Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation.

Macrophages exhibit a spectrum of activation states ranging from classical to alternative activation1. Alternatively, activated macrophages are involved in diverse pathophysiological processes such as confining tissue parasites2, improving insulin sensitivity3 or promoting an immune-tolerant microenvironment that facilitates tumour growth and metastasis4. Recently, the metabolic regulation of macrophage function has come into focus as both the classical and alternative activation programmes require specific regulated metabolic reprogramming5. While most of the studies regarding immunometabolism have focussed on the catabolic pathways activated to provide energy, little is known about the anabolic pathways mediating macrophage alternative activation. In this study, we show that the anabolic transcription factor sterol regulatory element binding protein 1 (SREBP1) is activated in response to the canonical T helper 2 cell cytokine interleukin-4 to trigger the de novo lipogenesis (DNL) programme, as a necessary step for macrophage alternative activation. Mechanistically, DNL consumes NADPH, partitioning it away from cellular antioxidant defences and raising reactive oxygen species levels. Reactive oxygen species serves as a second messenger, signalling sufficient DNL, and promoting macrophage alternative activation. The pathophysiological relevance of this mechanism is validated by showing that SREBP1/DNL is essential for macrophage alternative activation in vivo in a helminth infection model.

Macrophage beta2-adrenergic receptor is dispensable for the adipose tissue inflammation and function.

OBJECTIVE: Neuroimmune interactions between the sympathetic nervous system (SNS) and macrophages are required for the homeostasis of multiple tissues, including the adipose tissue. It has been proposed that the SNS maintains adipose tissue macrophages (ATMs) in an anti-inflammatory state via direct norepinephrine (NE) signaling to macrophages. This study aimed to investigate the physiological importance of this paradigm by utilizing a mouse model in which the adrenergic signaling from the SNS to macrophages, but not to other adipose tissue cells, was disrupted. METHODS: We generated a macrophage-specific B2AR knockout mouse (Adrb2ΔLyz2) by crossing Adrb2fl/fl and Lyz2Cre/+ mice. We have previously shown that macrophages isolated from Adrb2ΔLyz2 animals do not respond to NE stimulation in vitro. Herein we performed a metabolic phenotyping of Adrb2ΔLyz2 mice on either chow or high-fat diet (HFD). We also assessed the adipose tissue function of Adrb2ΔLyz2 animals during fasting and cold exposure. Finally, we transplanted Adrb2ΔLyz2 bone marrow to low-density lipoprotein receptor (LDLR) knockout mice and investigated the development of atherosclerosis during Western diet feeding. RESULTS: We demonstrated that SNS-associated ATMs have a transcriptional profile indicative of activated beta-2 adrenergic receptor (B2AR), the main adrenergic receptor isoform in myeloid cells. However, Adrb2ΔLyz2 mice have unaltered energy balance on a chow or HFD. Furthermore, Adrb2ΔLyz2 mice show similar levels of adipose tissue inflammation and function during feeding, fasting, or cold exposure, and develop insulin resistance during HFD at the same rate as controls. Finally, macrophage-specific B2AR deletion does not affect the development of atherosclerosis on an LDL receptor-null genetic background. CONCLUSIONS: Overall, our data suggest that the SNS does not directly modulate the phenotype of adipose tissue macrophages in either lean mice or mouse models of cardiometabolic disease. Instead, sympathetic nerve activity exerts an indirect effect on adipose tissue macrophages through the modulation of adipocyte function.

Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. METHODS AND RESULTS: Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. CONCLUSIONS: Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.

Peroxisome Proliferator-Activated Receptor γ2 Controls the Rate of Adipose Tissue Lipid Storage and Determines Metabolic Flexibility.

One understudied function of white adipose tissue (AT) is its role in postprandial lipid buffering. In this study, we demonstrate that mice lacking the adipose tissue-specific transcription factor peroxisome proliferator-activated receptor γ2 (PPARγ2) exhibit a defect in their rate of adipose tissue lipid storage. Impaired adipose tissue storage rate reduces metabolic flexibility, without compromising fasted glucose tolerance or insulin sensitivity, even following prolonged high-fat feeding. However, acutely overfeeding PPARγ2-KO mice caused a 10-fold increase in insulin levels compared with controls. Although impaired adipose tissue storage rate did not result in insulin resistance in young mice, 1-year-old PPARγ2-KO mice developed skeletal muscle insulin resistance. Our data indicate that failed adipose tissue storage may occur prior to defects in glucose handling and that overfeeding protocols may uncover genes controlling adipose tissue storage rate, as opposed to capacity, and act as a diagnostic test for early-stage human metabolic disease.

Adipose tissue fatty acid chain length and mono-unsaturation increases with obesity and insulin resistance.

The non-essential fatty acids, C18:1n9, C16:0, C16:1n7, C18:0 and C18:1n7 account for over 75% of fatty acids in white adipose (WAT) triacylglycerol (TAG). The relative composition of these fatty acids (FA) is influenced by the desaturases, SCD1-4 and the elongase, ELOVL6. In knock-out models, loss of SCD1 or ELOVL6 results in reduced Δ9 desaturated and reduced 18-carbon non-essential FA respectively. Both Elovl6 KO and SCD1 KO mice exhibit improved insulin sensitivity. Here we describe the relationship between WAT TAG composition in obese mouse models and obese humans stratified for insulin resistance. In mouse models with increasing obesity and insulin resistance, there was an increase in scWAT Δ9 desaturated FAs (SCD ratio) and FAs with 18-carbons (Elovl6 ratio) in mice. Data from mouse models discordant for obesity and insulin resistance (AKT2 KO, Adiponectin aP2-transgenic), suggested that scWAT TAG Elovl6 ratio was associated with insulin sensitivity, whereas SCD1 ratio was associated with fat mass. In humans, a greater SCD1 and Elovl6 ratio was found in metabolically more harmful visceral adipose tissue when compared to subcutaneous adipose tissue.

Brown Adipose Tissue Thermogenic Capacity Is Regulated by Elovl6.

Although many transcriptional pathways regulating BAT have been identified, the role of lipid biosynthetic enzymes in thermogenesis has been less investigated. Whereas cold exposure causes changes in the fatty acid composition of BAT, the functional consequences of this remains relatively unexplored. In this study, we demonstrate that the enzyme Elongation of Very Long Chain fatty acids 6 (Elovl6) is necessary for the thermogenic action of BAT. Elovl6 is responsible for converting C16 non-essential fatty acids into C18 species. Loss of Elovl6 does not modulate traditional BAT markers; instead, it causes reduced expression of mitochondrial electron transport chain components and lower BAT thermogenic capacity. The reduction in BAT activity appears to be counteracted by increased beiging of scWAT. When beige fat is disabled by thermoneutrality or aging, Elovl6 KO mice gain weight and have increased scWAT mass and impaired carbohydrate metabolism. Overall, our study suggests fatty acid chain length is important for BAT function.

Spatial distribution and deployment of community-based distributors implementing integrated community case management (iCCM): Geographic information system (GIS) mapping study in three South Sudan states.

AIM: In late 2012 and in conjunction with South Sudan's Ministry of Health - National Malaria Control Program, PSI (Population Services International) conducted a comprehensive mapping exercise to assess geographical coverage of its integrated community case management (iCCM) program and consider scope for expansion. The operational research was designed to provide evidence and support for low-cost mapping and monitoring systems, demonstrating the use of technology to enhance the quality of programming and to allow for the improved allocation of resources through appropriate and need-based deployment of community-based distributors (CBDs). METHODS: The survey took place over the course of three months and program staff gathered GPS (global positioning system) data, along with demographic data, for over 1200 CBDs and 111 CBD supervisors operating in six counties in South Sudan. Data was collated, cleaned and quality assured, input into an Excel database, and subsequently uploaded to geographic information system (GIS) for spatial analysis and map production. RESULTS: The mapping results showed that over three-quarters of CBDs were deployed within a five kilometer radius of a health facility or another CBD, contrary to program planning and design. Other characteristics of the CBD and CBD supervisor profiles (age, gender, literacy) were more closely matched with other regional programs. CONCLUSIONS: The results of this mapping exercise provided a valuable insight into the contradictions found between a program "deployment plan" and the realities observed during field implementation. It also highlighted an important need for program implementers and national-level strategy makers to consider the natural and community-driven diffusion of CBDs, and take into consideration the strength of the local health facilities when developing a deployment plan.

A new role for lipocalin prostaglandin d synthase in the regulation of brown adipose tissue substrate utilization.

In this study, we define a new role for lipocalin prostaglandin D synthase (L-PGDS) in the control of metabolic fuel utilization by brown adipose tissue (BAT). We demonstrate that L-PGDS expression in BAT is positively correlated with BAT activity, upregulated by peroxisome proliferator-activated receptor γ coactivator 1α or 1ß and repressed by receptor-interacting protein 140. Under cold-acclimated conditions, mice lacking L-PGDS had elevated reliance on carbohydrate to provide fuel for thermogenesis and had increased expression of genes regulating glycolysis and de novo lipogenesis in BAT. These transcriptional differences were associated with increased lipid content in BAT and a BAT lipid composition enriched with de novo synthesized lipids. Consistent with the concept that lack of L-PGDS increases glucose utilization, mice lacking L-PGDS had improved glucose tolerance after high-fat feeding. The improved glucose tolerance appeared to be independent of changes in insulin sensitivity, as insulin levels during the glucose tolerance test and insulin, leptin, and adiponectin levels were unchanged. Moreover, L-PGDS knockout mice exhibited increased expression of genes involved in thermogenesis and increased norepinephrine-stimulated glucose uptake to BAT, suggesting that sympathetically mediated changes in glucose uptake may have improved glucose tolerance. Taken together, these results suggest that L-PGDS plays an important role in the regulation of glucose utilization in vivo.

Lipocalin prostaglandin D synthase and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism in vivo.

Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism.

BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions.

Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b(-/-) mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b(-/-) mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT.

Two human ARFGAPs associated with COP-I-coated vesicles.

ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.

Multipoint laser vibrometer for modal analysis.

Experimental modal analysis of multifrequency vibration requires a measurement system with appropriate temporal and spatial resolution to recover the mode shapes. To fully understand the vibration it is necessary to be able to measure not only the vibration amplitude but also the vibration phase. We describe a multipoint laser vibrometer that is capable of high spatial and temporal resolution with simultaneous measurement of 256 points along a line at up to 80 kHz. The multipoint vibrometer is demonstrated by recovering modal vibration data from a simple test object subject to transient excitation. A practical application is presented in which the vibrometer is used to measure vibration on a squealing rotating disk brake.

The alpha- and beta'-COP WD40 domains mediate cargo-selective interactions with distinct di-lysine motifs.

Coatomer is required for the retrieval of proteins from an early Golgi compartment back to the endoplasmic reticulum. The WD40 domain of alpha-COP is required for the recruitment of KKTN-tagged proteins into coatomer-coated vesicles. However, lack of the domain has only minor effects on growth in yeast. Here, we show that the WD40 domain of beta'-COP is required for the recycling of the KTKLL-tagged Golgi protein Emp47p. The protein is degraded more rapidly in cells with a point mutation in the WD40 domain of beta'-COP (sec27-95) or in cells lacking the domain altogether, whereas a point mutation in the Clathrin Heavy Chain Repeat (sec27-1) does not affect the turnover of Emp47p. Lack of the WD40 domain of beta'-COP has only minor effects on growth of yeast cells; however, absence of both WD40 domains of alpha- and beta'-COP is lethal. Two hybrid studies together with our analysis of the maturation of KKTN-tagged invertase and the turnover of Emp47p in alpha- and beta'-COP mutants suggest that the two WD40 domains of alpha- and beta'-COP bind distinct but overlapping sets of di-lysine signals and hence both contribute to recycling of proteins with di-lysine signals.