Identification of a Distal GLUT4 Trafficking Event Controlled by Actin Polymerization

The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking, and fusion of GLUT4 vesicles with the plasma membrane (PM) represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the PM. In this study we have identified a previously uncharacterized role for cortical actin in the distal trafficking of GLUT4. Using high-frequency total internal reflection fluorescence microscopy (TIRFM) imaging, we show that insulin increases actin polymerization near the PM and that disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion, we found that defective actin remodeling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM, but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process.     

Drosophila Adiponectin Receptor in Insulin Producing Cells Regulates Glucose and Lipid Metabolism by Controlling Insulin Secretion

Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.     

Control of triglyceride storage by a WD40/TPR-domain protein

Obesity is a metabolic disorder related to improper control of energy uptake and expenditure, which results in excessive accumulation of body fat. Initial insights into the genetic pathways that regulate energy metabolism have been provided by a discrete number of obesity-related genes that have been identified in mammals. Here, we report the identification of the adipose (adp) gene, the mutation of which causes obesity in Drosophila. Loss of adp activity promotes increased fat storage, which extends the lifespan of mutant flies under starvation conditions. By contrast, adp gain-of-function causes a specific reduction of the fat body in Drosophila. adp encodes an evolutionarily conserved WD40/tetratricopeptide-repeat-domain protein that is likely to represent an intermediate in a novel signalling pathway.     

Role of Fat Body Lipogenesis in Protection against the Effects of Caloric Overload in Drosophila

The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.     

Understanding the distinct subcellular trafficking of CD36 and GLUT4 during the development of myocardial insulin resistance

CD36 and GLUT4 are the main cardiac trans-sarcolemmal transporters for long-chain fatty acids and glucose, respectively. Together they secure the majority of cardiac energy demands. Moreover, these transporters each represent key governing kinetic steps in cardiac fatty acid and glucose fluxes, thereby offering major sites of regulation. The underlying mechanism of this regulation involves a perpetual vesicle-mediated trafficking (recycling) of both transporters between intracellular stores (endosomes) and the cell surface. In the healthy heart, CD36 and GLUT4 translocation to the cell surface is under short-term control of the same physiological stimuli, most notably increased contraction and insulin secretion. However, under chronic lipid overload, a condition that accompanies a Western lifestyle, CD36 and GLUT4 recycling are affected distinctly, with CD36 being expelled to the sarcolemma while GLUT4 is imprisoned within the endosomes. Moreover, the increased CD36 translocation towards the cell surface is a key early step, setting the heart on a route towards insulin resistance and subsequent contractile dysfunction. Therefore, the proteins making up the trafficking machinery of CD36 need to be identified with special focus to the differences with the protein composition of the GLUT4 trafficking machinery. These proteins that are uniquely dedicated to either CD36 or GLUT4 traffic may offer targets to rectify aberrant substrate uptake seen in the lipid-overloaded heart. Specifically, CD36-dedicated trafficking regulators should be inhibited, whereas such GLUT4-dedicated proteins would need to be activated. Recent advances in the identification of CD36-dedicated trafficking proteins have disclosed the involvement of vacuolar-type H⁺-ATPase and of specific vesicle-associated membrane proteins (VAMPs). In this review, we summarize these recent findings and sketch a roadmap of CD36 and GLUT4 trafficking compatible with experimental findings.     

Why clone flies? Using Cloned Drosophila to Monitor Epigenetic Defects

The birth of Dolly the sheep in 1996 transformed nuclear transplantation from an arcane technique to a technology at the core of medical biotechnology. Transplantation of nuclei allows the generation of new individuals and is being exploited to create genetically tailored stem cells for replacement tissues and organs. The list of cloned animals reared to adulthood currently includes the frog, sheep, mouse, cow, goat, pig, rabbit, cat, zebrafish, mule, horse, rat and dog. The addition of Drosophila to this elite bestiary of cloned animals has prompted the question – why clone flies? Organisms generated by nuclear transplantation suffer from a high rate of associated defects. This review summarizes recent literature that demonstrates that the majority of these defects, in both mammals and flies, arise from mis-regulation of imprinted genes. The regulation of imprinted genes relies on a suite of highly conserved chromatin-modifying genes, first identified in Drosophila. Thus, Drosophila can be used to rapidly assess epigenetic dysfunction in cloned animals and could be used to screen for genes that allow the production of healthy clones.     

Subcellular localization and trafficking of the GLUT4 glucose transporter isoform in insulin-responsive cells

The rate-limiting step in the uptake and metabolism of Dglucose by insulin target cells is thought to be glucose transport mediated by glucose transporters (primarily the GLUT4 isoform) localized to the plasma membrane. However, subcellular fractionation, photolabelling and immunocytochemical studies have shown that the pool of GLUT4 present in the plasma membrane is only one of many subcellular pools of this protein. GLUT4 has been found in occluded vesicles at the plasma membrane, clathrin-coated pits and vesicles, early endosomes, and tubulo-vesicular structures; the latter are analogous to known specialized secretory compartments. Tracking the movement of GLUT4 through these compartments, and defining the mechanism and site of action of insulin in stimulating this subcellular trafficking, are major topics of current investigation. Recent evidence focuses attention on the exocytosis of GLUT4 as the major site of insulin action. Increased exocytosis may be due to decreased retention of glucose transporters in an intracellular pool, or possibly to increased assembly of a vesicle docking and fusion complex. Although details are unknown, the presence in GLUT4 vesicles of a synaptobrevin homologue leads us to propose that a process analogous to that occurring in synaptic vesicle trafficking is involved in the assembly of GLUT4 vesicles into a form suitable for fusion with the plasma membrane. Evidence that the pathways of signalling from the insulin receptor and of GLUT4 vesicle exocytosis may converge at the level of the key signalling enzyme, phosphatidylinositol 3-kinase, is discussed.     

The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster

Most associations between animals and their gut microbiota are dynamic, involving sustained transfer of food-associated microbial cells into the gut and shedding of microorganisms into the external environment with feces, but the interacting effects of host and microbial factors on the composition of the internal and external microbial communities are poorly understood. This study on laboratory cultures of the fruit fly Drosophila melanogaster reared in continuous contact with their food revealed time-dependent changes of the microbial communities in the food that were strongly influenced by the presence and abundance of Drosophila. When germfree Drosophila eggs were aseptically added to nonsterile food, the microbiota in the food and flies converged to a composition dramatically different from that in fly-free food, showing that Drosophila has microbiota-independent effects on the food microbiota. The microbiota in both the flies that developed from unmanipulated eggs (bearing microorganisms) and the associated food was dominated by the bacteria most abundant on the eggs, demonstrating effective vertical transmission via surface contamination of eggs. Food coinoculated with a four-species defined bacterial community of Acetobacter and Lactobacillus species revealed the progressive elimination of Lactobacillus from the food bearing few or no Drosophila, indicating the presence of antagonistic interactions between Acetobacter and Lactobacillus. Drosophila at high densities ameliorated the Acetobacter/Lactobacillus antagonism, enabling Lactobacillus to persist. This study with Drosophila demonstrates how animals can have major, coordinated effects on the composition of microbial communities in the gut and immediate environment.     

Exploring the whereabouts of GLUT4 in skeletal muscle (review)

The glucose transporter GLUT4 is expressed in muscle, fat cells, brain and kidney. In contrast to other glucose transporters, GLUT4 in unstimulated cells is mostly intracellular. Stimuli such as insulin and muscle contractions then cause the translocation of GLUT4 to the cell surface. Questions related to GLUT4 storage compartments, trafficking to the surface membrane, and nature of the intracellular pools, have kept many groups busy for the past 20 years. Yet, one of the main questions in the field remains the universality of GLUT4 features. Can one extrapolate work done on fat cells to muscle or brain? Or vice-versa? Can one use cultures to predict GLUT4 behaviour in fully differentiated tissues? This review summarizes the authors' knowledge of GLUT4 biology in skeletal muscle, which is the predominant tissue for glucose homeostasis. The results are compared to those obtained with the fat cell system, and an attempt is made to assess the universality principle.     

Activation of the glucose transporter GLUT4 by insulin.

The transport of glucose into cells and tissues is a highly regulated process, mediated by a family of facilitative glucose transporters (GLUTs). Insulin-stimulated glucose uptake is primarily mediated by the transporter isoform GLUT4, which is predominantly expressed in mature skeletal muscle and fat tissues. Our recent work suggests that two separate pathways are initiated in response to insulin: (i) to recruit transporters to the cell surface from intracellular pools and (ii) to increase the intrinsic activity of the transporters. These pathways are differentially inhibited by wortmannin, demonstrating that the two pathways do not operate in series. Conversely, inhibitors of p38 mitogen-activated protein kinase (MAPK) imply that p38 MAPK is involved only in the regulation of the pathway leading to the insulin-stimulated activation of GLUT4. This review discusses the evidence for the divergence of GLUT4 translocation and activity and proposed mechanisms for the regulation of GLUT4.     

GLUT4 Defects in Adipose Tissue Are Early Signs of Metabolic Alterations in Alms1GT/GT,a Mouse Model for Obesity and Insulin Resistance

Dysregulation of signaling pathways in adipose tissue leading to insulin resistance can contribute to the development of obesity-related metabolic disorders. Alström Syndrome, a recessive ciliopathy, caused by mutations in ALMS1, is characterized by progressive metabolic alterations such as childhood obesity, hyperinsulinemia, and type 2 diabetes. Here we investigated the role of Alms1 disruption in AT expansion and insulin responsiveness in a murine model for Alström Syndrome. A gene trap insertion in Alms1 on the insulin sensitive C57BL6/Ei genetic background leads to early hyperinsulinemia and a progressive increase in body weight. At 6 weeks of age, before the onset of the metabolic disease, the mutant mice had enlarged fat depots with hypertrophic adipocytes, but without signs of inflammation. Expression of lipogenic enzymes was increased. Pre-adipocytes isolated from mutant animals demonstrated normal adipogenic differentiation but gave rise to mature adipocytes with reduced insulin-stimulated glucose uptake. Assessment of whole body glucose homeostasis revealed glucose intolerance. Insulin stimulation resulted in proper AKT phosphorylation in adipose tissue. However, the total amount of glucose transporter 4 (SLC4A2) and its translocation to the plasma membrane were reduced in mutant adipose depots compared to wildtype littermates. Alterations in insulin stimulated trafficking of glucose transporter 4 are an early sign of metabolic dysfunction in Alström mutant mice, providing a possible explanation for the reduced glucose uptake and the compensatory hyperinsulinemia. The metabolic signaling deficits either reside downstream or are independent of AKT activation and suggest a role for ALMS1 in GLUT4 trafficking. Alström mutant mice represent an interesting model for the development of metabolic disease in which adipose tissue with a reduced glucose uptake can expand by de novo lipogenesis to an obese state.     

Neuronal function and dysfunction of Drosophila dTDP

Background

TDP-43 is an RNA- and DNA-binding protein well conserved in animals including the mammals, Drosophila, and C. elegans. In mammals, the multi-function TDP-43 encoded by the TARDBP gene is a signature protein of the ubiquitin-positive inclusions (UBIs) in the diseased neuronal/glial cells of a range of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U).

Methodology/Principal Findings

We have studied the function and dysfunction of the Drosophila ortholog of the mammalian TARDBP gene, dTDP, by genetic, behavioral, molecular, and cytological analyses. It was found that depletion of dTDP expression caused locomotion defect accompanied with an increase of the number of boutons at the neuromuscular junctions (NMJ). These phenotypes could be rescued by overexpression of Drosophila dTDP in the motor neurons. In contrast, overexpression of dTDP in the motor neurons also resulted in reduced larval and adult locomotor activities, but this was accompanied by a decrease of the number of boutons and axon branches at NMJ. Significantly, constitutive overexpression of dTDP in the mushroom bodies caused smaller axonal lobes as well as severe learning deficiency. On the other hand, constitutive mushroom body-specific knockdown of dTDP expression did not affect the structure of the mushroom bodies, but it impaired the learning ability of the flies, albeit moderately. Overexpression of dTDP also led to the formation of cytosolic dTDP (+) aggregates.

Conclusion/Significance

These data together demonstrate the neuronal functions of dTDP, and by implication the mammalian TDP-43, in learning and locomotion. The effects of mis-expression of dTDP on Drosophila NMJ suggest that eukaryotic TDP-43 guards against over development of the synapses. The conservation of the regulatory pathways of functions and dysfunctions of Drosophila dTDP and mammalian TDP-43 also shows the feasibility of using the flies as a model system for studying the normal TDP-43 function and TDP-43 proteinopathies in the vertebrates including human.     

The Drosophila homolog of methionine sulfoxide reductase A extends lifespan and increases nuclear localization of FOXO

Methionine sulfoxide reductase A (msrA) was previously found to increase resistance to oxidative stress and longevity in animals. We identified Drosophila msrA (dmsrA), a Drosophila homolog of human msrA, as a downstream effector of forkhead box O (FOXO) signaling in Drosophila, which enhances resistance to oxidative stress and increases survival under stressed conditions. Additionally, overexpression of dmsrA in neurons extended the lifespan of flies. Moreover, overexpression of dmsrA in fat body cells caused FOXO to translocate to the nucleus, implying that this possible positive feedback loop between dmsrA and FOXO could potentiate the antioxidant activity of dmsrA and increase the lifespan in Drosophila.     

Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation

Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.     

Drice restrains Diap2-mediated inflammatory signalling and intestinal inflammation

The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. We have found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. We have found that this complex formation induces its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, we show that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine.Subject terms: Ubiquitins, Gene regulation, Antimicrobial responses, Chronic inflammation, Signal transduction     

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.