Adipose Triglyceride Lipase Is Implicated in Fuel- and Non-fuel-stimulated Insulin Secretion

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous β-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL^−/− mice indicated the presence of other TG lipase(s) in the β-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K_ATP-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL^−/− mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL^−/− mice. Accordingly, isolated islets from ATGL^−/− mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL^−/− islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.Free fatty acids (FFA)⁵ and other lipid molecules are important for proper glucose-stimulated insulin secretion (GSIS) by β-cells. Thus, deprivation of fatty acids (FA) in vivo (1) diminishes GSIS, whereas a short term exposure to FFA enhances it (1–3). In contrast, a sustained provision of FA, particularly in the presence of high glucose in vitro, is detrimental to β-cells in that it reduces insulin gene expression (4) and secretion (5) and induces β-cell apoptosis (6). The FA supply to the β-cells can be from exogenous sources, such as plasma FFAs and lipoproteins, or endogenous sources, such as intracellular triglyceride (TG) stores. Studies from our laboratory (7–10) and others (11, 12) support the concept that the hydrolysis of endogenous TG plays an important role in fuel-induced insulin secretion because TG depletion with leptin (13) or inhibition of TG lipolysis by lipase inhibitors such as 3,5-dimethylpyrazole (7) or orlistat (11, 12) markedly curtail GSIS in rat islets. Furthermore, mice with β-cell-specific knock-out of hormone-sensitive lipase (HSL), which hydrolyzes both TG and diacylglycerol (DAG), show defective first phase GSIS in vivo and in vitro (14).Lipolysis is an integral part of an essential metabolic pathway, the TG/FFA cycle, in which FFA esterification onto a glycerol backbone leading to the synthesis of TG is followed by its hydrolysis with the release of the FFA that can then be re-esterified. Intracellular TG/FFA cycling is known to occur in adipose tissue of rats and humans (15, 16) and also in liver and skeletal muscle (17). It is generally described as a “futile cycle” as it leads to the net hydrolysis of ATP with the generation of heat (18). However, several studies have shown that this cycle has important functions in the cell. For instance, in brown adipose tissue, it contributes to overall thermogenesis (17, 19). In islets from the normoglycemic, hyperinsulinemic, obese Zucker fatty rat, increased GSIS is associated with increased glucose-stimulated lipolysis and FA esterification, indicating enhanced TG/FFA cycling (10). Stimulation of lipolysis by glucose has also been observed in isolated islets from normal rats (12) and HSL^−/− mice (8) indicating the presence of glucose-responsive TG/FFA cycling in pancreatic β-cells.The identity of the key lipases involved in the TG/FFA cycle in pancreatic islets is uncertain. HSL is expressed in islets (20), is up-regulated by long term treatment with elevated glucose (21), and is associated with insulin secretory granules (22). In addition, our earlier results suggested that elevated HSL expression correlates with augmented TG/FFA cycling in islets of Zucker fatty rats (10). However, it appears that other lipases may contribute to lipolysis and the regulation of GSIS in islet tissue. Thus, results from studies using HSL^−/− mice showed unaltered GSIS (8, 23), except in fasted male mice (8, 9) in which lipolysis was decreased but not abolished. Furthermore, HSL^−/− mice show residual TG lipase activity (8) indicating the presence of other TG lipases.Recently, adipocyte triglyceride lipase (ATGL; also known as Desnutrin, TTS-2, iPLA₂-ζ, and PNPLA2) (24–26) was found to account for most if not all of the residual lipolysis in HSL^−/− mice (26, 27). Two homologues of ATGL, Adiponutrin and GS2, have been described in adipocytes (24). All three enzymes contain a patatin-like domain with broad lipid acyl-hydrolase activity. However, it is not known if adiponutrin and GS2 are actually TG hydrolases. An additional lipase, TG hydrolase or carboxylesterase-3, has been identified in rat adipose tissue (28, 29). Although the hydrolysis of TG is catalyzed by all these lipases, HSL can hydrolyze both TG and DAG, the latter being a better substrate (30).In this study, we observed that besides HSL, ATGL (31), adiponutrin, and GS2 are expressed in rat islets and INS832/13 cells, with ATGL being the most abundant. We then focused on the role of ATGL in fuel-stimulated insulin secretion in two models, INS832/13 β-cells in which ATGL expression was reduced by RNA interference-knockdown (ATGL-KD) and ATGL^−/− mice.     

Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Lipolysis in hMADS Adipocytes

Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes.Adipose tissue fat stores in humans are mainly dependent upon fatty acid (FA)² supply, FA esterification to triglycerides (TG), and TG breakdown, or lipolysis. Adipose tissue lipolysis is governed by three lipases. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) both have the capacity to initiate TG degradation by cleaving the first ester bond, but HSL is unique in its capacity to break down the second ester bond, converting diglycerides (DG) to monoglycerides (1–3). The non-rate-limiting monoglyceride lipase completes lipolysis by cleaving the last ester bond from a monoglyceride molecule, leading to glycerol release (4). Adipose tissue lipolysis has received much attention over the past 10 years because of its altered regulation in obesity (5).HSL resides freely in the cytosol and can associate with lipid droplets (LD). It is regulated by hormones such as catecholamines, insulin, and natriuretic peptides. Catecholamines bind to β-adrenoceptors on adipocyte cell membranes and activate cyclic AMP-dependent protein kinase. Similarly, natriuretic peptides bind to type A receptors and activate cyclic GMP-dependent protein kinase (6). The protein kinase action in stimulated lipolysis is 2-fold: 1) phosphorylation of HSL, leading to its translocation from the cytosol to LD (7, 8), and 2) phosphorylation of perilipin A (6, 9, 10), the predominant perilipin isoform in adipocytes, enhancing interaction between HSL and LD. The importance of HSL activity in stimulating complete lipolysis is indisputable, particularly given its unique capacity to hydrolyze DG. However, lipolysis is not exclusively dependent upon HSL because HSL null mice revealed residual TG lipase activity in adipose tissue (2, 11). Another adipose tissue lipase was identified (3, 12, 13). ATGL, also known as desnutrin or patatin-like phospholipase domain-containing protein 2, shows affinity toward TG only (3, 14). ATGL is activated by CGI-58, an esterase/thioesterase/lipase subfamily protein devoid of TG hydrolase enzymatic activity (15, 16). The role of HSL and ATGL has been investigated in murine fat cell lipolysis, but the relative importance of these lipases in basal and protein kinase A-stimulated human fat cell lipolysis has remained elusive.Increased fat mass is associated with defects in adipose tissue metabolism. In obesity, resistance to catecholamine-induced lipolysis is observed (17–19). This inhibition of lipolysis may be naturally occurring as an adaptive protective mechanism to minimize FA release and its deleterious consequences on metabolism. Indeed, decreased expression of HSL and ATGL has been observed in isolated adipocytes and differentiated preadipocytes of obese subjects and adipose tissue of insulin-resistant subjects, respectively (20–23). However, by virtue of its mass, adipose tissue basal lipolysis elevates circulating levels of FAs in obese subjects, thereby increasing the risk of insulin resistance. Therefore, the use of pharmacological lipid-lowering agents that act through inhibition of lipolysis has been a promising research avenue leading to the development of several series of HSL inhibitors (24).Herein, we sought to examine the respective contributions of HSL and ATGL to lipolysis and re-esterification in fat cells derived from human adipose tissue derived-multipotent stem cells (termed hMADS cells). These cells, which exhibit at a clonal level normal karyotype, self-renewal ability, and no tumorigenicity, are able to differentiate into functional adipocytes (25, 26). We investigated the localization of HSL and ATGL in basal and stimulated lipolytic conditions and studied lipase activities and whole cell lipolysis in adipocytes with altered expression levels of HSL, ATGL, and its coactivator CGI-58. Our results provide novel insights into ATGL localization and its critical role with coactivator CGI-58 in DG provision to HSL during basal and stimulated lipolysis.     

Heparan Sulfate Proteoglycans Are Receptors for the Cell-surface Trafficking and Biological Activity of Transglutaminase-2

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.Transglutaminase type 2 (TG2,² EC 2.3.2.13) is the most widespread member of a large family of enzymes that catalyze the Ca²⁺-dependent post-translational modification of proteins leading to intra- or intermolecular Nϵ(γ-glutamyl)lysine bonds (1, 2). Unlike other family members, TG2 is uniquely exported through a yet to be elucidated non-conventional pathway. Once secreted, TG2 finds in the extracellular compartment the ideal conditions of high Ca²⁺ and low GTP concentration for the activation of its intrinsic transamidation activity (cross-linking) (2, 3). Intracellularly, GTP binding suppresses the Ca²⁺-dependent cross-linking activity and determines the additional GTPase activity of TG2 (4, 5), which is responsible for signal transduction (6). Once externalized, TG2 remains tightly bound to the cell surface and to the extracellular matrix (ECM) (7, 8), and it is rarely found free in the conditioned medium, unless overexpressed by cell transfection (9).Extracellular TG2 activity is involved in the cross-linking of the ECM, conferring resistance to matrix metalloproteinase and promoting cell-matrix interactions via cross-linking of fibronectin (FN) and collagen (1, 7, 11, 12). TG2 has an additional non-enzymatic role in the matrix as an integrin-β₁ co-receptor (8) by supporting RGD-independent cell adhesion to FN (8, 13, 14).Extracellular cross-linking and TG2-mediated adhesion facilitate the repair process in many tissue compartments (1, 2, 15, 16). On the other hand, uncontrolled cross-linking as a consequence of chronic cell insult and secretion of TG2 has been implicated in a number of pathological conditions, including kidney, liver, and pulmonary fibrosis (17–20).Understanding how TG2 is exported and targeted to the cell surface is critical for limiting its cellular secretion and extracellular action. Although a key trigger for TG2 export is cell stress (2, 21, 22), TG2 is not unspecifically released, because extracellular trafficking occurs in the absence of leakage of intracellular components and cells remain viable (23). We know that TG2 requires the tertiary structure of its active site region to be secreted (9); moreover, TG2 is acetylated on the N terminus (24), a process reported to affect membrane targeting of non-conventional secreted proteins (25). Two main binding partners for TG2, FN and integrin-β1, have both been attributed a possible role in the transport of TG2 to the cell surface (8, 26). FN was shown to co-localize with TG2 once released (26), and integrin-β1 to co-associate with TG2 in cells induced to differentiate (8).TG2 has also long been known to have some affinity for heparin (27, 28), a highly sulfated analogue of heparan sulfate (HS) glycosaminoglycan chains, which are abundant constituents of the cell surface/ECM. HS chains are linear polysaccharides consisting of alternating N-acetylated or N-sulfated glucosamine units (GlcNAc or GlcNS), and uronic acids (glucuronic acid GlcA or iduronic acid IdoA residues) (29), which only exist covalently bound to the core protein of cell-surface proteoglycans (syndecans and glypicans) and secreted proteoglycans (29). Heparin binding is a property common to many ECM proteins (29), but the level of affinity has never been established for TG2, which makes it difficult to estimate the real biological significance of this interaction. Heparan sulfate proteoglycans (HSPG) bind ECM ligands through the HS chains, influencing their biological activity, trafficking, and secretion. Among the HSPG subfamilies, the syndecans act as co-receptors for both ECM components and soluble ligands (30), and syndecan-4 has overlapping roles with extracellular TG2 in wound healing and fibrosis (31, 32). In this study, we show that TG2 has a surprisingly high affinity for heparin and HS, raising the hypothesis that HSPG are involved in its biological activity. We demonstrate that HSPGs are essential for the transamidating activity of TG2 at the cell surface and that syndecan-4 acts as a receptor for TG2, which is involved in the trafficking and cell-surface localization, and thus activity of TG2.     

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.Caspases, a family of evolutionarily conserved proteases, mediate apoptosis, inflammation, proliferation, and differentiation by cleaving many cellular substrates (1–3). The apoptotic initiator caspases (caspase-8, -9, and -10) are activated in large signaling platforms and propagate the death signal by cleavage-induced activation of executioner caspase-3 and -7 (4, 5). Most of the cleavage events occurring during apoptosis have been attributed to the proteolytic activity of these two executioner caspases, which can act on several hundreds of proteins (2, 3, 6, 7). The substrate degradomes of the two main executioner caspases have not been determined but their identification is important to gaining greater insight in their cleavage specificity and biological functions.The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries (8), proteome-derived peptide libraries (9), and sets of individual peptide substrates (10, 11). The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspase-activated DNase, keratin 18, PARP,1 protein-disulfide isomerase, and Rho kinase I (for reviews, see Refs. 2, 3, and 7). This propagated the view that these two caspases have completely redundant functions during apoptosis. Surprisingly, mice deficient in one of these caspases (as well as mice deficient in both) have distinct phenotypes. Depending on the genetic background of the mice, caspase-3-deficient mice either die before birth (129/SvJ) or develop almost normally (C57BL/6J) (12–14). This suggests that dynamics in the genetic background, such as increased caspase-7 expression, compensate for the functional loss of caspase-3 (15). In the C57BL/6J background, caspase-7 single deficient mice are also viable, whereas caspase-3 and -7 double deficient mice die as embryos, further suggesting redundancy (12–14). However, because caspase-3 and -7 probably arose from gene duplication between the Cephalochordata-Vertebrata diversion (16), they might have acquired different substrate specificities during evolution. Caspase-3 and -7 do exhibit different activities on a few arbitrarily identified natural substrates, including BID, X-linked inhibitor of apoptosis protein, gelsolin, caspase-6, ataxin-7, and co-chaperone p23 (17–20). In addition, caspase-3 generally cleaves more substrates during apoptosis than caspase-7 and therefore appears to be the major executioner caspase. Moreover, a recent report describing caspase-1-dependent activation of caspase-7, but not of caspase-3, in macrophages in response to microbial stimuli supports the idea of a non-redundant function for caspase-7 downstream of caspase-1 (21).Commercially available “caspase-specific” tetrapeptide substrates are widely used for specific caspase detection, but they display substantial promiscuity and cannot be used to monitor individual caspases in cells (22, 23). Detecting proteolysis by measuring the release of C-terminal fluorophores, such as 7-amino-4-methylcoumarin (amc), restricts the specificity of these peptide substrates to non-prime cleavage site residues, which may have hampered the identification of specific cleavage events. To address this limitation, a recently developed proteomics technique, called proteomic identification of protease cleavage sites, was used to map both non-prime and prime preferences for caspase-3 and -7 on a tryptic peptide library (9). However, no clear distinction in peptide recognition motifs between caspase-3 and -7 could be observed (9). Because not all classical caspase cleavage sites are processed (7), structural or post-translational higher order constraints are likely involved in steering the cleavage site selectivity. Peptide-based approaches generally overlook such aspects.We made use of the COFRADIC N-terminal peptide sorting methodology (24–26) to profile proteolytic events of caspase-3 and -7 in a macrophage proteome labeled by triple stable isotope labeling by amino acids in cell culture (SILAC), which allowed direct comparison of peak intensities in peptide MS spectra and consequent quantification of N termini that are equally, preferably, or exclusively generated by the action of caspase-3 or -7 (26, 27). We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites.     

Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

Dual 12/15- and 5-Lipoxygenase Deficiency in Macrophages Alters Arachidonic Acid Metabolism and Attenuates Peritonitis and Atherosclerosis in ApoE Knock-out Mice

Lipoxygenase (LO) enzymes catalyze the conversion of arachidonic acid (AA) into biologically active lipid mediators. Two members, 12/15-LO and 5-LO, regulate inflammatory responses and have been studied for their roles in atherogenesis. Both 12/15-LO and 5-LO inhibitors have been suggested as potential therapy to limit the development of atherosclerotic lesions. Here we used a genetic strategy to disrupt both 12/15-LO and 5-LO on an apolipoprotein E (apoE) atherosclerosis-susceptible background to study the impact of dual LO blockade in atherosclerosis and inflammation. Resident peritoneal macrophages are the major cell type that expresses both LO enzymes, and we verified their absence in dual LO-deficient mice. Examination of AA conversion by phorbol myristate acetate-primed and A23187-challenged macrophages from dual LO-deficient mice revealed extensive accumulation of AA with virtually no diversion into the most common cyclooxygenase (COX) products measured (prostaglandin E₂ and thromboxane B₂). Instead the COX-1 by-products 11-hydroxy-eicosatetraenoic acid (HETE) and 15-HETE were elevated. The interrelationship between the two LO pathways in combination with COX-1 inhibition (SC-560) also revealed striking patterns of unique substrate utilization. 5-LO- and dual LO-deficient mice exhibited an attenuated response to zymosan-induced peritoneal inflammation, emphasizing roles for 5-LO in regulating vascular permeability. We observed gender-specific attenuation of atheroma formation at 6 months of age at both the aortic root and throughout the entire aorta in chow-fed female dual LO-deficient mice. We propose that some of the inconsistent data obtained with single LO-deficient mice could be attributable to macrophage-specific patterns of altered AA metabolism.Lipoxygenase (LO)² enzymes are an important source of lipid mediators throughout the plant and animal kingdoms (1, 2). In mammals, these mediators are predominantly formed from arachidonic acid (AA) and act in various physiological and pathological contexts (1–3). Accordingly 5-LO and 12/15-LO are two members of the LO family involved in cardiovascular and inflammatory diseases expressed to variable degrees in several cell types of the myeloid lineage, and their expression is strictly regulated and incompletely understood (2, 4, 5). Despite considerable structural homology between 5-LO and 12/15-LO, both enzymes generate distinct products. The 5-LO metabolite leukotriene (LT) A₄ is precursor to the proinflammatory LTB₄ and cysteinyl LTs, which regulate leukocyte subset-specific chemotaxis (LTB₄) and vascular permeability (cysteinyl LTs), both crucial events during acute peritonitis (1, 6, 7). 12- and 15-HETE, end products synthesized by 12/15-LO, play potential roles in cellular chemotaxis, cancer growth, and inflammation (2, 8). Transcellular interaction products derived from both 12/15-LO and 5-LO, such as lipoxins and maresins, indicate that these enzymes can possess anti-inflammatory activities in innate immunity and the resolution of inflammation (9, 10).In mice, only one cell type is known to express substantial quantities of both 5-LO and 12/15-LO, the peritoneal macrophage (PMΦ) (2, 11, 12). However, differences in subcellular localization, trafficking, and activation (8, 12–16) of these two LOs indicate that they are independently regulated and not functionally coupled. Tissue-resident MΦ (such as PMΦ) represent the first line of defense against invading pathogens and activate the immunological and inflammatory response (17). These phagocytes are capable of elaborating a wide spectrum of bioactive lipid mediators from the LO and cyclooxygenase (COX) pathways. Little is known about the regulation and putative interdependence of these pathways. Some insight was gained using mice lacking 12/15-LO where substrate shunting from the 12/15-LO into the 5-LO pathway was observed (12).The generation of knock-out mice for 12/15-LO (12) and 5-LO (18) has enabled the study of these lipid mediator pathways in models of health and disease. Because 12/15-LO and 5-LO are primarily expressed in distinct hematopoietic cells, their implication in various inflammatory disorders and models of host defense mechanisms have been investigated (2, 3). Atherosclerosis, an inflammatory disease prevalent in societies with high dietary fat intake, is initiated by low density lipoprotein (LDL) retention in the vascular wall (19) and subsequent oxidative modification. This process greatly enhances the LDL atherogenic potential, and intriguingly 12/15-LO can contribute to lipoprotein oxidation (11, 20). Initial studies using 12/15-LO- and 5-LO-deficient mice indicated proatherogenic roles for these enzymes (20, 21). Additionally mice lacking the LTB₄ receptor BLT-1 exhibit protection in early atherogenesis (22), but subsequent data from our laboratory using 5-LO-deficient mouse models have not supported an involvement of 5-LO in atherogenesis (3, 23, 24). Here we studied the consequences of simultaneous 12/15-LO and 5-LO knock-out on peritoneal inflammation and atherosclerosis in apoE-deficient mice and surmised whether some of the capricious results in atherosclerotic lesion studies could be attributable to variable eicosanoid profiles.     

Differential virus replication, cytokine production, and antigen-presenting function by microglia from susceptible and resistant mice infected with Theiler's virus

Infection with Theiler''s murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) causes an immune system-mediated demyelinating disease similar to human multiple sclerosis in susceptible but not resistant strains of mice. To understand the underlying mechanisms of differential susceptibility, we analyzed viral replication, cytokine production, and costimulatory molecule expression levels in microglia and macrophages in the CNS of virus-infected resistant C57BL/6 (B6) and susceptible SJL/J (SJL) mice. Our results indicated that message levels of TMEV, tumor necrosis factor alpha, beta interferon, and interleukin-6 were consistently higher in microglia from virus-infected SJL mice than in those from B6 mice. However, the levels of costimulatory molecule expression, as well as the ability to stimulate allogeneic T cells, were significantly lower in TMEV-infected SJL mice than in B6 mice. In addition, microglia from uninfected naïve mice displayed differential viral replication, T-cell stimulation, and cytokine production, similar to those of microglia from infected mice. These results strongly suggest that different levels of intrinsic susceptibility to TMEV infection, cytokine production, and T-cell activation ability by microglia contribute to the levels of viral persistence and antiviral T-cell responses in the CNS, which are critical for the differential susceptibility to TMEV-induced demyelinating disease between SJL and B6 mice.BeAn and DA are members of Theiler''s original subgroup of Theiler''s murine encephalitis virus (TMEV) (52). Intracerebral inoculation of susceptible mice, such as SJL/J (SJL) mice, with either of these viruses results in a biphasic disease characterized by early encephalitis and late chronic demyelination (24). Infection of susceptible mice with these viruses results in a chronic, white matter-demyelinating disease similar to human multiple sclerosis (24). In susceptible strains, infection of the central nervous system (CNS) with TMEV leads to a chronic immune response to viral antigens, which eventually leads to autoimmune responses against myelin autoantigens (29). In contrast, resistant mouse strains, such as C57BL/6 (B6), rapidly clear virus from the CNS and do not develop demyelinating disease, suggesting that viral persistence in these mice corresponds to susceptibility to disease (26, 42, 45). Demyelination in susceptible mice is considered to be immunity mediated, as removal of immune components reduces the clinical onset and severity of demyelinating disease (9, 25, 44, 47).In particular, infiltration of proinflammatory CD4⁺ Th1-type cells has been associated with tissue destruction and demyelination (41, 56). A number of CD4⁺ T cells specific for TMEV during the course of disease in SJL mice recognize four predominant viral capsid epitopes (VP1_233-250, VP2_74-86, VP3_24-37, and VP4_51-70), with one each on the external and internal capsid proteins (10, 19, 55, 56). The external capsid epitopes appear to account for the majority (∼80%) of major histocompatibility complex (MHC) class II-restricted T-cell responses to TMEV capsid proteins (55, 57). Recently, viral capsid epitopes recognized by CNS-infiltrating CD4⁺ T cells from TMEV-infected B6 mice have also been identified (18). When levels of virus capsid-specific CD4⁺ T cells in the CNS are compared between B6 and SJL mice at early stages of viral infection, significantly higher levels are found in the CNS of resistant B6 mice (30), suggesting that virus-specific CD4⁺ T cells are important for protection from demyelinating disease during initial immune responses (2). Similarly, levels of CNS-infiltrating virus-specific CD8⁺ T cells in the CNS are as much as threefold higher in resistant mice at the same time point (28). Therefore, it appears that levels of both initial CD4⁺ and CD8⁺ T-cell responses to the virus are critically important in setting the stage of viral persistence/clearance and consequent susceptibility or resistance to inflammatory demyelinating disease.In order to further understand the potential mechanisms of differences in susceptibility and antiviral immunity levels between SJL and B6 mice, the properties of resident microglial cells and infiltrating macrophages in the CNS during the early stage of viral infection in these mouse strains were investigated. It has previously been established that nonprofessional antigen-presenting cells (APCs; mainly microglial cells and astrocytes) isolated from the CNS of TMEV-infected SJL mice are capable of presenting antigens to both TMEV- and CNS autoantigen-specific T-cell hybridomas and clones (21, 33, 37). Furthermore, microglial cells and/or infiltrating macrophages in the CNS are known to be a major cell population supporting viral persistence during chronic infection (4). Hence, these cells support the replication of TMEV and the activation and/or differentiation of CD4⁺ and CD8⁺ T cells infiltrating the CNS of virus-infected mice. Therefore, CNS APCs involved in triggering T-cell responses and harboring viral persistence may ultimately determine susceptibility/resistance to TMEV-IDD via their effects on virus clearance/persistence as well as on target tissue inflammation.In this study, we compared the potential roles of microglia and macrophages from TMEV-infected susceptible SJL and resistant B6 mice in the innate and adaptive immune responses affecting viral persistence and ultimate disease susceptibility. Our results indicate that viral replication and cytokine production levels are consistently higher in microglia from TMEV-infected SJL mice than in those from B6 mice. In addition, the expression of costimulatory molecules is significantly higher in resistant B6 mice throughout the course of viral infection, suggesting more efficient T-cell activation in resistant B6 mice. On the other hand, both virus replication and type I interferon (IFN) production in microglia from naïve SJL mice are significantly higher than those in such cells from naïve B6 mice. Therefore, differences in the intrinsic properties of microglia in viral replication and virus-induced innate cytokine production are likely to contribute significantly to viral persistence, cellular infiltration to the CNS, and consequent inflammation, leading to the development of demyelinating disease.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.     

Integrative Proteomic Profiling of Protein Activity and Interactions Using Protein Arrays

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.Proteomics is the large-scale analysis of whole proteins and their role in biological systems. Abundance-based proteomics assigns protein functions in normal and pathological processes by quantification of global differences in protein expression levels (1). This classic approach identifies functional biomarkers by comparing samples from healthy individuals and patients. However, this abundance-based approach provides only indirect information about protein function (2). The abundance of a protein is not necessarily correlated with its activity because protein activities are predominantly regulated by a series of post-translational modifications (1, 2). Activity-based proteomics (activity-based protein profiling) is therefore considered an alternative approach to assigning protein functions in biological processes of interest (3). In this approach, specific activity-based probes using fluorescent, radioactive, and affinity tags are usually designed for detection of protein activity (2, 4–6). Activity-based proteomics identifies markers by comparative analyses of activity profiles between healthy and diseased cells and tissues (3, 7, 8). This approach is also used for profiling enzyme inhibitors, for developing therapeutic reagents, and for diagnosis (2, 5). Another functional proteomic approach using large-scale analysis is interactomics or interaction proteomics, which is a useful method for understanding the regulation of proteins in biological systems (9). To elucidate bioactive protein interactions with proteins or ligands, a number of technologies are currently used including the yeast two-hybrid system, affinity purification and mass spectrometry, the protein fragment complementation assay, the luminescence-based mammalian interactome, and protein arrays (9–13). Global differences in the dynamics of the interactome between healthy and diseased individuals provide new insights into causes of disease and can be used for biomarker identification and drug discovery (14–16). Thus, combinatory analyses of abundance, activity, and interaction have great potential in revealing regulation mechanisms and functions of proteins, although such an integrated proteomic approach has not been widely used.These proteomic methods have been coupled with various detection methods including one- or two-dimensional gel electrophoresis, one- or two-dimensional liquid chromatography and tandem mass spectrometry, surface plasmon resonance, and fluorometric assays for analyses of the proteome (9, 11). In combination with specific probes, colorimetric and fluorometric assays using multiwell plates have been extensively used for the determination of the abundance and activity of various proteins. Although often limited by the amount of sample, these methods nonetheless facilitate real-time measurement of changes in protein activity and high-throughput analyses of protein abundances and activities (17). Surface plasmon resonance, a method that does not necessitate labeling of proteins, has also been used for analysis of protein abundance, activity, and binding affinity (18–20). Using only very small amounts of sample, the microarray combined with fluorometric probes is a promising technology for the rapid analysis of a wide variety of biomolecular interactions, protein abundances, and activities. This approach has been used for serodiagnosis and identification of biomarkers by abundance-based protein profiling in human sera (21–25). It has also been used for kinetic studies of carbohydrate-protein (17) and peptide-protein interactions (26, 27). In addition, this technology has been used for the rapid determination of enzyme activities and for the identification of enzyme substrates and inhibitors (24, 28–33). However, combinatory profiling of protein activities and interactions based on array technology has yet to be reported.Using protein arrays, we propose as a model system an integrative proteomic approach for simultaneous profiling of the transamidating activity and interactions of transglutaminase 2 (TG2) with TG2-related proteins. TG2, known as tissue transglutaminase, is a member of the calcium-dependent transglutaminase family. Its activity and interactions are associated with a wide variety of diseases and cellular events (34). TG2 is implicated in the pathogenesis of a wide variety of diseases including inflammatory diseases such as celiac sprue, neurodegenerative disorders such as Huntington''s, Alzheimer''s, and Parkinson''s disease, as well as cancers, cardiovascular diseases, and diabetes (34–36). TG2 is also involved in various cellular events including cell growth, cell differentiation, cell adhesion, extracellular matrix crosslinking, and apoptosis (34, 37, 38). In the present study, the transamidating activity and binding affinity of TG2 for 16 proteins were simultaneously monitored using Cy5-conjugated TG2 and protein arrays (Fig. 1). Using this large-scale analysis, we constructed a quantitative activity-interaction (AI)¹ map to describe the quantitative interaction of TG2 with its related proteins. Thus, this integrative proteomic approach can be used to characterize functions and regulation mechanisms of a target protein in many biological processes of interest.Open in a separate windowFig. 1.Schematic diagram for the simultaneous analysis of transamidating activity and interaction of TG2 with TG2-related proteins. BAPA, 5-(biotinamido)pentylamine; Pr, protein; SA, streptavidin; TG2, transglutaminase 2.     

Adipose Triglyceride Lipase Deficiency Causes Tissue-specific Changes in Insulin Signaling

Triacylglycerol accumulation in insulin target tissues is associated with insulin resistance. Paradoxically, mice with global targeted deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triacylglycerol hydrolysis, display improved glucose tolerance and insulin sensitivity despite triacylglycerol accumulation in multiple tissues. To determine the molecular mechanisms for this phenotype, ATGL-deficient (ATGL^−/−) and wild-type mice were injected with saline or insulin (10 units/kg, intraperitoneally), and then phosphorylation and activities of key insulin-signaling proteins were determined in insulin target tissues (liver, adipose tissue, and muscle). Insulin signaling and/or glucose transport was also evaluated in isolated adipocytes and skeletal muscle ex vivo. In ATGL^−/− mice, insulin-stimulated phosphatidylinositol 3-kinase and Akt activities as well as phosphorylation of critical residues of IRS1 (Tyr(P)-612) and Akt (Ser(P)-473) were increased in skeletal muscle in vivo. Insulin-stimulated phosphatidylinositol 3-kinase activity and total insulin receptor and insulin receptor substrate 1, but not other parameters, were also increased in white adipose tissue in vivo. In contrast, in vivo measures of insulin signaling were decreased in brown adipose tissue and liver. Interestingly, the enhanced components of insulin signaling identified in skeletal muscle and white adipose tissue in vivo and their expected downstream effects on glucose transport were not present ex vivo. ATGL deficiency altered intramyocellular lipids as well as serum factors known to influence insulin sensitivity. Thus, skeletal muscle, rather than other tissues, primarily contributes to enhanced insulin sensitivity in ATGL^−/− mice in vivo despite triacylglycerol accumulation, and both local and systemic factors contribute to tissue-specific effects of global ATGL deficiency on insulin action.Triacylglycerols (TAGs)⁴ are the predominant form of energy storage in animals. The ability to store and release this energy in response to variable energy availability requires a carefully regulated balance between TAG synthesis and hydrolysis. In the setting of chronic energy excess, however, TAGs and other lipid metabolites accumulate in adipose tissue as well as in metabolically relevant non-adipose tissues where they have been proposed to contribute to cellular dysfunction via a process known as lipotoxicity (1–3). Indeed, intracellular TAG accumulation has been repeatedly associated with metabolic dysfunction, a relationship that is particularly strong for insulin resistance (1–3). Despite this strong association, however, intracellular TAG accumulation is not always associated with insulin resistance (4) and may even be associated with insulin sensitivity, as is the case with highly trained endurance athletes (the so-called “athlete paradox”) (5). Thus, the contribution of intracellular TAGs and TAG metabolism per se to lipotoxicity remains controversial. What is clear is that lipid-induced insulin resistance is a major risk factor for morbidity and mortality from a variety of causes, including overt diabetes mellitus, nonalcoholic fatty liver disease, and cardiovascular disease. Hence, understanding the mechanisms by which dysregulated TAG metabolism contributes to steatosis, lipotoxicity, and insulin resistance is essential to understanding and treating these increasingly prevalent disorders.Although no mechanistic data have been identified directly linking intracellular TAGs per se to insulin resistance, lipotoxicity may occur when the capacity of the lipid droplets to effectively store TAGs is exceeded. Several other lipid metabolites that are products of TAG hydrolysis (i.e. diacylglyerols (DAGs), fatty acids (FAs), fatty acyl-CoAs (FA-CoAs), and ceramides) have been shown to directly or indirectly interfere with insulin signaling and glucose transport via a variety of mechanisms (6–9). Under normal physiological circumstances, insulin binds to the insulin receptor (IR), thereby triggering its intrinsic protein-tyrosine kinase activity. The subsequent autophosphorylation of several IR tyrosine residues promotes the recruitment and tyrosine phosphorylation of IR substrates (IRSs) followed by activation of phosphatidylinositol 3-kinase (PI3K) and Akt, which in turn promote the pleiotrophic downstream effects of insulin. The above lipid metabolites have been shown to increase serine/threonine phosphorylation and decrease tyrosine phosphorylation of IRS1, decrease serine/threonine phosphorylation of Akt, decrease IRS1-associated PI3K activity and Akt activity, and decrease Glut4 translocation (6–9). Possible mechanisms by which these lipid metabolites may influence glucose homeostasis and insulin action include competition for substrate oxidation, interference with cellular energy sensing, regulation of gene expression, promotion of oxidative stress and mitochondrial dysfunction, and activation of inflammatory and apoptotic pathways (6–9). However, most studies evaluating the role of lipotoxicity in insulin resistance have focused on cellular lipid uptake or oxidation, both of which produce unidirectional changes in intracellular TAGs and other intracellular lipid metabolites and hence do not adequately address the role of intracellular TAGs and TAG metabolism per se to this process.Understanding the role of TAG metabolism in lipotoxicity and insulin resistance has been further complicated by the fact that the rate-limiting enzyme for TAG hydrolysis, adipose triglyceride lipase (ATGL), has only recently been identified (10–12). ATGL has been most extensively studied in adipose tissue where it mediates the hydrolysis of long chain fatty acyl TAGs (10). ATGL is also expressed in other tissues, including liver, muscle, and pancreas (13), where its contribution to tissue-specific and systemic metabolism is less well understood. Mice with global targeted deletion of ATGL (ATGL^−/− mice) have severe defects in TAG hydrolysis, leading to TAG accumulation in virtually all tissues (14). Surprisingly, despite increased adiposity and “ectopic” TAG accumulation, which are characteristically associated with insulin resistance, ATGL^−/− mice paradoxically exhibit enhanced glucose tolerance and insulin sensitivity (14). This finding has largely been attributed to the effect of reduced systemic FA delivery on energy substrate availability (14). However, the contribution of altered tissue-specific insulin action to this phenotype has not been evaluated.ATGL^−/− mice represent a unique model for examining the contribution of intracellular TAG accumulation to glucose homeostasis and insulin action because intracellular TAG accumulation is dissociated from systemic FA delivery, and presumably also from the production/accumulation of other intracellular lipid metabolites. In addition, ATGL^−/− mice differ from the other models in which increased adiposity is paradoxically associated with insulin sensitivity in that enhanced expansion of adipose tissue mass and reduced systemic FA delivery do not protect against ectopic lipid deposition in ATGL^−/− mice (15, 16). The aims of this study were to evaluate the mechanisms by which impaired TAG hydrolysis and intracellular TAG accumulation because of global ATGL deficiency promote whole-body glucose tolerance and insulin sensitivity and to define the contribution of tissue-specific changes in insulin action to this phenotype. Here we demonstrate that global ATGL deficiency in mice not only reduces energy substrate availability but also produces tissue-specific changes in insulin action.