Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

The phosphatidic acid-binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3.

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes.

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the ß-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that ß-adrenergic-mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase-Akt-mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.

Inhibited insulin signaling in mouse hepatocytes is associated with increased phosphatidic acid but not diacylglycerol.

Although an elevated triacylglycerol content in non-adipose tissues is often associated with insulin resistance, the mechanistic relationship remains unclear. The data support roles for intermediates in the glycerol-3-phosphate pathway of triacylglycerol synthesis: diacylglycerol (DAG), which may cause insulin resistance in liver by activating PKCϵ, and phosphatidic acid (PA), which inhibits insulin action in hepatocytes by disrupting the assembly of mTOR and rictor. To determine whether increases in DAG and PA impair insulin signaling when produced by pathways other than that of de novo synthesis, we examined primary mouse hepatocytes after enzymatically manipulating the cellular content of DAG or PA. Overexpressing phospholipase D1 or phospholipase D2 inhibited insulin signaling and was accompanied by an elevated cellular content of total PA, without a change in total DAG. Overexpression of diacylglycerol kinase-θ inhibited insulin signaling and was accompanied by an elevated cellular content of total PA and a decreased cellular content of total DAG. Overexpressing glycerol-3-phosphate acyltransferase-1 or -4 inhibited insulin signaling and increased the cellular content of both PA and DAG. Insulin signaling impairment caused by overexpression of phospholipase D1/D2 or diacylglycerol kinase-θ was always accompanied by disassociation of mTOR/rictor and reduction of mTORC2 kinase activity. However, although the protein ratio of membrane to cytosolic PKCϵ increased, PKC activity itself was unaltered. These data suggest that PA, but not DAG, is associated with impaired insulin action in mouse hepatocytes.

Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation.

Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1-3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.

The lipin gene family encodes a class of Mg(2+)-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.

Glycerol-3-phosphate acyltransferase-4-deficient mice are protected from diet-induced insulin resistance by the enhanced association of mTOR and rictor.

Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might contribute to the development of hepatic insulin resistance. Compared with control mice fed a high fat diet, Gpat4(-/-) mice were more glucose tolerant and were protected from insulin resistance. Overexpression of GPAT4 in mouse hepatocytes impaired insulin-suppressed gluconeogenesis and insulin-stimulated glycogen synthesis. Impaired glucose homeostasis was coupled to inhibited insulin-stimulated phosphorylation of Akt(Ser47³) and Akt(Thr³°8). GPAT4 overexpression inhibited rictor's association with the mammalian target of rapamycin (mTOR), and mTOR complex 2 (mTORC2) activity. Compared with overexpressed GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest decrease in 16:0-PA species. Compared with controls, liver and skeletal muscle from Gpat4(-/-)-deficient mice fed a high-fat diet were more insulin sensitive and had a lower hepatic content of di16:0-PA. Taken together, these data demonstrate that a GPAT4-derived lipid signal, likely di16:0-PA, impairs insulin signaling in mouse liver and contributes to hepatic insulin resistance.

Spatial and Seasonal Variation of dissolved organic carbon (DOC) concentrations in Irish streams: importance of soil and topography characteristics.

Dissolved organic carbon (DOC) concentrations have increased in many sites in Europe and North America in recent decades. High DOC concentrations can damage the structure and functions of aquatic ecosystems by influencing water chemistry. This study investigated the spatial and seasonal variation of DOC concentrations in Irish streams across 55 sites at seven time occasions over 1 year (2006/2007). The DOC concentrations ranged from 0.9 to 25.9 mg/L with a mean value of 6.8 and a median value of 5.7 mg/L and varied significantly over the course of the year. The DOC concentrations from late winter (February: 5.2 ± 3.0 mg/L across 55 sites) and early spring (April: 4.5 ± 3.5 mg/L) had significantly lower DOC concentrations than autumn (October: mean 8.3 ± 5.6 mg/L) and early winter (December: 8.3 ± 5.1 mg/L). The DOC production sources (e.g., litterfall) or the accumulation of DOC over dry periods might be the driving factor of seasonal change in Irish stream DOC concentrations. Analysis of data using stepwise multiple linear regression techniques identified the topographic index (TI, an indication of saturation-excess runoff potential) and soil conditions (organic carbon content and soil drainage characteristics) as key factors in controlling DOC spatial variation in different seasons. The TI and soil carbon content (e.g., soil organic carbon; peat occurrence) are positively related to DOC concentrations, while well-drained soils are negatively related to DOC concentrations. The knowledge of spatial and seasonal variation of DOC concentrations in streams and their drivers are essential for optimum riverine water resources management.

Assembly of high molecular weight complexes of lipin on a supported lipid bilayer observed by atomic force microscopy.

Lipins are phosphatidic acid phosphatases involved in the biosynthesis of triacylglycerols and phospholipids. They are associated with the endoplasmic reticulum but can also travel into the nucleus and alter gene expression. Previous studies indicate lipins in solution form high molecular weight complexes, possibly tetramers. This study was undertaken to determine if lipins form complexes on membranes as well. Murine lipin 1b was applied to a supported bilayer of phosphatidylcholine, phosphatidylserine, and cholesterol and examined by atomic force microscopy (AFM) over time. Lipin on bare mica appeared as a symmetric particle with a volume consistent with the size of a monomer. On the bilayer, lipin initially bound as asymmetric, curved particles that sometimes assembled into circular structures with an open center. Subsequently, lipin assemblies grew into large, symmetric particles with an average volume 12 times that of the monomer. Over time, some of the lipin assemblies were removed from the bilayer by the AFM probe leaving behind "footprints" composed of complex patterns that may reflect the substructure of the lipin assemblies. The lipin complexes appeared very flat, with a diameter 20 times their height. The footprints had a similar diameter, providing confirmation of the extensive deformation of the protein under the AFM probe. The ability of lipin to form large complexes on membranes may have significant implications for the local concentrations of the product, diacylglycerol, formed during hydrolysis of phosphatidic acid and for cooperative hormonal regulation of lipin activity through phosphorylation of one or more monomers in the complexes.

Protection of the membrane permeability barrier by annexins.

Biological membranes are exposed to a number of chemical and physical stresses that may alter the structure of the lipid bilayer in such a way that the permeability barrier to hydrophilic molecules and ions is degraded. These stresses include amphiphilic molecules involved in metabolism and signaling, highly charged polyamines, membrane-permeating peptides, and mechanical and osmotic stresses. As annexins are known to bind to lipid headgroups in the presence of calcium and increase the order of the bilayer lipids, this study addressed whether this activity of annexins provides a potential benefit to the membrane by protecting the bilayer against disruptions of this nature or can promote restoration of the permeability barrier after damage by such agents. The release of carboxyfluorescein from large unilamellar vesicles composed of lipids characteristically present in the inner leaflet of cell membranes (phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and cholesterol) was used to measure membrane permeability. It was determined that in the presence of calcium, annexin A5 reduced the level of baseline leakage from vesicles and reduced or reversed damage due to arachidonic acid, lysophosphatidic acid, lysophosphatidylcholine, diacylglycerol, monoacylglycerol, spermidine, amyloid-ß, amylin, and osmotic shock. Annexin A6 was also able to provide membrane protection in many but not all of these cases. In a cell, it is likely annexins would move to sites of breakdown of the permeability barrier because of the calcium-dependent promotion of the binding of annexins to membranes at sites of calcium entry. Because of the fundamental importance to life of maintaining the permeability barrier of the cell membrane, it is proposed here that this property of annexins may represent a critical, primordial activity that explains their great evolutionary conservation and abundant expression in most cells.

Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity.

Rhabdomyolysis is an acute syndrome due to extensive injury of skeletal muscle. Recurrent rhabdomyolysis is often caused by inborn errors in intermediary metabolism, and recent work has suggested that mutations in the human gene encoding lipin 1 (LPIN1) may be a common cause of recurrent rhabdomyolysis in children. Lipin 1 dephosphorylates phosphatidic acid to form diacylglycerol (phosphatidic acid phosphohydrolase; PAP) and acts as a transcriptional regulatory protein to control metabolic gene expression. Herein, a 3-year-old boy with severe recurrent rhabdomyolysis was determined to be a compound heterozygote for a novel c.1904T>C (p.Leu635Pro) substitution and a previously reported genomic deletion of exons 18-19 (E766-S838_del) in LPIN1. Western blotting with patient muscle biopsy lysates demonstrated a marked reduction in lipin 1 protein, while immunohistochemical staining for lipin 1 showed abnormal subcellular localization. We cloned cDNAs to express recombinant lipin 1 proteins harboring pathogenic mutations and showed that the E766-S838_del allele was not expressed at the RNA or protein level. Lipin 1 p.Leu635Pro was expressed, but the protein was less stable, was aggregated in the cytosol, and was targeted for proteosomal degradation. Another pathogenic single amino acid substitution, lipin 1 p.Arg725His, was well expressed and retained its transcriptional regulatory function. However, both p.Leu635Pro and p.Arg725His proteins were found to be deficient in PAP activity. Kinetic analyses demonstrated a loss of catalysis rather than diminished substrate binding. These data suggest that loss of lipin 1-mediated PAP activity may be involved in the pathogenesis of rhabdomyolysis in lipin 1 deficiency.