Cecilia Holm

Identification of Novel Phosphorylation Sites in Hormone-sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitrophosphorylation of HSL containing Ser → Ala mutations in residues 563 and 565 (S563A,S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Molecular mechanisms regulating hormone-sensitive lipase and lipolysis

HSL (hormone-sensitive lipase) is a key enzyme in the mobilization of fatty acids from acylglycerols in adipocytes as well as non-adipocytes. In adipocytes, catecholamines stimulate lipolysis mainly through PKA (protein kinase A)-mediated phosphorylation of HSL and perilipin, a protein coating the lipid droplet. The anti-lipolytic action of insulin is mediated mainly via lowered cAMP levels, accomplished through activation of phosphodiesterase 3B. Phosphorylation of HSL by PKA occurs at three sites, the serines 563, 659 and 660, both in vitro and in primary rat adipocytes. Phosphorylation of Ser-659 and -660 is required for in vitro activation as well as translocation from the cytosol to the lipid droplet, whereas the role of the third PKA site remains elusive. Adipocytes isolated from homozygous HSL-null mice, generated in our laboratory, exhibit completely blunted catecholamine-induced glycerol release and reduced fatty acid release, suggesting the presence of additional, although not necessarily hormone-activatable, triacylglycerol lipase(s). Basal hyperinsulinaemia, release of exaggerated amounts of insulin during glucose challenges and retarded glucose disposal during insulin tolerance tests suggest that HSL-null mice are insulin resistant. Liver, adipose tissue and skeletal muscle appear all to be sites of impaired insulin sensitivity in HSL-null mice.

cDNA Cloning, Tissue Distribution, and Identification of the Catalytic Triad of Monoglyceride Lipase EVOLUTIONARY RELATIONSHIP TO ESTERASES, LYSOPHOSPHOLIPASES, AND HALOPEROXIDASES

Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.

Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle.

The enzymic regulation of triacylglycerol breakdown in skeletal muscle is poorly understood. Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying demonstrated the presence of immunoreactive hormone-sensitive lipase (HSL), with the concentrations in soleus and diaphragm being more than four times the concentrations in extensor digitorum longus and epitrochlearis muscles. Neutral lipase activity determined under conditions optimal for HSL varied directly with immunoreactivity. Expressed relative to triacylglycerol content, neutral lipase activity in soleus muscle was about 10 times that in epididymal adipose tissue. In incubated soleus muscle, both neutral lipase activity against triacylglycerol (but not against a diacylglycerol analogue) and glycogen phosphorylase activity increased in response to adrenaline (epinephrine). The lipase activation was completely inhibited by anti-HSL antibody and by propranolol. The effect of adrenaline could be mimicked by incubation of crude supernatant from control muscle with the catalytic subunit of cAMP-dependent protein kinase, while no effect of the kinase subunit was seen with supernatant from adrenaline-treated muscle. The results indicate that HSL is present in skeletal muscle and is stimulated by adrenaline via beta-adrenergic activation of cAMP-dependent protein kinase. The concentration of HSL is higher in oxidative than in glycolytic muscle, and the enzyme is activated in parallel with glycogen phosphorylase.

Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue

A polyclonal rabbit antibody was used to detect hormone-sensitive lipase in rat organs other than white adipose tissue. Inhibition of tissue diacylglycerol lipase activity by the anti-hormone-sensitive lipase, and by NaF, Hg2+ and diisopropyl fluorophosphate, known inhibitors of the hormone-sensitive lipase, demonstrated its presence in the adrenals, ovaries, testes, heart and skeletal muscle, but not in the liver and kidneys. After enrichment by immunoprecipitation an immunoreactive protein, corresponding to the adipose tissue hormone-sensitive lipase 84 kDa subunit, and some additional, higher Mrapp proteins, were detected by Western blotting in the same tissues. The adipose tissue contained greater than 80% of the total hormone-sensitive lipase, with 5-10- and 50-100-fold lower specific activity in the steroid-producing and the muscle tissues, respectively.

Adipocyte hormone-sensitive lipase: a major regulator of lipid metabolism

Le tissu adipeux joue un r81e important dans le contrde de la balance CnergCtique. La mobilisation des triacylglycCrols par la lipase hormono-sensible (EC 3.1.1.3; LHS) est soumis a un contrBle direct par les hormones et neurotransmetteurs qui modulent les concentrations intracellulaires d’AMP cyclique (AMPc). L’hydrolyse des triacylglycCrols par la LHS constitue l’etape limitante de la lipolyse. La LHS est phosphorylCe sur le site rkgulateur (Ser552 dans la LHS humaine) par la p r o t h e kinase dependante de 1’AMPc (EC 2.7.1.37) lorsque les concentrations intracellulaires d’ AMPc augmentent. Cette phosphorylation conduit B l’activation de l’enzyme. Une deuxikme site de phosphorylation (Ser5.54 dans la LHS humaine) dCnommC site basal est la cible de la protCine kinase activCe par I’AMP. La phosphorylation du site basal ne conduit pas B l’activation de la LHS et empkche la phosphorylation sur le site regulateur. La phosphorylation et I’activation de la protCine kinase activke par 1’AMP constitue donc un mecanisme antilipolytique qui est fonctionnel sur cellules isolCes mais dont l’importance physiologique n’est pas connue. Les ADN complkmentaires de la LHS de plusieurs espkces ont CtC clones et la structure des gbnes de LHS de l’homme et de la souris sont connus. Differents domaines fonctionnels de la protCine ont CtC proposes. Une rCgion d’homologie de sequences en amont de la serine 424 du site catalytique avec cinq enzymes d’organismes procaryotes et une enzyme humaine a ete decrite. La localisation chromosomique du gbne de la LHS est connue chez l’homme (chromosome 19, region q13-1+13.2), le porc et la souris. La mise en evidence de marqueurs polymorphiques dans le g&ne devrait permettre de tester l’hypothkse d’une implication de la LHS dans certaines maladies hereditaires du metabolisme lipidique. L’expression de la LHS varie selon la localisation anatomique du tissu adipeux chez le rat. Cette expression subit Cgalement des variations durant la gestation chez le rat et le cycle annuel chez les mammifbres hibernants. Chez l’homme, les taux d’ARN messagers de la LHS sont diminuCs dans le tissu adipeux de certains patients atteints de cancer. L‘activitC enzymatique totale est diminuee chez les patients atteints d’hyperlipidemie familiale combinCe mais pas chez les patients atteints du syndrome metabolique bien que, dans les deux cas, la lipolyse adipocytaire maximale soit diminuke. Les mkcanismes molCculaires de contr6le de l’expression de la LHS sont pratiquement inconnus.

Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase.

Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL). It is a canonical α/β hydrolase with two insertions forming the substrate binding pocket. The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.

Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins

Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.

Endosperm and whole grain rye breads are characterized by low post-prandial insulin response and a beneficial blood glucose profile

BackgroundRye products have previously been shown to induce comparatively low post-prandial insulin responses; irrespectively of their glycaemic indices (GI). However, the mechanism behind this lowered insulin demand remains unknown. An improved insulin economy might contribute to the benefits seen in epidemiological studies with whole grain diets on metabolic risk factors and weight regulation. The objective of this study was to explore the mechanism for a reduced post-prandial insulin demand with rye products.Methods12 healthy subjects were given flour based rye products made from endosperm, whole grain or bran, produced with different methods (baking, simulated sour-dough baking and boiling) as breakfasts in random order in a cross-over design. White wheat bread (WWB) was used as a reference. Blood glucose, serum insulin, plasma ghrelin and subjective satiety were measured during 180 minutes. To evaluate the course of post-meal glycaemia, a measure of the glycaemic profile (GP) was introduced defined as the duration for the incremental post-prandial blood glucose response divided with the blood glucose incremental peak (min/mM).ResultsThe study shows that whole grain rye breads and endosperm rye products induced significantly (p < 0.05) lower insulinaemic indices (IIs) than WWB. Rye bran bread (RBB) produced significantly higher II compared with all the other rye products. Furthermore, the acute insulin response showed better correlations with the GP than with the GI of the products. The endosperm rye bread and the whole grain rye bread with lactic acid induced a significantly higher GP than RBB, WWB, white wheat- and whole grain rye porridge, respectively. A low insulin incremental peak was associated with less severe late post-prandial hypoglycaemia (r = 0.38, p < 0.001), and hypoglycaemia was negatively correlated to subjective satiety at 180 min (r = -0.28, p < 0.05). A low insulin incremental peak was also associated with a milder recovery of plasma ghrelin in the late post-prandial phase (180 min, r = 0.34, p < 0.01).ConclusionOur study shows that endosperm and wholegrain rye products induce low acute insulinaemic responses and improved glycaemic profiles. The results also suggest that the rye products possess beneficial appetite regulating properties. Further studies are needed to identify the unknown property or bioactive component(s) responsible for these beneficial metabolic features of rye.

Mutational Analysis of the Hormone-sensitive Lipase Translocation Reaction in Adipocytes

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093–1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.