Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORγt+IL-17+ Tregs and improve metabolism.

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration. Intake of the Methylococcu-based diet markedly boosts Parabacteroides abundances in a manner depending on adaptive immunity, and upregulates triple positive (Foxp3+RORγt+IL-17+) regulatory T cells in the small and large intestine. Collectively, these data point to the potential for leveraging the use of McB lysates to improve immunometabolic homeostasis.

Genetic and functional characterization of clonally derived adult human brown adipocytes.

Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and its activation to a state of increased energy expenditure is believed to protect against the development of obesity. Even though the existence of BAT in adult humans has been widely appreciated, its cellular origin and molecular identity remain elusive largely because of high cellular heterogeneity within various adipose tissue depots. To understand the nature of adult human brown adipocytes at single cell resolution, we isolated clonally derived adipocytes from stromal vascular fractions of adult human BAT from two individuals and globally analyzed their molecular signatures. We used RNA sequencing followed by unbiased genome-wide expression analyses and found that a population of uncoupling protein 1 (UCP1)-positive human adipocytes possessed molecular signatures resembling those of a recruitable form of thermogenic adipocytes (that is, beige adipocytes). In addition, we identified molecular markers that were highly enriched in UCP1-positive human adipocytes, a set that included potassium channel K3 (KCNK3) and mitochondrial tumor suppressor 1 (MTUS1). Further, we functionally characterized these two markers using a loss-of-function approach and found that KCNK3 and MTUS1 were required for beige adipocyte differentiation and thermogenic function. The results of this study present new opportunities for human BAT research, such as facilitating cell-based disease modeling and unbiased screens for thermogenic regulators.

Transcriptome profiling of brown adipose tissue during cold exposure reveals extensive regulation of glucose metabolism.

We applied digital gene expression profiling to determine the transcriptome of brown and white adipose tissues (BAT and WAT, respectively) during cold exposure. Male C57BL/6J mice were exposed to cold for 2 or 4 days. A notable induction of genes related to glucose uptake, glycolysis, glycogen metabolism, and the pentose phosphate pathway was observed in BAT from cold-exposed animals. In addition, glycerol-3-phosphate dehydrogenase 1 expression was induced in BAT from cold-challenged mice, suggesting increased synthesis of glycerol from glucose. Similarly, expression of lactate dehydrogenases was induced by cold in BAT. Pyruvate dehydrogenase kinase 2 (Pdk2) and Pdk4 were expressed at significantly higher levels in BAT than in WAT, and Pdk2 was induced in BAT by cold. Of notice, only a subset of the changes detected in BAT was observed in WAT. Based on changes in gene expression during cold exposure, we propose a model for the intermediary glucose metabolism in activated BAT: 1) fluxes through glycolysis and the pentose phosphate pathway are induced, the latter providing reducing equivalents for de novo fatty acid synthesis; 2) glycerol synthesis from glucose is increased, facilitating triacylglycerol synthesis/fatty acid re-esterification; 3) glycogen turnover and lactate production are increased; and 4) entry of glucose carbon into the tricarboxylic acid cycle is restricted by PDK2 and PDK4. In summary, our results demonstrate extensive and diverse gene expression changes related to glucose handling in activated BAT.

ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue.

Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis.

Discovery of a novel selective PPARγ ligand with partial agonist binding properties by integrated in silico/in vitro work flow.

Full agonists to the peroxisome proliferator-activated receptor (PPAR)γ, such as Rosiglitazone, have been associated with a series of undesired side effects, such as weight gain, fluid retention, cardiac hypertrophy, and hepatotoxicity. Nevertheless, PPARγ is involved in the expression of genes that control glucose and lipid metabolism and is an important target for drugs against type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In an effort to identify novel PPARγ ligands with an improved pharmacological profile, emphasis has shifted to selective ligands with partial agonist binding properties. Toward this end we applied an integrated in silico/in vitro workflow, based on pharmacophore- and structure-based virtual screening of the ZINC library, coupled with competitive binding and transactivation assays, and adipocyte differentiation and gene expression studies. Hit compound 9 was identified as the most potent ligand (IC50 = 0.3 µM) and a relatively poor inducer of adipocyte differentiation. The binding mode of compound 9 was confirmed by molecular dynamics simulation, and the calculated free energy of binding was -8.4 kcal/mol. A novel functional group, the carbonitrile group, was identified to be a key substituent in the ligand-protein interactions. Further studies on the transcriptional regulation properties of compound 9 revealed a gene regulatory profile that was to a large extent unique, however functionally closer to that of a partial agonist.

High-glycemic index carbohydrates abrogate the antiobesity effect of fish oil in mice.

Fish oil rich in n-3 polyunsaturated fatty acids is known to attenuate diet-induced obesity and adipose tissue inflammation in rodents. Here we aimed to investigate whether different carbohydrate sources modulated the antiobesity effects of fish oil. By feeding C57BL/6J mice isocaloric high-fat diets enriched with fish oil for 6 wk, we show that increasing amounts of sucrose in the diets dose-dependently increased energy efficiency and white adipose tissue (WAT) mass. Mice receiving fructose had about 50% less WAT mass than mice fed a high fish oil diet supplemented with either glucose or sucrose, indicating that the glucose moiety of sucrose was responsible for the obesity-promoting effect of sucrose. To investigate whether the obesogenic effect of sucrose and glucose was related to stimulation of insulin secretion, we combined fish oil with high and low glycemic index (GI) starches. Mice receiving the fish oil diet containing the low-GI starch had significantly less WAT than mice fed high-GI starch. Moreover, inhibition of insulin secretion by administration of nifedipine significantly reduced WAT mass in mice fed a high-fish oil diet in combination with sucrose. Our data show that the macronutrient composition of the diet modulates the effects of fish oil. Fish oil combined with sucrose, glucose, or high-GI starch promotes obesity, and the reported anti-inflammatory actions of fish oil are abrogated. In conclusion, our data indicate that glycemic control of insulin secretion modulates metabolic effects of fish oil by demonstrating that high-GI carbohydrates attenuate the antiobesity effects of fish oil.

Analysis of SOX2 expression in developing human testis and germ cell neoplasia.

The transcriptional regulators of pluripotency, POU5F1 (OCT4), NANOG and SOX2, are highly expressed in embryonal carcinoma (EC). In contrast to OCT4 and NANOG, SOX2 has not been demonstrated in the early human germ cell lineage or carcinoma in situ (CIS), the precursor for testicular germ cell tumours (TGCTs). Here, we have analysed SOX2 expression in CIS and overt TGCTs, as well as normal second and third trimester fetal, prepubertal and adult testes by in situ hybridisation and immunohistochemistry using three different antibodies. In contrast to earlier studies, we detected SOX2 mRNA in most CIS cells. We also detected speckled nuclear SOX2 immunoreactivity in CIS cells with one primary antibody, which was not apparent with other primary antibodies. The results demonstrate SOX2 gene expression in CIS for the first time and raise the possibility of post-transcriptional regulation, most likely sumoylation as a mechanism for limiting SOX2 action in these cells.

Improved gene expression signature of testicular carcinoma in situ.

The carcinoma in situ (CIS) stage is the common precursor of testicular germ cell tumours (TGCTs) that arise in young adults. Within the past decade genome wide gene expression tools have been developed and have greatly advanced the insight into the biology of TGCTs. Two independent data sets on global gene expression in testicular CIS have been previously published. We have merged the two data sets on CIS samples (n = 6) and identified the shared gene expression signature in relation to expression in normal testis. Among the top-20 highest expressed genes, one-third was transcription factors and the list included some 'novel' CIS markers (i.e. DOCK11 and ANXA3). Genes related to biological terms 'nucleic acid binding' and 'translational activity' (e.g. transcription factors and ribosomal proteins, respectively) were consistently and significantly over-represented. Some of the significantly over-expressed genes in CIS cells were selected for validation by RT-PCR (IFI16, DOCK11, and ANXA3), immunohistochemistry (HLXB9), or in situ hybridization (IFI16). High-level analysis utilizing the Ingenuity pathway analysis tool indicated that networks relating to 'gene expression in cancer' and 'embryonic development' were significantly altered and could collectively affect cellular pathways like the WNT signalling cascade, which thus may be disrupted in testicular CIS. The merged CIS data from two different microarray platforms, to our knowledge, provide the most precise CIS gene expression signature to date.

CDH1 (E-cadherin) in testicular germ cell neoplasia: suppressed translation of mRNA in pre-invasive carcinoma in situ but increased protein levels in advanced tumours.

E-cadherin (CDH1) is a transmembrane glycoprotein involved in cellular adhesion. In our recent microarray studies of testicular germ cell tumours (TGCTs) and the common precursor carcinoma in situ (CIS), CDH1 mRNA was highly expressed in CIS and embryonal carcinoma. It has previously been reported that the CDH1 protein is not expressed in CIS. To resolve the discrepancy, we performed a detailed analysis of the expression of CDH1 mRNA and protein in a series of normal and neoplastic testes. High expression of CDH1 mRNA in CIS was confirmed by real-time PCR and in situ hybridisation. At the protein level, however, CDH1 was only detected with one of three tested antibodies, but Western blotting analysis with this antibody showed additional bands, suggesting unspecific staining. The levels of a CDH1 protein fragment in serum samples from 58 patients with TGCTs were analysed by ELISA; we found significantly higher levels in patients with advanced disease (stage II/III) when compared to healthy individuals and patients with stage I TGCT. In conclusion, despite high mRNA levels, the CDH1 protein is not expressed in CIS, suggesting translational suppression of CDH1 protein expression. CDH1 serum levels may be a serological marker for staging of TGCT patients.