Adipose tissue-selective ablation of ADAM10 results in divergent metabolic phenotypes following long-term dietary manipulation.

A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10), is involved in several metabolic and inflammatory pathways. We speculated that ADAM10 plays a modulatory role in adipose tissue inflammation and metabolism. To this end, we studied adipose tissue-specific ADAM10 knock-out mice (aKO). While young, regular chow diet-fed aKO mice showed increased insulin sensitivity, following prolonged (33 weeks) high-fat diet (HFD) exposure, aKO mice developed obesity and insulin resistance. Compared to controls, aKO mice showed less inflammatory adipokine profile despite the significant increase in adiposity. In brown adipose tissue, aKO mice on HFD had changes in CD8+ T cell populations indicating a lesser inflammatory pattern. Following HFD, both aKO and control littermates demonstrated decreased adipose tissue pro-inflammatory macrophages, and increased anti-inflammatory accumulation, without differences between the genotypes. Collectively, our observations indicate that selective deletion of ADAM10 in adipocytes results in a mitigated inflammatory response, leading to increased insulin sensitivity in young mice fed with regular diet. This state of insulin sensitivity, following prolonged HFD, facilitates energy storage resulting in increased fat accumulation which ultimately leads to the development of a phenotype of obesity and insulin resistance. In conclusion, the data indicate that ADAM10 has a modulatory effect of inflammation and whole-body energy metabolism.

Selective adipocyte loss of Angiopoietin-2 prompts female-specific obesity and metabolic syndrome.

Thermogenic fat differentiation and function can be promoted through multiple pathways, resulting in a common cell phenotype characterized by the expression of Uncoupling Protein-1 and the ability to dissipate energy, but local and systemic stimuli are necessary to promote adequate thermogenic fat vascularization, which is a precondition for the transport of substrate and the dissipation of heat. Angiopoietin-2 is an important driver of vascularization, and its transcription is in part promoted by estrogen signaling. In this study we demonstrate that adipose tissue-specific knock out of Angiopoietin-2 causes a female-specific reduced thermogenic fat differentiation and function, resulting in obesity and impaired glucose tolerance with end-organ features consistent with metabolic syndrome. In humans, angiopoietin-2 levels are higher in females than in males, and are inversely correlated with adiposity and age more strongly in pre-menopause when compared to post-menopause. Collectively, these data indicate a novel and important role for estrogen-mediated Angiopoietin-2 adipose tissue production in the protection against calorie overload in females, and potentially in the development of postmenopausal weight gain.

Isolation of the Stromal Vascular Fraction from Adipose Tissue and Subsequent Differentiation into White or Beige Adipocytes.

Non-alcoholic steatohepatitis (NASH) is linked to adipose tissue dysfunction, with weight loss being the only treatment shown to reverse it. Due to this correlation with obesity, the study of adipose tissue and adipocytes is an important step in understanding the pathogenesis of this disease. Here, we describe the isolation process of the stromal vascular fraction (SVF) of adipose tissue. The SVF contains the foundational cells that will differentiate into adipocytes. These cells can be isolated and subsequently differentiated in vitro into white and beige adipocytes. We outline the in vitro differentiation of pre-adipocytes into cultured white and beige adipocytes using both human and mouse adipose tissue.

Metabolic Phenotyping in Mice with NASH Using Indirect Calorimetry.

Obesity caused by caloric overload has assumed epidemic proportions. Obesity is frequently associated with metabolic dysfunctions, such as type 2 diabetes, non-alcoholic steatohepatitis (NASH), cardiovascular diseases, and cancer. Metabolic phenotyping is a set of techniques for studying metabolic dysfunction and behavior information including energy expenditure, body weight gain, glucose homeostasis, and lipid profile. Among different metabolic phenotyping methods, indirect calorimetry is an indispensable tool for quantifying the energy balance/imbalance in various mouse models, which enables researchers to probe the development of disease and to evaluate the therapeutic benefit from different interventions. In this chapter, we will describe the procedures of metabolic phenotyping using indirect calorimetry in db/db mouse, a metabolic disorder mouse model which develops NASH.

Fast SARS-CoV-2 Variant Detection Using Snapback Primer High-Resolution Melting.

SARS-CoV-2, the virus responsible for COVID-19, emerged in late 2019 and has since spread throughout the world, infecting over 200 million people. The fast spread of SARS-CoV-2 showcased the need for rapid and sensitive testing methodologies to help track the disease. Over the past 18 months, numerous SARS-CoV-2 variants have emerged. Many of these variants are suggested to be more transmissible as well as less responsive to neutralization by vaccine-induced antibodies. Viral whole-genome sequencing is the current standard for tracking these variants. However, whole-genome sequencing is costly and the technology and expertise are limited to larger reference laboratories. Here, we present the feasibility of a fast, inexpensive methodology using snapback primer-based high-resolution melting to test for >20 high-consequence SARS-CoV-2 spike mutations. This assay can distinguish between multiple variant lineages and be completed in roughly 2 h for less than $10 per sample.

Extraction-Free Rapid Cycle Quantitative RT-PCR and Extreme RT-PCR for SARS-CoV-2 Virus Detection.

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. This study explored the kinetic limitations of extraction-free rapid cycle quantitative real-time RT-PCR for SARS-CoV-2 virus detection using the commercially available capillary-based LightCycler. After optimizing for time and reaction conditions, a protocol for sensitive and specific quantitative RT-PCR of SARS-CoV-2 RNA from nasopharyngeal swabs in <20 minutes was developed, with minimal hands-on time requirements. This protocol improves detection speed while maintaining the sensitivity and specificity of hydrolysis probe-based detection. Percentage agreement between the developed assay and previously tested positive patient samples was 97.6% (n = 40/41), and negative patient samples was 100% (40/40). The study further demonstrates that using purified RNA, SARS-CoV-2 testing using extreme RT-PCR, and product verification by melting can be completed in <3 minutes. Overall, these studies provide a framework for increasing the speed of SARS-CoV-2 and other infectious disease testing.

Identification of the transgene insertion site for an adipocyte-specific adiponectin-cre model and characterization of the functional consequences.

The Adipoq-Cre transgenic mouse is widely used in the development of adipocyte-specific genetic manipulations for the study of obesity and type 2 diabetes. In the process of developing a new mouse model utilizing the adipocyte selective Adipoq-Cre transgenic mouse, strong genetic linkage between a gene of interest, Adam10, and the Adipoq-Cre transgene was discovered. Whole-genome sequencing of the Adipoq-Cre transgenic mouse model identified the genomic insertion site within the Tbx18 gene locus on chromosome 9 and this insertion causes a significant decrease in Tbx18 gene expression in adipose tissue. Insertion of genes Kng2, Kng1, Eif4a2 and Rfc4 also occurred in the Adipoq-Cre transgenic mouse, and these passenger genes may have functional consequences in various tissues.

An appraisal of whole-room indirect calorimeters and a metabolic cart for measuring resting and active metabolic rates.

Whole-room indirect calorimeters (WRICs) have traditionally been used for real-time resting metabolic rate (RMR) measurements, while metabolic rate (MR) during short-interval exercises has commonly been measured by metabolic carts (MCs). This study aims to investigate the feasibility of incorporating short-interval exercises into WRIC study protocols by comparing the performance of WRICs and an MC. We assessed the 40-min RMR of 15 subjects with 2-day repeats and the 10-15 min activity MR (AMR) of 14 subjects at three intensities, using a large WRIC, a small WRIC, and an MC. We evaluated the biases between the instruments and quantified sources of variation using variance component analysis. All three instruments showed good agreement for both RMR (maximum bias = 0.07 kcal/min) and AMR assessment (maximum bias = 0.53 kcal/min). Moreover, the majority of the variability was between-subject and between-intensity variation, whereas the types of instrument contributed only a small amount to total variation in RMR (2%) and AMR (0.2%) data. In Conclusion, the good reproducibility among the instruments indicates that they may be used interchangeably in well-designed studies. Overall, WRICs can serve as an accurate and versatile means of assessing MR, capable of integrating RMR and short-interval AMR assessments into a single protocol.

Adipocyte ADAM17 plays a limited role in metabolic inflammation.

The role of ADAM17, its substrates, and its natural inhibitor has been well studied in the context of inflammation, including metabolic inflammation, with mixed results. Previous studies examining global Adam17 knockdown models and ADAM17 inhibition using overexpression of endogenous ADAM17 inhibitors have shown improved metabolic health and decreased metabolic inflammation. However, there have been no studies examining the role of adipocyte ADAM17 using in vivo models. In this study, we developed an adipocyte-specific Adam17 knockout model using Adipoq-Cre-expressing mice crossed with Adam17-floxed mice. Using this model, we show that loss of adipocyte ADAM17 plays no evident role in baseline metabolic responses. Surprisingly, in a state of metabolic stress using high-fat diet (HFD), we observed that adipocyte ADAM17 had little effect overall on the metabolic phenotype as well as inflammatory cell populations. Using whole-body metabolic phenotyping, we show that loss of ADAM17 has no effect on energy utilization both at a baseline state as well as following HFD. However, lastly, using high-parameter flow cytometry, we show that loss of adipocyte ADAM17 alters macrophage and eosinophil populations following HFD. Overall, the studies presented here give more insight into the role of ADAM17 in metabolic responses and metabolic inflammation, specifically in adipocytes.

E0771 and 4T1 murine breast cancer cells and interleukin 6 alter gene expression patterns but do not induce browning in cultured white adipocytes.

Breast cancer remains a substantial clinical problem worldwide, and cancer-associated cachexia is a condition associated with poor prognosis in this and other malignancies. Adipose tissue is involved in the development and progression of cancer-associated cachexia, but its various roles and mechanisms of action are not completely defined, especially as it relates to breast cancer. Interleukin 6 has been implicated in several mechanisms contributing to increased breast cancer tumorigenesis, as well as a net-negative energy balance and cancer-associated cachexia via adipose tissue remodeling in other models of cancer; however, its potential role in breast cancer-associated white adipose browning has not been explored. In this study, we demonstrate localized white adipose tissue browning in a spontaneous model of murine mammary cancer. We then used an in vitro murine adipocyte culture system with the E0771 and 4T1 cell lines as models of breast cancer. We demonstrate that while the E0771 and 4T1 secretomes and cross-talk with white adipocytes alter white adipocyte mRNA expression, they do not directly induce white adipocyte browning. Additionally, we show that neither exogenous administration of interleukin 6 alone or with its soluble receptor directly induce white adipocyte browning. Together, these results demonstrate that neither the E0771 or 4T1 murine breast cancer cell lines, nor interleukin 6, directly cause browning of cultured white adipocytes. This suggests that their roles in adipose tissue remodeling are more complex and indirect in nature.

STAT3 suppresses Wnt/ß-catenin signaling during the induction phase of primary Myf5+ brown adipogenesis.

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1 (UCP1). We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. The inability of STAT3-/- preadipocytes to differentiate can be rescued using Wnt ligand secretion inhibitors when applied during the induction stage. Through chemical inhibition and RNAi, we show that it is the canonical ß-catenin pathway that is responsible for the block in differentiation; inhibition or knockdown of ß-catenin can fully rescue adipogenesis and UCP1 expression in the STAT3-/- adipocytes. During the induction stage, Wnts 1, 3a, and 10b have increased expression in the STAT3-/- adipocytes, potentially explaining the increased levels and activity of ß-catenin. Our results for the first time point towards an interaction between the JAK/STAT pathway and the Wnt/ß-catenin pathway during the early stages of in-vitro adipogenesis.

A novel role for PTK2B in cultured beige adipocyte differentiation.

Adipocyte differentiation is a tightly regulated process which requires the sequential and organized expression of numerous genes and proteins. Phosphorylation of cytoplasmic proteins and key transcription factors represents a critical regulatory mechanism of the process leading to adipocyte maturation and modulation of associated metabolic pathways. Despite the recognition of the importance of protein phosphorylation in adipocyte biology, relatively little is known about the role of specific kinases in thermogenic (brown or beige) adipocyte differentiation and function. In this study, we demonstrate that the non-receptor protein tyrosine kinase 2 beta (PTK2B) plays a critical role in murine beige adipocyte differentiation. We observed that PTK2B protein expression is associated with beige adipocyte differentiation in cultured, immortalized, inguinal stromal vascular fraction cells. CRISPR/Cas9-mediated knock-out of Ptk2b results in non-differentiating white adipocytes, and differentiated beige adipocytes with significantly reduced thermogenic gene and protein expression, enlarged lipid droplet size, and altered mitochondrial respiration. Together, our data in a cell culture system provides evidence for a role of PTK2B in the differentiation of murine beige adipocytes.

Metabolic Effects of FGF-21: Thermoregulation and Beyond.

Fibroblast growth factor (FGF)-21, a member of the FGF family, is a novel hormone involved in the control of metabolism by modulating glucose homeostasis, insulin sensitivity, ketogenesis, and promoting adipose tissue "browning." Recent studies demonstrated that brown adipose tissue is not only a target for FGF-21, but is also a potentially important source of systemic FGF-21. These findings support the hypothesis that FGF-21 plays a physiologic role in thermogenesis and thermogenic recruitment of white adipose tissue by an autocrine-paracrine axis. This review examines the role of FGF-21 in thermogenesis from the perspective of cell-based, animal model, and human studies. We also present recent advances in the characterization of FGF-21's regulation of metabolism.

Thyroid Hormone Mediated Modulation of Energy Expenditure.

Thyroid hormone (TH) has diverse effects on mitochondria and energy expenditure (EE), generating great interest and research effort into understanding and harnessing these actions for the amelioration and treatment of metabolic disorders, such as obesity and diabetes. Direct effects on ATP utilization are a result of TH's actions on metabolic cycles and increased cell membrane ion permeability. However, the majority of TH induced EE is thought to be a result of indirect effects, which, in turn, increase capacity for EE. This review discusses the direct actions of TH on EE, and places special emphasis on the indirect actions of TH, which include mitochondrial biogenesis and reduced metabolic efficiency through mitochondrial uncoupling mechanisms. TH analogs and the metabolic actions of T2 are also discussed in the context of targeted modulation of EE. Finally, clinical correlates of TH actions on metabolism are briefly presented.

Extreme PCR: efficient and specific DNA amplification in 15-60 seconds.

BACKGROUND: PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS: We developed prototype instruments to temperature cycle 1- to 5-µL samples in 0.4-2.0 s at annealing/extension temperatures of 62 °C-76 °C and denaturation temperatures of 85 °C-92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS: Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15-60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any "hot start" technique. Amplification efficiencies were 91.7%-95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS: The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4-2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

Microfluidic genotyping by rapid serial PCR and high-speed melting analysis.

BACKGROUND: Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS: We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.*97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platform's liquid-handling system automated PCR and HSM. RESULTS: PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS: We demonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis.

Snapback primer genotyping of the Gilbert syndrome UGT1A1 (TA)(n) promoter polymorphism by high-resolution melting.

BACKGROUND: Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine-adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)(n) promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)(5) and (TA)(8) alleles. METHODS: An unlabeled 5' extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)(n) polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans. RESULTS: Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)(n) polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments. CONCLUSIONS: In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5' extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.