Featured ArticlesVolume 6 | No. 4 | April 2017
|Changes in adipose tissue macrophage and T cells after sleeve gastrectomyBariatric surgery is currently the most successful treatment for obesity and is the only therapeutic option that also causes sustained substantial reduction of type 2 diabetes. Frikke-Schmidt and colleagues found that vertical sleeve gastrectomy (VSG) in mice altered the immune cell populations residing in the epididymal white adipose tissue in a weight-independent manner. The CD11c- adipose tissue macrophage population increased in number, while different T cell populations expanded 3-fold. These strong, weight-independent changes in immune populations after surgery suggest that the immune system could play a significant role in the weight-independent adipose metabolic improvements observed after VSG in rodents.|
Abstract | PDF
Objective: In addition to adipocytes, adipose tissue contains large numbers of immune cells. A wide range of evidence links the activity of these cells to regulation of adipocyte and systemic metabolic function. Bariatric surgery improves several aspects of metabolic derangements and at least some of these effects occur in a weight-loss independent manner. We sought to investigate the impact of vertical sleeve gastrectomy (VSG) on adipose immune cell frequencies.
Methods: We analyzed the frequencies of immune cells within distinct adipose tissue depots in obese mice that had VSG or sham surgery with a portion of the latter group pair-fed such that their body mass was matched to the VSG animals.
Results: We demonstrate that VSG induced a shift in the epididymal adipose tissue leukocyte profile including increased frequencies of CD11c− macrophages, increased frequencies of T cells (CD4+, CD8+, and CD4−/CD8− T cells all increased), but a significantly decreased frequency of adipose tissue dendritic cells (ATDC) that, despite the continued high fat feeding of the VSG group, dropped below control diet levels.
Conclusions: These results indicate that VSG induces substantial changes in the immune populations residing in the adipose depots independent of weight loss.[Hide abstract]
|DNA methylation contributes to pathophysiology in mice fed a high fat dietGenetics and environment contribute to the development of obesity and its associated disorders. The interactions between epigenetic changes and obesity in metabolically relevant tissues, such as liver, however, are not well understood. One particularly important epigenetic mark is DNA methylation, associated with repressed chromatin states. Zhang et al. show that a high fat diet alters hepatic DNA methylation, transcription, and gene expression patterns, which contributes to the pathophysiology of obesity.|
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Objective: Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity.
Methods: RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in vivo knockdown of an identified driver gene, Phlda1, were used to validate the results.
Results: A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype.
Conclusions: We conclude that a high fat diet alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity.[Hide abstract]
|A role for Elovl2 in glucose-induced insulin secretionHigh fat-high sucrose (HFHS) diet-fed mice become severely glucose intolerant, and insulin resistance progressively worsens with time. However, the development and severity of the diabetic phenotype depends on several factors, including the choice of genetic background. Cruciani-Guglielmacci, Bellini et al. analyzed in parallel the effect of a HFHS diet on six commonly used laboratory mouse strains using carefully controlled housing and experimental conditions. The strains show marked differences in susceptibility to diet-induced obesity and β-cell failure. This approach provides evidence that Elongase of very long-chain fatty acids 2 (Elovl2), an enzyme involved in very long chain fatty acid synthesis, is a key player in regulating glucose-stimulated insulin secretion in the context of β-cell dysfunction.|
Abstract | PDF
Objective: In type 2 diabetes (T2D), pancreatic β cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress.
Methods: Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis.
Results: A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2, encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human β cell lines.
Conclusions: Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of β cell failure under metabolic stress.[Hide abstract]
|mRNA sequencing of skeletal muscle: search for exercise-regulated myokines Proteins and peptides produced by and released from skeletal muscles are termed myokines, and several myokines play important roles in muscle physiology as well as in tissue cross talk. Pourteymour and colleagues used mRNA sequencing to identify numerous transcripts that were regulated in skeletal muscle after acute and/or long-term exercise. These transcripts encode potential myokines, which may play key roles in local and systemic adaptations to exercise. Furthermore, they identified CSF1 as a novel myokine, which was increased after acute and long-term exercise and secreted from cultured human myotubes in response to electrical pulse stimulation.|
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Objective: Skeletal muscle is an important secretory organ, producing and releasing numerous myokines, which may be involved in mediating beneficial health effects of physical activity. More than 100 myokines have been identified by different proteomics approaches, but these techniques may not detect all myokines. We used mRNA sequencing as an untargeted approach to study gene expression of secreted proteins in skeletal muscle upon acute as well as long-term exercise.
Methods: Twenty-six middle-aged, sedentary men underwent combined endurance and strength training for 12 weeks. Skeletal muscle biopsies from m. vastus lateralis and blood samples were taken before and after an acute bicycle test, performed at baseline as well as after 12 weeks of training intervention. We identified transcripts encoding secretory proteins that were changed more than 1.5-fold in muscle after exercise. Secretory proteins were defined based on either curated UniProt annotations or predictions made by multiple bioinformatics methods.
Results: This approach led to the identification of 161 candidate secretory transcripts that were up-regulated after acute exercise and 99 that where increased after 12 weeks exercise training. Furthermore, 92 secretory transcripts were decreased after acute and/or long-term physical activity. From these responsive transcripts, we selected 17 candidate myokines sensitive to short- and/or long-term exercise that have not been described as myokines before. The expression of these transcripts was confirmed in primary human skeletal muscle cells during in vitro differentiation and electrical pulse stimulation (EPS). One of the candidates we identified was macrophage colony-stimulating factor-1 (CSF1), which influences macrophage homeostasis. CSF1 mRNA increased in skeletal muscle after acute and long-term exercise, which was accompanied by a rise in circulating CSF1 protein. In cultured muscle cells, EPS promoted a significant increase in the expression and secretion of CSF1.
Conclusions: We identified 17 new, exercise-responsive transcripts encoding secretory proteins. We further identified CSF1 as a novel myokine, which is secreted from cultured muscle cells and up-regulated in muscle and plasma after acute exercise.[Hide abstract]
|Astrocyte signaling is required for obesity and inflammationObesity and excessive dietary consumption promote upregulation of the master inflammatory NF-κB pathway in important brain regions including the hypothalamus.
While several studies have implicated microglia in the generation of diet-induced inflammatory signals and metabolic dysfunction, a similar role for astrocytes remains unclear. To address this, Douglass et al. developed a mouse model with an inducible astrocyte-specific deletion of IKKβ. Using this approach, they demonstrate that reduction of astrocyte inflammatory signaling protects mice from high fat diet-induced hypothalamic inflammation and reduces susceptibility to diet induced obesity and glucose intolerance.
Abstract | PDF
Objective: Obesity and high fat diet (HFD) consumption in rodents is associated with hypothalamic inflammation and reactive gliosis. While neuronal inflammation promotes HFD-induced metabolic dysfunction, the role of astrocyte activation in susceptibility to hypothalamic inflammation and diet-induced obesity (DIO) remains uncertain.
Methods: Metabolic phenotyping, immunohistochemical analyses, and biochemical analyses were performed on HFD-fed mice with a tamoxifen-inducible astrocyte-specific knockout of IKKβ (GfapCreERIkbkbfl/fl, IKKβ-AKO), an essential cofactor of NF-κB-mediated inflammation.
Results: IKKβ-AKO mice with tamoxifen-induced IKKβ deletion prior to HFD exposure showed equivalent HFD-induced weight gain and glucose intolerance as Ikbkbfl/fl littermate controls. In GfapCreERTdTomato marker mice treated using the same protocol, minimal Cre-mediated recombination was observed in the mediobasal hypothalamus (MBH). By contrast, mice pretreated with 6 weeks of HFD exposure prior to tamoxifen administration showed substantially increased recombination throughout the MBH. Remarkably, this treatment approach protected IKKβ-AKO mice from further weight gain through an immediate reduction of food intake and increase of energy expenditure. Astrocyte IKKβ deletion after HFD exposure—but not before—also reduced glucose intolerance and insulin resistance, likely as a consequence of lower adiposity. Finally, both hypothalamic inflammation and astrocytosis were reduced in HFD-fed IKKβ-AKO mice.
Conclusions: These data support a requirement for astrocytic inflammatory signaling in HFD-induced hyperphagia and DIO susceptibility that may provide a novel target for obesity therapeutics.[Hide abstract]
|Cannabinoid receptor-containing axons innervate NPY/AgRP neurons Cannabinoid type 1 receptors (CB1R) mediate the effects of cannabinoids on energy metabolism. Neurons in the hypothalamic arcuate nucleus (ARC) that coexpress Agouti-related protein (AgRP) and neuropeptide Y (NPY) also affect whole body energy metabolism. Morozov et al. used transgenic mouse models and methods of electron microscopy combined with 3D reconstruction to evaluate the morphological substrate of presynaptic CB1R dependent control of AgRP/NPY neurons in the ARC. They find that eCBs and cannabis drugs may conduct prandial mechanisms through retrograde synaptic dis-inhibition and dis-excitation in the hypothalamus.|
Abstract | PDF
Objective: Phytocannabinoids, such as THC and endocannabinoids, are well known to promote feeding behavior and to control energy metabolism through cannabinoid type 1 receptors (CB1R). However, the underlying mechanisms are not fully understood. Generally, cannabinoid-conducted retrograde dis-inhibition of hunger-promoting neurons has been suggested to promote food intake, but so far it has not been demonstrated due to technical limitations.
Methods: We applied immunohistochemical labeling of CB1R for light microscopy and electron microscopy combined with three-dimensional reconstruction from serial sections in CB1R-expressing and CB1R-null mice, which served as a negative control. Hunger-promoting neurons expressing Agouti-related protein and neuropeptide Y (AgRP/NPY) in the hypothalamic arcuate nucleus were identified in NPY-GFP and NPY-hrGFP mice.
Results: Using three-dimensional reconstruction from serial sections we demonstrated numerous discontinuous segments of anti-CB1R labeling in the synaptic boutons and axonal shafts in the arcuate nucleus. We observed CB1R in the symmetric, presumed GABAergic, synaptic boutons innervating AgRP/NPY neurons. We also detected CB1R-containing axons producing symmetric and asymmetric synapses onto AgRP/NPY-negative neurons. Furthermore, we identified CB1R in close apposition to the endocannabinoid (2-arachidonoylglycerol)-synthesizing enzyme diacylglycerol lipase-alpha at AgRP/NPY neurons.
Conclusions: Our immunohistochemical and ultrastructural study demonstrates the morphological substrate for cannabinoid-conducted feeding behavior via retrograde dis-inhibition of hunger-promoting AgRP/NPY neurons.[Hide abstract]