Featured ArticlesVolume 24 | June 2019
|Lipid metabolism biomarkers of BAT agingAging is a negative regulator of brown adipose tissue (BAT) formation and function. There is evidence that the effects of various lipid species on adipose tissue play a central role in the emergence of metabolic pathologies. However, very little is known about age-related changes of lipid metabolites in BAT, their involvement in BAT dysfunction and the contribution of this process to metabolic disorders. To identify functional lipid biomarkers of brown adipose tissue aging, Gohlke et al. combined metabolomic and proteomic analyses. These revealed that changes of several lipid classes and individual metabolite species correlate with adipose tissue aging. Among these, sphingolipids are significantly induced in aged BAT.|
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Objective: Aging is accompanied by loss of brown adipocytes and a decline in their thermogenic potential, which may exacerbate the development of adiposity and other metabolic disorders. Presently, only limited evidence exists describing the molecular alterations leading to impaired brown adipogenesis with aging and the contribution of these processes to changes of systemic energy metabolism.
Methods: Samples of young and aged murine brown and white adipose tissue were used to compare age-related changes of brown adipogenic gene expression and thermogenesis-related lipid mobilization. To identify potential markers of brown adipose tissue aging, non-targeted proteomic and metabolomic as well as targeted lipid analyses were conducted on young and aged tissue samples. Subsequently, the effects of several candidate lipid classes on brown adipocyte function were examined.
Results: Corroborating previous reports of reduced expression of uncoupling protein-1, we observe impaired signaling required for lipid mobilization in aged brown fat after adrenergic stimulation. Omics analyses additionally confirm the age-related impairment of lipid homeostasis and reveal the accumulation of specific lipid classes, including certain sphingolipids, ceramides, and dolichols in aged brown fat. While ceramides as well as enzymes of dolichol metabolism inhibit brown adipogenesis, inhibition of sphingosine 1-phosphate receptor 2 induces brown adipocyte differentiation.
Conclusions: Our functional analyses show that changes in specific lipid species, as observed during aging, may contribute to reduced thermogenic potential. They thus uncover potential biomarkers of aging as well as molecular mechanisms that could contribute to the degradation of brown adipocytes, thereby providing potential treatment strategies of age-related metabolic conditions.[Hide abstract]
|Lipocalin 2 overexpression protects against decline in thermogenesis and metabolic deteriorationAging is the greatest risk factor for perturbations in metabolism including insulin resistance, dyslipidemia, and inflammation. Lipocalin 2 (Lcn2) is a circulating factor secreted from adipose tissue in response to obesity, inflammation, and nutrient/growth signals. Deis, Guo, et al. found that overexpression of Lcn2 in adipose tissue led to improved cold adaptation, altered whole-body energy expenditure with a trend towards fat oxidation, increased oxidative gene expression, decreased adipose tissue weight and adipocyte size, and decreased glucose/TG levels. Their results also suggest that overexpression of Lcn2 preserves adipose tissue function and prevents age-related glucose intolerance and liver lipid accumulation.|
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Objective: Aging increases the risk for development of adipose tissue dysfunction, insulin resistance, dyslipidemia, and liver steatosis. Lipocalin 2 (Lcn2) deficient mice are more prone to diet-induced obesity and metabolic dysfunction, indicating a protective role for Lcn2 in younger mice. In this study, we determined whether overexpressing Lcn2 in adipose tissue can protect against age-related metabolic deterioration.
Methods: We developed ap2-promoter-driven Lcn2 transgenic (Tg) mice and aged Lcn2 Tg mice for the metabolic assessments.
Results: We found decreased adipocyte size in inguinal white adipose tissue (iWAT) from 10-month-old Lcn2 Tg mice relative to WT. This was accompanied by increased markers of adipogenesis in iWAT and attenuation of the age-related decline in AMP-activated protein kinase (AMPK) phosphorylation in adipose tissue depots. In addition to improvements in adipose tissue function, whole-body metabolic homeostasis was maintained in aged Lcn2 Tg mice. This included improved glucose tolerance and reduced serum triglycerides in older Lcn2 Tg mice relative to WT mice. Further, liver morphology and liver lipid levels were improved in older Lcn2 Tg mice, alongside a decrease in markers of liver inflammation and fibrosis.
Conclusions: We demonstrate that overexpression of Lcn2 in adipose tissue not only preserves adipose tissue function during aging but also promotes maintenance of glucose tolerance, decreases dyslipidemia, and prevents liver lipid accumulation and steatosis.[Hide abstract]
|Heterogeneity in the perirenal region suggests dormant brown adipose tissuePerirenal fat surrounds the kidney as well as the adrenal gland, which secretes epinephrine and norepinephrine in response to sympathetic activation. Jespersen, Feizi, et al. hypothesized that multilocular brown adipose tissue (BAT) would accumulate close to the adrenal gland and aimed to characterize the molecular differences between this fat compared to perirenal fat more distant from sources of norepinephrine. They demonstrate that most of the perirenal fat in adult humans consists of dormant BAT, while small amounts of adipocytes with a multilocular morphology and gene expression signature of active BAT, are present near regions where sympathetic activity is expected to be high.|
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Objective: Increasing the amounts of functionally competent brown adipose tissue (BAT) in adult humans has the potential to restore dysfunctional metabolism and counteract obesity. In this study, we aimed to characterize the human perirenal fat depot, and we hypothesized that there would be regional, within-depot differences in the adipose signature depending on local sympathetic activity.
Methods: We characterized fat specimens from four different perirenal regions of adult kidney donors, through a combination of qPCR mapping, immunohistochemical staining, RNA-sequencing, and pre-adipocyte isolation. Candidate gene signatures, separated by adipocyte morphology, were recapitulated in a murine model of unilocular brown fat induced by thermoneutrality and high fat diet.
Results: We identified widespread amounts of dormant brown adipose tissue throughout the perirenal depot, which was contrasted by multilocular BAT, primarily found near the adrenal gland. Dormant BAT was characterized by a unilocular morphology and a distinct gene expression profile, which partly overlapped with that of subcutaneous white adipose tissue (WAT). Brown fat precursor cells, which differentiated into functional brown adipocytes were present in the entire perirenal fat depot, regardless of state. We identified SPARC as a candidate adipokine contributing to a dormant BAT state, and CLSTN3 as a novel marker for multilocular BAT.
Conclusions: We propose that perirenal adipose tissue in adult humans consists mainly of dormant BAT and provide a data set for future research on factors which can reactivate dormant BAT into active BAT, a potential strategy for combatting obesity and metabolic disease.[Hide abstract]
|Deletion of Tcf7l2 promotes adipocyte hypertrophy and impaired glucose metabolism TCF7L2 is a component of the Wnt signaling pathway that is known to regulate metabolic disease in humans. Polymorphisms in Tcf7l2 are associated with type 2 diabetes. Geoghegan, Simcox, and colleagues conditionally deleted Tcf7l2 in adipocytes. They found that this leads to adipocyte hypertrophy and impaired glucose handling on a high-fat diet. This highlights that TCF7L2 has a role beyond development that extends to metabolic control in adipocytes.|
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Objective: Activation of the Wnt-signaling pathway is known to inhibit differentiation in adipocytes. However, there is a gap in our understanding of the transcriptional network regulated by components of the Wnt-signaling pathway during adipogenesis and in adipocytes during postnatal life. The key intracellular effectors of the Wnt-signaling pathway occur through TCF transcription factors such as TCF7L2 (transcription factor-7-like 2). Several genetic variants in proximity to TCF7L2 have been linked to type 2 diabetes through genome-wide association studies in various human populations. Our work aims to functionally characterize the adipocyte specific gene program regulated by TCF7L2 and understand how this program regulates metabolism.
Methods: We generated Tcf7l2F/F mice and assessed TCF7L2 function in isolated adipocytes and adipose specific knockout mice. ChIP-sequencing and RNA-sequencing was performed on the isolated adipocytes with control and TCF7L2 knockout cells. Adipose specific TCF7L2 knockout mice were challenged with high fat diet and assessed for body weight, glucose tolerance, and lipolysis.
Results: Here we report that TCF7L2 regulates adipocyte size, endocrine function, and glucose metabolism. Tcf7l2 is highly expressed in white adipose tissue, and its expression is suppressed in genetic and diet-induced models of obesity. Genome-wide distribution of TCF7L2 binding and gene expression analysis in adipocytes suggests that TCF7L2 directly regulates genes implicated in cellular metabolism and cell cycle control. When challenged with a high-fat diet, conditional deletion of TCF7L2 in adipocytes led to impaired glucose tolerance, impaired insulin sensitivity, promoted weight gain, and increased adipose tissue mass. This was accompanied by reduced expression of triglyceride hydrolase, reduced fasting-induced free fatty acid release, and adipocyte hypertrophy in subcutaneous adipose tissue.
Conclusions: Together our studies support that TCF7L2 is a central transcriptional regulator of the adipocyte metabolic program by directly regulating the expression of genes involved in lipid and glucose metabolism.[Hide abstract]
|Ferritin regulates energy balance and thermogenesisIntracellular Fe redox activity is controlled by ferritin, a multimeric protein complex composed of variable ratios of ferritin light chain (FTL) and ferritin heavy chain (FTH). Blankenhaus, Braza, et al. report that global Fth deletion in adult mice leads to a profound disruption of organismal Fe metabolism and redox homeostasis, associated with failure to sustain energy expenditure and thermal homeostasis. Ferritin acts as a gatekeeper of organismal Fe metabolism, coupling nutritional Fe supply to organismal energy expenditure and thermoregulation.|
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Objective: The ferritin heavy/heart chain (FTH) gene encodes the ferroxidase component of the iron (Fe) sequestering ferritin complex, which plays a central role in the regulation of cellular Fe metabolism. Here we tested the hypothesis that ferritin regulates organismal Fe metabolism in a manner that impacts energy balance and thermal homeostasis.
Methods: We developed a mouse strain, referred herein as FthR26 fl/fl, expressing a tamoxifen-inducible Cre recombinase under the control of the Rosa26 (R26) promoter and carrying two LoxP (fl) sites: one at the 5′end of the Fth promoter and another the 3' end of the first Fth exon. Tamoxifen administration induces global deletion of Fth in adult FthR26Δ/Δ mice, testing whether FTH is required for maintenance of organismal homeostasis.
Results: Under standard nutritional Fe supply, Fth deletion in adult FthR26Δ/Δ mice led to a profound deregulation of organismal Fe metabolism, oxidative stress, inflammation, and multi-organ damage, culminating in death. Unexpectedly, Fth deletion was also associated with a profound atrophy of white and brown adipose tissue as well as with collapse of energy expenditure and thermogenesis. This was attributed mechanistically to mitochondrial dysfunction, as assessed in the liver and in adipose tissue.
Conclusions: The FTH component of ferritin acts as a master regulator of organismal Fe homeostasis, coupling nutritional Fe supply to organismal redox homeostasis, energy expenditure and thermoregulation.[Hide abstract]
|Point mutations in the PDX1 transactivation domain impair human β-cell development and functionThe transcription factor pancreas/duodenum homeobox protein 1 (PDX1) is one of the master regulators of pancreas development and β-cell function. In humans, several missense coding mutations in the PDX1 gene such as the P33T and C18R mutations in the transactivation domain have been associated with an increased risk for diabetes. The exact mechanisms by which these mutations contribute to diabetes predisposition are not understood. Wang et al. used patient-derived induced pluripotent stem cells to reveal mechanistic details of how these common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and β-cell function.
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Objective: Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown.
Methods: In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/−).
Results: Using an in vitro β-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/− and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion.
Conclusions: Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and β-cell function and contribute to the predisposition for diabetes.[Hide abstract]
|Novel eQTLs associated with type 2 diabetes Genome-wide association studies (GWAS) meta-analyses have revealed more than 200 loci associated with type 2 diabetes (T2D). Combining GWAS with expression data to produce expression quantitative trait loci (eQTLs) has become a powerful tool to shed light into the causative mechanisms of these genetic associations. Khamis, Canouil, and colleagues tested a total of 203 islet samples from European subjects with and without diabetes and provide a catalogue of cis-eQTLs associated with T2D.|
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Objective: Genome wide association studies (GWAS) for type 2 diabetes (T2D) have identified genetic loci that often localise in non-coding regions of the genome, suggesting gene regulation effects. We combined genetic and transcriptomic analysis from human islets obtained from brain-dead organ donors or surgical patients to detect expression quantitative trait loci (eQTLs) and shed light into the regulatory mechanisms of these genes.
Methods: Pancreatic islets were isolated either by laser capture microdissection (LCM) from surgical specimens of 103 metabolically phenotyped pancreatectomized patients (PPP) or by collagenase digestion of pancreas from 100 brain-dead organ donors (OD). Genotyping (> 8.7 million single nucleotide polymorphisms) and expression (> 47,000 transcripts and splice variants) analyses were combined to generate cis-eQTLs.
Results: After applying genome-wide false discovery rate significance thresholds, we identified 1,173 and 1,021 eQTLs in samples of OD and PPP, respectively. Among the strongest eQTLs shared between OD and PPP were CHURC1 (OD p-value=1.71 × 10-24; PPP p-value = 3.64 × 10–24) and PSPH (OD p-value = 3.92 × 10−26; PPP p-value = 3.64 × 10−24). We identified eQTLs in linkage-disequilibrium with GWAS loci T2D and associated traits, including TTLL6, MLX and KIF9 loci, which do not implicate the nearest gene. We found in the PPP datasets 11 eQTL genes, which were differentially expressed in T2D and two genes (CYP4V2 and TSEN2) associated with HbA1c but none in the OD samples.
Conclusions: eQTL analysis of LCM islets from PPP led us to identify novel genes which had not been previously linked to islet biology and T2D. The understanding gained from eQTL approaches, especially using surgical samples of living patients, provides a more accurate 3-dimensional representation than those from genetic studies alone.[Hide abstract]
|Discovering metabolic disease gene interactions by correlated effects on cellular morphology A plethora of disease relevant genetic loci have been identified by genetic association studies. Jiao and colleagues hypothesized that perturbing these loci/genes in adipocytes in vitro and assessing the effect on morphologic features would enable disease relevant functional annotation. Here, they demonstrate the utility of this approach in metabolic disease. They ablated 125 genes in human pre-adipocytes using CRISPR/CAS9 and profiled the effect on cellular morphology using morphologic similarity to identify mechanistic interactions between genes. They demonstrate that this morphometric approach is capable of surveying diverse cellular mechanisms by validating both a protein-protein interaction on the lipid droplet surface and a transcriptional regulatory interaction in the DNA.|
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Objective: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease–gene interactions during adipocyte differentiation.
Methods: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene.
Results: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein–protein and gene regulatory interactions.
Conclusions: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.[Hide abstract]
|Dopamine neuronal protection by GHSR is independent of electric activityParkinson’s Disease (PD) is a neurodegenerative disease that appears when levels of dopamine (DA) in the substantia nigra (SN) display a 70-80% reduction. The etiology of PD is still elusive. Dln101 is a potential neuroprotective compound that targets the growth hormone secretagogue 1 receptor (GHSR). Stutz et al. evaluated Dln101 in comparison with ghrelin and investigated its GHSR-mediated effects that increase SN DA resilience. Although Dln101-mediated neuroprotection of SN DA cells was equivalent to that of ghrelin, its effect on electrical activity was very distinct despite the requirement of GHSR in mediating effects of both molecules.|
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Objective: Dopamine neurons in the Substantia nigra (SN) play crucial roles in control of voluntary movement. Extensive degeneration of this neuronal population is the cause of Parkinson's disease (PD). Many factors have been linked to SN DA neuronal survival, including neuronal pacemaker activity (responsible for maintaining basal firing and DA tone) and mitochondrial function. Dln-101, a naturally occurring splice variant of the human ghrelin gene, targets the ghrelin receptor (GHSR) present in the SN DA cells. Ghrelin activation of GHSR has been shown to protect SN DA neurons against 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP) treatment. We decided to compare the actions of Dln-101 with ghrelin and identify the mechanisms associated with neuronal survival.
Methods: Histologial, biochemical, and behavioral parameters were used to evaluate neuroprotection. Inflammation and redox balance of SN DA cells were evaluated using histologial and real-time PCR analysis. Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology was used to modulate SN DA neuron electrical activity and associated survival. Mitochondrial dynamics in SN DA cells was evaluated using electron microscopy data.
Results: Here, we report that the human isoform displays an equivalent neuroprotective factor. However, while exogenous administration of mouse ghrelin electrically activates SN DA neurons increasing dopamine output, as well as locomotion, the human isoform significantly suppressed dopamine output, with an associated decrease in animal motor behavior. Investigating the mechanisms by which GHSR mediates neuroprotection, we found that dopamine cell-selective control of electrical activity is neither sufficient nor necessary to promote SN DA neuron survival, including that associated with GHSR activation. We found that Dln101 pre-treatment diminished MPTP-induced mitochondrial aberrations in SN DA neurons and that the effect of Dln101 to protect dopamine cells was dependent on mitofusin 2, a protein involved in the process of mitochondrial fusion and tethering of the mitochondria to the endoplasmic reticulum.
Conclusions: Taken together, these observations unmasked a complex role of GHSR in dopamine neuronal protection independent on electric activity of these cells and revealed a crucial role for mitochondrial dynamics in some aspects of this process.[Hide abstract]
|Dietary calories and lipids synergistically shape adipose tissue cellularityThe quantity and anatomical distribution of adipose tissue are key drivers of the metabolic syndrome. Obesity and abdominal visceral adiposity in particular are major risk factors for type 2 diabetes and cardiovascular disorders. Accumulating evidence suggests that childhood and adolescence are critical periods for shaping adipose tissue properties. However, the control of adipose progenitor proliferation and hyperplasia by dietary components are poorly understood. Meln, Wolff, et al. have investigated the role of excess calories and dietary lipids in adipose tissue proliferative growth in juvenile mice and found that both differentially and synergistically drive adipose tissue proliferative growth and the programming of the metabolic syndrome in childhood.|
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Objective: The susceptibility to abdominal obesity and the metabolic syndrome is determined to a substantial extent during childhood and adolescence, when key adipose tissue characteristics are established. Although the general impact of postnatal nutrition is well known, it is not clear how specific dietary components drive adipose tissue growth and how this relates to the risk of metabolic dysfunction in adulthood.
Methods: Adipose tissue growth including cell proliferation was analyzed in juvenile mice upon dietary manipulation with in vivo nucleotide labeling. The proliferative response of progenitors to specific fatty acids was assayed in primary cultures. Long-term metabolic consequences were assessed through transient dietary manipulation post-weaning with a second obesogenic challenge in adulthood.
Results: Dietary lipids stimulated adipose tissue progenitor cell proliferation in juvenile mice independently of excess caloric intake and calorie-dependent adipocyte hypertrophy. Excess calories increased mitogenic IGF-1 levels systemically, whereas palmitoleic acid was able to enhance the sensitivity of progenitors to IGF-1, resulting in synergistic stimulation of proliferation. Early transient consumption of excess lipids promoted hyperplastic adipose tissue expansion in response to a second dietary challenge in adulthood and this correlated with abdominal obesity and hyperinsulinemia.
Conclusions: Dietary lipids and calories differentially and synergistically drive adipose tissue proliferative growth and the programming of the metabolic syndrome in childhood.[Hide abstract]
|Weight loss induces reactive astrogliosisAstrocytes and microglia respond to noxious stimuli by a process known as reactive gliosis (RG). RG is characterized by morphological changes such as increased cell size, enlarged, lengthened processes, and an increase in proliferation. Previously, in mice fed a high-fat diet (HFD) the number of reactive astrocytes and microglia in the arcuate nucleus (ARC) was increased. Harrison et al. have found that mice which are chronically obese after 22 weeks of HFD feeding display RG levels in the ARC comparable to levels seen in age-matched chow fed mice. Furthermore, profound weight loss results in an increase in ARC related astrocytic RG. However, whether or not reactive gliosis plays a role in chronic obesity, or rather in the acute adaptation to the dietary environment, remains to be fully understood.|
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Objective: Obesity has been linked to an inflammation like state in the hypothalamus, mainly characterized by reactive gliosis (RG) of astrocytes and microglia. Here, using two diet models or pharmacological treatment, we assessed the effects of mild and drastic weight loss on RG, in the context of high-fat diet (HFD) induced obesity.
Methods: We subjected HFD-induced obese (DIO) male C57BL/6J mice to a weight loss intervention with a switch to standard chow, calorie restriction (CR), or treatment with the Glp1 receptor agonist Exendin-4 (EX4). The severity of RG was estimated by an ordinal scoring system based on fluorescence intensities of glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1 positive (Iba1), cell numbers, and morphological characteristics.
Results: In contrast to previous reports, DIO mice fed chronically with HFD showed no differences in microglial or astrocytic RG, compared to chow controls. Moreover, mild or profound weight loss had no impact on microglial RG. However, astrocyte RG was increased in CR and EX4 groups compared to chow fed animals and strongly correlated to body weight loss. Profound weight loss by either CR or EX4 was further linked to increased levels of circulating non-esterified free fatty acids.
Conclusions: Overall, our data demonstrate that in a chronically obese state, astrocyte and microglial RG is indifferent from that observed in age-matched chow controls. Nonetheless, profound acute weight loss can induce astrocyte RG in the hypothalamic arcuate nucleus, possibly due to increased circulating NEFAs. This suggests that astrocytes may sense acute changes to both the dietary environment and body weight.[Hide abstract]