The Ras family G-proteins RalA and RalB make critical non-overlapping contributions to the generation of a tumorigenic regulatory network, supporting bypass of the normal restraints on both cell proliferation and survival. The Sec6/8 complex, or exocyst, has emerged as a principal direct effector complex for Ral GTPases. Here, we show that RalA and RalB support mitotic progression through mobilization of the exocyst for two spatially and kinetically distinct steps of cytokinesis. RalA is required to tether the exocyst to the cytokinetic furrow in early cytokinesis. RalB is then required for recruitment of the exocyst to the midbody of this bridge to drive abscission and completion of cytokinesis. The collaborative action of RalA and RalB is specified by discrete subcellular compartmentalization and unique pairs of RalGEF proteins that provide inputs from both Ras-family protein-dependent and protein-independent regulatory cues. This suggests that Ral GTPases integrate diverse upstream signals to choreograph multiple roles for the exocyst in mitotic progression.
ObjectiveNon-shivering thermogenesis in brown adipose tissue (BAT) plays a central role in energy homeostasis. Thioesterase superfamily member 1 (Them1), a BAT-enriched long chain fatty acyl-CoA thioesterase, is upregulated by cold and downregulated by warm ambient temperatures. Them1−/− mice exhibit increased energy expenditure and resistance to diet-induced obesity and diabetes, but the mechanistic contribution of Them1 to the regulation of cold thermogenesis remains unknown.MethodsThem1−/− and Them1+/+ mice were subjected to continuous metabolic monitoring to quantify the effects of ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C) on energy expenditure, core body temperature, physical activity and food intake. The effects of Them1 expression on O2 consumption rates, thermogenic gene expression and lipolytic protein activation were determined ex vivo in BAT and in primary brown adipocytes.ResultsThem1 suppressed thermogenesis in mice even in the setting of ongoing cold exposure. Without affecting thermogenic gene transcription, Them1 reduced O2 consumption rates in both isolated BAT and primary brown adipocytes. This was attributable to decreased mitochondrial oxidation of endogenous but not exogenous fatty acids.ConclusionsThese results show that Them1 may act as a break on uncontrolled heat production and limit the extent of energy expenditure. Pharmacologic inhibition of Them1 could provide a targeted strategy for the management of metabolic disorders via activation of brown fat.
Orally delivered drugs and nutrients must diffuse through mucus to enter the circulatory system, but the barrier properties of mucus and their modulation by physiological factors are generally poorly characterized. The main objective of this study was to examine the impact of physicochemical changes occurring upon food ingestion on gastrointestinal (GI) mucus barrier properties. Lipids representative of postprandial intestinal contents enhanced mucus barriers, as indicated by a 10 – 142-fold reduction in the transport rate of 200 nm microspheres through mucus, depending on surface chemistry. Physiologically relevant increases in [Ca2+] resulted in a 2 - 4-fold reduction of transport rates, likely due to enhanced cross-linking of the mucus gel network. Reduction of pH from 6.5 to 3.5 also affected mucus viscoelasticity, reducing particle transport rates approximately 5 – 10-fold. Macroscopic visual observation and micro-scale lectin staining revealed mucus gel structural changes, including clumping into regions into which particles did not penetrate. Histological examination indicated food ingestion can prevent microsphere contact with and endocytosis by intestinal epithelium. Taken together, these results demonstrate that GI mucus barriers are significantly altered by stimuli associated with eating and potentially dosing of lipid-based delivery systems; these stimuli represent broadly relevant variables to consider upon designing oral therapies.
Objective Obesity associates with increased numbers of inflammatory cells in adipose tissue (AT), including T cells, but the mechanism of T cell recruitment remains unknown. This study tested the hypothesis that the chemokine receptor CXCR3 participates in T-cell accumulation in AT of obese mice, and thus in the regulation of local inflammation and systemic metabolism. Approach/Results Obese wild-type mice exhibited higher mRNA expression of CXCR3 in peri-epididymal AT-derived stromal vascular cells, compared to lean mice. We evaluated the function of CXCR3 in AT inflammation in vivo using CXCR3-deficient and wild-type control mice that consumed a high-fat diet (HFD). Peri-epididymal AT from obese CXCR3-deficient mice contained fewer T cells than obese controls after 8 and 16 weeks on HFD, as assessed by flow cytometry. Obese CXCR3-deficient mice had greater glucose tolerance than obese controls after 8 weeks, but not after 16 weeks. CXCR3-deficient mice fed HFD had reduced mRNA expression of pro-inflammatory mediators such as MCP-1 and RANTES, and of anti-inflammatory genes such as Foxp3, IL-10, and arginase-1 in peri-epididymal AT, compared to obese controls. Conclusions These results demonstrate that CXCR3 contributes to T-cell accumulation in peri-epididymal AT of obese mice. Our results also suggest that CXCR3 regulates the accumulation of distinct subsets of T cells, and that the ratio between these functional subsets across time likely modulates local inflammation and systemic metabolism.
Edited by Jeffrey E. Pessin In mammals, leptin production in adipocytes is up-regulated by feeding and insulin. Although this regulatory connection is central to all physiological effects of leptin, its molecular mechanism remains unknown. Here, we show that the transcription factor early growth response 1, Egr1, is rapidly but transiently induced by insulin in adipose cells both in vitro and in vivo, and its induction is followed by an increase in leptin transcription. ChIP and luciferase assays demonstrate that Egr1 directly binds to and activates the leptin promoter. Interestingly, the lipid droplet protein FSP27 may work as a co-factor for Egr1 in regulating leptin expression. By using siRNA-mediated knockout of Egr1 along with its overexpression in adipocytes, we demonstrate that Egr1 is both necessary and sufficient for the stimulatory effect of insulin on leptin transcription.
The escort factor Scap is essential in mammalian cells for regulated activation of sterol regulatory element binding proteins (SREBPs). SREBPs are membrane-bound transcription factors. Cells lacking Scap cannot activate SREBP. They are therefore deficient in the transcription of numerous genes involved in lipid synthesis and uptake; they cannot survive in the absence of exogenous lipid. Here we report that, in contrast to mammalian cells, Drosophila completely lacking dscap are viable. Flies lacking dscap emerge at $70% of the expected rate and readily survive as homozygous stocks. These animals continue to cleave dSREBP in some tissues. Transcription of dSREBP target genes in dscap mutant larvae is reduced compared to wild type. It is greater than in mutants lacking dSREBP and remains responsive to dietary lipids in dscap mutants. Flies lacking dscap do not require the caspase Drice to activate dSREBP. This contrasts with ds2p mutants. ds2p encodes a protease that releases the transcription factor domain of dSREBP from the membrane. Larvae doubly mutant for dscap and ds2p exhibit phenotypes similar to those of ds2p single mutants. Thus, dScap and dS2P, essential components of the SREBP activation machinery in mammalian cells, are dispensable in Drosophila owing to different compensatory mechanisms.
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