Fatty Liver Dystrophy Research Articles

Abstract P237: Elucidation of Blood Pressure Control Function of Essential Hypertension Candidate Gene Lpin1

We have found that the LPIN1 is the candidate gene for essential hypertension (EHT) by genome-wide association analysis in 2007. Mice characterized by loss of body fat, fatty liver, insulin resistance and peripheral neuropathy were discovered in nature and named fatty liver dystrophy (fld) mice in 2000. Recently, the fld mice were shown that they exhibit few LPIN1 genes, and the LPIN1 gene was proved as the cause of phenotype of fld mice. However, the blood pressure (BP) and the alteratios of the vasoactive factors in fld mice have not yet been elucidated. Therefore, to clarify the BP and the effects of LPIN1 gene defeciency, we analyzed the BP and regulatory factors of the fld mice. Furthermore, we transplanted the fat to fld mice, and clarify the importance of adipocytokine on BP in the model mice. The BP were measured by the tail cuff method and the telemetry method. We performe fat grafting or sham surgery on fld mice, and systolic BP (SBP) was measured by tail cuff method 8 weeks after surgery. We confirmed that the SBP of fld mice were higher than that of CO by the tail cuff method( fld 134±5 vs CO 111±4 mmHg P<0.05). Furthermore, SBP of the fld mice were higher than that of CO (fld 167.7±9.8 vs CO 133.5±10.9 mmHg P<0.01) by the telemetry method. It was confirmed that the adrenalin and noradrenalin excretions in the urine of fld mice were higher than those of CO(fld 0.133±0.067 vs CO 0.038±0.013 μg/day P<0.01 )(fld 0.980 ±0.313 vs CO 0.364±0.080 μg/day P<0.001). There is no significant difference in plasma aldosterone concentration between two groups( fld 434±276.7 vs CO 781±469.5 pg/mL n.s).The SBP tended to decrease with the fat trasplantation in fld mice ( fat transplanted 103.7±4.41 vs sham 114.7±7.67 mmHg P=0.056). We confirmed that fld mouse exhibits higher SBP by both tailcuff method and telemetry method. Because the fld mice exhibit higher PR and high levels of CA concentration and excretion in urine, it was suggested that the sympathetic nervous systems of fld mice were activated. Sympathetic nervous system activity may be related to an increase in BP of fld mouse, and further research is under way. Since it showed that BP decreased by fat grafting, the visceral fat are essential for fld mice to BP control. The implications of adipokine and other fats are under consideration.

An anesthesiologist’s view of problems in HIV-positive patients

Currently, HIV infection is becoming epidemic. More than 42% of HIV-infected people in the world are women of childbearing age. With the onset of antiretroviral therapy, chemoprophylaxis during pregnancy, and planned cesarean section, the risk of perinatal HIV transmission decreases to 1–2%. However, various side effects such as anemia, neutropenia, thrombocytopenia, fatty liver dystrophy, toxic hepatitis, acute pancreatitis, increased serum transaminases, changes in biochemical parameters of liver function, impaired glucose tolerance, and hypocoagulation may occur in the context of antiretroviral therapy. This poses to the anesthesiologist a number of new tasks to ensure patient safety during the perioperative period.

Lipid status and vascular bloodstream of servicemen with initial liver fibrosis

Frequency of violations of nutritional status, lipid metabolism, cardiovascular status and large vessels endothelium changes in servicemen with fatty liver dystrophy and early fibrosis are assessed. It is revealed that the prevalence of hepatic pathology with dysmetabolic aetiology in this category has vary from 41 to 67%, increasing in combination with subclinical atherosclerosis. According to the results of the study, it is found out that the fibrotic changes in the liver of servicemen associated with a higher frequency of abdominal obesity (higher body mass index and waist circumference), dyslipidemia and structural atherogenic changes of common carotid arteries intima-media complex by type of subclinical atherosclerosis. Have been determined that a violation of the liver elasticity revealed during conducting of transient elastography on servicemen with initial fibrosis meets I-II stage. In the same group with biochemical study increased hepatic biochemical parameters of cytolysis and cholestasis that may indicate activation of inflammatory reactions and transition from fatty hepatosis to more severe stage of steatohepatitis are revealed. A possible mechanism of relationship of obesity, liver fibrosis and atherosclerotic changes of endothelium is proposed. A primary role of fibrosis in nonalcoholic fatty liver disease as a risk factor for the progression of hepatic and cardiovascular pathology in young men was determined.

Гістологічні зміни в котів за хламідіозу

The article presents the results of the study of histological changes in organs and tissues of cats for chlamydial infection. The histopathological examination of the postmortem cases of cats (n = 8) of different breeds between the ages of 3 to 6 years old, who lives with the laboratory methods have been diagnosed and identified the pathogen Chlamydia felis. According to historical data from sick animals were recorded various nature and degree of conjunctivitis and pronounced signs of a lesion of the respiratory tract (rhinitis, bronchitis, pneumonia). The made histological sections were stained with hematoxylin and eosin according to routine standard methods. The general histological structure and microstructural changes in histological preparations were studied under a light microscope. The histopathological examination were confirmed and specified the pathoanatomical diagnoses, established after autopsy of dead animals. It is shown that the most pronounced damage and characteristic changes all dead cats fixed contact in lung tissues and regional lymph nodes (mediastinal and bronchial), and in the spleen. The morphological criteria of chlamydial infection in the studied dead animals at the microscopic level were as follows:1) interstitial pneumonia; 2) pulmonary fibrosis; 3) fibrinous-purulent pleuropneumonia; 4) hyperplasia and serous lymphadenitis of the mediastinal and bronchial lymph nodes; 5) hyperplasia of lymphoid nodules of the spleen; 6) passive venous congestion of the liver and kidneys; 7) fatty and granular liver dystrophy. The results of our study, the pathomorphological diagnosis of chlamydial infection in most of the cats that died was based on changes characteristic of interstitial pneumonia followed pneumosclerosis or fibrinous-purulent pleuropneumonia, with deep affection of the bronchial epithelium. Consequently, the features of chlamydial infection are such that the clinical picture does not always correspond to the severity of morphological manifestations and complications of infection. Weak clinical manifestations can be combined with significant destructive, degenerative and necrotic changes in organs and tissues.

The Relationship of Obesity to the Character of Nutrition

The aim of the investigation was to study pathogenesis of abdominal obesity on the model of experimental fatty dystrophy in rats; to assess the state of lipid and protein metabolism and to elucidate morpho-biochemical interrelations between pathogenesis of fatty liver dystrophy and character of nutrition. Male Wistar rats (n = 42, weight 210 g) were object of the research. Three groups of animals were formed: rats of group 1 (control group, n = 15) received standard vivarium diet; rats of group 2 (n = 15) received hypercaloric fatty diet with allyl alcohol; rats of group 3 (with alloxan diabetes, n = 12) also received hypercaloric fatty diet with allyl alcohol. Rats were maintained on these diets for 90 days and were controlled every 30 days. Dynamics of body mass of animals after 90 days of feeding was: group 1--from 200 ± 10 to 230 ± 10, group 2--from 200 ± 10 to 385 ± 5, group 3--from 200 ± 10 to 400 ± 10 g. Dynamic development of obesity and liver dystrophy is caused by disturbances of lipid and protein metabolism at the background of imbalance of alimentary fat and metabolic changes. Lipid disorganization is caused by the deficiency of the hepatrophic factors.

Histomorphological structure of the liver in roach (Rutilus rutilus) and pike (Esox lucius) from lakes with different levels of anthropogenic impact

Using histological techniques, changes in histomorphological characteristics of the liver were revealed in two fish species from lakes with different levels of anthropogenic impact. The most significant changes in the liver of roach from Kostomukshskoe Lake (used as a tailing dump of the Kostomuksha Mining and Ore-processing Plant) included initial stages of parenchymal protein dystrophy and, to a lesser extent, fatty dystrophy; in pike, fatty liver dystrophy prevailed. Compared to roach, pike proved to be more sensitive to ecological conditions in Kostomukshskoe lake, including increased water mineralization. In both fish species from Koivas Lake (20 km from the pollution source), only the initial stages of local dystrophic changes could be detected. The fish from clean Kamennoe Lake (located in a protected area) had no pathological changes in the liver.

Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. PA phosphatase contains five tryptophan residues and exhibited inherit fluorescence that increased upon interaction with phosphatidylcholine liposomes. The interaction was enhanced by inclusion of other phospholipids and especially the substrate PA. Interaction was dependent on both the concentration of phosphatidylcholine-PA liposomes as well as the surface concentration of PA in liposomes. Mg(2+) ions, which were required for catalysis, did not affect PA phosphatase interaction with phosphatidylcholine-PA liposomes. PA phosphatase was a substrate for protein kinase A, protein kinase C, and casein kinase II, and these phosphorylations decreased PA phosphatase interaction with phosphatidylcholine-PA liposome membranes.

Relationship of glucose and oleate metabolism to cardiac function in lipin-1 deficient (fld) mice

Lipin-1 is the major phosphatidate phosphatase (PAP) in the heart and a transcriptional coactivator that regulates fatty acid (FA) oxidation in the liver. As the control of FA metabolism is essential for maintaining cardiac function, we investigated whether lipin-1 deficiency affects cardiac metabolism and performance. Cardiac PAP activity in lipin-1 deficient [fatty liver dystrophy (fld)] mice was decreased by >80% compared with controls. Surprisingly, oleate oxidation and incorporation in triacylglycerol (TG), as well as glucose oxidation, were not significantly different in perfused working fld hearts. Despite this, [³H]oleate accumulation in phosphatidate and phosphatidylinositol was increased in fld hearts, reflecting the decreased PAP activity. Phosphatidate accumulation was linked to increased cardiac mammalian target of rapamycin complex 1 (mTORC1) signaling and endoplasmic reticulum (ER) stress. Transthoracic echocardiography showed decreased cardiac function in fld mice; however, cardiac dysfunction was not observed in ex vivo perfused working fld hearts. This showed that changes in systemic factors due to the global absence of lipin-1 could contribute to the decreased cardiac function in vivo. Collectively, this study shows that fld hearts exhibit unchanged oleate esterification, as well as oleate and glucose oxidation, despite the absence of lipin-1. However, lipin-1 deficiency increases the accumulation of newly synthesized phosphatidate and induces aberrant cell signaling.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Liver regeneration is impaired in lipodystrophic fatty liver dystrophy mice

We previously reported that mice subjected to partial hepatectomy exhibit rapid development of hypoglycemia followed by transient accumulation of fat in the early regenerating liver. We also showed that disrupting these metabolic alterations results in impaired liver regeneration. The studies reported here were undertaken to further characterize and investigate the functional importance of changes in systemic adipose metabolism during normal liver regeneration. The results showed that a systemic catabolic response is induced in each of two distinct, commonly used experimental models of liver regeneration (partial hepatectomy and carbon tetrachloride treatment), and that this response occurs in proportion to the degree of induced hepatic insufficiency. Together, these observations suggest that catabolism of systemic adipose stores may be essential for normal liver regeneration. To test this possibility, we investigated the hepatic regenerative response in fatty liver dystrophy (fld) mice, which exhibit partial lipodystrophy and have diminished peripheral adipose stores. The results showed that the development of hypoglycemia and hepatic accumulation of fat was attenuated and liver regeneration was impaired following partial hepatectomy in these animals. The fld mice also exhibited increased hepatic p21 expression and diminished plasma levels of the adipose-derived hormones adiponectin and leptin, which have each been implicated as regulators of liver regeneration. These data suggest that the hypoglycemia that develops after partial hepatectomy induces systemic lipolysis followed by accumulation of fat derived from peripheral stores in the early regenerating liver, and that these events may be essential for initiation of normal liver regeneration.

Insulin-stimulated Interaction with 14-3-3 Promotes Cytoplasmic Localization of Lipin-1 in Adipocytes

Lipin-1 is a bifunctional protein involved in lipid metabolism and adipogenesis. Lipin-1 plays a role in the biosynthesis of triacylglycerol through its phosphatidate phosphatase activity and also acts as a transcriptional co-activator of genes involved in oxidative metabolism. Lipin-1 resides in the cytoplasm and translocates to the endoplasmic reticulum membrane to catalyze the phosphatidate phosphatase reaction. It also possesses a nuclear localization signal, which is required for its translocation to the nucleus and may therefore be important for lipin-1 co-activator function. Thus, subcellular localization may be an important factor in the regulation of this protein. Here, we show that the nuclear localization signal alone is not sufficient for lipin-1 nuclear localization, and identify lipin-1 interaction with 14-3-3 as a determinant of its subcellular localization. We demonstrate that lipin-1 interacts with 14-3-3 proteins and that overexpression of 14-3-3 promotes the cytoplasmic localization of lipin-1 in 3T3-L1 adipocytes. The effect of 14-3-3 is mediated through a serine-rich domain in lipin-1. Functional mapping of the 14-3-3-interacting region within the serine-rich domain indicates redundancy and cooperativity among several sites, including five phosphorylated serine and threonine residues. Insulin stimulation of 3T3-L1 adipocytes results in increased lipin-1 phosphorylation, enhanced interaction with 14-3-3, and predominantly cytoplasmic localization. In summary, our studies suggest that insulin may modulate the cellular function of lipin-1 by regulating its subcellular localization through interactions with 14-3-3 proteins.

The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

LPIN1 genetic variation is associated with rosiglitazone response in type 2 diabetic patients

Lipin1 protein, a product of the LPIN1 gene, is required for normal adipose tissue development and metabolism. Lipin1 deficiency results in immature adipocyte development in cases of mouse fatty liver dystrophy and human lipodystrophy. Recently, pioglitazone has been reported to increase human adipocyte lipin1 expression. We evaluated the effects of LPIN1 polymorphisms on rosiglitazone response in patients with type 2 diabetes (T2DM). A total of 262 patients were treated with 12 weeks of rosiglitazone (4 mg/day) in addition to their previous drug regimen medications. Six single nucleotide polymorphisms (SNPs) at the LPIN1 locus were genotyped: rs11693809, rs10192566, rs2278513, rs2577262, rs2716610, and rs1050800. Because rs11693809, rs10192566, and rs2278513 are in nearly complete linkage disequilibrium (D′ > 0.958, r 2 > 0.882), we analyzed rs10192566, rs2577262, rs2716610, and rs1050800. Rs10192566 was significantly associated with rosiglitazone treatment response. Patients with the G allele in rs10192566 had a larger decrease in fasting plasma glucose, 2-h postprandial glucose, and HbA1c than those without. This genetic effect remained significant after adjustment for age, sex, and initial body weight. No other SNPs were associated with response. These data suggest that LPIN1 genetic variations can affect rosiglitazone treatment response in T2DM.

Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.

The G80R mutation in the yeast lipin homolog Pah1p abolishes phosphatidate phosphatase activity

The yeast lipin homolog Pah1p is a Mg2+‐dependent phosphatidate (PA) phosphatase enzyme. Gly80 that is found in the NLIP domain of Pah1p is conserved in mammalian lipin proteins. A spontaneous G84R mutation in lipin 1 causes lipodystrophy and fatty liver in the fld2J (fatty liver dystrophy) mouse at birth. To examine the importance of Gly80 to the enzymatic function of yeast Pah1p, we constructed a mutant PAH1HA allele that contained arginine in place of the conserved glycine residue. While the G80R mutation did not affect the expression of Pah1p in yeast (as determined by immunoblot analysis using anti‐HA antibodies), the mutation had a major effect on its phosphatidate phosphatase activity. The overexpression of the G80R mutant protein did not have a significant effect on the level of PA phosphatase activity in pah1Δ mutant cells when compared with cells that overexpressed the wild type PAH1HA allele. Moreover, an immune complex containing the G80R mutant protein that was isolated from pah1Δ cells expressing the G80R PAH1HA allele had essentially no PA phosphatase activity. A His6‐tagged G80R mutant protein was expressed and purified from E. coli. The Vmax of the G80R mutant enzyme was 56‐fold lower than that of the wild type enzyme. The G80R mutation also caused a 2.5‐fold increase in the cooperativity respect to PA, but little effect on the Km value for PA. Supported by NIH grant GM 28140.

Association of Lipin 1 Gene Polymorphisms with Measures of Energy and Glucose Metabolism

To examine the importance of lipin 1 (LPIN1) gene variation in energy and glucose metabolism. Transgenic animal models have shown that lipin, a protein encoded by the LPIN1 gene, promotes fat synthesis and storage in adipose tissue while decreasing energy expenditure and lipid oxidation in skeletal muscle. Lpin1 was identified as the mutated gene in the fatty liver dystrophy mouse, which exhibits lipin deficiency and features of human lipodystrophy. We genotyped five LPIN1 polymorphisms and tested for association with resting metabolic rate (RMR), fat oxidation, fasting plasma insulin and glucose concentration, and obesity-related phenotypes, including BMI, body fat percentage, sum of six skinfolds, and waist circumference in 712 subjects of the Quebec Family Study. The strongest results were generation-specific. In parents, RMR of the G/G IVS13 + 3333A>G homozygotes was 107 kcal/d higher than in A/A homozygotes and 39 kcal/d higher than in A/G heterozygotes (p = 0.0003). In offspring, carriers of the C allele of the IVS18 + 181C>T variant had significantly higher (p < 0.0003) insulin levels than T/T homozygotes. These associations remained significant after adjusting for multiple testing. Several other associations between body composition measures and the IVS18 + 181C>T variant were significant (p = 0.05 to 0.003), suggesting a strong pattern of relationships. These findings support the hypothesis that sequence variation in the LPIN1 gene contributes to variation in RMR and obesity-related phenotypes potentially in an age-dependent manner.

Three Mammalian Lipins Act as Phosphatidate Phosphatases with Distinct Tissue Expression Patterns

We previously identified mutations in the Lpin1 gene, encoding lipin-1, as the underlying cause of lipodystrophy in the fatty liver dystrophy (fld) mutant mouse. Lipin-1 is normally expressed at high levels in adipose tissue and skeletal muscle, and deficiency in the fld mouse causes impaired adipose tissue development, insulin resistance, and altered energy expenditure. We also identified two additional lipin protein family members of unknown function, lipin-2 and lipin-3. Han et al. (Han, G. S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218) recently demonstrated that the single lipin homolog in yeast, Smp2, exhibits phosphatidate phosphatase type-1 (PAP1) activity, which has a key role in glycerolipid synthesis. Here we demonstrate that lipin-1 accounts for all of the PAP1 activity in white and brown adipose tissue and skeletal muscle. However, livers of lipin-1-deficient mice exhibited normal PAP1 activity, indicating that other members of the lipin protein family could have PAP1 activity. Consistent with this possibility, recombinant lipin-2 and lipin-3 possess PAP1 activity. Each of the three lipin family members showed Mg2+-dependent activity that was specific for phosphatidate under the conditions employed. The different lipins showed distinct tissue expression patterns. Our results establish the three mammalian lipin proteins as PAP1 enzymes and explain the biochemical basis for lipodystrophy in the lipin-1-deficient fld mouse.

Insulin Controls Subcellular Localization and Multisite Phosphorylation of the Phosphatidic Acid Phosphatase, Lipin 1

Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

Lipin Deficiency Impairs Diurnal Metabolic Fuel Switching

Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy (fld) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction.

Alternatively Spliced Lipin Isoforms Exhibit Distinct Expression Pattern, Subcellular Localization, and Role in Adipogenesis

We recently identified mutations in the Lpin1 (lipin) gene to be responsible for lipodystrophy in the fatty liver dystrophy (fld) mouse strain. Previous studies revealed that lipin plays a critical role in adipogenesis, explaining the adipose-deficient phenotype of the fld mouse. In the current study, we demonstrate that alternative mRNA splicing generates two lipin isoforms, lipin-alpha and lipin-beta, which are differentially expressed during adipocyte differentiation. Lipin-alpha expression peaks at day 2 of 3T3-L1 cell differentiation, after which its levels gradually decrease. In contrast, lipin-beta expression is transiently elevated at 10 h, followed by a drop to background levels at 20 h and a gradual increase between days 2 and 6 of differentiation. The two lipin isoforms also exhibit differences in subcellular localization. Lipin-alpha is predominantly nuclear, whereas lipin-beta is primarily located in the cytoplasm of 3T3-L1 adipocytes, suggesting distinct cellular functions. Using primary mouse embryonic fibroblasts expressing either lipin-alpha or lipin-beta, we demonstrate functional differences between the two isoforms. Whereas lipin-alpha is required for adipocyte differentiation, the predominant effect of lipin-beta expression is the induction of lipogenic genes. In vivo, overexpression of lipin-beta specifically in mature adipocytes leads to elevated expression of lipogenic genes and adipocyte hypertrophy, confirming a role of lipin-beta in the regulation of lipogenesis. In conclusion, our data suggest that the two lipin isoforms have distinct, but complementary, functions in adipogenesis, with lipin-alpha playing a primary role in differentiation and lipin-beta being predominantly involved in lipogenesis.