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OBJECTIVE The lateral parabrachial nucleus (lPBN) in the brainstem has emerged as a key area involved in feeding control that is targeted by several circulating anorexigenic hormones. Here, the objective was to determine whether the lPBN is also a relevant site for the orexigenic hormone ghrelin, inspired by studies in mice and rats showing that there is an abundance of ghrelin receptors in this area. METHODS This study first explored whether iPBN cells respond to ghrelin involving Fos mapping and electrophysiological studies in rats. Next, rats were injected acutely with ghrelin, a ghrelin receptor antagonist, or vehicle into the lPBN to investigate feeding-linked behaviors. RESULTS Curiously, ghrelin injection (intracerebroventricular or intravenous) increased Fos protein expression in the lPBN yet the predominant electrophysiological response was inhibitory. Intra-lPBN ghrelin injection increased chow or high-fat diet intake, whereas the antagonist decreased chow intake only. In a choice paradigm, intra-lPBN ghrelin increased intake of chow but not lard or sucrose. Intra-lPBN ghrelin did not alter progressive ratio lever pressing for sucrose or conditioned place preference for chocolate. CONCLUSIONS The lPBN is a novel locus from which ghrelin can alter consummatory behaviors (food intake and choice) but not appetitive behaviors (food reward and motivation).

Aim: MicroRNAs play an important role in the maintenance of cellular functions by fine-tuning gene expression levels. The aim of the current study was to identify genetically caused changes in microRNA expression which associate with islet dysfunction in diabetic mice. Methods: To identify novel microRNAs involved in islet dysfunction, transcriptome and miRNome analyses were performed in islets of obese, diabetes-susceptible NZO and diabetes-resistant B6-ob/ob mice and results combined with quantitative trait loci (QTL) and functional in vitro analysis. Results: In islets of NZO and B6-ob/ob mice, 94 differentially expressed microRNAs were detected, of which 11 are located in diabetes QTL. Focusing on conserved microRNAs exhibiting the strongest expression difference and which have not been linked to islet function, miR-205-5p was selected for further analysis. According to transcriptome data and target prediction analyses, miR-205-5p affects genes involved in Wnt and calcium signalling as well as insulin secretion. Over-expression of miR-205-5p in the insulinoma cell line INS-1 increased insulin expression, left-shifted the glucose-dependence of insulin secretion and supressed the expression of the diabetes gene TCF7L2. The interaction between miR-205-5p and TCF7L2 was confirmed by luciferase reporter assay. Conclusion: MiR-205-5p was identified as relevant microRNA involved in islet dysfunction by interacting with TCF7L2. (Less)

Summary: An insufficient adaptive beta-cell compensation is a hallmark of type 2 diabetes (T2D). Primary cilia function as versatile sensory antennae regulating various cellular processes, but their role on compensatory beta-cell replication has not been examined. Here, we identify a significant enrichment of downregulated, cilia-annotated genes in pancreatic islets of diabetes-prone NZO mice as compared with diabetes-resistant B6-ob/ob mice. Among 327 differentially expressed mouse cilia genes, 81 human orthologs are also affected in islets of diabetic donors. Islets of nondiabetic mice and humans show a substantial overlap of upregulated cilia genes that are linked to cell-cycle progression. The shRNA-mediated suppression of KIF3A, essential for ciliogenesis, impairs division of MIN6 beta cells as well as in dispersed primary mouse and human islet cells, as shown by decreased BrdU incorporation. These findings demonstrate the substantial role of cilia-gene regulation on islet function and T2D risk. : Kluth et al. identify a significant enrichment of cilia-annotated genes that are differentially expressed in pancreatic islets of obese mice. Many of the genes were also linked to human T2D, suggesting that dysregulation of cilia-associated genes may participate in T2D risk. Keywords: beta-cell proliferation, cilia genes, human pancreatic islets, islet transcriptomics, New Zealand Obese mouse, pathway enrichment analysis, primary cilia, type 2 diabetes

Type 2 diabetes (T2D) is a complex metabolic disease regulated by an interaction of genetic predisposition and environmental factors. To understand the genetic contribution in the development of diabetes, mice varying in their disease susceptibility were crossed with the obese and diabetes-prone New Zealand obese (NZO) mouse. Subsequent whole-genome sequence scans revealed one major quantitative trait loci (QTL), Nidd/DBA on chromosome 4, linked to elevated blood glucose and reduced plasma insulin and low levels of pancreatic insulin. Phenotypical characterization of congenic mice carrying 13.6 Mbp of the critical fragment of DBA mice displayed severe hyperglycemia and impaired glucose clearance at week 10, decreased glucose response in week 13, and loss of β-cells and pancreatic insulin in week 16. To identify the responsible gene variant(s), further congenic mice were generated and phenotyped, which resulted in a fragment of 3.3 Mbp that was sufficient to induce hyperglycemia. By combining transcriptome analysis and haplotype mapping, the number of putative responsible variant(s) was narrowed from initial 284 to 18 genes, including gene models and non-coding RNAs. Consideration of haplotype blocks reduced the number of candidate genes to four (Kti12, Osbpl9, Ttc39a, and Calr4) as potential T2D candidates as they display a differential expression in pancreatic islets and/or sequence variation. In conclusion, the integration of comparative analysis of multiple inbred populations such as haplotype mapping, transcriptomics, and sequence data substantially improved the mapping resolution of the diabetes QTL Nidd/DBA. Future studies are necessary to understand the exact role of the different candidates in β-cell function and their contribution in maintaining glycemic control.

BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association.; METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated invivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2.; RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage.; CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets.; LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease. Copyright © 2020 European Association for the Study of the Liver. All rights reserved.

To identify novel disease genes for type 2 diabetes (T2D) we generated two backcross populations of obese and diabetes-susceptible New Zealand Obese (NZO/HI) mice with the two lean mouse strains 129P2/OlaHsd and C3HeB/FeJ. Subsequent whole-genome linkage scans revealed 30 novel quantitative trait loci (QTL) for T2D-associated traits. The strongest association with blood glucose [12 cM, logarithm of the odds (LOD) 13.3] and plasma insulin (17 cM, LOD 4.8) was detected on proximal chromosome 7 (designated Nbg7p, NZO blood glucose on proximal chromosome 7) exclusively in the NZOxC3H crossbreeding, suggesting that the causal gene is contributed by the C3H genome. Introgression of the critical C3H fragment into the genetic NZO background by generating recombinant congenic strains and metabolic phenotyping validated the phenotype. For the detection of candidate genes in the critical region (30–46 Mb), we used a combined approach of haplotype and gene expression analysis to search for C3H-specific gene variants in the pancreatic islets, which appeared to be the most likely target tissue for the QTL. Two genes, Atp4a and Pop4, fulfilled the criteria from our candidate gene approaches. The knockdown of both genes in MIN6 cells led to decreased glucose-stimulated insulin secretion, indicating a regulatory role of both genes in insulin secretion, thereby possibly contributing to the phenotype linked to Nbg7p. In conclusion, our combined- and comparative-cross analysis approach has successfully led to the identification of two novel diabetes susceptibility candidate genes, and thus has been proven to be a valuable tool for the discovery of novel disease genes.

Objective: Changes to the microbial community in the human gut have been proposed to promote metabolic disturbances that also occur after short periods of sleep loss (including insulin resistance). However, whether sleep loss affects the gut microbiota remains unknown. Methods: In a randomized within-subject crossover study utilizing a standardized in-lab protocol (with fixed meal times and exercise schedules), we studied nine normal-weight men at two occasions: after two nights of partial sleep deprivation (PSD; sleep opportunity 02:45–07:00 h), and after two nights of normal sleep (NS; sleep opportunity 22:30–07:00 h). Fecal samples were collected within 24 h before, and after two in-lab nights, of either NS or PSD. In addition, participants underwent an oral glucose tolerance test following each sleep intervention. Results: Microbiota composition analysis (V4 16S rRNA gene sequencing) revealed that after two days of PSD vs. after two days of NS, individuals exhibited an increased Firmicutes:Bacteroidetes ratio, higher abundances of the families Coriobacteriaceae and Erysipelotrichaceae, and lower abundance of Tenericutes (all P

Obesity has become a worldwide health problem, but we still do not understand the molecular mechanisms that contribute to overeating and low expenditure of energy. Leptin has emerged as a major regulator of energy balance through its actions in the hypothalamus. Importantly, obese people exhibit high circulating levels of leptin, yet the hypothalamus no longer responds normally to this hormone to suppress appetite or to increase energy expenditure. Several well-known hypotheses have been proposed to explain impaired central responsiveness to the effects of leptin in obesity, including defective transit across the blood-brain barrier at the arcuate nucleus, hypothalamic endoplasmic reticulum stress, maladaptive sterile inflammation in the hypothalamus, and overexpression of molecules that may inhibit leptin signaling. We also discuss a new explanation that is based on our group's recent discovery of a signaling pathway that we named "NSAPP" after its five main protein components. The NSAPP pathway consists of an oxide transport chain that causes a transient, targeted burst in intracellular hydrogen peroxide (H2O2) to inactivate redox-sensitive members of the protein tyrosine phosphatase gene family. The NSAPP oxide transport chain is required for full activation of canonical leptin signaling in neurons but fails to function normally in states of overnutrition. Remarkably, leptin and insulin both require the NSAPP oxide transport chain, suggesting that a defect in this pathway could explain simultaneous resistance to the appetite-suppressing effects of both hormones in obesity.

Objective Obesity and type 2 diabetes (T2D) arise from the interplay between genetic, epigenetic, and environmental factors. The aim of this study was to combine bioinformatics and functional studies to identify miRNAs that contribute to obesity and T2D. Methods A computational framework (miR-QTL-Scan) was applied by combining QTL, miRNA prediction, and transcriptomics in order to enhance the power for the discovery of miRNAs as regulative elements. Expression of several miRNAs was analyzed in human adipose tissue and blood cells and miR-31 was manipulated in a human fat cell line. Results In 17 partially overlapping QTL for obesity and T2D 170 miRNAs were identified. Four miRNAs (miR-15b, miR-30b, miR-31, miR-744) were recognized in gWAT (gonadal white adipose tissue) and six (miR-491, miR-455, miR-423-5p, miR-132-3p, miR-365-3p, miR-30b) in BAT (brown adipose tissue). To provide direct functional evidence for the achievement of the miR-QTL-Scan, miR-31 located in the obesity QTL Nob6 was experimentally analyzed. Its expression was higher in gWAT of obese and diabetic mice and humans than of lean controls. Accordingly, 10 potential target genes involved in insulin signaling and adipogenesis were suppressed. Manipulation of miR-31 in human SGBS adipocytes affected the expression of GLUT4, PPARγ, IRS1, and ACACA. In human peripheral blood mononuclear cells (PBMC) miR-15b levels were correlated to baseline blood glucose concentrations and might be an indicator for diabetes. Conclusion Thus, miR-QTL-Scan allowed the identification of novel miRNAs relevant for obesity and T2D., Highlights • A computational biology approach identified ten miRNAs relevant for obesity/T2D. • miR-31 is upregulated in adipose tissue of obese and diabetic subjects. • miR-31 targets genes of the insulin signaling pathway and adipogenesis.

Aims/hypothesis Obesity results from a constant and complex interplay between environmental stimuli and predisposing genes. Recently, we identified the IFN-activated gene Ifi202b as the most likely gene responsible for the obesity quantitative trait locus Nob3 (New Zealand Obese [NZO] obesity 3). The aim of this study was to evaluate the effects of Ifi202b on body weight and adipose tissue biology, and to clarify the functional role of its human orthologue IFI16. Methods The impact of Ifi202b and its human orthologue IFI16 on adipogenesis was investigated by modulating their respective expression in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes. Furthermore, transgenic mice overexpressing IFI202b were generated and characterised with respect to metabolic traits. In humans, expression levels of IFI16 in adipose tissue were correlated with several variables of adipocyte function. Results In mice, IFI202b overexpression caused obesity (Δ body weight at the age of 30 weeks: 10.2 ± 1.9 g vs wild-type mice) marked by hypertrophic fat mass expansion, increased expression of Zfp423 (encoding the transcription factor zinc finger protein [ZFP] 423) and white-selective genes (Tcf21, Tle3), and decreased expression of thermogenic genes (e.g. Cidea, Ucp1). Compared with their wild-type littermates, Ifi202b transgenic mice displayed lower body temperature, hepatosteatosis and systemic insulin resistance. Suppression of IFI202b/IFI16 in pre-adipocytes impaired adipocyte differentiation and triacylglycerol storage. Humans with high levels of IFI16 exhibited larger adipocytes, an enhanced inflammatory state and impaired insulin-stimulated glucose uptake in white adipose tissue. Conclusions/interpretation Our findings reveal novel functions of Ifi202b and IFI16, demonstrating their role as obesity genes. These genes promote white adipogenesis and fat storage, thereby facilitating the development of obesity-associated insulin resistance. Electronic supplementary material The online version of this article (10.1007/s00125-018-4571-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Obesity is characterized as an excess accumulation of body fat resulting from a positive energy balance. It is the major risk factor for type 2 diabetes (T2D). The evidence for familial aggregation of obesity and its associated metabolic diseases is substantial. To date, about 150 genetic loci identified in genome-wide association studies (GWAS) are linked with obesity and T2D, each accounting for only a small proportion of the predicted heritability. However, the percentage of overall trait variance explained by these associated loci is modest (~5-10% for T2D, ~2% for BMI). The lack of powerful genetic associations suggests that heritability is not entirely attributable to gene variations. Some of the familial aggregation as well as many of the effects of environmental exposures, may reflect epigenetic processes. This review summarizes our current knowledge on the genetic basis to individual risk of obesity and T2D, and explores the potential role of epigenetic contribution.

Glucagon-like peptide 1 (GLP-1), produced in the intestine and the brain, can stimulate insulin secretion from the pancreas and alleviate type 2 diabetes. The cytokine interleukin-6 (IL-6) may enhance insulin secretion from β-cells by stimulating peripheral GLP-1 production. GLP-1 and its analogs also reduce food intake and body weight, clinically beneficial actions that are likely exerted at the level of the CNS, but otherwise are poorly understood. The cytokines IL-6 and interleukin 1β (IL-1β) may exert an anti-obesity effect in the CNS during health. Here we found that central injection of a clinically used GLP-1 receptor agonist, exendin-4, potently increased the expression of IL-6 in the hypothalamus (11-fold) and the hindbrain (4-fold) and of IL-1β in the hypothalamus, without changing the expression of other inflammation-associated genes. Furthermore, hypothalamic and hindbrain interleukin-associated intracellular signals [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and suppressor of cytokine signaling-1 (SOCS1)] were also elevated by exendin-4. Pharmacologic disruption of CNS IL-1 receptor or IL-6 biological activity attenuated anorexia and body weight loss induced by central exendin-4 administration in a rat. Simultaneous blockade of IL-1 and IL-6 activity led to a more potent attenuation of exendin-4 effects on food intake. Mice with global IL-1 receptor gene knockout or central IL-6 receptor knockdown showed attenuated decrease in food intake and body weight in response to peripheral exendin-4 treatment. GLP-1 receptor activation in the mouse neuronal Neuro2A cell line also resulted in increased IL-6 expression. These data outline a previously unidentified role of the central IL-1 and IL-6 in mediating the anorexic and body weight loss effects of GLP-1 receptor activation.

We investigated five methylation markers recently linked to body mass index, for their role in the neuropathology of obesity. In neuroimaging experiments, our analysis involving 23 participants showed that methylation levels for the cg07814318 site, which lies within the KLF13 gene, correlated with brain activity in the claustrum, putamen, cingulate gyrus and frontal gyri, some of which have been previously associated to food signaling, obesity or reward. Methylation levels at cg07814318 also positively correlated with ghrelin levels. Moreover, expression of KLF13 was augmented in the brains of obese and starved mice. Our results suggest the cg07814318 site could be involved in orexigenic processes, and also implicate KLF13 in obesity. Our findings are the first to associate methylation levels in blood with brain activity in obesity-related regions, and further support previous findings between ghrelin, brain activity and genetic differences.

Long-distance pollen transport can substantially raise local pollen levels, but their relative contribution has not yet been quantified temporally or spatially in ragweed infested regions. Using common ragweed (Ambrosia artemisiifolia) pollen accumulation at a ragweed infested area, Szeged, Hungary as a test case, this study attempted to: (1) identify, using cluster analysis, biogeographical regions that contribute to long-range transport of ragweed pollen to Szeged; (2) quantify the relative contribution of ragweed pollen from these regions; (3) determine the relative contribution of “local” and “transported” pollen for Szeged. Using the HYSPLIT model, three-dimensional backward trajectories were produced daily over a 5-year period, 2009–2013 for ragweed pollen accumulation at Szeged. A k-means clustering algorithm using the Mahalanobis distance was applied in order to develop trajectory types. Nine back-trajectory clusters were identified. Cluster 1 (direction: from the Channel area south of Great Britain) and cluster 5 (direction: from Northern Mediterranean) were found the most relevant potential long-distance sources for Ambrosia pollen transport to Szeged. Potential source contribution function (PSCF) and concentration weighted trajectory (CWT) values indicated additional potential source areas including the central and eastern part of France, the northern part of Italy and the Carpathian Basin. For Szeged on non-rainy days, medium-range transport is important, while on rainy days the two transport ranges have equal weights. Based on the Granger causality, annual pollen amount transported by the atmospheric circulation is 27.8% of the annual total pollen at Szeged. From this quantity, 7.5% is added to (due to transport), while 20.3% is subtracted from (e.g. because of wash-out by frontal rainfalls going towards Szeged) local sources.

The discovery of small RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi-associated RNAs (piRNAs) has led to new challenges in the selective detection of RNAs. Many noncoding RNAs act as post-translational regulators of gene expression and are involved in the regulation of cell proliferation or apoptosis, but are difficult to amplify, label, and detect. Standard microarray detection procedures involve pre-hybridization labeling or enzymatic 3'-labeling by polymerase-catalyzed extension. Dual labeling would improve the fidelity of detection, but no polymerases for 5'-extension are known. Here we report a novel labeling method for RNAs bearing natural 5'-phosphate groups, such as miRNAs, based on enzyme-free ligation of a biotin- or fluorophore-labeled oligonucleotide to the 5' termini. The method uses in situ activation of the natural 5'-phosphate groups in these RNAs and was optimized to give near-quantitative conversion in solution. With use of biotin- or fluorophore-bearing labeling strands, different miRNA sequences were detected on microarrays with little background fluorescence. In combination with an established method of enzymatic on-chip labeling at the 3' termini, highly selective detection of related miRNAs was achieved by dual recognition at both termini, even in the case of miRNAs differing in only one nucleotide.

Denileukin Diftitox (ONTAK(®), DAB(389) IL-2) is a recombinant DNA-derived fusion protein depleting cells that express high-affinity IL-2 receptor. Important cell targets are CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(reg)). Elimination of immunosuppressive T(reg) by Denileukin Diftitox may provide a way to modulate immune tolerance following stem cell transplantation. Here, we combined T(reg) depletion with a vaccination approach to induce donor-specific immune reactions. To investigate this approach we chose the mixed chimerism canine stem cell transplantation model which represents a high state of tolerance between two hematopoietic systems. The aim was therefore to induce a graft versus hematopoiesis effect thereby converting mixed to full donor chimerism. Dog leukocyte antigen identical siblings that had developed a stable mixed chimerism after non-myeloablative stem cell transplantation received a single dose of Denileukin Diftitox (18μg/kg, i.v.) followed by several cell-lysate vaccinations. Host peripheral blood mononuclear cell lysates combined with CpG-ODN, and Montanide(®) ISA 51 were locally applied. In vitro studies demonstrated that canine T(reg) are a target of Denileukin Diftitox. The suppression of T-cell proliferation by T(reg) was abolished by addition of Denileukin Diftitox (10nM). An increase of proliferation of median 300% (range: 200%-425%) was observed. No change in donor chimerism was observed after administration of Denileukin Diftitox and vaccination. This study highlights that application of Denileukin Diftitox resulted in a depletion of T(reg) followed by an increase of immune response in vitro. This effect could not be confirmed in vivo even if the immune system was stimulated by vaccinations.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

Hadi Al-Hasani and colleagues report that a frameshift deletion in Tbc1d1 in lean SJL mice underlies a quantitative trait locus conferring protection against high-fat diet-induced obesity. TBC1D1 seems to influence metabolism by regulating fatty acid uptake and oxidation in skeletal muscle cells. We previously identified Nob1 as a quantitative trait locus for high-fat diet–induced obesity and diabetes in genome-wide scans of outcross populations of obese and lean mouse strains. Additional crossbreeding experiments indicated that Nob1 represents an obesity suppressor from the lean Swiss Jim Lambert (SJL) strain. Here we identify a SJL-specific mutation in the Tbc1d1 gene that results in a truncated protein lacking the TBC Rab–GTPase-activating protein domain. TBC1D1, which has been recently linked to human obesity, is related to the insulin signaling protein AS160 and is predominantly expressed in skeletal muscle. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, whereas overexpression of TBC1D1 had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation and reduced glucose uptake in isolated skeletal muscle. Our data strongly suggest that mutation of Tbc1d1 suppresses high-fat diet–induced obesity by increasing lipid use in skeletal muscle.

Background DNA methylation is an essential nuclear process associated with genomic functions such as transcription factor binding and the regulation of gene expression. DNA methylation patterns can also serve as potential biomarkers for disease progression and response to therapy. However, the full dynamics of DNA methylation across daily physiologic events have not been fully elucidated. Objective We sought to study how ingesting a standardized meal acutely affects peripheral blood DNA methylation. Design We performed an observational study in healthy men (n = 26) on DNA methylation and gene expression in whole blood before and 160 min after the ingestion of a standardized meal. Cytosine-phosphate-guanine (CpG) methylation was assayed on the HumanMethylation450k microarray, and gene expression was measured with the Human Gene 2.1 ST Array. Results Differential methylation after food intake was detected in 13% of the analyzed probes (63,207 CpG probes) at a 5% false discovery rate (FDR). This effect was driven by changes in leukocyte fractions as estimated from comparisons against methylation datasets generated from sorted leukocytes. When methylation values were adjusted for estimated leukocyte fractions, 541 probes were observed to be altered in the postprandial state (5% FDR). Conclusions Apparent alterations in DNA methylation 160 min after meal ingestion mainly reflect changes in the estimated leukocyte population in whole blood. These results have major methodologic implications for genome-wide methylation studies because they highlight the strong underlying effects of changes in leukocyte fractions on CpG methylation patterns as well as the potential importance of meal-standardized sampling procedures for future investigations when alterations in white blood cell fractions are unavailable. This trial was registered at clinicaltrials.gov as LSF008786.

Context: Shift workers are at increased risk of metabolic morbidities. Clock genes are known to regulate metabolic processes in peripheral tissues, eg, glucose oxidation. Objective: This study aimed to investigate how clock genes are affected at the epigenetic and transcriptional level in peripheral human tissues following acute total sleep deprivation (TSD), mimicking shift work with extended wakefulness. Intervention: In a randomized, two-period, two-condition, crossover clinical study, 15 healthy men underwent two experimental sessions: x sleep (2230–0700 h) and overnight wakefulness. On the subsequent morning, serum cortisol was measured, followed by skeletal muscle and subcutaneous adipose tissue biopsies for DNA methylation and gene expression analyses of core clock genes (BMAL1, CLOCK, CRY1, PER1). Finally, baseline and 2-h post-oral glucose load plasma glucose concentrations were determined. Main Outcome Measures: In adipose tissue, acute sleep deprivation vs sleep increased methylation in the promoter of CRY1 (+4%; P = .026) and in two promoter-interacting enhancer regions of PER1 (+15%; P = .036; +9%; P = .026). In skeletal muscle, TSD vs sleep decreased gene expression of BMAL1 (−18%; P = .033) and CRY1 (−22%; P = .047). Concentrations of serum cortisol, which can reset peripheral tissue clocks, were decreased (2449 ± 932 vs 3178 ± 723 nmol/L; P = .039), whereas postprandial plasma glucose concentrations were elevated after TSD (7.77 ± 1.63 vs 6.59 ± 1.32 mmol/L; P = .011). Conclusions: Our findings demonstrate that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in key metabolic tissues. Tissue-specific clock alterations could explain why shift work may disrupt metabolic integrity as observed herein.

Birch trees produce large amounts of highly allergenic pollen grains that are distributed by wind and impact human health by causing seasonal hay fever, pollen-related asthma, and other allergic diseases. Traditionally, pollen forecasts are based on conventional microscopic counting techniques that are labor-intensive and limited in the reliable identification of species. Molecular biological techniques provide an alternative approach that is less labor-intensive and enables identification of any species by its genetic fingerprint. A particularly promising method is quantitative Real-Time polymerase chain reaction (qPCR), which can be used to determine the number of DNA copies and thus pollen grains in air filter samples. During the birch pollination season in 2010 in Mainz, Germany, we collected air filter samples of fine (

In several rodent strains such as the New Zealand Obese (NZO) mouse, the incidence of obesity-associated diabetes mellitus is much higher in males than in females. In the present study, we investigated the effects of ovariectomy on glucose homeostasis in female NZO mice in order to elucidate the mechanism of their diabetes resistance. NZO females were ovariectomized at the age of 4 weeks, received a high-fat diet and body weight, body fat, glucose and insulin tolerance were investigated in comparison to sham-operated mice. In a second experiment, operated mice were fed a carbohydrate-free diet up to the age of 19 weeks before they received the high-fat diet. In comparison with a sham-operated control group, ovariectomized female NZO mice exhibited similar body weights, a reduced glucose tolerance, developed significantly higher blood glucose levels, lost insulin producing β-cells, which finally resulted in a diabetes prevalence of 73% at the age of 16 weeks vs. 25% in controls. Similar to male NZO mice, ovariectomized females presented a more severe insulin resistance in the insulin tolerance test than sham-operated controls. Furthermore, the more severe insulin resistance in ovariectomized mice preceded the development of diabetes and pancreatic insulin depletion that was caused by a dietary regimen of carbohydrate restriction and subsequent re-exposure. In summary our data demonstrate that estrogen protects NZO females from β-cell loss and obesity-associated diabetes mellitus. This effect is due to a reduced insulin resistance and possibly also to a reduced sensitivity of β-cells to glucolipotoxic conditions.

A region on mouse distal chromosome 1 (Chr. 1) that is highly enriched in quantitative trait loci (QTLs) controlling neural and behavioral phenotypes overlaps with the peak region of a major obesity QTL (Nob3.38), which we identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6). By positional cloning we recently identified a microdeletion within this locus causing the disruption of Ifi202b that protects from adiposity by suppressing expression of 11β-Hsd1. Here we show that the Nob3.38 segment also corresponds with the QTL rich region (Qrr1) on Chr. 1 and associates with increased voluntary running wheel activity, Rota-rod performance, decreased grip strength, and anxiety-related traits. The characterization of a subcongenic line carrying 14.2 Mbp of Nob3.38 with a polymorphic region of 4.4 Mbp indicates that the microdeletion and/or other polymorphisms in its proximity alter body weight, voluntary activity, and exploration. Since 27 out of 32 QTL were identified in crosses with B6, we hypothesized that the microdeletion and or adjacent SNPs are unique for B6 mice and responsible for some of the complex Qrr1-mediated effects. Indeed, a phylogenic study of 28 mouse strains revealed a NZO-like genotype for 22 and a B6-like genotype for NZW/LacJ and 4 other C57BL strains. Thus, we suggest that a Nob3.38 interval (173.0–177.4 Mbp) does not only modify adiposity but also neurobehavioral traits by a haplotype segregating with C57BL strains.

Nob3 is a major obesity quantitative trait locus (QTL) identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6), and by introgression of its 38 Mbp peak region into B6 (B6.NZO-Nob3.38). B6.NZO-Nob3.38 mice carrying the NZO allele exhibited markedly increased body weight, fat mass, lean mass and a lower energy expenditure, than the corresponding B6 allele carriers. For positional cloning of the responsible obesity gene, five additional congenic lines (RCS) were generated and characterized, allowing to define a critical genomic interval comprising 43 genes. mRNA profiling and western blotting indicated that Ifi202b, a member of the Ifi200 family of interferon inducible transcriptional modulators, was expressed in NZO-allele carriers but was undetectable in tissues of homozygous B6-allele carriers due to a microdeletion, including the first exon and the 5'-flanking region of Ifi202b in B6. Transcriptome analysis of adipose tissue of RCS revealed a marked induction of 11β-hydroxysteroid dehydrogenase type 1 (11β-Hsd1) expression in mice expressing Ifi202b. Furthermore, siRNA-mediated Ifi202b suppression in 3T3-L1 adipocytes resulted in a significant inhibition of 11β-Hsd1 expression, whereas an adenoviral-mediated overexpression of Ifi202b increased 11β-Hsd1 mRNA levels. Expression of human IFI orthologues was significantly increased in visceral adipose tissue of obese subjects. We suggest that the disruption of Ifi202b in B6 is responsible for the effects of the obesity QTL Nob3, and that Ifi202b modulates fat accumulation through expression of adipogenic genes such as 11β-Hsd1.

Dysregulation of fatty acid oxidation plays a pivotal role in the pathophysiology of obesity and insulin resistance. Medium- and short-chain-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (SCHAD) (gene name, hadh) catalyze the third reaction of the mitochondrial beta-oxidation cascade, the oxidation of 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, for medium- and short-chain fatty acids. We identified hadh as a putative obesity gene by comparison of two genome-wide scans, a quantitative trait locus analysis previously performed in the polygenic obese New Zealand obese mouse and an earlier described small interfering RNA-mediated mutagenesis in Caenorhabditis elegans. In the present study, we show that mice lacking SCHAD (hadh(-/-)) displayed a lower body weight and a reduced fat mass in comparison with hadh(+/+) mice under high-fat diet conditions, presumably due to an impaired fuel efficiency, the loss of acylcarnitines via the urine, and increased body temperature. Food intake, total energy expenditure, and locomotor activity were not altered in knockout mice. Hadh(-/-) mice exhibited normal fat tolerance at 20 C. However, during cold exposure, knockout mice were unable to clear triglycerides from the plasma and to maintain their normal body temperature, indicating that SCHAD plays an important role in adaptive thermogenesis. Blood glucose concentrations in the fasted and postprandial state were significantly lower in hadh(-/-) mice, whereas insulin levels were elevated. Accordingly, insulin secretion in response to glucose and glucose plus palmitate was elevated in isolated islets of knockout mice. Therefore, our data indicate that SCHAD is involved in thermogenesis, in the maintenance of body weight, and in the regulation of nutrient-stimulated insulin secretion. (Endocrinology 152: 4641-4651, 2011)

Type 2 diabetes is a polygenic disease resulting from a combination of different disease alleles reflecting obesity, insulin resistance, and hyperglycemia. Using a positional cloning strategy with different inbred strains of mice, we mapped a disease locus for obesity-associated diabetes on chromosome 4. We analyzed all genes in this region and identified distinct differences in the expression levels of the transcription factor Zfp69. The expression of this gene mediated diabetes progression in a leptin-deficient congenic mouse line. The animals developed a disease pattern of hyperglycemia, reduced gonadal fat mass, and increased plasma and liver triglycerides, resembling a potential defect in triglyceride storage . In order to elucidate the impact of the human ortholog of Zfp69 in the development of type 2 diabetes, we tested its mRNA expression in human white adipose tissue. Consistent with the mouse data, mRNA-expression was significantly higher in diabetic subjects than in unaffected controls.

Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1) contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO) mice. A critical interval of distal chromosome 4 (2.1 Mbp) conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT–PCR, and RACE–PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a) in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box) and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lepob background, the diabetogenic Zfp69SJL allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes., Author Summary Type 2 diabetes in humans as well as in obese mice is caused by a combination of adipogenic and diabetogenic gene variants. We have identified a gene that appears to be involved in the pathogenesis of hyperglycaemia in obese mice: in some mouse strains, the gene Zfp69 is disrupted by a retroviral transposon (IAPLTR1a), which generates a truncated mRNA. Disruption of the gene was associated with a reduced susceptibility for diabetes, whereas the normal allele enhanced hyperglycaemia in obese mice. Zfp69 encodes a transcription factor which appears to interfere with lipid storage in adipose tissue, and thereby enhances lipid deposition in liver. In humans with type 2 diabetes, mRNA levels of the human orthologue of Zfp69 (ZNF642) were increased in adipose tissue. Thus, the transcription factor ZFP69/ZNF642 may be involved in the pathogenesis of obesity-associated diabetes.

New Zealand obese (NZO) mice present a metabolic syndrome of obesity, insulin resistance, and diabetes. To identify chromosomal segments associated with these traits, we intercrossed NZO mice with the lean and diabetes-resistant C57BL/6J (B6) strain. Obesity and hyperglycemia in the (NZO×B6)F2 intercross population were predominantly due to a broad quantitative trait locus (QTL) on chromosome 1 ( Nob3; logarithm of the odds score 16.1, 16.0, 4.0 for body weight, body fat, and blood glucose, respectively), producing a difference between genotypes of 12.7 or 5.2 g of body weight and 12.0 or 4.0 g of body fat in females or males, respectively. In addition, significant QTL on chromosomes 3 and 13 and suggestive QTL on chromosomes 4, 6, 9, 12, 14, and 19 contributed to the obese phenotype. Distal chromosome 5 was significantly linked with plasma cholesterol (LOD score 10.7). Introgression of two segments of Nob3 into B6 confirmed the adipogenic effect of the QTL and suggested the presence of at least one causal gene. Haplotype mapping reduced the critical region of the distal part of the QTL to 31 Mbp containing the potential candidates Nr1i3, Apoa2, Atp1a2, Prox1, and Hsd11b1. We conclude that obesity and hyperglycemia of NZO is to a large part caused by variant genes located in Nob3 on chromosome 1. Since these exert robust effects on a B6 background, the QTL Nob3 is a prime target for identification of a novel diabesity gene.

The comprehensive model system LM-ART was extended by a parameterisation which describes the emission flux of pollen grains. With this extension it is possible to simulate the dispersion of these airborne particles on the regional scale. LM is the operational forecast model of the German Weather Service. Therefore, it possible to run the whole model system in an operational mode. The implemented emission flux depends on plant specific parameters and on meteorological parameters such as friction velocity and temperature. The model was applied to an alder pollen episode which occurred in Germany in February 2004. To overcome the problem that the exact spatial distribution of alder is unknown, a map of the potential alder vegetation was used. The results of the simulation were compared with measured daily mean values of the stations of the Polleninformationsdienst. The comparison shows a good agreement with respect to the order of magnitude but larger differences for the spatial distributions. That is an implication of the lack of exact input data.

Recent discoveries indicate an important role for ghrelin in drug and alcohol reward and an ability of ghrelin to regulate mesolimbic dopamine activity. The role of dopamine in novelty seeking, and the association between this trait and drug and alcohol abuse, led us to hypothesize that ghrelin may influence novelty seeking behavior. To test this possibility we applied several complementary rodent models of novelty seeking behavior, i.e. inescapable novelty-induced locomotor activity (NILA), novelty-induced place preference and novel object exploration, in rats subjected to acute ghrelin receptor (growth hormone secretagogue receptor; GHSR) stimulation or blockade. Furthermore we assessed the possible association between polymorphisms in the genes encoding ghrelin and GHSR and novelty seeking behavior in humans. The rodent studies indicate an important role for ghrelin in a wide range of novelty seeking behaviors. Ghrelin-injected rats exhibited a higher preference for a novel environment and increased novel object exploration. Conversely, those with GHSR blockade drastically reduced their preference for a novel environment and displayed decreased NILA. Importantly, the mesolimbic ventral tegmental area selective GHSR blockade was sufficient to reduce the NILA response indicating that the mesolimbic GHSRs might play an important role in the observed novelty responses. Moreover, in untreated animals, a striking positive correlation between NILA and sucrose reward behavior was detected. Two GHSR single nucleotide polymorphisms (SNPs), rs2948694 and rs495225, were significantly associated with the personality trait novelty seeking, as assessed using the Temperament and Character Inventory (TCI), in human subjects. This study provides the first evidence for a role of ghrelin in novelty seeking behavior in animals and humans, and also points to an association between food reward and novelty seeking in rodents.

Common ragweed (Ambrosia artemisiifolia L.) is a highly allergenic plant that is spreading throughout Europe. Ragweed pollen can be transported over large distances by the wind. Even low pollen concentrations of less than 10 pollen m(-3) can lead to health problems in sensitive persons. Therefore, forecasting the airborne concentrations of ragweed pollen is becoming more and more important for public health. The question remains whether distant pollen sources need to be considered in reliable forecasts. We used the extended numerical weather prediction system COSMO-ART to simulate the release and transport of ragweed pollen in central Europe. A pollen episode (September 12-16, 2006) in north-eastern Germany was modeled in order to find out where the pollen originated. For this purpose, several different source regions were taken into account and their individual impact on the daily mean pollen concentration and the performance of the forecast were studied with the means of a 2 × 2 contingency table and skill scores. It was found that the majority of the pollen originated in local areas, but up to 20% of the total pollen load came from distant sources in Hungary. It is concluded that long-distance transport should not be neglected when predicting pollen concentrations.

Obesity has reached global epidemic proportions and creating an urgent need to understand mechanisms underlying excessive and uncontrolled food intake. Ghrelin, the only known circulating orexigenic hormone, potently increases food reward behavior. The neurochemical circuitry that links ghrelin to the mesolimbic reward system and to the increased food reward behavior remains unclear.Here we examine whether VTA-NAc dopaminergic signaling is required for the effects of ghrelin on food reward and intake. In addition, we examine the possibility of endogenous ghrelin acting on the VTA-NAc dopamine neurons. A D1-like or a D2 receptor antagonist was injected into the NAc in combination with ghrelin microinjection into the VTA to investigate whether this blockade attenuates ghrelin-induced food reward behavior. VTA injections of ghrelin produced a significant increase in food motivation/reward behavior, as measured by sucrose-induced progressive ratio operant conditioning, and chow intake. Pretreatment with either a D1-like or D2 receptor antagonist into the NAc, completely blocked the reward effect of ghrelin, leaving chow intake intact. We also found that this circuit is potentially relevant for the effects of endogenously released ghrelin as both antagonists reduced fasting (a state of high circulating levels of ghrelin) elevated sucrose-motivated behavior but not chow hyperphagia.Taken together our data identify the VTA to NAc dopaminergic projections, along with D1-like and D2 receptors in the NAc, as essential elements of the ghrelin responsive circuits controlling food reward behavior. Interestingly results also suggest that food reward behavior and simple intake of chow are controlled by divergent circuitry, where NAc dopamine plays an important role in food reward but not in food intake.