Genetic Architecture and Selection of Chinese Cattle Revealed by Whole Genome Resequencing.

The bovine genetic resources in China are diverse, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Chinese cattle, we analyzed the whole genomes of 46 cattle from six phenotypically and geographically representative Chinese cattle breeds, together with 18 Red Angus cattle genomes, 11 Japanese black cattle genomes and taurine and indicine genomes available from previous studies. Our results showed that Chinese cattle originated from hybridization between Bos taurus and Bos indicus. Moreover, we found that the level of genetic variation in Chinese cattle depends upon the degree of indicine content. We also discovered many potential selective sweep regions associated with domestication related to breed-specific characteristics, with selective sweep regions including genes associated with coat color (ERCC2, MC1R, ZBTB17, and MAP2K1), dairy traits (NCAPG, MAPK7, FST, ITFG1, SETMAR, PAG1, CSN3, and RPL37A), and meat production/quality traits (such as BBS2, R3HDM1, IGFBP2, IGFBP5, MYH9, MYH4, and MC5R). These findings substantially expand the catalogue of genetic variants in cattle and reveal new insights into the evolutionary history and domestication traits of Chinese cattle.

KDEL peptide gold nanoconstructs: promising nanoplatforms for drug delivery.

Gold nanoparticles (AuNPs) have been widely investigated as potential nanocarriers for drug delivery. In the present study, AuNPs were conjugated to a peptide that has a C-terminal Lys-Asp-Glu-Leu (KDEL) motif. In a pulse-chase study, time-course sampling revealed that AuNP-delivered KDEL peptides were rapidly localized to the endoplasmic reticulum (ER) in 5 to 15 min, and after 1h the majority of peptides were localized to the ER. Clathrin-coated vesicles and Golgi apparatus were also involved during the intracellular trafficking of KDEL peptide gold (AuNP-KDEL) nanoconstructs. Furthermore, overexpression of KDEL receptor (KDELR) significantly enhanced KDEL peptide uptake in both free and AuNP-conjugated forms. These data indicate that the AuNP-KDEL nanoconstructs are internalized via a clathrin-mediated pathway and trafficked to the ER via a retrograde transport pathway, bypassing the lysosomal degradation pathway. Thus, this novel approach to development of nanoconstruct-based drug delivery has the potential to evade intracellular degradation, enhancing drug efficacy. FROM THE CLINICAL EDITOR: In this study, gold nanoparticles were conjugated to a peptide with KDEL motif, resulting in internalization via a clathrin-mediated pathway and trafficking to the ER via retrograde transport meanwhile bypassing the lysosomal degradation pathway. This method results in a potential evasion of intracellular degradation, and enhanced drug efficacy.

Garcinia buchananii stem bark extract and its bioactive constituents manniflavanone, GB-2 and buchananiflavanone attenuate intestinal inhibitory neuromuscular transmission.

Garcinia buchananii stem bark extract (GBB), commonly used for treating diarrhea in Africa, triggers ectopic aboral contractions, causing inhibition of propulsive motility in the colon ex vivo. To determine whether or not these effects were associated with decreased inhibitory neuromuscular transmission, the responsible constituent compounds, and mechanisms of action, we studied the effects of GBB and specific fractions and flavanones isolated from GBB on intestinal motility using pellet propulsion assays in guinea pig distal colons. In addition, microelectrode recordings were used to measure the effects on the inhibitory junction potentials (IJPs) in the porcine ileum and descending colon smooth muscle. Psychoactive Drug Screening Program secondary receptor functional assays were used to determine whether or not GBB and its constituent compounds act via purinergic (P2Y) and muscarinic receptors. GBB inhibited propulsive motility, but (2R,3S,2″R,3″R)-manniflavanone (MNF), (2R,3S,2″R,3″R)-GB-2 (GB-2) and (2R,3S,2″S)-buchananiflavanone (BNF), the main ingredients of GBB, did not affect motility. We discovered that, in the porcine descending colon, IJPs contained purinergic, nitrergic, and nonpurinergic nonnitrergic components. Furthermore, ileal IJPs were purely purinergic. GBB blocked all components of IJPs, while MNF and GB-2 inhibited purinergic IJPs only. BNF inhibited the purinergic and nonpurinergic components of IJPs. MRS2365, a Y1 (P2Y) agonist, did not evoke sustained membrane hyperpolarization in the presence of GBB. However, GBB, MNF, GB-2 and BNF did not affect P2Y or muscarinic receptors. In conclusion, inhibitory neuromuscular transmission in the porcine descending colon involves all components of IJPs. GBB decreases inhibitory neuromuscular transmission, likely by the actions of MNF, GB-2 and BNF. These effects do not involve P2Y or muscarinic receptors.

Adipocyte differentiation-specific gene transcriptional response to C18 unsaturated fatty acids plus insulin.

Adipocyte differentiation (AD) and AD-specific gene expression was studied in 3T3-L1 cells in response to oleic acid (OA) or linoleic acid (LA) alone and in combination with insulin. This system facilitated the study of key regulators of adipogenesis PPARγ and C/EBPα and other AD-specific genes, in the absence of dexamethasone (DEX) and isobutyl-1-methyl xanthine (IBMX) (components of the traditional AD medium, DMI). Lipid accumulation and expression levels of AD-specific genes were enhanced by both OA and LA in the presence of insulin but not by OA or LA alone. Gene expression levels of PPARγ, C/EBPα, FABP4, and SREBP1c induced by OA plus insulin, were comparable to DMI medium, by study day 10. The response to long-chain fatty acids (LCFA) plus insulin in the presence or absence of LY294002 demonstrated that the insulin-induced PI 3-kinase pathway regulates AD and AD-specific gene expression levels. Insulin treatment in the presence or absence of genistein suggested that genistein invoked inhibition of AD and AD-specific gene expression. In contrast when LCFA were also included with insulin, the presence of genistein invoked a pronounced and opposite effect on AD to that in the absence of LCFA. This effect may be modulated via C/EBPα as C/EBPα but not PPARγ expression patterns closely reflected the changes in AD. DMI invoked a rapid expression of all genes studied, and LCFA plus insulin invoke more gradual increases in gene expression, to similar levels to those invoked by DMI. The model system is valuable for study of transactivators and response elements of PPARγ and C/EBPα genes.

Gold nanoparticles: the importance of physiological principles to devise strategies for targeted drug delivery.

Nanotechnology and its promise for clinical translation to targeted drug delivery with limited accompanying toxicity provide exciting research opportunities that demand multidisciplinary approaches. To make rapid progress in the design of nano-platforms for drug delivery and toward their use in the clinic, basic and mechanistic studies must first be tested in vitro and then progress to in vivo studies in animal models, incorporating an understanding of body functioning. Recently, gold nanoparticles (Au NPs) have gained much attention as model drug delivery platforms because of their advantageous surface characteristics that allow easy functionalization with chemical and biological molecules and also due to their apparently low toxicity. In this study we review recent in vitro and in vivo research progress with Au NPs as drug delivery platforms and suggest experimental strategies for future studies for efficacious, targeted delivery.

Gold-peptide nanoconjugate cellular uptake is modulated by serum proteins.

Gold nanoparticles (Au NPs, 20 nm) were conjugated with two different cysteine-terminated peptides. Radio-ligand binding studies were conducted to characterize Au NP-peptide binding, suggesting both covalent and noncovalent interactions. The interactions of serum proteins with Au NP-peptide nanoconjugates were determined using gel electrophoresis and dynamic light scattering. Serum proteins rapidly bound the nanoconjugates (15 minutes). The cellular uptake of free peptides and nanoconjugates into mouse myogenic (Sol8) cells was investigated in the absence or presence of serum. In the absence of serum, peptides presented as nanoconjugates showed significantly higher intracellular fluorescence signals compared to those in the presence of serum (P < 0.05), suggesting that serum proteins inhibit Au NP-mediated peptide delivery. The cellular uptake of nanoconjugates was also confirmed using transmission electron microscopy. These data suggest that Au NP-peptide nanoconjugates are a useful platform for intracellular delivery of therapeutics. However, a deeper understanding of the mechanisms regulating their uptake and intracellular trafficking is needed.

A qualitative exploration of mental health services provided in community pharmacies.

The burden of mental health problems continues to grow worldwide. Community pharmacists', as part of the primary care team, optimise care for people living with mental illness. This study aims to examine the factors that support or hinder the delivery of mental health services delivered in Australian community pharmacies and proposes ideas for improvement. A qualitative study was conducted comprising focus groups with community pharmacists and pharmacy staff across metropolitan, regional, and rural areas of New South Wales, Australia. Data were collected in eight focus groups between December 2020 and June 2021. Qualitative data were analysed using thematic analysis. Thirty-three community pharmacists and pharmacy staff participated in an initial round of focus groups. Eleven community pharmacists and pharmacy staff participated in a second round of focus groups. Twenty-four factors that enable or hinder the delivery of mental health services in community pharmacy were identified. Participant's perception of a lack of recognition and integration of community pharmacy within primary care were identified as major barriers, in addition to consumers' stigma and lack of awareness regarding service offering. Suggestions for improvement to mental health care delivery in community pharmacy included standardised practice through the use of protocols, remuneration and public awareness. A framework detailing the factors moderating pharmacists, pharmacy staff and consumers' empowerment in mental health care delivery in community pharmacy is proposed. This study has highlighted that policy and funding support for mental health services is needed that complement and expand integrated models, promote access to services led by or are conducted in collaboration with pharmacists and recognise the professional contribution and competencies of community pharmacists in mental health care. The framework proposed may be a step to strengthening mental health support delivered in community pharmacies.

Mental health training programs for community pharmacists, pharmacy staff and students: A systematic review.

BACKGROUND: Primary care is often the first point of contact for people living with mental disorders. Community pharmacists, pharmacy staff and students are increasingly being trained to deliver mental health care. However, there is still a gap in the literature exploring the characteristics of all available mental health training programs and their components and their influence on pharmacists, pharmacy staff and students' outcomes. OBJECTIVES: To summarize the evidence evaluating mental health training programs completed by community pharmacists, pharmacy staff and students. More specifically, to explore the components of mental health training programs and identify those that facilitate significant improvements in outcomes. METHODS: A systematic review was conducted following the Cochrane handbook and reported according to PRISMA guidelines. A search for published literature was conducted in three databases (PubMed, Scopus, and Web of Science) in July 2021. Eligible studies were included if they described and evaluated the impact of mental health training programs delivered to community pharmacists, pharmacy staff and pharmacy students regardless of design or comparator. The methodological quality of included studies was appraised using both the NIH quality assessment, to evaluate studies with an uncontrolled pre-post design, and the Cochrane EPOC risk of bias assessment, to evaluate studies with a controlled (randomized and non-randomized) study design. RESULTS: Thirty-three studies were included. Most of the identified mental health training programs contained knowledge-based components and active learning activities. Changes in participants' attitudes, stigma, knowledge, confidence and skills were frequently assessed. An extensive range of self-assessment and observational instruments used to evaluate the impact of the training programs were identified. Positive improvements in participants' attitudes, knowledge and stigma were frequently identified following participation in training programs. CONCLUSIONS: This systematic review highlights the importance of mental health training programs in increasing pharmacists', pharmacy staff and pharmacy students' skills and confidence to deliver mental health care in community pharmacy. Future research should build upon this basis and further focus on finding the most efficient measures to evaluate these training programs and assess their long-term effectiveness, allowing comparison between programs.

A Systematic Review of Campylobacter jejuni Vaccine Candidates for Chickens.

Campylobacter jejuni infection linked to the consumption of contaminated poultry products is one of the leading causes of human enteric illness worldwide. Vaccination of chickens is one of the potential strategies that could be used to control C. jejuni colonization. To date, various C. jejuni vaccines using potential antigens have been evaluated, but a challenge in identifying the most effective formulation is the wide variability in vaccine efficacies reported. A systematic review was undertaken to compare C. jejuni vaccine studies. Based upon specific selection criteria eligible papers were identified and included in the analysis. Vaccine efficacy reported from different C. jejuni antigens, vaccine types, and vaccination regimens reported in these papers were reviewed. Our analysis shows that total outer membrane proteins and cysteine ABC transporter substrate-binding protein were among the most efficacious vaccine antigen candidates reported. This review also highlights the importance of the need for increased consistency in the way C. jejuni vaccine studies in poultry are designed and reported in order to be able to undertake a robust comparison of C. jejuni vaccine candidates.

Investigation of Campylobacter colonization in three Australian commercial free-range broiler farms.

Campylobacter spp. contaminated poultry products are strongly associated with foodborne illnesses worldwide. Development of effective management strategies to reduce contamination by Campylobacter spp. requires an improved understanding of the numerous factors that drive these contamination processes. Currently, chicken farms are using more free-range chicken meat production systems in response to consumer preferences. However, Campylobacter spp. colonization has rarely been investigated on free-range broiler farms. The present study investigated the temporal and environmental factors influencing Campylobacter spp. colonization of free-range broilers as well as potential sources and genetic diversity of Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) in commercial free-range broiler farms. Genetic linkages among the isolates were analyzed using flaA amplicon analysis. Campylobacter coli was first detected in fecal samples of a commercial free-range broiler flock on day 10 of rearing. Multiple genotypes of C. jejuni and C. coli were identified in this study. The farm environment was identified as a potential source of C. jejuni and C. coli colonization of free-range broilers. The dominant Campylobacter genotype varied between free-range broiler farms over time, with C. jejuni being the most frequently isolated species. These findings enhance the understanding of C. jejuni and C. coli colonization in free-range broiler farms and could inform the development of more effective intervention strategies to help control this important foodborne pathogen.

Glowing locked nucleic acids: brightly fluorescent probes for detection of nucleic acids in cells.

Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2'-N-(pyren-1-yl)carbonyl-2'-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (Φ(F) = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

In vitro proliferating cell models to study cytotoxicity of silica nanowires.

Proliferating cells representing two disease models (HeLa and Panc 10.05 cells) and a more physiologically relevant cell model (3T3-L1 cells) were used to study the acute toxicologic effects of silica nanowires (NWs). Cellular responses to NW effects were determined over a 4- to 20-hour exposure time-course. Proliferation, viability, metabolic activity, and toxicologic mechanism (apoptosis vs. necrosis) data showed the following: 3 x 10(4) NWs per cell inhibited cell proliferation. The effect was rapid in HeLa cells, but 3T3-L1 and Panc 10.05 cells appeared to be more tolerant to NWs, effects being significant only after 20 or 16 hours, respectively. Cells of all three cell lines showed a significant reduction in cellular metabolic activity after 20 hours of treatment with NWs. Assay of NW-invoked mechanism of cell death (caspase 3/7 activity) indicated that apoptosis was minimally induced. Small but significant effects of NWs were detected in 3T3-L1 and HeLa cells after 20-hour treatment. No NW-induction of caspase 3/7 activity was detected for Panc 10.05 cells. Proliferating cells provide a sensitive model to study treatment with silica NWs. Silica NWs appeared to be well tolerated by these three cell lines at the doses tested. When effects were detected, cell necrosis and not apoptosis was the main mechanism of silica NW-induced cell death. FROM THE CLINICAL EDITOR: In this study, three relevant cell culture models were used to study the acute toxicological effects of silica nanowires (NW). All cell lines cells showed a significant reduction in cellular metabolic activity after 20 h of treatment with NW. Overall, silica NW appeared to be well-tolerated by these cell lines at the tested doses. Cell necrosis as opposed to apoptosis was the main mechanism of silica NW-induced cell death.

Potential Role of Phenolic Extracts of Mentha in Managing Oxidative Stress and Alzheimer's Disease.

With an increase in the longevity and thus the proportion of the elderly, especially in developed nations, there is a rise in pathological conditions that accompany ageing, such as neurodegenerative disorders. Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive and memory decline. The pathophysiology of the disease is poorly understood, with several factors contributing to its development, such as oxidative stress, neuroinflammation, cholinergic neuronal apoptotic death, and the accumulation of abnormal proteins in the brain. Current medications are only palliative and cannot stop or reverse the progression of the disease. Recent clinical trials of synthetic compounds for the treatment of AD have failed because of their adverse effects or lack of efficacy. Thus, there is impetus behind the search for drugs from natural origins, in addition to the discovery of novel, conventional therapeutics. Mints have been used traditionally for conditions relevant to the central nervous system. Recent studies showed that mint extracts and/or their phenolic constituents have a neuroprotective potential and can target multiple events of AD. In this review, we provide evidence of the potential role of mint extracts and their derivatives as possible sources of treatments in managing AD. Some of the molecular pathways implicated in the development of AD are reviewed, with focus on apoptosis and some redox pathways, pointing to mechanisms that may be modulated for the treatment of AD, and the need for future research invoking knowledge of these pathways is highlighted.

Neuroprotective Activity of Mentha Species on Hydrogen Peroxide-Induced Apoptosis in SH-SY5Y Cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with an unclear cause. It appears that multiple factors participate in the process of neuronal damage including oxidative stress and accumulation of the protein amyloid ß (Aß) in the brain. The search for a treatment for this disorder is essential as current medications are limited to alleviating symptoms and palliative effects. The aim of this study is to investigate the effects of mint extracts on selected mechanisms implicated in the development of AD. To enable a thorough investigation of mechanisms, including effects on ß-secretase (the enzyme that leads to the formation of Aß), on Aß aggregation, and on oxidative stress and apoptosis pathways, a neuronal cell model, SH-SY5Y cells, was selected. Six Mentha taxa were investigated for their in vitro ß-secretase (BACE) and Aß-aggregation inhibition activities. Moreover, their neuroprotective effects on H2O2-induced oxidative stress and apoptosis in SH-SY5Y cells were evaluated through caspase activity. Real-time PCR and Western blot analysis were carried out for the two most promising extracts to determine their effects on signalling pathways in SH-SY5Y cells. All mint extracts had strong BACE inhibition activity. M. requienii extracts showed excellent inhibition of Aß-aggregation, while other extracts showed moderate inhibition. M. diemenica and M. requienii extracts lowered caspase activity. Exposure of SH-SY5Y cells to M. diemenica extracts resulted in a decrease in the expression of pro-apoptotic protein, Bax, and an elevation in the anti-apoptotic protein, Bcl-xL, potentially mediated by down-regulation of the ASK1-JNK pathway. These results indicate that mint extracts could prevent the formation of Aß and also could prevent their aggregation if they had already formed. M. diemenica and M. requienii extracts have potential to suppress apoptosis at the cellular level. Hence, mint extracts could provide a source of efficacious compounds for a therapeutic approach for AD.

Effect of starvation on transcriptomes of brain and liver in adult female zebrafish (Danio rerio).

We used microarray and quantitative real-time PCR (qRT-PCR) analyses in adult female zebrafish (Danio rerio) to identify metabolic pathways regulated by starvation in the liver and brain. The transcriptome of whole zebrafish brain showed little response to 21 days of starvation. Only agouti-related protein 1 (agrp1) significantly responded, with increased expression in brains of starved fish. In contrast, a 21-day period of starvation significantly downregulated 466 and upregulated 108 transcripts in the liver, indicating an overall decrease in metabolic activity, reduced lipid metabolism, protein biosynthesis, proteolysis, and cellular respiration, and increased gluconeogenesis. Starvation also regulated expression of many components of the unfolded protein response, the first such report in a species other than yeast (Saccharomyces cerevisiae) and mice (Mus musculus). The response of the zebrafish hepatic transcriptome to starvation was strikingly similar to that of rainbow trout (Oncorhynchus mykiss) and less similar to mouse, while the response of common carp (Cyprinus carpio) differed considerably from the other three species.

Antibodies as molecular mimics of biomolecules: roles in understanding physiological functions and mechanisms.

Physiologists have routinely used understanding of the immune system to generate antibodies against regulatory molecules, growth factors, plasma membrane receptors, and other mammalian molecules in the development of analytical tools and assays. In taking this notion further, antibodies have been used in vivo to modulate physiological systems and to improve our understanding of their molecular interactions. To develop antibodies with physiological activity (efficacy), physiologists have worked with immunologists in developing interdisciplinary insights, requiring basic knowledge of immune system function in designing strategies to generate antibodies that interact with endogenous molecules of physiological interest, in vivo. Antibodies in different physiological systems have been shown to enhance or inhibit endogenous molecular functions. Two approaches have been used: passive and active immunization. Antibodies in these contexts have provided tools to develop further insights into molecular physiological mechanisms. Perhaps surprisingly, enhancing antibodies have been developed against a diverse set of target molecules including several members of the growth hormone/insulin-like growth factor-I axes and those of the beta(2)-adrenoceptor axis. Antibodies that inhibit the actions of somatostatin have also been developed. A further novel approach has been the development of antibodies that interact with adipose cells in vivo. These have the potential to be used in therapeutic antiobesity approaches. Antibodies with efficacy in vivo have provided new insights into molecular physiological mechanisms, enhancing our understanding of these complex processes.

(2R,3S,2″R,3″R)-Manniflavanone Protects Proliferating Skeletal Muscle Cells against Oxidative Stress and Stimulates Myotube Formation.

We investigated the antioxidative properties of (2R,3S,2″R,3″R)-manniflavanone (MF) using in vitro assays and examined its effects on myogenesis and lactate-induced oxidative stress in C2C12 cells. MF was purified from Garcinia buchananii stem bark. H2O2 and oxygen radical absorbance capacity assays demonstrated that MF is a powerful antioxidant. This finding was supported by diphenylpicrylhydrazine radical scavenging activity of MF. MF was less cytotoxic to C2C12 cells compared to ascorbic acid and myricetin. Moreover, MF accelerated myotube formation in the differentiated C2C12 cells by up-regulating myogenic proteins such as MyoG and myosin heavy chain. Furthermore, MF rescued late differentiation of myoblast suppressed by lactate treatment and up-regulated the expression levels of Nrf2 in lactate-induced oxidative stress, indicating that MF stimulates antioxidative activity inside C2C12 cells. Collectively, MF is a potent antioxidant with a higher safety profile than ascorbic acid and myricetin. It reduces oxidative stress-induced delaying of skeletal muscle differentiation by scavenging reactive oxygen species and regulating myogenic proteins factors.

Early developmental expression of two insulins in zebrafish (Danio rerio).

We have cloned a second insulin gene in zebrafish and studied temporal and spatial expression of two zebrafish insulin genes. Zebrafish insulin-a (insa) and -b (insb) mRNAs are derived from two different DNA contigs on chromosomes 5 and 14, respectively, representing two different insulin genes. Real-time PCR studies suggest that insa is a maternal and also a postzygotic transcript. insa was observed at 1 h postfertilization (hpf) and was rapidly degraded by 6 and 12 hpf but induced at 24 hpf (i.e., after pancreas formation). Expression levels at 24 hpf were approximately 220-fold higher than at 6 hpf and were significantly different from earlier time points. At 72 hpf (at time of hatching), zebrafish insa mRNA levels tended to be higher than at 24 hpf and were approximately 727-fold higher compared with 6 hpf. This further increase in insa expression may be one of the many rapid physiological changes associated with hatching. insb expression was observed from 1 hpf and was significantly decreased from 12 hpf onward. Its expression levels at 12 and 24 hpf were approximately twofold and sixfold lower, respectively, compared with expression at 6 hpf. insb expression levels at 48 hpf were significantly lower than at 24 hpf but not different from 72 hpf. Expression levels at 72 hpf were approximately 61-fold lower than at 6 hpf. In situ hybridization studies showed insb expression in proliferating blastomeres at 3 and 4 hpf. At later time points, insb expression was restricted to the brain and pancreas (24 and 48 hpf). insa expression was observed in the pancreas at 24 and 48 hpf. Expression of insb in blastomeres and head suggests that insb could be acting as a pro-growth, survival, and neurotrophic factor during development. Pancreatic insa and insb may both be involved in regulation of glucose homeostasis as in mammals.

Early signaling interactions between the insulin and leptin pathways in bovine myogenic cells.

Cross-talk between hormone signaling pathways provides mechanisms to facilitate flexibility in the cellular response to extracellular conditions. One function of insulin is to signal high extracellular glucose, while leptin may signal the abundance of extracellular lipid, both energy sources being readily utilized by muscle. The present study reports early signaling events in the insulin and leptin cascades in primary bovine myogenic cells (BMC). BMC were treated with insulin, or leptin for 1, 10, 30 and 120 min, or pretreated with leptin for 10 min followed by insulin for 1, 10, 30 and 120 min. BMC were insulin resistant, showing a significant inhibition of IRS-1 association with the insulin receptor (IR) following insulin stimulation, a corresponding increase in PI 3-kinase association with the IR, and a slow and modest increase in GLUT4 recruitment to the plasma membrane. Pretreatment of BMC for 10 min leptin, followed by insulin time-course, caused IRS-1 recruitment to be unresponsive, but evoked a rapid, phasic response of PI 3-kinase recruitment to the IR and abrogated the response of GLUT4 translocation to the plasma membrane evoked by insulin alone. The lack of insulin response was independent of IR abundance or affinity. JAK-2 association with the ObR and JAK-2 tyrosine phosphorylation were responsive to all three treatments. Insulin alone down-regulated the leptin signaling pathway, JAK-2 association with ObR decreased at all time-points, and JAK-2 phosphorylation decreased similarly. Leptin alone also appeared to down-regulate JAK-2 association with the ObR, but stimulated the down-regulated pathway to signal, JAK-2 tyrosine phosphorylation being increased at later time-points. Pretreatment with leptin followed by insulin time-course showed marked up-regulation of the early leptin signaling pathway, JAK-2 association with the ObR being increased by insulin while JAK-2 tyrosine phosphorylation was also increased. The contrasting responses of BMC to insulin alone, leptin alone and the sequential leptin-insulin treatment may point to the ability of these cells to respond to energy substrate availability, as bovine muscle has evolved to utilize lipids and fatty acids in response to a metabolism which provides only limited glucose. This cross-talk between insulin and leptin signaling pathways points to a better understanding of the mechanisms driving energy substrate utilization in ruminant muscle and may provide a useful model for greater understanding of the molecular mechanisms underlying the development of insulin resistance and Type 2 diabetes in man.

Early insulin signaling cascade in a model of oxidative skeletal muscle: mouse Sol8 cell line.

Cell models provide important tools to investigate the mechanisms modulating the insulin-signaling cascade. Insulin interaction and subsequent signaling of cells is complex and regulated at multiple levels: receptor abundance, binding dynamics, phosphorylation/dephosphorylation of tyrosine and serine/threonine residues, and subsequent interactions of key intracellular messengers. We report early insulin signaling events in the mouse Sol8 myogenic cell line. Sol8 cells responded to insulin by increasing total IRS-1, p85 PI3-kinase and tyrosine phosphorylated IRS-1 (pY-IRS-1) at 10 min (P<0.05), but not at 1 min of insulin stimulation. The dose-response relationships at 10-min insulin (10 to 300 nM) stimulation showed that IRS-1 and pY-IRS-1 responded to 100 and 300 nM insulin, and the p85 PI3-kinase response peaked at 30 nM insulin. PI3-kinase appeared to be present in high abundance and, in response to insulin, recruitment to the insulin receptor tyrosine kinase (IR) of IRS-1 and PI3-kinase was observed. The increase in IRS-1 detected in IR immunoprecipitates was twofold, while the corresponding increase in PI3-kinase was threefold, suggesting direct recruitment of PI3-kinase to the IR. PI3-kinase detected in IRS-1 immunoprecipitates in response to insulin increased 1.7-fold. An ultimate target of this pathway, GLUT4 recruitment to the PM, was delayed (30 min), the increase in GLUT4 being of similar magnitude (1.6-fold) to the early signaling events. Saturation binding analysis indicated that IR in the plasma membrane was not down-regulated in response to insulin. The present study suggests that early signaling events in the insulin cascade are invoked in Sol8 myogenic cells and that this cell line provides a useful model to study insulin signaling.