Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

Tracing past human male movements in northern/eastern Africa and western Eurasia: new clues from Y-chromosomal haplogroups E-M78 and J-M12

Detailed population data were obtained on the distribution of novel biallelic markers that finely dissect the human Y-chromosome haplogroup E-M78. Among 6,501 Y chromosomes sampled in 81 human populations worldwide, we found 517 E-M78 chromosomes and assigned them to 10 subhaplogroups. Eleven microsatellite loci were used to further evaluate subhaplogroup internal diversification. The geographic and quantitative analyses of haplogroup and microsatellite diversity is strongly suggestive of a northeastern African origin of E-M78, with a corridor for bidirectional migrations between northeastern and eastern Africa (at least 2 episodes between 23.9-17.3 ky and 18.0-5.9 ky ago), trans-Mediterranean migrations directly from northern Africa to Europe (mainly in the last 13.0 ky), and flow from northeastern Africa to western Asia between 20.0 and 6.8 ky ago. A single clade within E-M78 (E-V13) highlights a range expansion in the Bronze Age of southeastern Europe, which is also detected by haplogroup J-M12. Phylogeography pattern of molecular radiation and coalescence estimates for both haplogroups are similar and reveal that the genetic landscape of this region is, to a large extent, the consequence of a recent population growth in situ rather than the result of a mere flow of western Asian migrants in the early Neolithic. Our results not only provide a refinement of previous evolutionary hypotheses but also well-defined time frames for past human movements both in northern/eastern Africa and western Eurasia.     

Development of a TIRF-based biosensor for sensitive detection of progesterone in bovine milk

A total internal reflectance fluorescence (TIRF)-based biosensor for progesterone in bovine milk was developed and tested by measuring the progesterone level in daily milk samples for 25 days, covering a whole estrus cycle. The detection is based on total internal reflectance fluorescence. The assay has been designed as a binding-inhibition test with a progesterone derivative covalently immobilized on the sensor surface and a monoclonal anti-progesterone antibody as biological recognition element. First an existing progesterone assay was optimized by reducing the assay time per measurement, resulting in an assay time of about 5 min and reaching a limit of detection (LOD) of 0.04 ng mL(-1) and a quantification limit (LOQ) of 0.34 ng mL(-1). After calibration the assay was tested by measuring the progesterone level in daily milk samples over several weeks. An estrus cycle of a cow could be measured. As results become available within minutes without any preparation or pre-concentration of the milk samples the fully automated TIRF-based biosensor for progesterone can be used in-line in the milking parlor and thus could be an important tool for reproductive management of dairy cattle detecting heat and predicting pregnancy, which are critical parameters in milk production.     

Fibroblast growth factor 21 is induced upon cardiac stress and alters cardiac lipid homeostasis

Fibroblast growth factor 21 (FGF21) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF21 mRNA expression. Here we show that FGF21 mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle. In line with this assumption, the induction of ER stress increases FGF21 mRNA expression in H9C2 cardiomyotubes. Cardiac FGF21 expression was also induced upon fasting of healthy mice, implicating a role of FGF21 in cardiac energy metabolism. To address this question, we generated and characterized mice with cardiac-specific overexpression of FGF21 (CM-Fgf21). FGF21 was efficiently secreted from cardiomyocytes of CM-Fgf21 mice, which moderately affected cardiac TG homeostasis, indicating a role for FGF21 in cardiac energy metabolism. Together, our results show that FGF21 expression is activated upon cardiac ER stress linked to defective lipolysis and that a persistent increase in circulating FGF21 levels interferes with cardiac and whole body energy homeostasis.     

The lipolytic proteome of mouse adipose tissue

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.     

Defective uptake of triglyceride-associated fatty acids in adipose tissue causes the SREBP-1c-mediated induction of lipogenesis

Lipoprotein lipase (LPL) is the only known enzyme in the capillary endothelium of peripheral tissues that hydrolizes plasma triglycerides and provides fatty acids (FAs) for their subsequent tissue uptake. Previously, we demonstrated that mice that express LPL exclusively in muscle develop essentially normal fat mass despite the absence of LPL and the deprivation of nutritionally derived FAs in adipose tissue (AT). Using this mouse model, we now investigated the metabolic response to LPL deficiency in AT that enables maintenance of normal AT mass. We show that the rate of FA production was 1.8-fold higher in LPL-deficient AT than in control AT. The levels of mRNA and enzymatic activities of important enzymes involved in FA and triglyceride biosynthesis were induced concomitantly. Increased plasma glucose clearing and (14)C-deoxyglucose uptake into LPL-deficient mouse fat pads indicated that glucose provided the carbon source for lipid synthesis. Leptin expression was decreased in LPL-deficient AT. Finally, the induction of de novo FA synthesis in LPL-deficient AT was associated with increased expression and processing of sterol regulatory element binding protein 1 (SREBP-1), together with an increase in INSIG-1 expression. These results suggest that in the absence of LPL in AT, lipogenesis is activated through increased SREBP-1 expression and processing triggered by decreased availability of nutrition-derived FAs, elevated insulin, and low leptin levels.     

FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling

Lipolysis is defined as the catabolism of triacylglycerols stored in cellular lipid droplets. Recent discoveries of essential lipolytic enzymes and characterization of numerous regulatory proteins and mechanisms have fundamentally changed our perception of lipolysis and its impact on cellular metabolism. New findings that lipolytic products and intermediates participate in cellular signaling processes and that "lipolytic signaling" is particularly important in many nonadipose tissues unveil a previously underappreciated aspect of lipolysis, which may be relevant for human disease.     

Adipose Triglyceride Lipase (ATGL) and Hormone-Sensitive Lipase (HSL) Deficiencies Affect Expression of Lipolytic Activities in Mouse Adipose Tissues

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patters of brown and white adipose tissue from ATGL (−/−) and HSL (−/−) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols.Excess lipids are stored as intracellular triacylglycerol and steryl ester deposits in animals, plant seeds, and fungi. In mammals adipose tissue is the body''s largest storage organ for triacylglycerols (TAG)¹ as the primary source of energy during periods of starvation and increased energy demand. Two types of adipose tissue, namely brown (BAT) and white (WAT) adipose tissue exist in mammals, localizing to anatomically distinct areas. BAT and WAT differ in almost all their structural and functional features. Whereas BAT develops prenatally, WAT is subject to maturation postnatally. The different appearance of brown and white adipose tissue is caused by differences in lipid content and the abundance of mitochondria in the constituent adipocytes. Brown fat cells contain several small multilocular lipid droplets and a high number of large mitochondria with numerous cristae. In addition, BAT is highly vascularized and highly innervated by the sympathetic nervous system. In contrast, white adipocytes, usually contain one major unilocular lipid droplet that fills most of the cytoplasm leaving space for only few mitochondria (1–3). WAT accumulates excess energy as triacylglycerols, whereas BAT dissipates energy through adaptive thermogenesis. The thermogenic activity of BAT is caused by the expression of one protein unique in brown adipocytes, the uncoupling protein 1 (UCP1). This polypeptide is a facultative proton transporter and localizes to the inner mitochondrial membrane. It generates heat instead of ATP by uncoupling oxidation in the respiratory chain (3).Lipolysis in WAT is the catabolic process responsible for the release of free fatty acids from triacylglycerol (4, 5). The balance of lipid storage and mobilization is tightly regulated to ensure whole body energy homeostasis. The mobilization of triacylglycerol stores by activation of lipolytic enzymes is specifically stimulated by hormones and chemical agents. In addition, a number of specific physiological conditions owing to exercise, aging, and nutritional status (feeding, fasting) also regulate degradation of TAGs (6). Impairment of lipolysis in adipocytes may be associated with clinical symptoms including obesity, insulin resistance, diabetes mellitus, and dyslipidaemia. All these conditions seem to have a common substrate called lipotoxicity (7–10).The sequential hydrolysis of triacylglycerols in adipocytes producing FFAs is catalyzed by a cascade of lipolytic enzymes, with different substrate preferences (11). The committed enzyme catalyzing the first step of TAG hydrolysis is ATGL, which was identified in three different laboratories in 2004 (12–14). Its activity appears to be largely dependent on association with CGI-58 (14, 15). HSL exhibits a much broader substrate spectrum, with preference for diacylglycerols as well as cholesteryl and retinyl esters (16, 17). In the final step of lipolysis, monoacylglycerol lipase (MGL) degrades MAG thereby generating free fatty acid and glycerol (18). ATGL is the major TAG lipase in adipose tissue. Expression in other tissues is rather low. Currently it cannot be excluded, that other lipases also exist that are important for lipid catabolism (19). Recent functional proteomic screens in various mouse tissues led to the identification of enzyme candidates that are currently subject to functional characterization (unpublished data).The intracellular degradation of triacylglycerols is catalyzed by a cascade of lipolytic enzymes. There appears to be an overlap of substrate preferences as well as a redundancy of lipases to ensure a proper function of this important catabolic process if individual lipase activities are reduced or entirely absent. This study aimed at identifying the effects of ATGL and HSL-deficiency on the expression of other lipolytic enzymes in adipose tissue. For this purpose, we compared the lipolytic proteomes of BAT and WAT from ATGL (−/−) and HSL (−/−) mice with the enzyme patterns of wt tissues using differential activity-based gel electrophoresis (DABGE) (20). This method is based on activity-recognition probes containing same substrate analogous structures but carrying different fluorophores for specific detection of the individual enzyme patterns of two different tissues. These inhibitors react with the nucleophilic serine in the active center of lipolytic enzymes thereby generating covalent bound lipid-protein complexes, which can be separated by gel electrophoresis. We found, that ATGL-deficiency in BAT had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in BAT. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in WAT. ATGL-deficiency led to a significant but not total reduction in the TAG hydrolyzing activity of adipose tissues. Obviously, there must be (an)other enzyme(s) compensating for the hydrolytic capacity of ATGL. Three proteins that responded to ATGL-deficiency in BAT were overexpressed and their activities on acylglycerols were analyzed. Among these proteins, Es1, Es10, and Es31-like emerged as novel lipase candidates in these studies.