Transcellular Barriers to Glucose Delivery in the Body.

Glucose is the universal fuel of most mammalian cells, and it is largely replenished through dietary intake. Glucose availability to tissues is paramount for the maintenance of homeostatic energetics and, hence, supply should match demand by the consuming organs. In its journey through the body, glucose encounters cellular barriers for transit at the levels of the absorbing intestinal epithelial wall, the renal epithelium mediating glucose reabsorption, and the tight capillary endothelia (especially in the brain). Glucose transiting through these cellular barriers must escape degradation to ensure optimal glucose delivery to the bloodstream or tissues. The liver, which stores glycogen and generates glucose de novo, must similarly be able to release it intact to the circulation. We present the most up-to-date knowledge on glucose handling by the gut, liver, brain endothelium, and kidney, and discuss underlying molecular mechanisms and open questions. Diseases associated with defects in glucose delivery and homeostasis are also briefly addressed. We propose that the universal problem of sparing glucose from catabolism in favor of translocation across the barriers posed by epithelia and endothelia is resolved through common mechanisms involving glucose transfer to the endoplasmic reticulum, from where glucose exits the cells via unconventional cellular mechanisms.

Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation.

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.

Nucleotides released from palmitate-activated murine macrophages attract neutrophils.

Obesity and elevation of circulating free fatty acids are associated with an accumulation and proinflammatory polarization of macrophages within metabolically active tissues, such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high-fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here we used a cell culture system as proof of concept to show that, upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we found that palmitate up-regulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, shown previously to allow transfer of nucleotides across membranes. These findings suggest that proinflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity.

Sphingolipid changes do not underlie fatty acid-evoked GLUT4 insulin resistance nor inflammation signals in muscle cells.

Ceramides contribute to obesity-linked insulin resistance and inflammation in vivo, but whether this is a cell-autonomous phenomenon is debated, particularly in muscle, which dictates whole-body glucose uptake. We comprehensively analyzed lipid species produced in response to fatty acids and examined the consequence to insulin resistance and pro-inflammatory pathways. L6 myotubes were incubated with BSA-adsorbed palmitate or palmitoleate in the presence of myriocin, fenretinide, or fumonisin B1. Lipid species were determined by lipidomic analysis. Insulin sensitivity was scored by Akt phosphorylation and glucose transporter 4 (GLUT4) translocation, while pro-inflammatory indices were estimated by IκBα degradation and cytokine expression. Palmitate, but not palmitoleate, had mild effects on Akt phosphorylation but significantly inhibited insulin-stimulated GLUT4 translocation and increased expression of pro-inflammatory cytokines Il6 and Ccl2 Ceramides, hexosylceramides, and sphingosine-1-phosphate significantly heightened by palmitate correlated negatively with insulin sensitivity and positively with pro-inflammatory indices. Inhibition of sphingolipid pathways led to marked changes in cellular lipids, but did not prevent palmitate-induced impairment of insulin-stimulated GLUT4 translocation, suggesting that palmitate-induced accumulation of deleterious lipids and insulin resistance are correlated but independent events in myotubes. We propose that muscle cell-endogenous ceramide production does not evoke insulin resistance and that deleterious effects of ceramides in vivo may arise through ancillary cell communication.

Insulin uptake and action in microvascular endothelial cells of lymphatic and blood origin.

Whereas the blood microvasculature constitutes a biological barrier to the action of blood-borne insulin on target tissues, the lymphatic microvasculature might act as a barrier to subcutaneously administrated insulin reaching the circulation. Here, we evaluate the interaction of insulin with primary microvascular endothelial cells of lymphatic [human dermal lymphatic endothelial cells (HDLEC)] and blood [human adipose microvascular endothelial cells (HAMEC)] origin, derived from human dermal and adipose tissues, respectively. HDLEC express higher levels of insulin receptor and signal in response to insulin as low as 2.5 nM, while HAMEC only activate signaling at 100 nM (a dose that blood vessels do not normally encounter). Low insulin acts specifically through the insulin receptor, while supraphysiological insulin acts through both the IR and insulin growth factor-1 receptor. At supraphysiological or injection site-compatible doses pertinent to lymphatic microvessels, insulin enters HAMEC and HDLEC via fluid-phase endocytosis. Conversely, at physiologically circulating doses (0.2 nM) pertinent to blood microvessels, insulin enters HAMEC through a receptor-mediated process requiring IR autophosphorylation but not downstream insulin signaling. At physiological doses, internalized insulin is barely degraded and is instead released intact to the extracellular medium. In conclusion, we document for the first time the mechanism of interaction of insulin with lymphatic endothelial cells, which may be relevant to insulin absorption during therapeutic injections. Furthermore, we describe distinct action and uptake routes for insulin at physiological and supraphysiological doses in blood microvascular endothelial cells, providing a potential explanation for previously conflicting studies on endothelial insulin uptake.

Electrical pulse stimulation induces GLUT4 translocation in C2C12 myotubes that depends on Rab8A, Rab13, and Rab14.

The signals mobilizing GLUT4 to the plasma membrane in response to muscle contraction are less known than those elicited by insulin. This disparity is undoubtedly due to lack of suitable in vitro models to study skeletal muscle contraction. We generated C2C12 myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4 HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4 HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GLUT4 HA (GLUT4 translocation) comparably to insulin or 5-amino imidazole-4-carboxamide ribonucleotide (AICAR). A myosin II inhibitor prevented EPS-stimulated myotube contraction and reduced surface GLUT4 by 56%. EPS stimulated AMPK and CaMKII phosphorylation, and EPS-stimulated GLUT4 translocation was reduced in part by small interfering (si)RNA-mediated AMPKα1/α2 knockdown, compound C, siRNA-mediated Ca2+/calmodulin-dependent protein kinase (CaMKII)δ knockdown, or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS160 and TBC1D1 were phosphorylated in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulin-stimulated GLUT4 translocation, suggesting regulation of GLUT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a, Rab13, or Rab14 reduced, in part, GLUT4 translocation induced by EPS, whereas only Rab8a, or Rab14 knockdown reduced the AICAR response. In conclusion, EPS involves Rab8a, Rab13, and Rab14 to elicit GLUT4 translocation but not Rab10; moreover, Rab10 and Rab13 are not engaged by AMPK activation alone. C2C12-GLUT4 HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.

Deconstructing metabolic inflammation using cellular systems.

Over the past years, we have embarked in a systematic analysis of the effect of obesity or fatty acids on circulating monocytes, microvascular endothelial cells, macrophages, and skeletal muscle cells. With the use of cell culture strategies, we have deconstructed complex physiological systems and then reconstructed "partial equations" to better understand cell-to-cell communication. Through these approaches, we identified that in high saturated fat environments, cell-autonomous proinflammatory pathways are activated in monocytes and endothelial cells, promoting monocyte adhesion and transmigration. We think of this as a paradigm of the conditions promoting immune cell infiltration into tissues during obesity. In concert, it is possible that muscle and adipose tissue secrete immune cell chemoattractants, and indeed, our tissue culture reconstructions reveal that myotubes treated with the saturated fatty acid palmitate, but not the unsaturated fatty acid palmitoleate, release nucleotides that attract monocytes and other compounds that promote proinflammatory classically activated "(M1)-like" polarization in macrophages. In addition, palmitate directly triggers an M1-like macrophage phenotype, and secretions from these activated macrophages confer insulin resistance to target muscle cells. Together, these studies suggest that in pathophysiological conditions of excess fat, the muscle, endothelial and immune cells engage in a synergistic crosstalk that exacerbates tissue inflammation, leukocyte infiltration, polarization, and consequent insulin resistance.

Rac1 governs exercise-stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice.

KEY POINT: Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. The GTPase Rac1 can be activated by muscle contraction and has been found to be necessary for insulin-stimulated glucose uptake, although its role in exercise-stimulated glucose uptake is unknown. We show that Rac1 regulates the translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle during exercise. We find that Rac1 knockout mice display significantly reduced glucose uptake in skeletal muscle during exercise. ABSTRACT: Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. Despite extensive efforts, the signalling mechanisms vital for glucose uptake during exercise are not yet fully understood, although the GTPase Rac1 is a candidate molecule. The present study investigated the role of Rac1 in muscle glucose uptake and substrate utilization during treadmill exercise in mice in vivo. Exercise-induced uptake of radiolabelled 2-deoxyglucose at 65% of maximum running capacity was blocked in soleus muscle and decreased by 80% and 60% in gastrocnemius and tibialis anterior muscles, respectively, in muscle-specific inducible Rac1 knockout (mKO) mice compared to wild-type littermates. By developing an assay to quantify endogenous GLUT4 translocation, we observed that GLUT4 content at the sarcolemma in response to exercise was reduced in Rac1 mKO muscle. Our findings implicate Rac1 as a regulatory element critical for controlling glucose uptake during exercise via regulation of GLUT4 translocation.

Contracting C2C12 myotubes release CCL2 in an NF-κB-dependent manner to induce monocyte chemoattraction.

Muscle inflammation following exercise is characterized by expression of inflammatory cytokines and chemokines. Exercise also increases muscle macrophages derived from circulating monocytes. However, it is unknown whether muscle cells themselves attract circulating monocytes, or what is the underlying mechanism. We used an in vitro system of electrical stimulation (ES) causing C2C12 myotube contraction to explore whether monocyte chemoattraction ensues and investigated the mediating chemoattractants. Conditioned medium from ES-contracted myotubes caused robust chemoattraction of THP-1 monocytes across Boyden chambers. Following ES, expression of several known monocyte chemokines [C-C motif ligand 2 (CCL2) and C-X-C motif ligand (CXCL)1, -2, and -5] was elevated, but of these, only recombinant CCL2 effectively reproduced monocyte migration. Electrically stimulated myotubes secreted CCL2, and neutralization of CCL2 in conditioned medium or antagonizing the CCL2 receptor (CCR2) in THP-1 monocytes inhibited ES-induced monocyte migration. N-benzyl-p-toluene sulfonamide (BTS), a myosin II-ATPase inhibitor, prevented ES-induced myotube contraction but not CCL2 gene expression and secretion. The membrane-permeant calcium chelator BAPTA-AM reduced ES-induced CCL2 secretion. Hence, electrical depolarization, rather than mechanical contraction, drives the rise in CCL2, with partial calcium input. ES activated the NF-κB pathway; NF-κB inhibitors reduced ES-induced CCL2 gene expression and secretion and repressed ES-induced THP-1 chemoattraction. Thus, electrically stimulated myotubes chemoattract monocytes through NF-κB-regulated CCL2 secretion.

Saturated fatty acids activate caspase-4/5 in human monocytes, triggering IL-1ß and IL-18 release.

Obesity is associated with metabolic tissue infiltration by monocyte-derived macrophages. Saturated fatty acids contribute to proinflammatory gene induction in tissue-embedded immune cells. However, it is unknown how circulating monocytes, the macrophage precursors, react to high-fat environments. In macrophages, saturated fatty acids activate inflammatory pathways and, notably, prime caspase-associated inflammasomes. Inflammasome-activated IL-1ß contributes to type 2 diabetes. We hypothesized that 1) human monocytes from obese patients show caspase activation, and 2) fatty acids trigger this response and consequent release of IL-1ß/IL-18. Human peripheral blood monocytes were sorted by flow cytometry, and caspase activity was measured with a FLICA dye-based assay. Blood monocytes from obese individuals exhibited elevated caspase activity. To explore the nature and consequence of this activity, human THP1 monocytes were exposed to saturated or unsaturated fatty acids. Caspase activity was revealed by isoform-specific cleavage and enzymatic activity; cytokine expression/release was measured by qPCR and ELISA. Palmitate, but not palmitoleate, increased caspase activity in parallel to the release of IL-1ß and IL-18. Palmitate induced eventual monocyte cell death with features of pyroptosis (an inflammation-linked cell death program involving caspase-4/5), scored through LDH release, vital dye influx, cell volume changes, and nuclear morphology. Notably, selective gene silencing or inhibition of caspase-4/5 reduced palmitate-induced release of IL-1ß and IL-18. In summary, monocytes from obese individuals present elevated caspase activity. Mechanistically, palmitate activates a pyroptotic program in monocytes through caspase-4/5, causing inflammatory cytokine release, additional to inflammasomes. These caspases represent potential, novel, therapeutic targets to taper obesity-associated inflammation.

Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue infiltration by immune cells.

Cross-talk between skeletal muscle and immune cells: muscle-derived mediators and metabolic implications.

Skeletal muscles contain resident immune cell populations and their abundance and type is altered in inflammatory myopathies, endotoxemia or different types of muscle injury/insult. Within tissues, monocytes differentiate into macrophages and polarize to acquire pro- or anti-inflammatory phenotypes. Skeletal muscle macrophages play a fundamental role in repair and pathogen clearance. These events require a precisely regulated cross-talk between myofibers and immune cells, involving paracrine/autocrine and contact interactions. Skeletal muscle also undergoes continuous repair as a result of contractile activity that involves participation of myokines and anti-inflammatory input. Finally, skeletal muscle is the major site of dietary glucose disposal; therefore, muscle insulin resistance is essential to the development of whole body insulin resistance. Notably, muscle inflammation is emerging as a potential contributor to insulin resistance. Recent reports show that inflammatory macrophage numbers within muscle are elevated during obesity and that muscle cells in vitro can mount autonomous inflammatory responses under metabolic challenge. Here, we review the nature of skeletal muscle inflammation associated with muscle exercise, damage, and regeneration, endotoxin presence, and myopathies, as well as the new evidence of local inflammation arising with obesity that potentially contributes to insulin resistance.

Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells.

Skeletal muscle is the primary site of dietary glucose disposal, a function that depends on insulin-mediated exocytosis of GLUT4 vesicles to its cell surface. In skeletal muscle and adipocytes, this response involves Akt signaling to the Rab-GAP (GTPase-activating protein) AS160/TBC1D4. Intriguingly, the AS160-targeted Rabs appear to differ, with Rab8A participating in GLUT4 exocytosis in muscle cells and Rab10 in adipocytes, and their activation by insulin is unknown. Rabs 8A, 10, and 13 belong to the same subfamily of Rab-GTPases. Here we show that insulin promotes GTP loading of Rab13 and Rab8A but not Rab10 in rat L6 muscle cells, Rab8A activation preceding that of Rab13. siRNA-mediated Rab13 knockdown blocked the insulin-induced increase of GLUT4 at the muscle cell surface that was rescued by a Rab13 ortholog but not by Rab8A. Constitutively active AS160 lowered basal and insulin-stimulated levels of surface GLUT4, effects that were reversed by overexpressing Rab8A or Rab13, suggesting that both Rabs are targets of AS160-GAP activity in the context of GLUT4 traffic. Rab13 had a broader intracellular distribution compared with the perinuclear restriction of Rab8A, and insulin promoted Rab13 colocalization with GLUT4 at the cell periphery. We conclude that Rab13 and Rab8A are Rab-GTPases activated by insulin, and that downstream of AS160 they regulate traffic of GLUT4 vesicles, possibly acting at distinct steps and sites. These findings close in on the series of events regulating muscle GLUT4 traffic in response to insulin, crucial for whole-body glucose homeostasis.

Measurement of GLUT4 Traffic to and from the Cell Surface in Muscle Cells.

Elevated blood glucose following a meal is cleared by insulin-stimulated glucose entry into muscle and fat cells. The hormone increases the amount of the glucose transporter GLUT4 at the plasma membrane in these tissues at the expense of preformed intracellular pools. In addition, muscle contraction also increases glucose uptake via a gain in GLUT4 at the plasma membrane. Regulation of GLUT4 levels at the cell surface could arise from alterations in the rate of its exocytosis, endocytosis, or both. Hence, methods that can independently measure these traffic parameters for GLUT4 are essential to understanding the mechanism of regulation of membrane traffic of the transporter. Here, we describe cell population-based assays to measure the steady-state levels of GLUT4 at the cell surface, as well as to separately measure the rates of GLUT4 endocytosis and endocytosis. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Measuring steady-state cell surface GLUT4myc Basic Protocol 2: Measuring steady-state cell surface GLUT4-HA Basic Protocol 3: Measuring GLUT4myc endocytosis Basic Protocol 4: Measuring GLUT4myc exocytosis.

Muscle cells challenged with saturated fatty acids mount an autonomous inflammatory response that activates macrophages.

Obesity is associated with chronic low-grade inflammation. Within adipose tissue of mice fed a high fat diet, resident and infiltrating macrophages assume a pro-inflammatory phenotype characterized by the production of cytokines which in turn impact on the surrounding tissue. However, inflammation is not restricted to adipose tissue and high fat-feeding is responsible for a significant increase in pro-inflammatory cytokine expression in muscle. Although skeletal muscle is the major disposer of dietary glucose and a major determinant of glycemia, the origin and consequence of muscle inflammation in the development of insulin resistance are poorly understood.We used a cell culture approach to investigate the vectorial crosstalk between muscle cells and macrophages upon exposure to physiological, low levels of saturated and unsaturated fatty acids. Inflammatory pathway activation and cytokine expression were analyzed in L6 muscle cells expressing myc-tagged GLUT4 (L6GLUT4myc) exposed to 0.2 mM palmitate or palmitoleate. Conditioned media thereof, free of fatty acids, were then tested for their ability to activate RAW264.7 macrophages.Palmitate -but not palmitoleate- induced IL-6, TNFα and CCL2 expression in muscle cells, through activation of the NF-κB pathway. Palmitate (0.2 mM) alone did not induce insulin resistance in muscle cells, yet conditioned media from palmitate-challenged muscle cells selectively activated macrophages towards a pro-inflammatory phenotype.These results demonstrate that low concentrations of palmitate activate autonomous inflammation in muscle cells to release factors that turn macrophages pro-inflammatory. We hypothesize that saturated fat-induced, low-grade muscle cell inflammation may trigger resident skeletal muscle macrophage polarization, possibly contributing to insulin resistance in vivo.

Dynamic glucose uptake, storage, and release by human microvascular endothelial cells.

Endothelia determine blood-to-tissue solute delivery, yet glucose transit is poorly understood. To illuminate mechanisms, we tracked [3H]-2-deoxyglucose (2-DG) in human adipose-tissue microvascular endothelial cells. 2-DG uptake was largely facilitated by the glucose transporters GLUT1 and GLUT3. Once in the cytosol, >80% of 2-DG became phosphorylated and ∼20% incorporated into glycogen, suggesting that transported glucose is readily accessible to cytosolic enzymes. Interestingly, a fraction of intracellular 2-DG was released over time (15-20% over 30 min) with slower kinetics than for uptake, involving GLUT3. In contrast to intracellular 2-DG, the released 2-DG was largely unphosphorylated. Glucose release involved endoplasmic reticulum-resident translocases/phosphatases and was stimulated by adrenaline, consistent with participation of glycogenolysis and glucose dephosphorylation. Surprisingly, the fluorescent glucose derivative 2-NBD-glucose (2-NBDG) entered cells largely via fluid phase endocytosis and exited by recycling. 2-NBDG uptake was insensitive to GLUT1/GLUT3 inhibition, suggesting poor influx across membranes. 2-NBDG recycling, but not 2-DG efflux, was sensitive to N-ethyl maleimide. In sum, by utilizing radioactive and fluorescent glucose derivatives, we identified two parallel routes of entry: uptake into the cytosol through dedicated glucose transporters and endocytosis. This reveals the complex glucose handling by endothelial cells that may contribute to glucose delivery to tissues.

DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling.

Cell proliferation is dependent on growth factors insulin and IGF1. We sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID), we detected 40 proteins displaying proximal interactions with IRS1, including DCAF7 and its interacting partners DYRK1A and DYRK1B. In HepG2 cells, DCAF7 knockdown attenuated cell proliferation by inducing cell cycle arrest at G2. DCAF7 expression was required for insulin-stimulated AKT phosphorylation, and its absence promoted nuclear localization of the transcription factor FOXO1. DCAF7 knockdown induced expression of FOXO1-target genes implicated in G2 cell cycle inhibition, correlating with G2 cell cycle arrest. In Drosophila melanogaster, wing-specific knockdown of DCAF7/wap caused smaller wing size and lower wing cell number; the latter recovered upon double knockdown of wap and dfoxo. We propose that DCAF7 regulates cell proliferation and cell cycle via IRS1-FOXO1 signaling, of relevance to whole organism growth.

Erratum: DCAF7 regulates cell proliferation through IRS1-FOXO1 signaling.

[This corrects the article DOI: 10.1016/j.isci.2022.105188.].

Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity.

During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes by in vivo and ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD in mice. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, Ly6Chigh skewing was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

PKCε regulates contraction-stimulated GLUT4 traffic in skeletal muscle cells.

The signaling pathways that stimulate glucose uptake in response to muscle contraction are not well defined. Recently, we showed that carbachol, an acetylcholine analog, stimulates contraction of C2C12 myotube cultures and the rapid arrival of myc-epitope tagged GLUT4 glucose transporters at the cell surface. Here, we explore a role for protein kinase C (PKC) in regulating GLUT4 traffic. Cell surface carbachol-induced GLUT4myc levels were partly inhibited by the conventional/novel PKC inhibitors GF-109203X, Gö6983, and Ro-31-8425 but not by the conventional PKC inhibitor Gö6976. C2C12 myotubes expressed several novel isoforms of PKC mRNA with PKCδ and PKCε in greater abundance. Carbachol stimulated phosphorylation of PKC isoforms and translocation of PKCδ and PKCε to membranes within 5 min. However, only a peptidic inhibitor of PKCε translocation (myristoylated-EAVSLKPT), but not one of PKCδ (myristoylated-SFNSYELGSL), prevented the GLUT4myc response to carbachol. Significant participation of PKCε in the carbachol-induced gain of GLUT4myc at the surface of C2C12 myotubes was further supported through siRNA-mediated PKCε protein knockdown. These findings support a role for novel PKC isoforms, especially PKCε, in contraction-stimulated GLUT4 traffic in muscle cells.