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.     

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.     

Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation

Endothelial lipase (EL) is a member of a subfamily of lipases that act on triglycerides and phospholipids in plasma lipoproteins, which also includes lipoprotein lipase and hepatic lipase. EL has a tropism for high density lipoprotein, and its level of phospholipase activity is similar to its level of triglyceride lipase activity. Inhibition or loss-of-function of EL in mice results in an increase in high density lipoprotein cholesterol, making it a potential therapeutic target. Although hepatic lipase and lipoprotein lipase have been shown to function as homodimers, the active form of EL is not known. In these studies, the size and conformation of the active form of EL were determined. Immunoprecipitation experiments suggested oligomerization. Ultracentrifugation experiments showed that the active form of EL had a molecular weight higher than the molecular weight of a simple monomer but less than a dimer. A construct encoding a covalent head-to-tail homodimer of EL (EL-EL) was expressed and had similar lipolytic activity to EL. The functional molecular weights determined by radiation inactivation were similar for EL and the covalent homodimer EL-EL. We previously showed that EL could be cleaved by proprotein convertases, such as PC5, resulting in loss of activity. In cells overexpressing PC5, the covalent homodimeric EL-EL appeared to be more stable, with reduced cleavage and conserved lipolytic activity. A comparative model obtained using other lipase structures suggests a structure for the head-to-tail EL homodimer that is consistent with the experimental findings. These data confirm the hypothesis that EL is active as a homodimer in head-to-tail conformation.Three members of the triglyceride lipase family, lipoprotein lipase (LPL),³ hepatic lipase (HL), and endothelial lipase (EL), contribute to lipoprotein catabolism in the plasma compartment. They are all secreted proteins that bind to heparan sulfate proteoglycans on the luminal side of endothelial cells where they interact with their lipoprotein substrates. They have different specificities for lipoproteins, and all hydrolyze triglycerides and phosphatidylcholine at the sn-1 position, albeit with widely differing efficiencies (1). The preferred lipoprotein substrates for LPL are the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoproteins; the triglyceride lipase activity of LPL is more than 100-fold greater than its phospholipase activity. The primary lipoprotein substrates for HL are chylomicron remnants, intermediate density lipoproteins, and large triglyceridase-enriched HDL; its triglyceride lipase activity is about 20-fold higher than its phospholipase activity. EL is much more active on HDL, and its phospholipase activity is quite similar to its triglyceride lipase activity.These three lipolytic enzymes share a number of structural features. By analogy to the crystal structure of pancreatic lipase (2), another member of the triglyceride lipase family, each has a clearly defined N-terminal and C-terminal structural domain, joined by a hinge region. They are all serine esterases with a catalytic triad of serine, aspartic acid, and histidine located in the N-terminal domain. The catalytic triad is covered by a lid domain that contributes to the preference for either triglyceride or phospholipid substrates (3–6). The C-terminal domain contributes to lipid binding and determines the preferences for binding to lipoproteins (7–12). The N-terminal portion of the EL molecule contains the active site of the enzyme. However, when this portion of the molecule is expressed without the C-terminal domain, it lacks enzymatic activity against phospholipid (13), triglyceride, and the more soluble micellar substrate tributyrin.⁴ Thus, the presence of the C-terminal domain is necessary for activity. All three enzymes are glycosylated to varying degrees, and two of the glycosylation sites (one each in the N- and C-terminal domains) are common to all three enzymes (14, 15). All three enzymes are also heparin-binding proteins, and regions that contribute to heparin binding are found in both the N- and C-terminal domains (16–18).EL and, to a lesser extent, LPL are subject to proteolytic cleavage by proprotein convertases at a prototypical RXKR site in the hinge region, but HL lacks the site and is not cleaved (19, 20). The catalytically active forms of both LPL and HL have been shown to be homodimers (18, 21–27), and in the case of LPL, the orientation of the subunits of the dimer has been shown to be head-to-tail (28–30). The present study tested the hypothesis that EL also functions as a head-to-tail dimer.     

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.     

Deorphanization of GPR109B as a Receptor for the ��-Oxidation Intermediate 3-OH-octanoic Acid and Its Role in the Regulation of Lipolysis

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the β-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to G_i-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC₅₀ values of about 4 and 8 μm, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased β-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in β-oxidation rates.Triacylglycerols stored in the white adipose tissue serve as the major energy reserve in higher eukaryotes (1). Although they are constantly turned over by lipolysis and re-esterification, their mobilization and storage are precisely balanced by various hormones and other factors depending on the nutritional state (2). The net rate of lipolysis is increased during fasting or periods of increased energy demand. Fatty acids generated via lipolysis undergo β-oxidation in the muscle and liver to serve directly as a source of energy or as a precursor for ketone bodies (3). The major intracellular regulator of lipolysis is cyclic AMP, which stimulates cAMP-dependent kinase to activate lipolytic enzymes (2, 4–6). This lipolytic pathway is induced, for example, via β-adrenergic receptors that couple to the G-protein G_s and thereby stimulate adenylyl cyclase (7, 8). To adjust lipolysis at the appropriate rate, the effects of prolipolytic stimuli are balanced by various antilipolytic influences. Besides insulin, which promotes the degradation of cAMP via activation of phosphodiesterase 3B (2, 5, 7), several antilipolytic stimuli decrease cAMP levels by activation of G_i-coupled receptors, which mediate an inhibition of adenylyl cyclase (5, 8). One of these receptors, GPR109A, has recently been shown to mediate the anti-lipolytic effects of high concentrations of the ketone body 3-OH-butyrate thereby providing a negative feedback mechanism during fasting (9, 10). GPR109A also binds nicotinic acid (11–13) and mediates the anti-lipolytic effects of this anti-dyslipidemic drug (12).GPR109B, a close relative of GPR109A, is the result of a recent gene duplication being present in humans but not in rodents and most other mammals (14). GPR109B differs from GPR109A in an extended C-terminal tail as well as in 16 amino acids (11, 13). Despite its high homology to GPR109A, GPR109B does not bind nicotinic acid or 3-OH-butyrate with reasonable affinity (10, 11, 13). Because GPR109A and GPR109B have very similar expression patterns (11, 13, 15) and are likely to have the same basic signaling properties, agonists of GPR109B are expected to have physiological and pharmacological effects comparable with those of the GPR109A agonist 3-OH-butyrate and nicotinic acid, respectively. Recently, several synthetic compounds as well as various aromatic d-amino acids have been shown to be selective agonists at GPR109B (16–18). However, endogenous physiological anti-lipolytic ligands of GPR109B are unknown.In this study we tested endogenous carboxylic acids for their ability to activate GPR109B. We found that the fatty acid β-oxidation intermediate 3-OH-octanoic acid is a highly specific agonist of GPR109B. 3-OH-octanoic acid has anti-lipolytic activity, and its plasma concentration in humans reflects the β-oxidation flux. Our data suggest that 3-OH-octanoic acid and GPR109B mediate a negative feedback regulation of adipocyte lipolysis.     

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]     

Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway

After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.The storage and mobilization of nutrients from specialized tissues requires the spatial organization of both signaling functions and energy stores. Nowhere is this more evident than in mammalian adipose tissue, which maintains the most efficient repository for readily available energy. Here, fuel is segregated into lipid droplets, once thought to be inert storehouses but now recognized as complex structures that represent a regulatable adaptation of a ubiquitous organelle (5, 40). The synthesis and maintenance of functional lipid droplets requires numerous proteins, not only fatty acid binding proteins and enzymes of lipid synthesis but also molecules critical to constitutive and specialized membrane protein trafficking (23).During times of nutritional need, triglycerides within the adipocyte lipid droplet are hydrolyzed into their components, fatty acids, acyl-glycerides, and, ultimately, glycerol. This process, termed lipolysis, is controlled dynamically by multiple hormonal signals that respond to the nutrient status of the organism. During fasting, catecholamines such as norepinephrine stimulate lipolysis via beta-adrenergic receptor activation, promoting adenylyl cyclase activity and the production of cyclic AMP (cAMP) (17). cAMP binds to the regulatory subunits of its major effector, protein kinase A (PKA), triggering the dissociation of these subunits and the subsequent activation of the catalytic subunits (62, 63). PKA is frequently sequestered into multiple parallel, intracellular signaling complexes, though such structures have not been studied in hormone-responsive adipocytes (68). Two targets of activated PKA important for lipolysis are hormone-sensitive lipase (HSL) and perilipin, the major lipid droplet coat protein (17). The phosphorylation of HSL on Ser 559/660 is crucial for its activation and translocation to the lipid droplet, where HSL catalyzes the hydrolysis of diglycerides to monoglycerides (26, 55). Another lipase, adipose triglyceride lipase (ATGL), carries out the initial cleavage of triglycerides to diglycerides and most likely is rate limiting for lipolysis, but it does not appear to be regulated directly via PKA phosphorylation (24, 73). Perilipin under basal conditions acts as a protective barrier against lipase activity; upon stimulation, the phosphorylation of least six PKA consensus sites triggers a conformational change in perilipin, permitting access to the lipid substrates in the droplet, the recruitment of HSL, and possibly the activation of ATGL (7, 8, 21, 41, 46, 58, 60, 61). Perilipin, therefore, possesses dual functions, both blocking lipolysis in the basal state as well as promoting lipolysis upon its phosphorylation (5, 58, 60).Following the ingestion of a meal, insulin stimulates the uptake of nutrients such as glucose into specialized tissues and also potently inhibits lipolysis in adipocytes (17). Insulin signaling in the adipocyte involves the activation of the insulin receptor tyrosine kinase, the phosphorylation of insulin receptor substrates, the activation of PI3K, and the subsequent production of specific phosphoinositides at the plasma membrane (59). These phosphoinositides then recruit Akt, via its pleckstrin homology domain, to the plasma membrane, where Akt becomes phosphorylated and activated by two upstream kinases. Akt stimulates the translocation of the glucose transporter GLUT4 to the plasma membrane, thereby promoting the uptake of glucose into the cell (2). The mechanism by which insulin inhibits lipolysis has been proposed to involve the reduction of cAMP levels and thus PKA activity. In this model, insulin signaling activates phosphodiesterase 3b (PDE3b) via the Akt-mediated phosphorylation of Ser273 (14, 32). Upon activation by Akt, PDE3b catalyzes the hydrolysis of cAMP to 5′AMP, thereby attenuating PKA activity and lipolysis. Recent studies of PDE3b knockout mice have highlighted the importance of PDE3b activity in the regulation of lipolysis but were uninformative regarding the mechanism of insulin action (12). Adipocytes isolated from these mice exhibit reduced responses to insulin with respect to lipolysis, but it is not clear whether this is due to the loss of the critical target enzyme or a normal mechanism being overwhelmed by supraphysiological concentrations of cAMP (12). Biochemical studies using dominant-inhibitory Akt have demonstrated that Akt can regulate PDE3b activity, and other studies also have suggested that Akt interacts directly with PDE3b, implying a direct connection to lipolysis regulation (1, 32). Nevertheless, the actual requirement for Akt in insulin action with regard to the lipolysis itself has not been demonstrated directly in, for example, genetic loss-of-function experiments.There now is substantial evidence implicating elevated free fatty acid levels as a consequence of inappropriate lipolysis as a major etiological factor for insulin resistance and type 2 diabetes mellitus (T2DM) (51). Conditions such as obesity and diabetes are characterized by a pathophysiological state in which these tissues become unresponsive to insulin, which contribute to the adverse long-term sequelae of diseases such as T2DM and the metabolic syndrome (4, 44). Thus, understanding in detail the mechanism by which insulin suppresses fat cell lipolysis is critical to identifying the underlying defect in resistant adipose tissue and ultimately developing effective therapeutics. In the present study, we investigated both Akt-dependent and -independent modes of insulin action toward lipolysis. We found the latter to predominate at low, physiological levels of adrenergic stimulation, acting via a pathway dependent on the preferential phosphorylation of downstream PKA substrates.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[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]     

Highly Conserved Cysteines within the Ly6 Domain of GPIHBP1 Are Crucial for the Binding of Lipoprotein Lipase

GPIHBP1, a glycosylphosphatidylinositol-anchored endothelial cell protein of the lymphocyte antigen 6 (Ly6) family, binds lipoprotein lipase (LPL) avidly and is required for the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 contains two key structural motifs, an acidic domain and an Ly6 motif (a three-fingered domain specified by 10 cysteines). The acidic domain is required for LPL binding, but the importance of the Ly6 domain is less clear. To explore that issue, we transfected cells with a wild-type GPIHBP1 expression vector or mutant GPIHBP1 vectors in which specific cysteines in the Ly6 domain were changed to alanine. The mutant GPIHBP1 proteins reached the cell surface, as judged by antibody binding studies and by the ability of a phosphatidylinositol-specific phospholipase C to release these proteins from the cell surface. However, cells expressing the cysteine mutants could not bind LPL. The acidic domain of the cysteine mutants appeared to remain accessible, as judged by binding studies with an antibody against the acidic domain. We also developed a cell-free assay of LPL binding. We created a rat monoclonal antibody against the carboxyl terminus of mouse GPIHBP1 and used that antibody to coat agarose beads. We then tested the ability of soluble forms of GPIHBP1 that had been immobilized on monoclonal antibody-coated beads to bind LPL. In this assay, wild-type soluble GPIHBP1 bound LPL avidly, but the cysteine mutants did not. Thus, our studies suggest that a structurally intact Ly6 domain (in addition to the acidic domain) is essential for LPL binding.Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)² is an endothelial cell protein that is required for the lipolytic processing of triglyceride-rich lipoproteins in the plasma (1). In the absence of GPIHBP1, lipolysis of plasma lipoproteins is virtually abolished, leading to severe hypertriglyceridemia (1). Expression of GPIHBP1 in cultured cells confers the ability to bind lipoprotein lipase (LPL) (1). That finding, along with the fact that GPIHBP1 is located in endothelial cells, led Beigneux et al. (1) to hypothesize that GPIHBP1 serves as an endothelial cell platform for lipolysis.The discovery of the role of GPIHBP1 in lipolysis prompted interest in defining which portions of GPIHBP1 are important for its function (e.g. for its ability to bind LPL). Mature GPIHBP1 is a relatively short protein with only two noteworthy structural domains (Fig. 1). First, the amino terminus of the protein contains a strongly acidic domain (amino acids 24–48 in the mouse sequence) with a large number of aspartates and glutamates (2). This negatively charged domain is absolutely critical for binding LPL, a protein that contains two well characterized positively charged “heparin-binding domains” (3–5). Second, GPIHBP1 contains a cysteine-rich Ly6 domain (amino acids 63–135 in the mouse sequence). The function of the Ly6 domain in LPL binding is less clearly defined.Open in a separate windowFIGURE 1.Schematic of human GPIHBP1, showing the location of the acidic domain and the 10 highly conserved cysteines of the Ly6 domain. The location of Gln¹¹⁵ is also shown; a Q115P mutation was identified in association with chylomicronemia in a young man (15). This figure is modified, with permission, from a figure published in Ref. 22.The Ly6 domain is an ancient motif containing either 8 or 10 cysteines that are organized in a highly characteristic spacing pattern (6, 7). Mammalian genomes contain ∼25–30 Ly6 proteins, of which the best studied are urokinase-type plasminogen activator receptor and CD59. In those proteins, as well as in other Ly6 proteins, crystallographic studies have shown that each of the cysteines is involved in a disulfide bond, producing a three-fingered structural motif (8–10).In the case of urokinase-type plasminogen activator receptor and CD59, the Ly6 motif is crucial for ligand binding (8–13). But in the case of GPIHBP1, one could argue that this domain might be dispensable, simply because it is plausible that the acidic domain would be sufficient for binding the positively charged domains within LPL. On the other hand, other considerations suggest that the GPIHBP1 Ly6 domain might actually be important for GPIHBP1 function. First, the cysteines that define the Ly6 domain are perfectly conserved in the GPIHBP1 of every mammalian species from platypus to human, suggesting that the three-fingered structure of this domain is important (14). Second, Beigneux et al. (15) found that a missense mutation adjacent to a conserved cysteine (Q115P) impaired the ability of GPIHBP1 to bind LPL. The latter observation led us to entertain the possibility that the Ly6 domain could be functionally important in LPL binding.To explore the functional relevance of the GPIHBP1 Ly6 domain, we decided to mutate the highly conserved cysteines in GPIHBP1 (Fig. 1), because those mutations would be predicted to disrupt the structure of the three-fingered motif. For other Ly6 proteins, mutation of conserved cysteines appears to impair protein function. Mutation of several (but not all) of the Ly6 cysteines in CD59 reduces the ability of that molecule to inhibit complement-mediated membrane attack (13). Also, missense mutations involving cysteines were found in a secreted Ly6 protein, SLURP1, in association with a recessive palmoplantar keratoderma (16). Unfortunately, the function and the binding partner for SLURP1 are unknown, so no one has been able to use biochemical assays to rigorously assess the impact of cysteine mutations on protein function. In contrast, GPIHBP1 has a well established function—binding LPL, and a cell-based system for assessing LPL binding has been developed (1, 15, 17).In the current study, we sought to assess the impact of Ly6 cysteine mutations on the LPL binding capacity of GPIHBP1. But as we embarked on these studies, we worried that the cell-based assay system might be inadequate, for the simple reason that cysteine mutations sometimes interfere with the ability of proteins to reach the cell surface (13, 18). Accordingly, we also developed a cell-free, monoclonal antibody-based assay system for assessing the ability of GPIHBP1 to bind LPL. Both the cell-based binding system and the cell-free system were used to test the possibility that the Ly6 domain of GPIHBP1, like the acidic domain, is critical for binding LPL.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[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]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[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]