Identification of a regulatory pathway inhibiting adipogenesis via RSPO2

Healthy adipose tissue remodeling depends on the balance between de novo adipogenesis from adipogenic progenitor cells and the hypertrophy of adipocytes. De novo adipogenesis has been shown to promote healthy adipose tissue expansion, which confers protection from obesity-associated insulin resistance. Here, we define the role and trajectory of different adipogenic precursor subpopulations and further delineate the mechanism and cellular trajectory of adipogenesis, using single-cell RNA-sequencing datasets of murine adipogenic precursors. We identify Rspo2 as a functional regulator of adipogenesis, which is secreted by a subset of CD142$^{+}$ cells to inhibit maturation of early progenitors through the receptor Lgr4. Increased circulating RSPO2 in mice leads to adipose tissue hypertrophy and insulin resistance and increased RSPO2 levels in male obese individuals correlate with impaired glucose homeostasis. Taken together, these findings identify a complex cellular crosstalk that inhibits adipogenesis and impairs adipose tissue homeostasis

Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis

To liberate fatty acids (FAs) from intracellular stores, lipolysis is regulated by the activity of the lipases adipose triglyceride lipase (ATGL), hormone-sensitive lipase and monoacylglycerol lipase. Excessive FA release as a result of uncontrolled lipolysis results in lipotoxicity, which can in turn promote the progression of metabolic disorders. However, whether cells can directly sense FAs to maintain cellular lipid homeostasis is unknown. Here we report a sensing mechanism for cellular FAs based on peroxisomal degradation of FAs and coupled with reactive oxygen species (ROS) production, which in turn regulates FA release by modulating lipolysis. Changes in ROS levels are sensed by PEX2, which modulates ATGL levels through post-translational ubiquitination. We demonstrate the importance of this pathway for non-alcoholic fatty liver disease progression using genetic and pharmacological approaches to alter ROS levels in vivo, which can be utilized to increase hepatic ATGL levels and ameliorate hepatic steatosis. The discovery of this peroxisomal β-oxidation-mediated feedback mechanism, which is conserved in multiple organs, couples the functions of peroxisomes and lipid droplets and might serve as a new way to manipulate lipolysis to treat metabolic disorders

GPR180 is a component of TGFβ signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGF beta signalling pathway and regulates the activity of the TGF beta receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGF beta signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism.Activation of thermogenic adipocytes is a strategy to combat metabolic diseases. Here the authors report that GPR180 is a component of TGF beta signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1, and contributes to the regulation of glucose and energy metabolism

Vimar suppresses neuronal necrosis and muscle degeneration induced by the <i>Pink1</i> mutant.

<p>(<b>A</b>) Effect of the <i>Drp1</i> mutant on neuronal necrosis. The micrographs showed the live images from larval chordotonal neurons. The control (<i>Appl>GFP;tub-Gal80</i>^<i>ts</i>) displays the cell bodies of the wild type chordotonal neurons, which form a cluster containing 6 neurons. In the <i>AGG</i> background, the wild type (+/+) flies showed swollen cell bodies, weakened GFP intensity and neuronal cell loss; and these defects were rescued under the <i>Drp1</i> mutant (<i>Drp1</i>^<i>1</i>) background. The right panel shows the quantification of the cell loss. For all quantification of neuronal necrosis, trial N = 5, with 10–15 flies were examined in each trial in this figure. (<b>B</b>) Effect of the <i>Drp1</i> mutant on the survival of the <i>AG</i> adult flies. For all quantification of <i>AG</i> lethality, trial N = 3, with 100–150 flies were examined for each trial. (<b>C</b>) Effect of <i>vimar</i> mutant on neuronal necrosis. (<b>D</b>) Effect of the <i>vimar</i> mutant on the survival of the <i>AG</i> flies. (<b>E</b>) Effect of <i>vimar</i> overexpression on neuronal necrosis. (<b>F</b>) Effect of <i>vimar</i> overexpression on the survival of the <i>AG</i> flies. (<b>G</b>) Effect of <i>Miro</i> overexpression on neuronal necrosis. The result showed that <i>Miro</i> overexpression enhanced neuronal necrosis; and the <i>vimar</i> mutant had no rescue effect on this defect. (<b>H</b>) Effect of <i>Miro</i> overexpression on the survival of the <i>AG</i> flies. (<b>I</b>) Effect of the <i>vimar</i> mutant (<i>vimar</i>^{<i>k16722</i>}) on <i>PINK1</i> mutant induced mitochondrial defect. The live image showed the mitochondrial morphology in the <i>PINK1</i> mutant (<i>PINK1</i>^<i>5</i>) and under the <i>vimar</i> mutant background. Ten thoraces were analyzed for each genotype. (<b>J</b>) Effect of the <i>vimar</i> mutant (<i>vimar</i>^{<i>k16722</i>}) on the wing posture defect of the <i>PINK1</i> mutant (<i>PINK1</i>^<i>5</i>). Trial N = 3, with 100–150 flies were examined in each trial.</p

Vimar Is a Novel Regulator of Mitochondrial Fission through Miro

<div><p>As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF). However, the GEF for Miro has not been identified. While studying mitochondrial morphology in <i>Drosophila</i>, we incidentally observed that the loss of <i>vimar</i>, a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro <i>in vitro</i>, we speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF) <i>vimar</i> mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF) <i>Miro</i> transgene; whereas a GOF <i>vimar</i> transgene enhanced <i>Miro</i> function. In addition, <i>vimar</i> lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, we found that mitochondrial fission played a functional role in high-calcium induced necrosis, and a LOF <i>vimar</i> mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miro’s effect on mitochondrial morphology is altered upon binding with calcium. In addition, a <i>PINK1</i> mutant, which induced mitochondrial enlargement and had been considered as a <i>Drosophila</i> model of Parkinson’s disease (PD), caused fly muscle defects, and the loss of <i>vimar</i> could rescue these defects. Furthermore, we found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional.</p></div

Interaction of vimar and Miro.

<p>(<b>A</b>) Co-Immunoprecipitation of vimar and Miro. The proteins were collected from the HEK293T cells that expressed both Flag-tagged Vimar (Flag-Vimar) and HA-tagged Miro (HA-Miro). Then, the proteins were precipitated with a HA (left panel) or Flag antibody (right panel). The control IgG is shown as a negative control. The total protein input is shown as the protein loading control. (<b>B</b>) An example of the defective wing posture. Compared to the control, overexpression of <i>Miro</i> in the adult flight muscle (<i>Mhc>Miro</i>) resulted in an upright fly wing posture. (<b>C</b>) Quantification of defective wing posture in the <i>Miro</i> overexpression background or in the <i>Mhc-Gal4</i> background. Trial N = 3, with 100–150 flies examined in each experiment. (<b>D</b>) Live imaging of the mitochondrial morphology in the fly flight muscle. The genotype of each fly muscle is labeled on the micrograph. Five thoraces were quantified for each genotype. (<b>E</b>) Quantification of defective wing posture in the Miro20V and Miro25N overexpression background. Trial N = 3, with 100–150 flies examined in each experiment. (<b>F</b>) Live imaging of the mitochondrial morphology in the fly flight muscle in the indicated genotypes. Five thoraces were quantified for each genotype.</p

The conserved role of RAP1GDS1 in mammalian cells.

<p>(<b>A</b>) Effect of RAP1GDS1 knock down on the mitochondrial morphology in HEK293T cells. The mitochondria in the cells that stably express the RAP1GDS1 shRNA are labeled with a transiently transfected MitoDsred expression vector. The cells were classified as tubular-shape or punctate-shape based on differences in their mitochondrial lengths. The ratio of punctate-shape mitochondria is shown in the right panel. The result showed that RAP1GDS1 shRNA had a trend to increase the punctate-shape mitochondria (not statistically different from the control shRNA). Trial N = 3, with 100 cells were quantified in each trial. (<b>B</b>) Effect of RAP1GDS1 knocking down on the mitochondrial fragmentation under calcium overload stress. The HEK293T cells were treated with 20 μM calcium ionophore (A23187) for 4 hours. The result showed that RAP1GDS1 shRNA reduced fragmented mitochondria upon calcium ionophore treatment. Trial N = 3, with 100 cells were quantified in each trial. (<b>C</b>) Effect of the RAP1GDS1 shRNA on calcium ionophore-induced necrosis. The HEK293T control and RAP1GDS1 shRNA stable cell lines were treated with 20 μM A23187 for 14 hours. Then, the cell death was quantified by the ATP assay. The result indicated that less cell death occurred in the RAP1GDS1 shRNA expressing cells. Trial N = 3. (<b>D</b>) Effect of the RAP1GDS1 shRNA on calcium ionophore-induced necrosis. The PI and DAPI staining patterns are shown. The red signals indicate the PI-positive cells and the blue channel indicates the DAPI staining. Trial N = 3. (<b>E</b>) Effect of the <i>Miro1</i> siRNA on calcium ionophore induced necrosis determined by the ATP assay. The <i>Miro1</i> siRNA was transiently transfected in HEK293T cells for 48 hours. Trial N = 3. (<b>F</b>) Effect of the <i>Miro1</i> siRNA on calcium ionophore induced necrosis determined by the PI staining assay. The PI and DAPI staining patterns are shown. The same result was observed as in <b>E</b>. Trial N = 3. (<b>G</b>) Co-Immunoprecipitation of RAP1GDS1 and Miro1. The proteins were collected from the HEK293T cells that expressed Flag-tagged RAP1GDS1 (Flag-RAP1GDS1) and HA-tagged Miro1 (HA-Miro1). The control IgG is shown as a negative control. The total protein input is shown as the protein loading control. Trial N = 3.</p

A schematic model of Miro/vimar function on mitochondrial morphology.

<p>In normal calcium conditions, the Miro/vimar complex promotes mitochondrial fission inhibition, and their GOF results in elongated mitochondria. Increased mitochondrial fusion is known to occur in the <i>PINK1</i> mutant flies, and this defect can be rescued by LOF Miro/vimar. In the high calcium state, the Miro/vimar complex promotes mitochondrial fragmentation, which accelerates neuronal necrosis. Regardless of the intracellular calcium level, vimar enhances the function of Miro, because vimar is likely the GEF to promote Miro's GTP/GDP exchange.</p

<i>Drosophila vimar</i> affects mitochondrial morphology under normal conditions.

<p>(<b>A</b>) <b>a-e</b>, Live imaging of the mitochondrial morphology in the flight muscle of adult flies. The mitochondria are labeled with <i>UAS-mitoGFP</i> driven by <i>Mhc-Gal4</i> (<i>Mhc>mitoGFP</i>). The genotype is indicated on each micrograph. <b>f</b>, To quantify the mitochondrial size, the averaged mitochondrial size of the control (+/+) is set as 1, and the relative ratios of the other genotypes to the control are shown. Five thoraces from each genotype were quantified. Bar graphs throughout all figures are means ± SD. The white bar represents the control, the gray bar represents no statistical different from the control, and the black bar represents significantly different from the control. * for p<0.05; ** for p<0.01; ***for p<0.001. (<b>B</b>) Mitochondrial distribution and morphology in larval oenocytes. <b>a</b>, The mitochondria are labeled with <i>PromE(800)> mitoGFP</i>. <b>b-e</b>, The effects of <i>Khc</i>, <i>Milton</i>, <i>Miro</i> and <i>vimar RNAi</i> are shown. The dotted red lines denote the cell boundaries, which were determined by the mitoGFP background. <b>a'-e'</b>, Enlarged view of the white box labeled area in the upper panel. <b>f</b>, To quantify the mitochondrial length, the averaged mitochondrial length of the control (+/+) is set as 1, and the relative ratios of the other genotypes to the control are shown. Mitochondrial length of five oenocytes was quantified per genotype and shown as means ± SD. (<b>C</b>) Live imaging of mitochondria in eye disc after knocking down <i>vimar</i> by <i>GMR>mitoGFP</i>. Three eye discs were analyzed for each genotype. (<b>D</b>) Effect of <i>vimar</i> on mitochondrial transport. The mitochondria are labeled with mitoGFP (<i>CCAP>mitoGFP</i>), and their movements in the axons were recorded and transformed into kymographs. Mitochondria motion in ten axons from five larvae was analyzed for each genotype. The quantification is shown on the bar graph. (<b>E</b>) Subcellular vimar protein distribution by protein fractionation. The proteins from adult thoraces (<i>Mhc>MitoGFP</i>) were separated into cytosolic and crude mitochondrial fractions. The vimar protein enrichment was analyzed by immunobloting with the anti-vimar antibody. The mitoGFP protein was detected by the anti-GFP antibody; and β-actin is a cytosolic protein.</p

Quantification of adipocyte numbers following adipose tissue remodeling

To analyze the capacity of white and brown adipose tissue remodeling, we developed two mouse lines to label, quantitatively trace, and ablate white, brown, and brite/beige adipocytes at different ambient temperatures. We show here that the brown adipocytes are recruited first and reach a peak after 1 week of cold stimulation followed by a decline during prolonged cold exposure. On the contrary, brite/beige cell numbers plateau after 3 weeks of cold exposure. At thermoneutrality, brown adipose tissue, in spite of being masked by a white-like morphology, retains its brown-like physiology, as Ucp1+ cells can be recovered immediately upon beta3-adrenergic stimulation. We further demonstrate that the recruitment of Ucp1+ cells in response to cold is driven by existing adipocytes. In contrast, the regeneration of the interscapular brown adipose tissue following ablation of Ucp1+ cells is driven by de novo differentiation.ISSN:2666-3864ISSN:2211-124