Mice with an adipocyte-specific lipin 1 separation-of-function allele reveal unexpected roles for phosphatidic acid in metabolic regulation.

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of Lpin1 deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific Lpin1 gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.

Combination of lipid metabolism alterations and their sensitivity to inflammatory cytokines in human lipin-1-deficient myoblasts.

Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes [peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2)] were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.

Hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice.

UNLABELLED: Lipin-1 regulates lipid metabolism by way of its function as an enzyme in the triglyceride synthesis pathway and as a transcriptional coregulatory protein and is highly up-regulated in alcoholic fatty liver disease. In the present study, using a liver-specific lipin-1-deficient (lipin-1LKO) mouse model, we aimed to investigate the functional role of lipin-1 in the development of alcoholic steatohepatitis and explore the underlying mechanisms. Alcoholic liver injury was achieved by pair feeding wild-type and lipin-1LKO mice with modified Lieber-DeCarli ethanol-containing low-fat diets for 4 weeks. Surprisingly, chronically ethanol-fed lipin-1LKO mice showed markedly greater hepatic triglyceride and cholesterol accumulation, and augmented elevation of serum liver enzymes accompanied by increased hepatic proinflammatory cytokine expression. Our studies further revealed that hepatic removal of lipin-1 in mice augmented ethanol-induced impairment of hepatic fatty acid oxidation and lipoprotein production, likely by way of deactivation of peroxisome proliferator-activated receptor γ coactivator-1 alpha, a prominent transcriptional regulator of lipid metabolism. CONCLUSIONS: Liver-specific lipin-1 deficiency in mice exacerbates the development and progression of experimental alcohol-induced steatohepatitis. Pharmacological or nutritional modulation of hepatic lipin-1 may be beneficial for the prevention or treatment of human alcoholic fatty liver disease.

Comparative clinical study for the efficacy and safety of two different hyaluronic acid-based fillers with Tri-Hyal versus Vycross technology: A long-term prospective randomized clinical trial.

BACKGROUND: Hyaluronic acid-based fillers have an immediate volumizing effect for the treatment of dermal contour deformities and to smooth dermal depressions formed by the loss of volume. A previous study on 2016-2018 has shown the efficacy and safety of the HA-based filler ART FILLER® Volume on the midface only, but not in a comparative manner. METHODS: In this context, an 18 months prospective randomized single-blind study of the non-inferiority of ART FILLER® Volume versus the reference product Juvéderm Voluma® was performed on the midface, temple, and jawline, and non-comparative study on the chin. The efficacy and the longevity were evaluated for the selected face areas via dedicated clinical scoring systems after a single filler injection without any re-touch or re-injection. The short- and long-term adverse effects were also recorded. RESULTS: The observations confirmed the non-inferiority of ART FILLER® Volume versus the reference product on the different injected areas. For both fillers, the beneficial effects on volumes restoration were maintained 18 months post-injection; however, these effects were diminished among the time. Furthermore, injections of Art Filler® Volume were well tolerated by the subjects and showed less acute side effects compared with the reference product that may be explained by a lower induction of inflammation.

Long-Term Efficacy and Tolerability of a Medium G' HA Filler with Tri-Hyal Technology on the Rejuvenation of the Mobile Facial Zone.

Purpose: Injectable hyaluronic acid-based fillers are commonly used for the correction of skin contour irregularities and to smooth skin depressions formed by volume loss during the aging process. These fillers are particularly efficient to restore perioral skin depressions/wrinkles or to correct topographical anomalies. The European directives require a continuous evaluation of the performance of these medical devices, particularly for CE marked products. Methods: An 18-month prospective randomized single-blind study for the efficacy and safety of ART FILLER Universal (AFU) was performed on the lips, the nasolabial folds, and the marionettes lines. The evaluations were performed on 153 subjects enrolled in this study. The efficacy, the longevity, and the safety were evaluated for the injected areas via area specific clinical scoring after a single injection with the filler and with no re-touch. Results: We showed here that filler injection induced potent improvements of volume restoration after a single injection on all the treated areas. These beneficial properties of the filler were significant 3 weeks after injection and during the whole study period. Moreover, injections of the filler were well tolerated by the subjects. The recorded adverse events are routinely seen with HA fillers for face volume corrections, and most of these local reactions resolved within 14 days. Conclusion: AFU was well tolerated and showed a continuous efficacy for at least 18 months, in exploratory analyses.

Long-term efficacy and safety of a hyaluronic acid dermal filler based on Tri-Hyal technology on restoration of midface volume.

INTRODUCTION: Art Filler Volume (AFV) is a hyaluronic acid (HA)-based filler formulated with "Tri-Hyal" technology, a unique combination of three sizes of HA chains. This study assessed AFV efficacy and safety over 18 months when used to restore midface volume. METHODS: During this open-label study, a maximum of 1.8 mL AFV was injected into each cheek area on Day 0 (D0). Subjects were evaluated at D21, when, if necessary, a retouch could be performed (maximum 1.2 mL per cheek). Subjects were evaluated at seven follow-up visits through to D540. The primary assessment was based on the evolution of the Medicis Midface Volume Scale (MMVS) grade on D21. Secondary outcomes were local and general adverse events, investigator- and subject-assessed Global Aesthetic Improvement Scale scores and changes in self-esteem. RESULTS: Of the 79 healthy Caucasians enrolled (mean age 54.8 years), 25 required a second injection. In the intention-to-treat population, mean overall MMVS scores improved significantly from D0 (3.2 ± 0.4) to D21 (1.8 ± 0.6) and D42 (1.7 ± 0.6) (all p < 0.0001). MMVS scores for each cheek also improved significantly, irrespective of retouch on D21: 22% of injections showed a persistent benefit at D540 without retouch. The most common adverse events were pain on palpation (19%), erythema (15%) and edema (13%); most were mild or moderate and resolved within 2 weeks. CONCLUSION: AFV produces a sustained objective and subjective midface volume restoration in female and male subjects, often without retouching, and was well tolerated.

A prospective multicenter clinical trial evaluating the efficacy and safety of a hyaluronic acid-based filler with Tri-Hyal technology in the treatment of lips and the perioral area.

BACKGROUND: Age-related changes of facial soft tissue cause clinical signs of facial aging such as lip atrophy, marionette lines, and an accentuated nasolabial fold. These changes can be modified using dermal fillers. AIMS: To evaluate efficacy, longevity, and safety of a cross-linked hyaluronic acid-based filler with Tri-Hyal technology in the treatment of lips, nasolabial folds, and marionette lines. MATERIALS AND METHODS: This prospective, multi-center trial evaluated injections of three different areas (lips, nasolabial fold alone, or with marionette wrinkles) with a soft tissue filler containing 25 mg/ml cross-linked hyaluronic acid and 0.3% lidocaine. Primary endpoint was the aesthetic correction 3 weeks after one injection session without touch-up. Follow-up was 18 months. Assessments were performed using the Global Aesthetic Score (GAS), clinical scoring based on photographic scales, high-frequency ultrasound imaging, and the Global Aesthetic Improvement Scale (GAIS). RESULTS: In total, 100 subjects were injected. GAS improved significantly for all treatment indications at 3 weeks (p < 0.0001). Success rates were highest for nasolabial folds (98.4%), followed by marionette lines (94.4%) and lips (73.5%). After 18 months post-injection, success was observed in 91%, 88%, and 33% of subjects injected into nasolabial folds, marionette lines, and lips, respectively. GAIS scored highest for nasolabial folds (SGAIS: 71%; IGAIS: 40%), followed by marionette lines (SGAIS: 56%; IGAIS: 33%) and lips (SGAIS: 30%; IGAIS: 22%) at 18 months follow-up. CONCLUSIONS: The filler demonstrated high efficacy and safety in all indications. Regional differences in longevity were evident. Thus, the necessity of regional retreatments should be discussed with patients before injection.

A hypomorphic mutation in Lpin1 induces progressively improving neuropathy and lipodystrophy in the rat.

The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critical role in lipid metabolism. In this study we describe the identification and characterization of a rat model with a mutated Lpin1 gene (Lpin1(1Hubr)), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin1(1Hubr) rats are characterized by hindlimb paralysis and mild lipodystrophy that are detectable from the second postnatal week. Sequencing of Lpin1 identified a point mutation in the 5'-end splice site of intron 18 resulting in mis-splicing, a reading frameshift, and a premature stop codon. As this mutation does not induce nonsense-mediated decay, it allows the production of a truncated Lipin 1 protein lacking PAP1 activity. Lpin1(1Hubr) rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1(fld/fld) mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular analyses revealed that these lesions improve in older Lpin1(1Hubr) rats as compared with young Lpin1(1Hubr) rats and Lpin1(fld/fld) mice. We observed activation of compensatory biochemical pathways substituting for missing PAP1 activity that, in combination with a possible non-enzymatic Lipin 1 function residing outside of its PAP1 domain, may contribute to the less severe phenotypes observed in Lpin1(1Hubr) rats as compared with Lpin1(fld/fld) mice. Although we are cautious in making a direct parallel between the presented rodent model and human disease, our data may provide new insight into the pathogenicity of recently identified human LPIN1 mutations.

Epineurial adipocytes are dispensable for Schwann cell myelination.

Previous clinical observations and data from mouse models with defects in lipid metabolism suggested that epineurial adipocytes may play a role in peripheral nervous system myelination. We have used adipocyte-specific Lpin1 knockout mice to characterize the consequences of the presence of impaired epineurial adipocytes on the myelinating peripheral nerve. Our data revealed that the capacity of Schwann cells to establish myelin, and the functional properties of peripheral nerves, were not affected by compromised epineurial adipocytes in adipocyte-specific Lpin1 knockout mice. To evaluate the possibility that Lpin1-negative adipocytes are still able to support endoneurial Schwann cells, we also characterized sciatic nerves from mice carrying epiblast-specific deletion of peroxisome proliferator-activated receptor gamma, which develop general lipoatrophy. Interestingly, even the complete loss of adipocytes in the epineurium of peroxisome proliferator-activated receptor gamma knockout mice did not lead to detectable defects in Schwann cell myelination. However, probably as a consequence of their hyperglycemia, these mice have reduced nerve conduction velocity, thus mimicking the phenotype observed under diabetic condition. Together, our data indicate that while adipocytes, as regulators of lipid and glucose homeostasis, play a role in nerve function, their presence in epineurium is not essential for establishment or maintenance of proper myelin.

SCAP is required for timely and proper myelin membrane synthesis.

Myelination requires a massive increase in glial cell membrane synthesis. Here, we demonstrate that the acute phase of myelin lipid synthesis is regulated by sterol regulatory element-binding protein (SREBP) cleavage activation protein (SCAP), an activator of SREBPs. Deletion of SCAP in Schwann cells led to a loss of SREBP-mediated gene expression involving cholesterol and fatty acid synthesis. Schwann cell SCAP mutant mice show congenital hypomyelination and abnormal gait. Interestingly, aging SCAP mutant mice showed partial regain of function; they exhibited improved gait and produced small amounts of myelin indicating a slow SCAP-independent uptake of external lipids. Accordingly, extracellular lipoproteins partially rescued myelination by SCAP mutant Schwann cells. However, SCAP mutant myelin never reached normal thickness and had biophysical abnormalities concordant with abnormal lipid composition. These data demonstrate that SCAP-mediated regulation of glial lipogenesis is key to the proper synthesis of myelin membrane, and provide insight into abnormal Schwann cell function under conditions affecting lipid metabolism.

A hyaluronic acid-based filler reduces lipolysis in human mature adipocytes and maintains adherence and lipid accumulation of long-term differentiated human preadipocytes.

The beneficial role of subcutaneous adipose tissue in skin rejuvenation derived from its capacity to fill the under-layer volumes but also from its ability to regulate the extracellular matrix production by dermis fibroblasts. Hyaluronic acid (HA), a major component of the extracellular matrix, is a commonly used injectable dermal filler showing excellent efficiencies to maintain tissue augmentation even after its biodegradation. To improve their stability, the HA molecules can also be "cross-linked" to each other. The effects of cross-linked HA-based fillers on the dermal structure are well known. For safety reasons, most of the physicians prefer to use the blunt cannula for injections. However, evidences showed that the cannula could not be located in the dermis, but it passes through immediate hypodermis and the long-lasting effect of cross-linked HA-based fillers may be related to its effects on adipose tissue. To test whether cross-linked HA has a direct effect on human adipocytes, we treated isolated adipocytes and precursors cells from human skin donors with cross-linked HA. Biochemical and cellular analysis demonstrated that treatment by cross-linked HA showed beneficial effects on differentiated cell adherence and survival as well as reduced basal and induced lipolysis in fully mature adipocytes. Taken together, these data showed that cross-linked HA promoted cell adherence and preserved the adipogenic capacity of preadipocytes during prolonged cell culture, bringing additional evidences of the beneficial role of cross-linked HA-based fillers in maintenance of the subcutaneous fat mass. This first study could defend a preventive approach to facial volume loss during natural aging.

Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

Cell autonomous lipin 1 function is essential for development and maintenance of white and brown adipose tissue.

Through analysis of mice with spatially and temporally restricted inactivation of Lpin1, we characterized its cell autonomous function in both white (WAT) and brown (BAT) adipocyte development and maintenance. We observed that the lipin 1 inactivation in adipocytes of aP2(Cre/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice resulted in lipodystrophy and the presence of adipocytes with multilocular lipid droplets. We further showed that time-specific loss of lipin 1 in mature adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice led to their replacement by newly formed Lpin1-positive adipocytes, thus establishing a role for lipin 1 in mature adipocyte maintenance. Importantly, we observed that the presence of newly formed Lpin1-positive adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice protected these animals against WAT inflammation and hepatic steatosis induced by a high-fat diet. Loss of lipin 1 also affected BAT development and function, as revealed by histological changes, defects in the expression of peroxisome proliferator-activated receptor alpha (PPARα), PGC-1α, and UCP1, and functionally by altered cold sensitivity. Finally, our data indicate that phosphatidic acid, which accumulates in WAT of animals lacking lipin 1 function, specifically inhibits differentiation of preadipocytes. Together, these observations firmly demonstrate a cell autonomous role of lipin 1 in WAT and BAT biology and indicate its potential as a therapeutical target for the treatment of obesity.

The glucocorticoid-induced leucine zipper (gilz/Tsc22d3-2) gene locus plays a crucial role in male fertility.

The glucocorticoid-induced leucine zipper (Tsc22d3-2) is a widely expressed dexamethasone-induced transcript that has been proposed to be important in immunity, adipogenesis, and renal sodium handling based on in vitro studies. To address its function in vivo, we have used Cre/loxP technology to generate mice deficient for Tsc22d3-2. Male knockout mice were viable but surprisingly did not show any major deficiencies in immunological processes or inflammatory responses. Tsc22d3-2 knockout mice adapted to a sodium-deprived diet and to water deprivation conditions but developed a subtle deficiency in renal sodium and water handling. Moreover, the affected animals developed a mild metabolic phenotype evident by a reduction in weight from 6 months of age, mild hyperinsulinemia, and resistance to a high-fat diet. Tsc22d3-2-deficient males were infertile and exhibited severe testis dysplasia from postnatal d 10 onward with increases in apoptotic cells within seminiferous tubules, an increased number of Leydig cells, and significantly elevated FSH and testosterone levels. Thus, our analysis of the Tsc22d3-2-deficient mice demonstrated a previously uncharacterized function of glucocorticoid-induced leucine zipper protein in testis development.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

Phosphatidic acid mediates demyelination in Lpin1 mutant mice.

Lipids play crucial roles in many aspects of glial cell biology, affecting processes ranging from myelin membrane biosynthesis to axo-glial interactions. In order to study the role of lipid metabolism in myelinating glial cells, we specifically deleted in Schwann cells the Lpin1 gene, which encodes the Mg2+-dependent phosphatidate phosphatase (PAP1) enzyme necessary for normal triacylglycerol biosynthesis. The affected animals developed pronounced peripheral neuropathy characterized by myelin degradation, Schwann cell dedifferentiation and proliferation, and a reduction in nerve conduction velocity. The observed demyelination is mediated by endoneurial accumulation of the substrate of the PAP1 enzyme, phosphatidic acid (PA). In addition, we show that PA is a potent activator of the MEK-Erk pathway in Schwann cells, and that this activation is required for PA-induced demyelination. Our results therefore reveal a surprising role for PA in Schwann cell fate determination and provide evidence of a direct link between diseases affecting lipid metabolism and abnormal Schwann cell function.