetyltransferase), but on the opposite strand. Comparison of the putative promoter regions of the peritrophin-55 and CG7714 genes revealed little similarity except for a small semi-conserved sequence that is suggestive of a common transcription factor-binding site possibly contributing to the highly restricted developmental and tissue-specific expression patterns of these genes.

Production of antibodies to recombinant antigens from Lucilia cuprina following cutaneous immunisation of sheep.

Immunological control of cutaneous myiasis of sheep caused by Lucilia cuprina larvae has been an elusive goal. Antibody to antigens derived from the peritrophic membrane can stunt or kill larvae in a dose dependent fashion. Thus efficacy of vaccines employing these antigens may be limited by the amount of antibody in skin available for ingestion by larvae. The potential for elevating antibody concentrations in skin by intradermal immunisation with the recombinant peritrophic membrane antigens peritrophin-44, peritrophin-48 and peritrophin-95 was therefore examined. Using within-animal comparisons, specific antibody was significantly higher in skin transudates from locally immunised sites than from adjacent adjuvant control sites. It was concluded that cutaneous immunisation may assist immunological control of blowfly larvae.

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin.

The most abundant protein present in Boophilus microplus eggs, vitellin, was isolated and purified as a non-covalent complex of six glyco-polypeptides of Mr 44-107kDa. The protein complex bound haem. Immuno-blots demonstrated that antibodies raised to vitellin recognised a 200kDa polypeptide in the haemolymph of adult female ticks. This is consistent with the general proposal that in arthropods vitellin is derived by proteolytic processing from a large precursor protein, vitellogenin. In parallel with this study, an 80kDa glycoprotein (GP80) was independently purified from larvae of B. microplus using efficacy in vaccination trials as an assay. Antibodies to GP80 also recognised a 200kDa protein in the haemolymph of ticks and a major 87kDa polypeptide present in the vitellin complex. Conversely, antibodies to purified vitellin recognised GP80. The amino-terminal amino acid sequences of the 87kDa vitellin polypeptide and GP80 were identical for at least the first 11 residues and internal peptide sequences from both polypeptides were co-located in a single but incomplete deduced amino sequence of B. microplus vitellogenin. Thus, GP80 is a processed product from vitellogenin and highly related to but not completely identical with the 87kDa vitellin polypeptide. Vaccination trials in the model host sheep were performed with purified vitellin and GP80. Sheep vaccinated with either purified vitellin or GP80 returned significantly reduced numbers of engorged female ticks with decreased weights and reduced oviposition. In contrast, sheep vaccinated with recombinant hexahis-GP80, which was incorrectly folded and not glycosylated showed no significant effects on ticks. It was concluded that vitellin and GP80 could induce immune responses that partially protect sheep from the tick, B. microplus. However, critical protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it.

Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana.

The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition.

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.

Role of oligosaccharides in the immune response of sheep vaccinated with Lucilia cuprina larval glycoprotein, peritrophin-95.

The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep. The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen. This protein induced an immune response in vaccinated sheep that inhibited larval growth. Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells. The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded. Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity. Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement. The activity was associated with IgG(1) and IgG(2) but not IgM. There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein. Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies. Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95. It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.

Secretion of the type 2 peritrophic matrix protein, peritrophin-15, from the cardia.

The midgut of most insects is lined with a peritrophic matrix, which is thought to facilitate digestion and protect the midgut digestive epithelial cells from abrasive damage and invasion by ingested micro-organisms. The type 2 peritrophic matrix is synthesised by a complex and highly specialised organ called the cardia typically located at the junction of the cuticle-lined foregut and midgut. Although the complex anatomy of this small organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2 peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and secretion of the intrinsic peritrophic matrix protein, peritrophin-15 has been followed in the cardia of Lucilia cuprina larvae using immuno-gold localisations. The protein is synthesised by cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with the plasma membrane at the base of the microvilli and release peritrophin-15 into the inter-microvilli spaces. The protein then becomes associated with the nascent peritrophic matrix, which lies along the tips of the epithelial cell microvilli. It is proposed that peritrophin-15 binds to the ends of chitin fibrils present in the nascent peritrophic matrix, thereby protecting the fibril from the action of exochitinases.

A novel family of chitin-binding proteins from insect type 2 peritrophic matrix. cDNA sequences, chitin binding activity, and cellular localization.

The peritrophic matrix is a prominent feature of the digestive tract of most insects, but its function, formation, and even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of the larval peritrophic matrices of the Old World screwworm fly, Chrysomya bezziana, and sheep blowfly, Lucilia cuprina. Their deduced amino acid sequences code for a 8-kDa secreted protein characterized by a highly conserved and novel register of six cysteines. Two Drosophila homologues have also been identified from unannotated genomic sequences. Recombinant peritrophin-15 binds strongly and specifically to chitin; however, the stoichiometry of binding is low (1:10,000 N-acetyl glucosamine). We propose that peritrophin-15 caps the ends of the chitin polymer. Immunogold studies localized peritrophin-15 to the peritrophic matrix and specific vesicles in cells of the cardia, the small organ of the foregut responsible for peritrophic matrix synthesis. The vesicular contents are disgorged at the base of microvilli underlying the newly formed peritrophic matrix. This is the first time that the process of synthesis and integration of a peritrophic matrix protein into the nascent peritrophic matrix has been observed.

Chitin is only a minor component of the peritrophic matrix from larvae of Lucilia cuprina.

The gut of most insects is lined with a peritrophic matrix that facilitates the digestive process and protects insects from invasion by micro-organisms and parasites. It is widely accepted that the matrix is composed of chitin, proteins and proteoglycans. Here we critically re-examine the chitin content of the typical type 2 peritrophic matrix from the larvae of the fly Lucilia cuprina using a range of techniques. Many of the histochemical and biochemical techniques indicate the presence of chitin, although they are often adversely influenced by the presence of highly glycosylated proteins, a principal component of the matrix. The alkali-stable fraction, which is used as an indicator of the maximum chitin content in a biological sample, is only 7.2% of the weight of the matrix. Larvae fed on the potent chitin synthase inhibitor polyoxin D or the chitin-binding agent Calcofluor White, showed strong concentration-dependent inhibition of larval weight and survival but no discernible effects on the matrix structure. A bacterial endochitinase fed to larvae had no effect on larval growth and no observable effect in vitro on the structure of isolated peritrophic matrix. RT-PCR did not detect a chitin synthase mRNA in cardia, the tissue from which PM originates. It is concluded that chitin is a minor structural component of the type 2 peritrophic matrix of this insect.

Insect chitin synthase cDNA sequence, gene organization and expression.

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.

Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep.

In mature male sheep, the level of nutrition acutely influences the secretion of reproductive hormones. The mechanism involved is not fully understood but findings in humans and laboratory rodents would suggest a major role for leptin that is secreted from adipose tissue and then travels via the circulation to the central nervous system. Before we can begin to test this hypothesis, we need to be able to measure leptin concentrations in blood plasma and cerebrospinal fluid. We have therefore developed a radioimmunoassay using antibodies raised against biologically active recombinant bovine-ovine leptin. Using this assay, we found that plasma concentrations of leptin were highly correlated to back-fat thickness and to the ratio of back-fat thickness to liveweight, in female and castrated male sheep. Plasma concentrations of leptin were higher in female sheep than in castrated or intact male sheep. Serial samples (every 5 min) suggested that the secretion of leptin in male sheep is episodic but it does not appear to show clear pulsatility, increases post-prandially, or a diurnal rhythm. Leptin concentrations in both plasma and cerebrospinal fluid increased within 5 days in male sheep fed a diet with a high content of energy and protein that also stimulates the secretion of LH pulses. These data suggest that in sheep, as in other species, leptin production is correlated with the mass of adipose tissue and that the hormone passes from the circulation to the cerebrospinal fluid and then to hypothalamic sites. There, it may affect appetite and perhaps GnRH secretion. The role of leptin in the link between nutrition and reproduction needs further investigation.

Adrenaline, insulin and glucagon do not have acute effects on plasma leptin levels in sheep: development and characterisation of an ovine leptin ELISA.

Leptin, a recently discovered hormone secreted mainly from adipose tissue, was first described as a regulator of adiposity, food intake and energy metabolism. It is now apparent that leptin physiology is much more complex and is likely to play an important role in many other systems including reproduction, haematopoiesis and immunity. Leptin levels have been shown to be well correlated with body fat in both humans and rodents, with administration of exogenous leptin to rats and mice resulting in loss of body fat. Leptin is, therefore, likely to be an important humoral signal to the central nervous system on body composition and regulation of food consumption. Due to the limited cross-reactivity of leptin from other species in the current assays for leptin, physiological research on leptin has, to a large extent, been restricted to rodents and humans. The aim of this study was to develop a leptin immunoassay suitable for use with sheep, enabling the investigation of the basic physiology of leptin in an animal larger than rats or mice, thus allowing repeated blood sampling. Using this assay we investigated the short-term effects of insulin, adrenaline and glucagon (all modulators of blood glucose) on plasma leptin levels. Antiserum to bovine recombinant leptin (brLeptin) raised in chickens was used to develop a competitive ELISA. Using brLeptin as standard, the assay has a sensitivity of 0. 5 ng/ml with inter- and intra-assay variation of 15% and 7% respectively. The cross-reactivity of human recombinant leptin was 36.5%, while mouse leptin showed no cross-reactivity. Plasma samples from ewes, male castrate animals and rams (n=4-5) diluted in parallel to the standard with mean leptin concentrations of 6.0+/-2. 9, 3.3+/-0.4 and 3.1+/-1.3 ng/ml respectively. Leptin levels in rams were significantly lower than in ewes. The non-significant difference in leptin levels between rams and male castrate animals suggests that testosterone may not be responsible for the lower levels of leptin. Four groups of 3-4 ewes were given intravenous insulin (1 iu/kg), adrenaline (65 microg/kg), glucagon (24 iu/kg) or saline. Blood samples were taken at 1, 3, 5, 10, 20, 30, 60, 90 and 120 min after injection. As expected, glucose levels declined within 10 min of the insulin injection and rose after 3 min following both adrenaline and glucagon injections. Leptin levels, however, remained relatively unchanged for the 2 h following the treatments. Finally, a bolus intravenous dose of glucose (240 mg/kg) was given and sequential blood samples taken. Despite plasma glucose levels rising to over 200 mg/dl, leptin levels did not significantly change over the three hours following treatment. These data indicate that plasma leptin levels in sheep, in contrast to rodents, are not responsive to short-term changes in blood glucose or insulin, as has been shown in humans.