Combining evidence from human genetic and functional screens to identify pathways altering obesity and fat distribution.

Overall adiposity and body fat distribution are heritable traits associated with altered risk of cardiometabolic disease and mortality. Performing rare variant (minor allele frequency<1%) association testing using exome-sequencing data from 402,375 participants in the UK Biobank (UKB) for nine overall and tissue-specific fat distribution traits, we identified 19 genes where putatively damaging rare variation associated with at least one trait (Bonferroni-adjusted P<1.58×10-7) and 52 additional genes at FDR≤1% (P≤4.37×10-5). These 71 genes exhibited higher (P=3.58×10-18) common variant prioritisation scores than genes not significantly enriched for rare putatively damaging variation, with evidence of monotonic allelic series (dose-response relationships) among ultra-rare variants (minor allele count≤10) in 22 genes. Five of the 71 genes have cognate protein UKB Olink data available; all five associated (P<3.80×10-6) with three or more analysed traits. Combining rare and common variation evidence, allelic series and proteomics, we selected 17 genes for CRISPR knockout in human white adipose tissue cell lines. In three previously uncharacterised target genes, knockout increased (two-sided t-test P<0.05) lipid accumulation, a cellular phenotype relevant for fat mass traits, compared to Cas9-empty negative controls: COL5A3 (fold change [FC]=1.72, P=0.0028), EXOC7 (FC=1.35, P=0.0096), and TRIP10 (FC=1.39, P=0.0157); furthermore, knockout of SLTM resulted in reduced lipid accumulation (FC=0.51, P=1.91×10-4). Integrating across population-based genetic and in vitro functional evidence, we highlight therapeutic avenues for altering obesity and body fat distribution by modulating lipid accumulation.

Genome-wide analyses identify 21 infertility loci and over 400 reproductive hormone loci across the allele frequency spectrum.

Genome-wide association studies (GWASs) may help inform treatments for infertility, whose causes remain unknown in many cases. Here we present GWAS meta-analyses across six cohorts for male and female infertility in up to 41,200 cases and 687,005 controls. We identified 21 genetic risk loci for infertility (P≤5E-08), of which 12 have not been reported for any reproductive condition. We found positive genetic correlations between endometriosis and all-cause female infertility (rg=0.585, P=8.98E-14), and between polycystic ovary syndrome and anovulatory infertility (rg=0.403, P=2.16E-03). The evolutionary persistence of female infertility-risk alleles in EBAG9 may be explained by recent directional selection. We additionally identified up to 269 genetic loci associated with follicle-stimulating hormone (FSH), luteinising hormone, oestradiol, and testosterone through sex-specific GWAS meta-analyses (N=6,095-246,862). While hormone-associated variants near FSHB and ARL14EP colocalised with signals for anovulatory infertility, we found no rg between female infertility and reproductive hormones (P>0.05). Exome sequencing analyses in the UK Biobank (N=197,340) revealed that women carrying testosterone-lowering rare variants in GPC2 were at higher risk of infertility (OR=2.63, P=1.25E-03). Taken together, our results suggest that while individual genes associated with hormone regulation may be relevant for fertility, there is limited genetic evidence for correlation between reproductive hormones and infertility at the population level. We provide the first comprehensive view of the genetic architecture of infertility across multiple diagnostic criteria in men and women, and characterise its relationship to other health conditions.

ACAD10 is not required for metformin's metabolic actions or for maintenance of whole-body metabolism in C57BL/6J mice.

AIM: Acyl-coenzyme A dehydrogenase family member 10 (ACAD10) is a mitochondrial protein purported to be involved in the fatty acid oxidation pathway. Metformin is the most prescribed therapy for type 2 diabetes; however, its precise mechanisms of action(s) are still being uncovered. Upregulation of ACAD10 is a requirement for metformin's ability to inhibit growth in cancer cells and extend lifespan in Caenorhabditis elegans. However, it is unknown whether ACAD10 plays a role in metformin's metabolic actions. MATERIALS AND METHODS: We assessed the role for ACAD10 on whole-body metabolism and metformin action by generating ACAD10KO mice on a C57BL/6J background via CRISPR-Cas9 technology. In-depth metabolic phenotyping was conducted in both sexes on a normal chow and high fat-high sucrose diet. RESULTS: Compared with wildtype mice, we detected no difference in body composition, energy expenditure or glucose tolerance in male or female ACAD10KO mice, on a chow diet or high-fat, high-sucrose diet (p ≥ .05). Hepatic mitochondrial function and insulin signalling was not different between genotypes under basal or insulin-stimulated conditions (p ≥ .05). Glucose excursions following acute administration of metformin before a glucose tolerance test were not different between genotypes nor was body composition or energy expenditure altered after 4 weeks of daily metformin treatment (p ≥ .05). Despite the lack of a metabolic phenotype, liver lipidomic analysis suggests ACAD10 depletion influences the abundance of specific ceramide species containing very long chain fatty acids, while metformin treatment altered clusters of cholesterol ester, plasmalogen, phosphatidylcholine and ceramide species. CONCLUSIONS: Loss of ACAD10 does not alter whole-body metabolism or impact the acute or chronic metabolic actions of metformin in this model.

Therapeutic blockade of ER stress and inflammation prevents NASH and progression to HCC.

The incidence of hepatocellular carcinoma (HCC) is rapidly rising largely because of increased obesity leading to nonalcoholic steatohepatitis (NASH), a known HCC risk factor. There are no approved treatments to treat NASH. Here, we first used single-nucleus RNA sequencing to characterize a mouse model that mimics human NASH-driven HCC, the MUP-uPA mouse fed a high-fat diet. Activation of endoplasmic reticulum (ER) stress and inflammation was observed in a subset of hepatocytes that was enriched in mice that progress to HCC. We next treated MUP-uPA mice with the ER stress inhibitor BGP-15 and soluble gp130Fc, a drug that blocks inflammation by preventing interleukin-6 trans-signaling. Both drugs have progressed to phase 2/3 human clinical trials for other indications. We show that this combined therapy reversed NASH and reduced NASH-driven HCC. Our data suggest that these drugs could provide a potential therapy for NASH progression to HCC.

Fructose stimulated de novo lipogenesis is promoted by inflammation.

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.

Adipocyte-specific deletion of IL-6 does not attenuate obesity-induced weight gain or glucose intolerance in mice.

It has been suggested that interleukin-6 (IL-6) produced by adipocytes in obesity leads to liver insulin resistance, although this hypothesis has never been definitively tested. Accordingly, we did so by generating adipocyte-specific IL-6-deficient (AdipoIL-6-/-) mice and studying them in the context of diet-induced and genetic obesity. Mice carrying two floxed alleles of IL-6 (C57Bl/6J) were crossed with Cre recombinase-overexpressing mice driven by the adiponectin promoter to generate AdipoIL-6-/- mice. AdipoIL-6-/- and floxed littermate controls were fed a standard chow or high-fat diet (HFD) for 16 wk and comprehensively metabolically phenotyped. In addition to a diet-induced obesity model, we also examined the role of adipocyte-derived IL-6 in a genetic model of obesity and insulin resistance by crossing the AdipoIL-6-/- mice with leptin-deficient (ob/ob) mice. As expected, mice on HFD and ob/ob mice displayed marked weight gain and increased fat mass compared with chow-fed and ob/+ (littermate control) animals, respectively. However, deletion of IL-6 from adipocytes in either model had no effect on glucose tolerance or fasting hyperinsulinemia. We concluded that adipocyte-specific IL-6 does not contribute to whole body glucose intolerance in obese mice.

Author Correction: Relieving ER stress to target NASH-driven hepatocellular carcinoma.

In the original version of this manuscript, the incorrect names for two proteins were given. S1P and S2P should have been defined as site-1 protease and site-2 protease. This has been corrected in the HTML and PDF versions of the manuscript.

Protein Kinase C Epsilon Deletion in Adipose Tissue, but Not in Liver, Improves Glucose Tolerance.

Protein kinase C epsilon (PKCɛ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCɛ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCɛ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCɛ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCɛ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCɛ activation in this tissue should be interpreted with caution. However, PKCɛ activity in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver.

Preclinical Models for Studying NASH-Driven HCC: How Useful Are They?

Hepatocellular carcinoma (HCC) is one of the most fatal and fastest-growing cancers. Recently, non-alcoholic steatohepatitis (NASH) has been recognized as a major HCC catalyst. However, it is difficult to decipher the molecular mechanisms underlying the pathogenesis of NASH and understand how it progresses to HCC by studying humans. Progress in this field depends on the availability of reliable preclinical models amenable to genetic and functional analyses and exhibiting robust NASH-to-HCC progression. Although numerous mouse models of NASH have been described, many do not faithfully mimic the human disease and few reliably progress to HCC. Here, we review current literature on the molecular etiology of NASH-related HCC and critically evaluate existing mouse models and their suitability for studying this malignancy. We also compare human transcriptomic and histopathological profiles with data from MUP-uPA mice, a reliable model of NASH-driven HCC that has been useful for evaluation of HCC-targeting immunotherapies.

Evidence that TLR4 Is Not a Receptor for Saturated Fatty Acids but Mediates Lipid-Induced Inflammation by Reprogramming Macrophage Metabolism.

Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation in vivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.

GeneXX: an online tool for the exploration of transcript changes in skeletal muscle associated with exercise.

Exercise stimulates a wide array of biological processes, but the mechanisms involved are incompletely understood. Many previous studies have adopted transcriptomic analyses of skeletal muscle to address particular research questions, a process that ultimately results in the collection of large amounts of publicly available data that has not been fully integrated or interrogated. To maximize the use of these available transcriptomic exercise data sets, we have downloaded and reanalyzed them and formulated the data into a searchable online tool, geneXX. GeneXX is highly intuitive and free and provides immediate information regarding the response of a transcript of interest to exercise in skeletal muscle. To demonstrate its utility, we carried out a meta-analysis on the included data sets and show transcript changes in skeletal muscle that persist regardless of sex, exercise mode, and duration, some of which have had minimal attention in the context of exercise. We also demonstrate how geneXX can be used to formulate novel hypotheses on the complex effects of exercise, using preliminary data already generated. This resource represents a valuable tool for researchers with interests in human skeletal muscle adaptation to exercise.

[Morphology of low-velocity impact stains produced from single drops of blood]. / Zur Morphologie langsam beschleunigter Blutauftropfspuren.

Systematic variation of blood droplet volume, the distance fallen and the surface (paper, wood, plastics, tiles) led to the conclusion that the size and the shape of the stains ("fingers", satellites) allowed to deduce the distance fallen but only if the actual surface structure was known. We found that detailed photography at the crime scene was necessary, yet experiments have to be performed due to the extreme influence of the actual surface texture on all characteristics (size, spines, peripheral spatter) of the blood stains.

A new simulation-based model for calculating post-mortem intervals using developmental data for Lucilia sericata (Dipt.: Calliphoridae).

Homicide investigations often depend on the determination of a minimum post-mortem interval (PMI(min)) by forensic entomologists. The age of the most developed insect larvae (mostly blow fly larvae) gives reasonably reliable information about the minimum time a person has been dead. Methods such as isomegalen diagrams or ADH calculations can have problems in their reliability, so we established in this study a new growth model to calculate the larval age of Lucilia sericata (MEIGEN, 1826). This is based on the actual non-linear development of the blow fly and is designed to include uncertainties, e.g. for temperature values from the crime scene. We used published data for the development of L. sericata to estimate non-linear functions describing the temperature dependent behavior of each developmental state. For the new model it is most important to determine the progress within one developmental state as correctly as possible since this affects the accuracy of the PMI estimation by up to 75%. We found that PMI calculations based on one mean temperature value differ by up to 65% from PMIs based on an 12-hourly time temperature profile. Differences of 2 degrees C in the estimation of the crime scene temperature result in a deviation in PMI calculation of 15-30%.

Use of Megaselia scalaris (Diptera: Phoridae) for post-mortem interval estimation indoors.

In forensic entomology, the determination of a minimum post-mortem interval often relies on the determination of the age of blow flies, since they are generally among the first colonisers of a corpse. In indoor cases, the blow flies might be delayed in arriving at the corpse. If the windows are closed, the attracting odour is confined and does not reach the flies, so that it takes longer for them to find and access the corpse. If blow flies are delayed or are unable to reach a corpse lying inside a room, much smaller flies (Phoridae) can enter and deposit their offspring. We present three indoor-case scenarios in which age determination of Megaselia scalaris gave much more accurate estimates of the minimum post-mortem interval than from larvae of Calliphoridae. In all cases, the estimated age of the blow fly larvae was between 10 and 20 days too short compared to the actual PMI. Estimation of the PMI using developmental times of Phoridae can be a good alternative to the determination of blow fly larval age, since Phoridae are found inside apparently enclosed environments (sealed plastic bags or rooms with closed doors and windows) and also at temperatures at which blow flies are inactive.

Molecular identification of forensically important blowfly species (Diptera: Calliphoridae) from Germany.

Forensic entomology applies knowledge about the behaviour and ecology of insects associated to corpses to homicide investigations. It is possible to calculate a minimum post-mortem interval by determining the age of the oldest blowfly larvae feeding on a corpse. The growth rate of the larvae is highly dependent on temperature and also varies between the different blowfly species infesting a corpse. It is, thus, crucial to correctly identify the species collected from a crime scene. To increase the quality of species identification, molecular methods were applied to 53 individuals of six different species sampled in Bonn, Germany: Calliphora vicina, Calliphora vomitoria, Lucilia caesar, Lucilia sericata, Lucilia illustris, and Protophormia terraenovae. We extracted DNA and checked a 229 bp sequence within the mitochondrial cytochrome oxidase subunit I. The sequences of the local flies were aligned to published data of specimens from other countries. We also studied the practical value of the analysed DNA region for their differentiation. All species were matched correctly by a Basic Local Alignment Search Tool (BLAST) search apart from L. caesar and L. illustris. Although molecular methods are very useful-especially if it is necessary to identify small fragments of insect material or very young larvae-we propose to use it only in addition to the conventional methods.

[An entomological case report during the winter months: estimation of the post-mortem interval considering the influence of cold temperatures on the development of the forensically important blowfly Calliphora vomitoria]. / Eine insektenkundliche Fallbetrachtung in den Wintermonaten: Leichenliegezeitbestimmung unter Berücksichtigung des Einflusses niedriger Temperaturen auf die Entwicklung der forensisch wichtigen Schmeissfliege Calliphora vomitoria.

The authors describe a case report with entomological estimation of the post-mortem interval in the winter months. In early December 2007, the body of a suicide was discovered not far from a lake near Bonn in North Rhine-Westphalia four weeks after the man had disappeared from a hospital. The corpse was very well preserved and did not show any signs of advanced putrefaction. The stage of decomposition did not allow a correct estimation of the time since death. Infestation of insect larvae of the species Calliphora vomitoria was detected in the oral cavity as well as in the self-inflicted deep cut to the throat responsible for death. The age of the larvae was determined by considering the specific minimum threshold of the species (minimum temperature necessary for development). To estimate the time until the blowflies detect the body and start to oviposit, the authors ran an experiment with a pig in a comparable environment with similar temperatures. Altogether, these investigations suggested that the man had committed suicide shortly after disappearing from the hospital. Without the entomological evaluation it would have been very difficult to narrow down the post-mortem interval correctly.

[Dumping of corpses in compost bins--two forensic entomological case reports]. / Leichen-"Entsorgung" in einer Biotonne--zwei forensisch-entomologische Fallberichte.

Two cases from the Ruhr Area in Western Germany are presented. In each case, the deceased had been wrapped in plastic bags and placed inside a large compost bin in the backyard of the property. In both cases, the relatives claimed that the decedent had died from a natural cause and that they had concealed the body to ensure the further payment of the nursing care and pension benefits. In the first case, the responsible person stated that the body had been inside the bin for three years; in case 2 the postmortem interval indicated was 6 months. In spite of the closed lid of the bin the insect infestation was extensive and rich in species: Empty pupal cases of several blowfly species were collected as well as histerids and pupal cases of Fannia scalaris in the first case. In case 2, phorids and larval skins of dermestids were also found.

fgf1 is required for normal differentiation of erythrocytes in zebrafish primitive hematopoiesis.

Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood cells. We here show that zebrafish fgf1 is involved in vivo in primitive hematopoiesis. Fibroblast growth factor-1 (FGF1) morpholino knockdown leads to abnormal accumulation of blood cells in the posterior intermediate cell mass at 32 hr postfertilization. Expression of the erythroid markers gata1 and ika, normally diminishing in differentiating erythrocytes at this stage, is maintained at abnormally high levels in primitive blood cells. The onset of erythrocyte differentiation as assessed by o-dianisidine staining is severely delayed. Most fgf1 morphants later recover to wild-type appearance, and primitive erythrocytes eventually differentiate. Zebrafish fgf1 is syntenic to human FGF1, which maps to a critically deleted region in human del(5q) syndrome posing an increased risk of leukemia to patients. As its knockdown in zebrafish changes expression of gata1, a gene involved in hematopoietic stem cell decisions, FGF1 should be considered to play a role in the pathogenesis of del(5q) syndrome.

Analysis of origin and growth of the thyroid gland in zebrafish.

The zebrafish thyroid gland shows a unique pattern of growth as a differentiated endocrine gland. Here, we analyze the onset of differentiation, the contribution of lineages, and the mode of growth of this gland. The expression of genes involved in hormone production and the establishment of epithelial polarity show that differentiation into a first thyroid follicle takes place early during embryonic development. Thyroid follicular tissue then grows along the pharyngeal midline, initially independently of thyroid stimulating hormone. Lineage analysis reveals that thyroid follicle cells are exclusively recruited from the pharyngeal endoderm. The ultimobranchial bodies that merge with the thyroid in mammals form separate glands in zebrafish as visualized by calcitonin precursor gene expression. Mosaic analysis suggests that the first thyroid follicle differentiating at 55 hours postfertilization corresponds later to the most anterior follicle and that new follicles are added caudally.