To the Editor: The Comment by Fisher, Cannon and Nedergaard [1] on our recent paper [2] advocates use of the term ‘anapyrexia,’ which connotes a biologically defended decrease of body temperature; e.g., the opposite of a fever, to describe the subnormal core temperature of leptin-deficient ob/ob mice. Another option is the conjunction ‘regulated hypothermia’ [3], based on the Glossary of Terms for Thermal Physiology [4], which notes that “hypothermia may be regulated (e.g., Torpor, Hibernation) or may be forced if heat loss exceeds the capacity for total heat production.” [Hide abstract]

The signaling mechanisms by which exercise improves muscle insulin sensitivity seem even harder to solve than to get people to exercise. Exercise (muscle contraction) has two diverse effects on muscle glucose metabolism. Firstly, acute exercise stimulates glucose uptake in skeletal muscles via translocation of GLUT4 translocation. This effect is insulin independent, and glucose uptake remains elevated a couple of hours after termination of exercise. Secondly, exercise increases insulin sensitivity in skeletal muscles. [Hide abstract]

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.

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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.

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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.

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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.

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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 (Gfap^CreERIkbkb^fl/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 Ikbkb^fl/fl littermate controls. In Gfap^CreERTdTomato 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.

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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.

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