Unique diagnostic signatures of concussion in the saliva of male athletes: the Study of Concussion in Rugby Union through MicroRNAs (SCRUM).

OBJECTIVE: To investigate the role of salivary small non-coding RNAs (sncRNAs) in the diagnosis of sport-related concussion. METHODS: Saliva was obtained from male professional players in the top two tiers of England's elite rugby union competition across two seasons (2017-2019). Samples were collected preseason from 1028 players, and during standardised head injury assessments (HIAs) at three time points (in-game, post-game, and 36-48 hours post-game) from 156 of these. Samples were also collected from controls (102 uninjured players and 66 players sustaining a musculoskeletal injury). Diagnostic sncRNAs were identified with next generation sequencing and validated using quantitative PCR in 702 samples. A predictive logistic regression model was built on 2017-2018 data (training dataset) and prospectively validated the following season (test dataset). RESULTS: The HIA process confirmed concussion in 106 players (HIA+) and excluded this in 50 (HIA-). 32 sncRNAs were significantly differentially expressed across these two groups, with let-7f-5p showing the highest area under the curve (AUC) at 36-48 hours. Additionally, a combined panel of 14 sncRNAs (let-7a-5p, miR-143-3p, miR-103a-3p, miR-34b-3p, RNU6-7, RNU6-45, Snora57, snoU13.120, tRNA18Arg-CCT, U6-168, U6-428, U6-1249, Uco22cjg1,YRNA_255) could differentiate concussed subjects from all other groups, including players who were HIA- and controls, immediately after the game (AUC 0.91, 95% CI 0.81 to 1) and 36-48 hours later (AUC 0.94, 95% CI 0.86 to 1). When prospectively tested, the panel confirmed high predictive accuracy (AUC 0.96, 95% CI 0.92 to 1 post-game and AUC 0.93, 95% CI 0.86 to 1 at 36-48 hours). CONCLUSIONS: SCRUM, a large prospective observational study of non-invasive concussion biomarkers, has identified unique signatures of concussion in saliva of male athletes diagnosed with concussion.

Differential Expression of Circulating Inflammatory Proteins Following Sport-Related Traumatic Brain Injury.

Sport-related traumatic brain injury (TBI) elicits a multifaceted inflammatory response leading to brain injury and morbidity. This response could be a predictive tool for the progression of TBI and to stratify the injury of which mild TBI is most prevalent. Therefore, we examined the differential expression of serum inflammatory markers overtime and identified novel markers in repetitively concussed athletes. Neuropsychological assessment by Wechsler Adult Intelligence Scale (WAIS) and Immediate Post Concussion Assessment and Cognitive Test (ImPACT) was performed on rugby players and serum was taken from healthy, concussed and repetitively concussed athletes. Serum was also obtained <1 week and >1 week after trauma and analyzed for 92 inflammatory protein markers. Fibroblast growth factor 21 (FGF21) and interleukin-7 (IL-7) differentiated repetitively concussed athletes. Macrophage chemotactic protein-1 (MCP-1), tumor necrosis factor superfamily member 14 (TNFSF14) were significantly reduced >1 week and chemokine (C-X3-C motif) ligand 1 (CX3CL1) upregulated <1 week after injury. FGF21 and MCP-1 negatively correlated with symptoms and their severity. We have identified dynamic changes in the inflammatory response overtime and in different classes of concussion correlating with disease progression. This data supports the use of inflammatory biomarkers as predictors of symptom development due to secondary complications of sport-related mTBI.

NF-Y-dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage.

Glutamate receptor subunit 4 (GluA4) is highly expressed by neural cells sensitive to excitotoxicity, and is the predominant subunit expressed by oligodendrocyte precursor cells (OPC) during a key period of vulnerability to hypoxic-ischemic injury. Therefore, transcriptional networks downstream of excitotoxic GluA4 activation represent a promising area for therapeutic intervention. In this work, we identify the CCAAT binding transcription factor NF-Yb as a novel transcriptional regulator of Gria4 (GluA4 gene), and a controller of excitotoxic death in the oligodendroglial lineage. We describe a novel regulatory region within Gria4 containing CCAAT sequences whose binding by NF-Yb is regulated by excitotoxicity. Excitotoxicity-induced alterations in NF-Yb binding are associated with changes in Gria4 transcription, while knockdown of NF-Yb alters the transcription of reporter constructs containing this regulatory region. Data from immortalized and primary OPC reveal that RNAi and pharmacological disruption of NF-Yb alter Gria4 transcription, with the latter inducing apoptosis and influencing a set of apoptotic genes similarly regulated during excitotoxicity. These data provide the first definition of a trans-acting mechanism regulating Gria4, and identify the NF-Y network as a potential source of pharmacological targets for promoting OPC survival.

Epigenetic changes in fetal hypothalamic energy regulating pathways are associated with maternal undernutrition and twinning.

Undernutrition during pregnancy is implicated in the programming of offspring for the development of obesity and diabetes. We hypothesized that maternal programming causes epigenetic changes in fetal hypothalamic pathways regulating metabolism. This study used sheep to examine the effect of moderate maternal undernutrition (60 d before to 30 d after mating) and twinning to investigate changes in the key metabolic regulators proopiomelanocortin (POMC) and the glucocorticoid receptor (GR) in fetal hypothalami. Methylation of the fetal hypothalamic POMC promoter was reduced in underfed singleton, fed twin, and underfed twin groups (60, 73, and 63% decrease, respectively). This was associated with reduced DNA methyltransferase activity and altered histone methylation and acetylation. Methylation of the hypothalamic GR promoter was decreased in both twin groups and in maternally underfed singleton fetuses (52, 65, and 55% decrease, respectively). This correlated with changes in histone methylation and acetylation and increased GR mRNA expression in the maternally underfed singleton group. Alterations in GR were hypothalamic specific, with no changes in hippocampi. Unaltered levels of OCT4 promoter methylation indicated gene-specific effects. In conclusion, twinning and periconceptional undernutrition are associated with epigenetic changes in fetal hypothalamic POMC and GR genes, potentially resulting in altered energy balance regulation in the offspring.

Co-Expression Analysis of microRNAs and Proteins in Brain of Alzheimer's Disease Patients.

Alzheimer's disease (AD) is the most common form of dementia globally; however, the aetiology of AD remains elusive hindering the development of effective therapeutics. MicroRNAs (miRNAs) are regulators of gene expression and have been of growing interest in recent studies in many pathologies including AD not only for their use as biomarkers but also for their implications in the therapeutic field. In this study, miRNA and protein profiles were obtained from brain tissues of different stage (Braak III-IV and Braak V-VI) of AD patients and compared to matched controls. The aim of the study was to identify in the late stage of AD, the key dysregulated pathways that may contribute to pathogenesis and then to evaluate whether any of these pathways could be detected in the early phase of AD, opening new opportunity for early treatment that could stop or delay the pathology. Six common pathways were found regulated by miRNAs and proteins in the late stage of AD, with one of them (Rap1 signalling) activated since the early phase. MiRNAs and proteins were also compared to explore an inverse trend of expression which could lead to the identification of new therapeutic targets. These results suggest that specific miRNA changes could represent molecular fingerprint of neurodegenerative processes and potential therapeutic targets for early intervention.

Determining the effect of ocular chemical injuries on topical drug delivery.

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E-05 cm.min-1 vs. Hanks' Balanced Salt Solution (HBSS) 1.64 ± 1.01E-06 cm.min-1) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E-04 cm.min-1 vs. HBSS 4.60 ± 0.972E-04 cm.min-1) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E-04 cm.min-1 vs. HBSS 2.69 ± 0.439E-04 cm.min-1). By contrast, H2SO4 decreased trans-corneal penetration of hydrophilic fluorescein (H2SO4 1.16 ± 14.2E-07 cm.min-1) and of hydrophobic dexamethasone (H2SO4 1.88 ± 0.646E-04 cm.min-1) and rhodamine B (H2SO4 4.60 ± 1.42E-05 cm.min-1). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.

ILB® resolves inflammatory scarring and promotes functional tissue repair.

Fibrotic disease is a major cause of mortality worldwide, with fibrosis arising from prolonged inflammation and aberrant extracellular matrix dynamics. Compromised cellular and tissue repair processes following injury, infection, metabolic dysfunction, autoimmune conditions and vascular diseases leave tissues susceptible to unresolved inflammation, fibrogenesis, loss of function and scarring. There has been limited clinical success with therapies for inflammatory and fibrotic diseases such that there remains a large unmet therapeutic need to restore normal tissue homoeostasis without detrimental side effects. We investigated the effects of a newly formulated low molecular weight dextran sulfate (LMW-DS), termed ILB®, to resolve inflammation and activate matrix remodelling in rodent and human disease models. We demonstrated modulation of the expression of multiple pro-inflammatory cytokines and chemokines in vitro together with scar resolution and improved matrix remodelling in vivo. Of particular relevance, we demonstrated that ILB® acts, in part, by downregulating transforming growth factor (TGF)ß signalling genes and by altering gene expression relating to extracellular matrix dynamics, leading to tissue remodelling, reduced fibrosis and functional tissue regeneration. These observations indicate the potential of ILB® to alleviate fibrotic diseases.

Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis.

BACKGROUND: Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. METHODS: Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. RESULTS: We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. CONCLUSION: We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

Rapid assessment of ocular drug delivery in a novel ex vivo corneal model.

Drug delivery by topical application has higher patient acceptance and lower morbidity than intraocular injection, but many ophthalmic treatments are unable to enter the eye or reach the posterior segment after topical application. The first stage towards posterior segment delivery after topical application is ocular surface penetration and existing models are in vivo or use large quantities of tissue. We therefore developed a novel ex vivo model using discs of porcine and human cornea and sclera (5 mm diameter) to assess penetration of a candidate neuroprotective siRNA. siRNA against caspase 2 or control solutions of known penetrance were applied to the corneal epithelial surface and trans-corneal penetration and corneal adsorbance measured at fixed time points. To demonstrate that leakage did not occur, we applied dextran blue, which should not penetrate the intact cornea and did not do so in our model. Fluorescein penetration (0.09%) was less than rhodamine B (6.98%) at 60 min. siCASP2 penetration was 0.01% by 60 min. When the applied siCASP2 was washed off after 2 min, (representing lacrimal drainage) 0.071% penetrated porcine cornea by 60 min and 0.0002% penetrated human cornea and 0.001% penetrated human sclera. Our ex vivo model rapidly and cost-effectively assesses transcorneal penetration of candidate topical therapies, allowing rates of trans-corneal penetration for potential therapies such as siRNA to be evaluated with small quantities of human or animal tissue.

Triamcinolone acetonide loaded-cationic nano-lipoidal formulation for uveitis: Evidences of improved biopharmaceutical performance and anti-inflammatory activity.

Topical administration of corticosteroids is the cornerstone treatment of anterior uveitis, but poor corneal penetration and retention cause hindrance in their therapeutic utility. The conventional eye drops are less valuable in conditions where inflammation reaches deeper regions of the eye. Therefore, there is a clear need for an effective drug delivery system, which can increase corticosteroid penetration after topical application. To address this, cationic nanostructured lipid carriers of the drug triamcinolone acetonide (cTA-NLC) were prepared. The cTA-NLC were prepared by a hot microemulsion method and evaluated for drug release, permeation, cell uptake, cytotoxicity, anti-inflammatory activity and ocular irritancy. The cTA-NLC are nanometric in size (< 200 nm), with a zeta potential of about +35 mv and % drug EE of 88 %. The nanocarriers exhibited slow and sustained release of around 84 % in 24 h and transcorneal drug permeation of 51 % in 8 h. The nanocarriers exhibited no cytotoxicity (% cell viability of>90 %). The cell uptake study showed that nanocarriers could retain inside the cells for 24 h. The developed formulation could significantly reduce the TNF-α level in LPS induced inflamed cells. The studies indicated that cTA-NLC could be a promising option for the topical treatment of uveitis.

Alkyltransferase-like proteins.

Recent in silico analysis has revealed the presence of a group of proteins in pro and lower eukaryotes, but not in Man, that show extensive amino acid sequence similarity to known O(6)-alkylguanine-DNA alkyltransferases, but where the cysteine at the putative active site is replaced by another residue, usually tryptophan. Here we review recent work on these proteins, which we designate as alkyltransferase-like (ATL) proteins, and consider their mechanism of action and role in protecting the host organisms against the biological effects of O(6)-alkylating agents, and their evolution. ATL proteins from Escherichia coli (eAtl, transcribed from the ybaz open reading frame) and Schizosaccharomyces pombe (Atl1) are able to bind to a range of O(6)-alkylguanine residues in DNA and to reversibly inhibit the action of the human alkyltransferase (MGMT) upon these substrates. Isolated proteins were not able to remove the methyl group in O(6)-methylguanine-containing DNA or oligonucleotides, neither did they display glycosylase or endonuclease activity. S. pombe does not contain a functional alkyltransferase and atl1 inactivation sensitises this organism to a variety of alkylating agents, suggesting that Atl1 acts by binding to O(6)-alkylguanine lesions and signalling them for processing by other DNA repair pathways. Currently we cannot exclude the possibility that ATL proteins arose through independent mutation of the alkyltransferase gene in different organisms. However, analyses of the proteins from E. coli and S. pombe, are consistent with a common function.

A novel DNA damage recognition protein in Schizosaccharomyces pombe.

Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N'nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.

Altered Decorin Biology in Proliferative Vitreoretinopathy: A Mechanistic and Cohort Study.

Purpose: To determine if vitreous levels of the pro-fibrotic cytokine transforming growth factor beta2 (TGF-ß2) and its opposing regulator decorin predict subsequent proliferative vitreoretinopathy (PVR) development in patients with rhegmatogenous retinal detachment (RRD). Methods: We examined the effect of TGF-ß2 and decorin on epithelial-mesenchymal transition (EMT) and collagen expression in vitro using ARPE-19 cells, and we analyzed extracellular matrix marker expression in PVR membrane and internal limiting membrane patient samples. We performed a prospective noninterventional cohort study, recruiting 125 patients undergoing vitrectomy for RRD and macular hole surgery, measured vitreous levels of TGF-ß2 and decorin by ELISA, and followed them up for 6 months. Patients who did not develop PVR were compared to those who did, in order to determine whether vitreous TGF-ß2 and decorin levels predicted PVR development. Results: In vitro, TGF-ß2 induced EMT and collagen production. Decorin strongly inhibited EMT and collagen production at high levels. PVR membranes expressed high levels of fibrosis-associated proteins, consistent with EMT. Vitreous TGF-ß2 levels were unchanged between patients with macular holes and RRD who did or did not subsequently develop PVR. Average decorin levels were higher in the vitreous of RRD patients who subsequently developed PVR compared to those who did not, but at the measured vitreous concentrations (1-2 µg/mL), decorin did not demonstrate an in vitro inhibitory effect on EMT. Conclusions: In vitro, high concentrations of decorin inhibited EMT and fibrosis. At the levels seen in human vitreous, decorin did not prevent fibrosis or EMT in vitro, and higher initial vitreous decorin levels were associated with the development of postoperative PVR after vitrectomy to treat RRD, but did not reliably predict the outcome.

Developmental programming of hypothalamic neuronal circuits: impact on energy balance control.

The prevalence of obesity in adults and children has increased globally at an alarming rate. Mounting evidence from both epidemiological studies and animal models indicates that adult obesity and associated metabolic disorders can be programmed by intrauterine and early postnatal environment- a phenomenon known as "fetal programming of adult disease." Data from nutritional intervention studies in animals including maternal under- and over-nutrition support the developmental origins of obesity and metabolic syndrome. The hypothalamic neuronal circuits located in the arcuate nucleus controlling appetite and energy expenditure are set early in life and are perturbed by maternal nutritional insults. In this review, we focus on the effects of maternal nutrition in programming permanent changes in these hypothalamic circuits, with experimental evidence from animal models of maternal under- and over-nutrition. We discuss the epigenetic modifications which regulate hypothalamic gene expression as potential molecular mechanisms linking maternal diet during pregnancy to the offspring's risk of obesity at a later age. Understanding these mechanisms in key metabolic genes may provide insights into the development of preventative intervention strategies.

Maternal undernutrition programs tissue-specific epigenetic changes in the glucocorticoid receptor in adult offspring.

Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

Epigenetic changes in the hypothalamic pro-opiomelanocortin gene: a mechanism linking maternal undernutrition to obesity in the offspring?

Maternal undernutrition is associated with programming of obesity in offspring. While previous evidence has linked programming to the hypothalamic, pituitary, and adrenal (HPA) axis it could also affect the hypothalamic neuropeptides which regulate food intake and energy balance. Alpha melanocyte stimulating hormone (αMSH), a key regulator of these neuronal pathways, is derived from pro-opiomelanocortin (POMC) which is therefore a prime target for the programming of obesity. Several models of maternal undernutrition have identified changes in POMC in hypothalami from foetuses or offspring at various ages. These models have also shown that the offspring go on to develop obesity and/or glucose intolerance. It is our hypothesis that programming leads to epigenetic changes in hypothalamic neuropeptide genes. Therefore when there is subsequent increased food availability, the epigenetic changes could cause dysfunctional transcriptional regulation of energy balance. We present evidence of epigenetic changes in the POMC gene promoter in foetal hypothalami after peri-conceptional undernutrition. In this model there are also epigenetic changes in the hypothalamic glucocorticoid receptor with consequent up-regulation of the receptor which could lead to alterations in the regulation of POMC and neuropeptide Y (NPY) in the hypothalamus. Thus maternal undernutrition could cause epigenetic changes in the POMC and glucocorticoid receptor genes, in the foetal hypothalamus, which may predispose the offspring to altered regulation of food intake, energy expenditure and glucose homeostasis, later in life.

Deletion of Lkb1 in pro-opiomelanocortin neurons impairs peripheral glucose homeostasis in mice.

OBJECTIVE: AMP-activated protein kinase (AMPK) signaling acts as a sensor of nutrients and hormones in the hypothalamus, thereby regulating whole-body energy homeostasis. Deletion of Ampkα2 in pro-opiomelanocortin (POMC) neurons causes obesity and defective neuronal glucose sensing. LKB1, the Peutz-Jeghers syndrome gene product, and Ca(2+)-calmodulin-dependent protein kinase kinase ß (CaMKKß) are key upstream activators of AMPK. This study aimed to determine their role in POMC neurons upon energy and glucose homeostasis regulation. RESEARCH DESIGN AND METHODS: Mice lacking either Camkkß or Lkb1 in POMC neurons were generated, and physiological, electrophysiological, and molecular biology studies were performed. RESULTS: Deletion of Camkkß in POMC neurons does not alter energy homeostasis or glucose metabolism. In contrast, female mice lacking Lkb1 in POMC neurons (PomcLkb1KO) display glucose intolerance, insulin resistance, impaired suppression of hepatic glucose production, and altered expression of hepatic metabolic genes. The underlying cellular defect in PomcLkb1KO mice involves a reduction in melanocortin tone caused by decreased α-melanocyte-stimulating hormone secretion. However, Lkb1-deficient POMC neurons showed normal glucose sensing, and body weight was unchanged in PomcLkb1KO mice. CONCLUSIONS: Our findings demonstrate that LKB1 in hypothalamic POMC neurons plays a key role in the central regulation of peripheral glucose metabolism but not body-weight control. This phenotype contrasts with that seen in mice lacking AMPK in POMC neurons with defects in body-weight regulation but not glucose homeostasis, which suggests that LKB1 plays additional functions distinct from activating AMPK in POMC neurons.

Epigenetic changes in the hypothalamic proopiomelanocortin and glucocorticoid receptor genes in the ovine fetus after periconceptional undernutrition.

Maternal food restriction is associated with the development of obesity in offspring. This study examined how maternal undernutrition in sheep affects the fetal hypothalamic glucocorticoid receptor (GR) and the appetite-regulating neuropeptides, proopiomelanocortin (POMC) and neuropeptide Y, which it regulates. In fetuses from ewes undernourished from -60 to +30 d around conception, there was increased histone H3K9 acetylation (1.63-fold) and marked hypomethylation (62% decrease) of the POMC gene promoter but no change in POMC expression. In the same group, acetylation of histone H3K9 associated with the hypothalamic GR gene was increased 1.60-fold and the GR promoter region was hypomethylated (53% decrease). In addition, there was a 4.7-fold increase in hypothalamic GR expression but no change in methylation of GR gene expression in the anterior pituitary or hippocampus. Interestingly, hypomethylation of both POMC and GR promoter markers in fetal hypothalami was also identified after maternal undernutrition from -60 to 0 d and -2 to +30 d. In comparison, the Oct4 gene, was hypermethylated in both control and underfed groups. Periconceptional undernutrition is therefore associated with marked epigenetic changes in hypothalamic genes. Increase in GR expression in the undernourished group may contribute to fetal programming of a predisposition to obesity, via altered GR regulation of POMC and neuropeptide Y. These epigenetic changes in GR and POMC in the hypothalamus may also predispose the offspring to altered regulation of food intake, energy expenditure, and glucose homeostasis later in life.