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.     

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.     

The Mechanism of nucleotide-assisted molybdenum insertion into molybdopterin. A novel route toward metal cofactor assembly

The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn(2+)-/Mg(2+)-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.     

Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.     

Promoter analysis of MADS-box genes in eudicots through phylogenetic footprinting

The MIKC MADS-box gene family has been shaped by extensive gene duplications giving rise to subfamilies of genes with distinct functions and expression patterns. However, within these subfamilies the functional assignment is not that clear-cut, and considerable functional redundancy exists. One way to investigate the diversity in regulation present in these subfamilies is promoter sequence analysis. With the advent of genome sequencing projects, we are now able to exert a comparative analysis of Arabidopsis and poplar promoters of MADS-box genes belonging to the same subfamily. Based on the principle of phylogenetic footprinting, sequences conserved between the promoters of homologous genes are thought to be functional. Here, we have investigated the evolution of MADS-box genes at the promoter level and show that many genes have diverged in their regulatory sequences after duplication and/or speciation. Furthermore, using phylogenetic footprinting, a distinction can be made between redundancy, neo/nonfunctionalization, and subfunctionalization.     

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.     

Analysis of Triclosan-Selected Salmonella enterica Mutants of Eight Serovars Revealed Increased Aminoglycoside Susceptibility and Reduced Growth Rates

The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPC_TRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10^-10 - 9.9 x 10^-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly₉₃→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly₉₃→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased susceptibility to triclosan.     

Factors Determining Patient-Prosthesis Mismatch after Aortic Valve Replacement – A Prospective Cohort Study

Objective

“Patient-prosthesis mismatch” (PPM) after aortic valve replacement (AVR) has been reported to increase morbidity and mortality. Although algorithms have been developed to avoid PPM, factors favouring its occurrence have not been well defined.

Design and Setting

This was a prospective cohort study performed at the Medical University of Vienna.

Patients

361 consecutive patients who underwent aortic valve replacement for isolated severe aortic stenosis were enrolled.

Main Outcome Measures

Patient- as well as prosthesis-related factors determining the occurrence of moderate and severe PPM (defined as effective orifice area indexed to body surface area ≤ 0.8 cm²/m²) were studied.

Results

Postoperatively, 172 patients (48%) were diagnosed with PPM. The fact that predominantly female patients were affected (58% with PPM diagnosis in women versus 36% in men, p<0.001) was explained by the finding that they had smaller aortic root diameters (30.5±4.7 mm versus 35.3±4.2 mm, p<0.0001) and a higher proportion of bioprosthetic valves (82% versus 62%, p<0.0001), both independent predictors of PPM (aortic root diameter: OR 0.009 [95% CI, 0.004;0.013]; p = 0.0003, presence of bioprosthetic valve: OR 0.126 [95% CI, 0.078;0.175]; p<0.0001).

Conclusions

The occurrence of PPM is determined by aortic root diameter and prosthesis type. Novel sutureless bioprostheses with optimized hemodynamic performance or transcatheter aortic valves may become a promising alternative to conventional bioprosthetic valves in the future.     

Functional Cardiac Lipolysis in Mice Critically Depends on Comparative Gene Identification-58

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues. ATGL gene mutations cause a severe phenotype especially in cardiac muscle leading to cardiomyopathy that can be lethal. In contrast, CGI-58 gene mutations provoke severe ichthyosis and hepatosteatosis in humans and mice, whereas the role of CGI-58 in muscle energy metabolism is less understood. Here we show that mice lacking CGI-58 exclusively in muscle (CGI-58KOM) developed severe cardiac steatosis and cardiomyopathy linked to impaired TG catabolism and mitochondrial fatty acid oxidation. The marked increase in ATGL protein levels in cardiac muscle of CGI-58KOM mice was unable to compensate the lack of CGI-58. The addition of recombinant CGI-58 to cardiac lysates of CGI-58KOM mice completely reconstituted TG hydrolytic activities. In skeletal muscle, the lack of CGI-58 similarly provoked TG accumulation. The addition of recombinant CGI-58 increased TG hydrolytic activities in control and CGI-58KOM tissue lysates, elucidating the limiting role of CGI-58 in skeletal muscle TG catabolism. Finally, muscle CGI-58 deficiency affected whole body energy homeostasis, which is caused by impaired muscle TG catabolism and increased cardiac glucose uptake. In summary, this study demonstrates that functional muscle lipolysis depends on both CGI-58 and ATGL.     

Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance

Monoglyceride lipase (MGL) influences energy metabolism by at least two mechanisms. First, it hydrolyzes monoacylglycerols (MG) into fatty acids and glycerol. These products can be used for energy production or synthetic reactions. Second, MGL degrades 2-arachidonoyl glycerol (2-AG), the most abundant endogenous ligand of cannabinoid receptors (CBR). Activation of CBR affects energy homeostasis by central orexigenic stimuli, by promoting lipid storage, and by reducing energy expenditure. To characterize the metabolic role of MGL in vivo, we generated an MGL-deficient mouse model (MGL-ko). These mice exhibit a reduction in MG hydrolase activity and a concomitant increase in MG levels in adipose tissue, brain, and liver. In adipose tissue, the lack of MGL activity is partially compensated by hormone-sensitive lipase. Nonetheless, fasted MGL-ko mice exhibit reduced plasma glycerol and triacylglycerol, as well as liver triacylglycerol levels indicative for impaired lipolysis. Despite a strong elevation of 2-AG levels, MGL-ko mice exhibit normal food intake, fat mass, and energy expenditure. Yet mice lacking MGL show a pharmacological tolerance to the CBR agonist CP 55,940 suggesting that the elevated 2-AG levels are functionally antagonized by desensitization of CBR. Interestingly, however, MGL-ko mice receiving a high fat diet exhibit significantly improved glucose tolerance and insulin sensitivity in comparison with wild-type controls despite equal weight gain. In conclusion, our observations implicate that MGL deficiency impairs lipolysis and attenuates diet-induced insulin resistance. Defective degradation of 2-AG does not provoke cannabinoid-like effects on feeding behavior, lipid storage, and energy expenditure, which may be explained by desensitization of CBR.