Christopher D. McMahon

Effects of melatonin on the yield and composition of milk from grazing dairy cows in New Zealand

The aim was to determine whether administration of melatonin would alter the yield and composition of milk from grazing dairy cows in summer. Twelve sets of spring-calving identical twin Friesian cows were used in the experiment. In late-November (late spring), one twin from each set was given slow-release melatonin implants behind the ears (108 mg melatonin/cow). Two further implantations occurred at 4-weekly intervals to maintain increased circulating concentrations of melatonin for 12 weeks. The other twin served as a control. Milk yield and composition were measured twice prior to treatment and then four times over the following 12 weeks. Concentrations of melatonin, prolactin and insulin-like growth factor 1 (IGF-1) were measured in blood plasma twice before treatment and then either seven (melatonin and prolactin) or three (IGF-1) further times during the experiment. Management procedures for all cows were similar and cows grazed a daily pasture allowance of approximately 30 kg DM/cow as their sole feed source. In melatonin-treated cows there was a decrease in mean concentrations of prolactin in plasma, but concentrations of IGF-1 did not change. Melatonin reduced milk yield by 6 weeks after treatment and by the end of the 12-week experimental period milk yield in melatonin-treated cows had fallen by 23%. Melatonin also reduced concentrations of lactose in milk, but increased concentrations of fat, protein and casein, changes that were broadly similar to those that occur in late lactation in seasonally calving dairy cows. Thus, the results suggest that some of the variation in the volume and quality of milk throughout the season in New Zealand dairy systems may be due to changes in photoperiod mediated by increased concentrations of plasma melatonin in association with decreased concentrations of plasma prolactin.

Central role of the melanocortin-4 receptors in appetite regulation after endotoxin.

Melanocortin-4 receptors (MC4R) are key factors in the depression of appetite during disease. This study was designed to determine the role of agouti-related protein (AgRP) in the effect of endotoxin (lipopolysaccharide, LPS) on appetite. Sheep received an intracerebroventricular injection of either saline or AgRP (0.5 nmol/kg of BW) 1 h before intravenous injection of either saline or LPS (0.6 microg/kg of BW) at time 0 and again at 4 h. Agouti-related protein prevented the reduction in feed intake due to LPS (P < 0.05). In a second experiment, AgRP gene expression was unaffected at 3 h and increased (P < 0.01) at 6 h after LPS. Immunohistochemical evidence indicated that there was an increase in the percentage of AgRP neurons with c-Fos immunoreactive nuclei 6 h after sheep were injected with LPS (P < 0.04) and a corresponding decrease in a-melanocyte-stimulating hormone neurons coexpressing c-Fos (P < 0.001). In situ hybridization provided evidence for an increase in AgRP gene expression and a decrease in proopiomelanocortin gene expression 6 h after LPS (P < 0.05). In a final experiment, physiological elevation of orexigenic agents by short-term fasting kept feed intake at the same level as controls, in spite of the presence of LPS, similar to the effects of AgRP in Exp. 1. The AgRP inhibition of the MC4R prevents appetite inhibition in response to LPS and well after LPS inhibition of feed intake, both AgRP and a-melanocyte-stimulating hormone may change in a pattern that favors appetite increases. These studies support the notion of the MC4R as a critical component of the mechanism for appetite suppression due to endotoxin.

A role for agouti-related protein in appetite regulation in a species with continuous nutrient delivery.

Knowledge of specific neurotransmitters as well as the pathways and mechanisms regulating appetite in ruminants that continually graze, such as sheep, is incomplete. Although fundamentally agouti-related protein (AGRP) has a similar function across species to increase food intake, the regulation of AGRP may vary across grazing and intermittent feeders. To investigate the role of orexigenic peptides in the regulation of feed intake, we first extracted messenger RNA from sheep that were fasted for 3 days, which was then used for PCR followed by cloning and sequencing to demonstrate the presence of hypothalamic AGRP expression. Ovine AGRP was closely related to the bovine, but contained sequence differences with human and mouse AGRP. Analysis of genomic DNA also revealed a similar gene structure to other published species. Secondly, using dual-labeled immunohistochemistry, we determined that there was both increased AGRP immunoreactivity and increased abundance of c-Fos immunoreactivity in AGRP neurons in the arcuate nucleus of fasted sheep. Because AGRP neurons are activated by fasting, we hypothesized that AGRP would stimulate feeding in this ruminant species. Sheep fed ad libitum were injected intracerebroventricularly with concentrations of AGRP at 0.2 and 2.0 nmol/kg. AGRP at 2.0 nmol/kg significantly increased food intake at 4, 6 and 12 h (p < 0.05). A 4th study was done to investigate the interactions of AGRP and neuropeptide Y (NPY) on food intake over a 24-hour period. Intracerebroventricular injections of either AGRP or NPY significantly increased cumulative food intake over saline controls. When AGRP and NPY were injected in combination, food intake was increased over saline controls; however, AGRP did not potentiate the effects of NPY. These results demonstrate that AGRP stimulates food intake in sheep and highlights the important differences between this species and rodent models.

Decreased expression of β1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands

The mechanisms regulating involution of mammary glands after weaning are not clear, but engorgement with milk is a key trigger. Many cell types require to be anchored to an extracellular matrix (ECM) as a prerequisite for survival and this is achieved via intregrins binding to specific motifs and signalling their attachment, intracellularly, via focal adhesion kinase (FAK). We sought to determine firstly, if expression of β1‐integrin and FAK is reduced during the first stage of involution. Expression of β1‐integrin and FAK was significantly reduced at 6 h after sealing teats and this was accompanied with a decreased abundance of cytochrome C in mitochondria. Secondly, we sought to determine if expression of β1‐integrin and FAK was restored during the first, partially reversible stage of involution (at 24 h), but not during the second irreversible stage, which occurs after 72 h. Re‐suckling restored full expression of the 80 kDa fragment of FAK, but not of the 125 kDa protein or β1‐integrin at 24 h after weaning. Re‐suckling did not restore expression of either peptide after 72 h. Changes in expression of cytochrome C and pro‐caspase‐3 (apoptotic markers) were similar to that of the 80 kDa fragment of FAK. These data suggest that epithelial cells can restore partial contact with their basement membrane during the first, reversible stage, but not during the second irreversible stage of involution. We speculate that decreased contact between epithelial cells and their basement membrane initiates apoptosis in mammary glands at weaning. This process begins within 6 h of pup withdrawal.

The decrease in mature myostatin protein in male skeletal muscle is developmentally regulated by growth hormone

Myostatin inhibits myogenesis and there is reduced abundance of the mature protein in skeletal muscles of adult male compared with female mice. This reduction probably occurs after translation, which suggests that it is a regulated mechanism to reduce the availability of myostatin in males. Reduced myostatin may, thereby, contribute to the development of sexually dimorphic growth of skeletal muscle. Our first objective was to determine if the decrease in mature myostatin protein occurs before the linear growth phase to aid growth, or afterwards to maintain the mass of adult muscle. Mice were killed from 2 to 32 weeks and the gastrocnemius muscle was excised. Myostatin mRNA increased from 2 to 32 weeks and was higher in males than females (P < 0.001). In contrast, mature protein decreased in males after 6 weeks (P < 0.001). Our second objective was to determine if growth hormone (GH) induces the decrease in mature myostatin protein. GH increased myostatin mRNA and decreased the abundance of mature protein in hypophysectomised mice (P < 0.05). Our final objective was to determine if the decrease in mature protein occurs in skeletal muscles of male Stat5b−/− mice (Stat5b mediates the actions of GH). As expected, mature myostatin protein was not reduced in Stat5b−/− males compared with females. However, myostatin mRNA remained higher in males than females irrespective of genotype. These data suggest that: (1) the decrease in mature myostatin protein is developmentally regulated, (2) GH acting via Stat5b regulates the abundance of mature myostatin and (3) GH acts via a non‐Stat5b pathway to regulate myostatin mRNA.

Deer antler--a novel model for studying organ regeneration in mammals.

Deer antler is the only mammalian organ that can fully grow back once lost from its pedicle - the base from which it grows. Therefore, antlers probably offer the most pertinent model for studying organ regeneration in mammals. This paper reviews our current understanding of the mechanisms underlying regeneration of antlers, and provides insights into the possible use for human regenerative medicine. Based on the definition, antler renewal belongs to a special type of regeneration termed epimorphic. However, histological examination failed to detect dedifferentiation of any cell type on the pedicle stump and the formation of a blastema, which are hallmark features of classic epimorphic regeneration. Instead, antler regeneration is achieved through the recruitment, proliferation and differentiation of the single cell type in the pedicle periosteum (PP). The PP cells are the direct derivatives of cells resident in the antlerogenic periosteum (AP), a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body. Both the AP and PP cells express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro and, therefore, they are termed antler stem cells, and antler regeneration is a stem cell-based epimorphic regeneration. Comparisons between the healing process on the stumps from an amputated mouse limb and early regeneration of antlers suggest that the stump of a mouse limb cannot regenerate because of the limited potential of periosteal cells in long bones to proliferate. If we can impart a greater potential of these periosteal cells to proliferate, we might at least be able to partially regenerate limbs lost from humans. Taken together, a greater understanding of the mechanisms that regulate the regeneration of antlers may provide a valuable insight to aid the field of regenerative medicine. This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.

Effect of botulinum toxin A-induced paralysis and exercise training on mechanosensing and signalling gene expression in juvenile rat gastrocnemius muscle

Intramuscular injections of the paralytic botulinum neurotoxin A (Btx) and physical exercise are used in the treatment of chronic spasticity in children with cerebral palsy. We tested whether Btx‐induced paralysis and/or exercise training would have differential effects on the expression of mechanosensing and signalling genes implicated in the adaptive remodelling of skeletal muscle. Juvenile (29‐day‐old) male rats were injected with Btx or saline (NoBtx) into the right gastrocnemius and housed in standard cages (NoEx) or with running wheels (Ex), for 3 weeks (n= 6 per group). The mRNA expression of nine sarcomere‐associated genes in the medial gastrocnemius was then determined by quantitative reverse transcriptase‐polymerase chain reaction. The Btx‐injected muscles weighed 50% less than NoBtx muscles, but Ex had no effect on the wet mass of Btx or NoBtx muscles. Atrogenic MuRF1, sarcomeric Titin and myogenic MyoD were upregulated (2‐fold) with the elimination of contractile activity in Btx muscle. Expression of CARP, Ankrd2 and MLP was increased with mechanical stimuli associated with Btx (5‐ to 10‐fold) or Ex (2‐ to 4‐fold). Expression of CARP and Ankrd2 increased synergistically in Btx–Ex muscle (≥20‐fold), indicating that these genes may be sensitive to passive stretch of the sarcomeric I‐band region of titin to which their proteins bind. Tcap, Myopalladin and Atrogin1 were not, or were no longer responsive to the altered mechanical stimuli after 3 weeks of Btx or Ex. The expression of Ankrd2, CARP and MLP may thus be enhanced by passive stretch within the Btx‐paralysed and/or exercising gastrocnemius and contribute to adaptations, other than muscle mass, in juvenile rats.

Botulinum toxin-induced paralysis leads to slower myosin heavy chain isoform composition and reduced titin content in juvenile rat gastrocnemius muscle.

Intramuscular injections of botulinum toxin A (Btx‐A) and exercise are used in the treatment of muscle spasticity in children with cerebral palsy. However, little is known about the biological changes within muscle subsequent to Btx‐A‐induced paralysis and how the combination of Btx‐A and exercise might affect the growing muscle. The wet mass, myosin heavy chain (MHC) composition, and titin content of the juvenile rat gastrocnemius muscle were determined 3 weeks after Btx‐A injections and subsequent voluntary wheel‐running exercise. Btx‐A increased the proportion of type IIa (+121%) and IIx (+65%) MHC while decreasing the proportion of type IIb MHC (−51%) and reducing the titin content (−18%). Exercise did not amplify or reduce the changes induced by Btx‐A. Thus, we conclude that although the sarcomeric stability of paralyzed muscle might be impaired, moderate mechanical loading does not seem to affect paralyzed muscle protein composition. Muscle Nerve, 2008

Proteomes and Signalling Pathways of Antler Stem Cells

As the only known example of complete organ regeneration in mammals, deer antler in the growing or velvet phase is of major interest in developmental biology. This regeneration event initiates from self-renewing antler stem cells that exhibit pluripotency. At present, it remains unclear how the activation and quiescence of antler stem cells are regulated. Therefore, in the present study proteins that were differentially expressed between the antler stem cells and somatic cells (facial periosteum) were identified by a gel-based proteomic technique, and analysed using Ingenuity Pathway Analysis software. Several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) were found to be activated during proliferation. Also expressed were the transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells. Expression of these proteins was confirmed in both cultured cells and fresh tissues by Western blot analysis. Therefore, the molecular pathways and transcription factors identified in the current study are common to embryonic and adult stem cells. However, expression of embryonic stem cell transcription factors would suggest that antler stem cells are, potentially, an intermediary stem cell type between embryonic and the more specialized tissue-specific stem cells like those residing in muscle, fat or from a hematopoietic origin. The retention of this embryonic, pluripotent lineage may be of fundamental importance for the subsequent regenerative capacity of antlers.

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice

The age‐related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin‐like growth factor‐1 (IGF‐1). We hypothesized that constitutively expressed IGF‐1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF‐1 Ea in skeletal muscles were compared with wild‐type littermates. Four‐month‐old mice were assigned to be sedentary, or had access to free‐running wheels, until 18 or 28 months of age. In wild‐type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF‐1 alone was ineffective, whereas the combination of exercise and IGF‐1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF‐1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF‐1 have a modest effect on reducing aged‐related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength.