A high-throughput chemical-genetics screen in murine adipocytes identifies insulin-regulatory pathways.

Pathways linking activation of the insulin receptor to downstream targets of insulin have traditionally been studied using a candidate gene approach. To elucidate additional pathways regulating insulin activity, we performed a forward chemical-genetics screen based on translocation of a glucose transporter 4 (Glut4) reporter expressed in murine 3T3-L1 adipocytes. To identify compounds with known targets, we screened drug-repurposing and natural product libraries. We identified, confirmed, and validated 64 activators and 65 inhibitors that acutely increase or rapidly decrease cell-surface Glut4 in adipocytes stimulated with submaximal insulin concentrations. These agents were grouped by target, chemical class, and mechanism of action. All groups contained multiple hits from a single drug class, and several comprised multiple structurally unrelated hits for a single target. Targets include the ß-adrenergic and adenosine receptors. Agonists of these receptors increased and inverse agonists/antagonists decreased cell-surface Glut4 independently of insulin. Additional activators include insulin sensitizers (thiazolidinediones), insulin mimetics, dis-inhibitors (the mTORC1 inhibitor rapamycin), cardiotonic steroids (the Na+/K+-ATPase inhibitor ouabain), and corticosteroids (dexamethasone). Inhibitors include heterocyclic amines (tricyclic antidepressants) and 21 natural product supplements and herbal extracts. Mechanisms of action include effects on Glut4 trafficking, signal transduction, inhibition of protein synthesis, and dissipation of proton gradients. Two pathways that acutely regulate Glut4 translocation were discovered: de novo protein synthesis and endocytic acidification. The mechanism of action of additional classes of activators (tanshinones, dalbergiones, and coumarins) and inhibitors (flavonoids and resveratrol) remains to be determined. These tools are among the most sensitive, responsive, and reproducible insulin-activity assays described to date.

High density lipoprotein promotes nascent apolipoprotein A-V secretion from mRNA transfected cells.

Despite its exceptionally low circulating concentration, apolipoprotein (apo) A-V is a potent modulator of plasma triacylglycerol levels. The secretion efficiency of nascent apoA-V was investigated in cultured cells transfected with mRNA. Following transfection of HepG2 cells with wild type apoA-V mRNA, apoA-V protein was detectable in cell lysates by 6 h. At 24 h post transfection, evidence of apoA-V secretion into media was obtained, although most apoA-V was recovered in the cell lysate fraction. By contrast, apoA-I was efficiently secreted into the culture medium. A positive correlation between culture medium fetal bovine serum content and the percentage of apoA-V recovered in conditioned media was observed. When transfected cells were cultured in serum-free media supplemented with increasing amounts of high density lipoprotein, a positive correlation with apoA-V secretion was observed. The data indicate that, following signal sequence cleavage, the bulk of nascent apoA-V remains cell associated. Transit of nascent apoA-V out of cultured cells is enhanced by the availability of extracellular lipid particle acceptors.

Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation.

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.

Rab14 limits the sorting of Glut4 from endosomes into insulin-sensitive regulated secretory compartments in adipocytes.

Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptor-related protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin.

Insulin-regulated Glut4 translocation: membrane protein trafficking with six distinctive steps.

The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min(-1) versus 0.6 min(-1)). Differentiation decreases Glut4 ken 40% (ken = 0.12 min(-1)). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025-0.038 min(-1)), not through the fast Tf receptor pathway (kex = 0.2 min(-1)). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min(-1)). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.

Characterization of trans-3-Methylglutaconyl CoA-Dependent Protein Acylation.

3-methylglutaconyl (3MGC) CoA hydratase (AUH) is the leucine catabolism pathway enzyme that catalyzes the hydration of trans-3MGC CoA to 3-hydroxy, 3-methylglutaryl (HMG) CoA. In several inborn errors of metabolism (IEM), however, metabolic dysfunction can drive this reaction in the opposite direction (the dehydration of HMG CoA). The recent discovery that trans-3MGC CoA is inherently unstable and prone to a series of non-enzymatic chemical reactions provides an explanation for 3MGC aciduria observed in these IEMs. Under physiological conditions, trans-3MGC CoA can isomerize to cis-3MGC CoA, which is structurally poised to undergo intramolecular cyclization with the loss of CoA, generating cis-3MGC anhydride. The anhydride is reactive and has two potential fates; (a) hydrolysis to yield cis-3MGC acid or (b) a reaction with lysine side-chain amino groups to 3MGCylate substrate proteins. An antibody elicited against a 3MGC hapten was employed to investigate protein acylation in incubations containing recombinant AUH, HMG CoA, and bovine serum albumin (BSA). The data obtained show that, as AUH dehydrates HMG CoA to trans-3MGC CoA, BSA is acylated. Moreover, α-3MGC IgG immunoblot signal intensity correlates with AUH concentration, HMG CoA substrate concentration, and incubation time. Thus, protein 3MGCylation may contribute to the phenotypic features associated with IEMs that manifest 3MGC aciduria.

Loss of AS160 Akt substrate causes Glut4 protein to accumulate in compartments that are primed for fusion in basal adipocytes.

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulin-mimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k(ex)). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k(ex)), endocytosis (k(en)), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k(ex) or k(en). Insulin increased k(ex) in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k(ex) and release from retention in control cells but only k(ex) in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulin-regulated step.

Kinetic evidence that Glut4 follows different endocytic pathways than the receptors for transferrin and alpha2-macroglobulin.

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and α(2)-macroglobulin (α-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k(ex)) for both Glut4 and Tf. However, the k(ex) of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k(en)) of Glut4 was also five times slower than Tf. Insulin did not affect the k(en) of either protein. In basal cells, the k(en) for α-2-M/LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k(en) for α-2-M/LRP-1 by 30%. In contrast, the k(ex) for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k(en) of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k(en) can be entirely accounted for by the well established increase in Glut4 k(ex).

Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.

Doxorubicin (DOX) is an aqueous soluble anthracycline therapeutic widely used in cancer treatment. Although DOX anti-cancer activity is dose-dependent, increased dosage enhances the risk of cardiotoxicity. Despite intensive investigation, the molecular basis of this undesirable side effect has yet to be established. In addition to serving as a DNA intercalation agent, DOX is known to bind to the signature mitochondrial phospholipid, cardiolipin (CL). Consistent with this, DOX associates with aqueous soluble nanoparticles, termed nanodisks (ND), comprised solely of CL and an apolipoprotein scaffold. Fluorescence microscopy analysis revealed that DOX uptake, and targeting to the nucleus of cultured hepatocarcinoma (HepG2) or breast cancer (MCF7) cells, was unaffected by its association with CL-ND. Subsequent studies revealed that free DOX and DOX-CL-ND were equivalent in terms of growth inhibition activity in both cell lines. By contrast, in studies with H9C2 cardiomyocytes, DOX-CL-ND induced a lesser concentration-dependent decline in cell viability than free DOX. Whereas incubation of H9C2 cardiomyocytes with free DOX caused a steep decline in maximal oxygen consumption rate, DOX-CL-ND treated cells were largely unaffected. The data indicate that association of DOX with CL-ND does not diminish its cancer cell growth inhibition activity yet confers protection to cardiomyocytes from DOX-induced effects on aerobic respiration. This study illustrates that interaction with CL plays a role in DOX-induced mitochondrial dysfunction and suggests CL-ND provide a tool for investigating the mechanistic basis of DOX-induced cardiotoxicity.

Role of non-enzymatic chemical reactions in 3-methylglutaconic aciduria.

3-Methylglutaconic (3MGC) aciduria occurs in numerous inborn errors associated with compromised mitochondrial energy metabolism. In these disorders, 3MGC CoA is produced de novo from acetyl CoA in three steps with the final reaction catalysed by 3MGC CoA hydratase (AUH). In in vitro assays, whereas recombinant AUH dehydrated 3-hydroxy-3-methylglutaryl (HMG) CoA to 3MGC CoA, free CoA was also produced. Although HMG CoA is known to undergo non-enzymatic intramolecular cyclisation, forming HMG anhydride and free CoA, the amount of free CoA generated increased when AUH was present. To test the hypothesis that the AUH-dependent increase in CoA production is caused by intramolecular cyclisation of 3MGC CoA, gas chromatography-mass spectrometry analysis of organic acids was performed. In the absence of AUH, HMG CoA was converted to HMG acid while, in the presence of AUH, 3MGC acid was also detected. To determine which 3MGC acid diastereomer was formed, immunoblot assays were conducted with 3MGCylated BSA. In competition experiments, when α-3MGC IgG was preincubated with trans-3MGC acid or cis-3MGC acid, the cis diastereomer inhibited antibody binding to 3MGCylated BSA. When an AUH assay product mix served as competitor, α-3MGC IgG binding to 3MGCylated BSA was also inhibited, indicating cis-3MGC acid is produced in incubations of AUH and HMG CoA. Thus, non-enzymatic isomerisation of trans-3MGC CoA drives AUH-dependent HMG CoA dehydration and explains the occurrence of cis-3MGC acid in urine of subjects with 3MGC aciduria. Furthermore, the ability of cis-3MGC anhydride to non-enzymatically acylate protein substrates may have deleterious pathophysiological consequences.

Insulin releases Glut4 from static storage compartments into cycling endosomes and increases the rate constant for Glut4 exocytosis.

In adipocytes, insulin triggers the redistribution of Glut4 from intracellular compartments to the plasma membrane. Two models have been proposed to explain the effect of insulin on Glut4 localization. In the first, termed dynamic exchange, Glut4 continually cycles between the plasma membrane and intracellular compartments in basal cells, and the major effect of insulin is through changes in the exocytic and endocytic rate constants, k(ex) and k(en). In the second model, termed static retention, Glut4 is packaged in specialized storage vesicles (GSVs) in basal cells and does not traffic through the plasma membrane or endosomes. Insulin triggers GSV exocytosis, increasing the amount of Glut4 in the actively cycling pool. Using a flow cytometry-based assay, we found that Glut4 is regulated by both static and dynamic retention mechanisms. In basal cells, 75-80% of the Glut4 is packaged in noncycling GSVs. Insulin increased the amount of Glut4 in the actively cycling pool 4-5-fold. Insulin also increased k(ex) in the cycling pool 3-fold. After insulin withdrawal, Glut4 is rapidly cleared from the plasma membrane (t((1/2)) of 20 min) by rapid adjustments in k(ex) and k(en) and recycled into static compartments. Complete recovery of the static pool required more than 3 h, however. We conclude that in fully differentiated confluent adipocytes, both the dynamic and static retention mechanisms are important for the regulation of plasma membrane Glut4 content. However, cell culture conditions affect Glut4 trafficking. For example, replating after differentiation inhibited the static retention of Glut4, which may explain differences in previous reports.

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4.

Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a ;reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.