Karim Nadra

Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor β/δ

ABSTRACT Mutation of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARβ/δ-null mutant embryos. While very little is known at present about the pathway governed by PPARβ/δ in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARβ/δ-null embryos is found in the giant cell layer. PPARβ/δ 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 PPARβ/δ is silenced. Conversely, exposure of Rcho-1 cells to a PPARβ/δ 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 PPARβ/δ 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, PPARβ/δ 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 PPARβ/δ-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 PPARβ/δ in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARβ/δ agonist as therapeutic agents of broad application.

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.

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.

PPARγ 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.

Cell Autonomous Lipin 1 Function Is Essential for Development and Maintenance of White and Brown Adipose Tissue

ABSTRACT 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 aP2Cre/+/LpfEx2-3/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 aP2Cre-ERT2/+/LpfEx2-3/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 aP2Cre-ERT2/+/LpfEx2-3/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.

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

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‐1alpha, 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. (Hepatology 2013; 58:1953–1963)

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.

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.

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 (Lpin11Hubr), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin11Hubr 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. Lpin11Hubr rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1fld/fld mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular analyses revealed that these lesions improve in older Lpin11Hubr rats as compared with young Lpin11Hubr rats and Lpin1fld/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 Lpin11Hubr rats as compared with Lpin1fld/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.