Renal injury in Seipin-deficient lipodystrophic mice and its reversal by adipose tissue transplantation or leptin administration alone: adipose tissue-kidney crosstalk.

Seipin deficiency is responsible for type 2 congenital generalized lipodystrophy with severe loss of adipose tissue (AT) and could lead to renal failure in humans. However, the effect of Seipin on renal function is poorly understood. Here we report that Seipin knockout (SKO) mice exhibited impaired renal function, enlarged glomerular and mesangial surface areas, renal depositions of lipid, and advanced glycation end products. Elevated glycosuria and increased electrolyte excretion were also detected. Relative renal gene expression in fatty acid oxidation and reabsorption pathways were impaired in SKO mice. Elevated glycosuria might be associated with reduced renal glucose transporter 2 levels. To improve renal function, AT transplantation or leptin administration alone was performed. Both treatments effectively ameliorated renal injury by improving all of the parameters that were measured in the kidney. The treatments also rescued insulin resistance and low plasma leptin levels in SKO mice. Our findings demonstrate for the first time that Seipin deficiency induces renal injury, which is closely related to glucolipotoxicity and impaired renal reabsorption in SKO mice, and is primarily caused by the loss of AT and especially the lack of leptin. AT transplantation and leptin administration are two effective treatments for renal injury in Seipin-deficient mice.-Liu, X.-J., Wu, X.-Y., Wang, H., Wang, S.-X., Kong, W., Zhang, L., Liu, G., Huang, W. Renal injury in Seipin-deficient lipodystrophic mice and its reversal by adipose tissue transplantation or leptin administration alone: adipose tissue-kidney crosstalk.

Activation of PPARγ Ameliorates Spatial Cognitive Deficits through Restoring Expression of AMPA Receptors in Seipin Knock-Out Mice.

A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Here, we show that seipin deficiency in hippocampal CA1 pyramidal cells caused the reduction of peroxisome proliferator-activated receptor gamma (PPARγ). Twelve-week-old systemic seipin knock-out mice and neuronal seipin knock-out (seipin-nKO) mice, but not adipose seipin knock-out mice, exhibited spatial cognitive deficits as assessed by the Morris water maze and Y-maze, which were ameliorated by the treatment with the PPARγ agonist rosiglitazone (rosi). In addition, seipin-nKO mice showed the synaptic dysfunction and the impairment of NMDA receptor-dependent LTP in hippocampal CA1 regions. The density of AMPA-induced current (IAMPA) in CA1 pyramidal cells and GluR1/GluR2 expression were significantly reduced in seipin-nKO mice, whereas the NMDA-induced current (INMDA) and NR1/NR2 expression were not altered. Rosi treatment in seipin-nKO mice could correct the decrease in expression and activity of AMPA receptor (AMPAR) and was accompanied by recovered synaptic function and LTP induction. Furthermore, hippocampal ERK2 and CREB phosphorylation in seipin-nKO mice were reduced and this could be rescued by rosi treatment. Rosi treatment in seipin-nKO mice elevated BDNF concentration. The MEK inhibitor U0126 blocked rosi-restored AMPAR expression and LTP induction in seipin-nKO mice, but the Trk family inhibitor K252a did not. These findings indicate that the neuronal seipin deficiency selectively suppresses AMPAR expression through reducing ERK-CREB activities, leading to the impairment of LTP and spatial memory, which can be rescued by PPARγ activation. SIGNIFICANCE STATEMENT: Congenital generalized lipodystrophy 2 (CGL2), caused by loss-of-function mutation of seipin gene, is characterized by mental retardation. By the generation of systemic or neuronal seipin knock-out mice, the present study provides in vivo evidence that neuronal seipin deficiency causes deficits in spatial memory and hippocampal LTP induction. Neuronal seipin deficiency selectively suppresses AMPA receptor expression, ERK-CREB phosphorylation with the decline of PPARγ. The PPARγ agonist rosiglitazone can ameliorate spatial cognitive deficits and rescue the LTP induction in seipin knock-out mice by restoring AMPA receptor expression and ERK-CREB activities.

Adipose tissue deficiency results in severe hyperlipidemia and atherosclerosis in the low-density lipoprotein receptor knockout mice.

Adipose tissue can store over 50% of whole-body cholesterol; however, the physiological role of adipose tissue in cholesterol metabolism and atherogenesis has not been directly assessed. Here, we examined lipoprotein metabolism and atherogenesis in a unique mouse model of severe lipodystrophy: the Seipin(-/-) mice, and also in mice deficient in both low-density lipoprotein receptor (Ldlr) and Seipin: the Ldlr(-/-)Seipin(-/-) mice. Plasma cholesterol was moderately increased in the Seipin(-/-) mice when fed an atherogenic diet. Strikingly, plasma cholesterol reached ~6000 mg/dl in the Seipin(-/-)Ldlr(-/-) mice on an atherogenic diet, as compared to ~1000 mg/dl in the Ldlr(-/-) mice on the same diet. The Seipin(-/-)Ldlr(-/-) mice also developed spontaneous atherosclerosis on chow diet and severe atherosclerosis on an atherogenic diet. Rosiglitazone treatment significantly reduced the hypercholesterolemia of the Seipin(-/-)Ldlr(-/-) mice, and also alleviated the severity of atherosclerosis. Our results provide direct evidence, for the first time, that the adipose tissue plays a critical role in the clearance of plasma cholesterol. Our results also reveal a previously unappreciated strong link between adipose tissue and LDLR in plasma cholesterol metabolism.

Seipin deficiency leads to defective parturition in mice.

Seipin is an integral endoplasmic reticulum membrane protein encoded by Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/Bscl2) gene. Seipin deficiency results in lipodystrophy, diabetes, muscle hypertrophy, and male infertility in both human and mouse. Seipin function in female reproduction is unknown. Bscl2-/- dams had normal embryo implantation and body weight gain during pregnancy but reduced delivery rates from 2nd to 4th pregnancies and reduced numbers of pups delivered from 1st to 4th pregnancies. Characterization of first pregnancy revealed increased gestation period and parturition problems, including uterine prolapse, difficulty in delivery, undelivered fetuses, and undelivered tissues in Bscl2-/- females. Bscl2-/- uterine weight was comparable to control at 3 weeks old but significantly increased with myometrial hypertrophy at 10 months old. In situ hybridization revealed relatively low level of Bscl2 mRNA expression in myometrium throughout pregnancy and postpartum but high level of expression in uterine luminal epithelium, suggesting that systemic effect (e.g. elevated glucose and insulin levels) rather than local seipin-deficiency in myometrium might be a main contributing factor to myometrial hypertrophy. On near-term gestation day 18.5 (D18.5), Bscl2-/- females had normal levels of serum progesterone and 17ß-estradiol, indicating functional ovary and placenta. Proliferating Cell Nuclear Antigen (PCNA) staining showed minimal myometrial cell proliferation in both D18.5 Bscl2+/+ and Bscl2-/- uteri. There was strong LC3 immunostaining in Bscl2+/+ and Bscl2-/- peripartum myometrium and increased LC3 staining in Bscl2-/- peripartum uterine luminal epithelium, suggesting a potential role of seipin in regulating autophagy in uterine luminal epithelium but not myometrium. This study demonstrates an association of seipin with myometrium and parturition.

Lack of testicular seipin causes teratozoospermia syndrome in men.

Obesity impairs male fertility, providing evidence for a link between adipose tissue and reproductive function; however, potential consequences of adipose tissue paucity on fertility remain unknown. Lack of s.c. fat is a hallmark of Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), which is caused by mutations in BSCL2-encoding seipin. Mice with a targeted deletion of murine seipin model BSCL2 with severe lipodystrophy, insulin resistance, and fatty liver but also exhibit male sterility. Here, we report teratozoospermia syndrome in a lipodystrophic patient with compound BSCL2 mutations, with sperm defects resembling the defects of infertile seipin null mutant mice. Analysis of conditional mouse mutants revealed that adipocyte-specific loss of seipin causes progressive lipodystrophy without affecting fertility, whereas loss of seipin in germ cells results in complete male infertility and teratozoospermia. Spermatids of the human patient and mice devoid of seipin in germ cells are morphologically abnormal with large ectopic lipid droplets and aggregate in dysfunctional clusters. Elevated levels of phosphatidic acid accompanied with an altered ratio of polyunsaturated to monounsaturated and saturated fatty acids in mutant mouse testes indicate impaired phospholipid homeostasis during spermiogenesis. We conclude that testicular but not adipose tissue-derived seipin is essential for male fertility by modulating testicular phospholipid homeostasis.

Lack of seipin in neurons results in anxiety- and depression-like behaviors via down regulation of PPARγ.

The Seipin gene was originally found to be responsible for type 2 congenital lipodystrophy and involved in lipid droplet formation. Seipin is highly expressed in the central nervous system as well. Seipin mutations have been identified in motor neuron diseases such as Silver syndrome and spastic paraplegia. In this study, we generated neuron-specific seipin knockout mice (seipin-nKO) to investigate the influence of seipin deficiency on locomotion and affective behaviors. In comparison with control mice, 8-week-old male seipin-nKO mice, but not female mice, displayed anxiety- and depression-like behaviors as assessed by open-field, elevated plus-maze, forced swim and tail suspension tests. However, neither male nor female seipin-nKO mice showed locomotion deficits in swimming tank and rotarod tests. Interestingly, the mRNA and protein levels of peroxisome proliferator-activated receptor gamma (PPARγ) in the hippocampus and cortex were lower in male seipin-nKO mice, but not female mice, than controls. In seipin-nKO mice, plasma levels of sex hormones including 17ß-estradiol (E2) in females and testosterone in males as well as corticosterone were not altered compared with controls. The treatment of male seipin-nKO mice with E2 ameliorated the anxiety- and depression-like behaviors and remarkably increased PPARγ levels. The PPARγ agonist rosiglitazone alleviated affective disorders in male seipin-nKO mice. Notably, anxiety- and depression-like behaviors appeared in female seipin-nKO mice after ovariectomy, which was associated with low PPARγ expression. Collectively, these results indicate that neuronal seipin deficiency causing reduced PPARγ levels leads to affective disorders in male mice that are rescued by E2-increased PPARγ expression.

Neuronal seipin knockout facilitates Aß-induced neuroinflammation and neurotoxicity via reduction of PPARγ in hippocampus of mouse.

BACKGROUND: A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Seipin is highly expressed in hippocampal pyramidal cells and astrocytes. Neuronal knockout of seipin in mice (seipin-KO mice) reduces the hippocampal peroxisome proliferator-activated receptor gamma (PPARγ) level without the loss of pyramidal cells. The down-regulation of PPARγ has gained increasing attention in neuroinflammation of Alzheimer's disease (AD). Thus, the present study focused on exploring the influence of seipin depletion on ß-amyloid (Aß)-induced neuroinflammation and Aß neurotoxicity. METHODS: Adult male seipin-KO mice were treated with a single intracerebroventricular (i.c.v.) injection of Aß25-35 (1.2 nmol/mouse) or Aß1-42 (0.1 nmol/mouse), generally a non-neurotoxic dose in wild-type (WT) mice. Spatial cognitive behaviors were assessed by Morris water maze and Y-maze tests, and hippocampal CA1 pyramidal cells and inflammatory responses were examined. RESULTS: The Aß25-35/1-42 injection in the seipin-KO mice caused approximately 30-35 % death of pyramidal cells and production of Hoechst-positive cells with the impairment of spatial memory. In comparison with the WT mice, the number of astrocytes and microglia in the seipin-KO mice had no significant difference, whereas the levels of IL-6 and TNF-α were slightly increased. Similarly, the Aß25-35/1-42 injection in the seipin-KO mice rather than the WT mice could stimulate the activation of astrocytes or microglia and further elevated the levels of IL-6 and TNF-α. Treatment of the seipin-KO mice with the PPARγ agonist rosiglitazone (rosi) could prevent Aß25-35/1-42-induced neuroinflammation and neurotoxicity, which was blocked by the PPARγ antagonist GW9962. In the seipin-KO mice, the level of glycogen synthase kinase-3ß (GSK3ß) phosphorylation at Tyr216 was elevated, while at Ser9, it was reduced compared to the WT mice, which were corrected by the rosi treatment but were unaffected by the Aß25-35 injection. CONCLUSIONS: Seipin deficiency in astrocytes increases GSK3ß activity and levels of IL-6 and TNF-α through reducing PPARγ, which can facilitate Aß25-35/1-42-induced neuroinflammation to cause the death of neuronal cells and cognitive deficits.

Heterotrimeric G protein subunit Gγ13 is critical to olfaction.

The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G-protein consisting of α, ß, and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its ß and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We, and others, have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knock-out mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice's olfactory epithelium. Intriguingly, Gαolf, ß1 subunits, Ric-8B and CEP290 proteins, were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G-protein with Gß1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G-protein's ßγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs.

Photoresponse diversity among the five types of intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate non-image-forming visual responses, including pupillary constriction, circadian photoentrainment and suppression of pineal melatonin secretion. Five morphological types of ipRGCs, M1-M5, have been identified in mice. In order to understand their functions better, we studied the photoresponses of all five cell types, by whole-cell recording from fluorescently labelled ipRGCs visualized using multiphoton microscopy. All ipRGC types generated melanopsin-based ('intrinsic') as well as synaptically driven ('extrinsic') light responses. The intrinsic photoresponses of M1 cells were lower threshold, higher amplitude and faster than those of M2-M5. The peak amplitudes of extrinsic light responses differed among the ipRGC types; however, the responses of all cell types had comparable thresholds, kinetics and waveforms, and all cells received rod input. While all five types exhibited inhibitory amacrine-cell and excitatory bipolar-cell inputs from the 'on' channel, M1 and M3 received additional 'off'-channel inhibition, possibly through their 'off'-sublamina dendrites. The M2-M5 ipRGCs had centre-surround-organized receptive fields, implicating a capacity to detect spatial contrast. In contrast, the receptive fields of M1 cells lacked surround antagonism, which might be caused by the surround of the inhibitory input nullifying the surround of the excitatory input. All ipRGCs responded robustly to a wide range of motion speeds, and M1-M4 cells appeared tuned to different speeds, suggesting that they might analyse the speed of motion. Retrograde labelling revealed that M1-M4 cells project to the superior colliculus, suggesting that the contrast and motion information signalled by these cells could be used by this sensorimotor area to detect novel objects and motion in the visual field.

Thiazolidinediones partially reverse the metabolic disturbances observed in Bscl2/seipin-deficient mice.

AIMS/HYPOTHESIS: Mutations in BSCL2/seipin cause Berardinelli-Seip congenital lipodystrophy (BSCL), a rare recessive disorder characterised by near absence of adipose tissue and severe insulin resistance. We aimed to determine how seipin deficiency alters glucose and lipid homeostasis and whether thiazolidinediones can rescue the phenotype. METHODS: Bscl2 (-/-) mice were generated and phenotyped. Mouse embryonic fibroblasts (MEFs) were used as a model of adipocyte differentiation. RESULTS: As observed in humans, Bscl2 (-/-) mice displayed an early depletion of adipose tissue, with insulin resistance and severe hepatic steatosis. However, Bscl2 (-/-) mice exhibited an unexpected hypotriglyceridaemia due to increased clearance of triacylglycerol-rich lipoproteins (TRL) and uptake of fatty acids by the liver, with reduced basal energy expenditure. In vitro experiments with MEFs demonstrated that seipin deficiency led to impaired late adipocyte differentiation and increased basal lipolysis. Thiazolidinediones were able to rescue the adipogenesis impairment but not the alteration in lipolysis in Bscl2 (-/-) MEFs. In vivo treatment of Bscl2 (-/-) mice with pioglitazone for 9 weeks increased residual inguinal and mesenteric fat pads as well as plasma leptin and adiponectin concentrations. Pioglitazone treatment increased energy expenditure and improved insulin resistance, hypotriglyceridaemia and liver steatosis in these mice. CONCLUSIONS/INTERPRETATION: Seipin plays a key role in the differentiation and storage capacity of adipocytes, and affects glucose and lipid homeostasis. The hypotriglyceridaemia observed in Bscl2 (-/-) mice is linked to increased uptake of TRL by the liver, offering a new model of liver steatosis. The demonstration that the metabolic complications associated with BSCL can be partially rescued with pioglitazone treatment opens an interesting therapeutic perspective for BSCL patients.

Functional characterization of bitter-taste receptors expressed in mammalian testis.

Mammalian spermatogenesis and sperm maturation are susceptible to the effects of internal and external factors. However, how male germ cells interact with and respond to these elements including those potentially toxic substances is poorly understood. Here, we show that many bitter-taste receptors (T2rs), which are believed to function as gatekeepers in the oral cavity to detect and innately prevent the ingestion of poisonous bitter-tasting compounds, are expressed in mouse seminiferous tubules. Our in situ hybridization results indicate that Tas2r transcripts are expressed postmeiotically. Functional analysis showed that mouse spermatids and spermatozoa responded to both naturally occurring and synthetic bitter-tasting compounds by increasing intracellular free calcium concentrations, and individual male germ cells exhibited different ligand-activation profiles, indicating that each cell may express a unique subset of T2r receptors. These calcium responses could be suppressed by a specific bitter-tastant blocker or abolished by the knockout of the gene for the G protein subunit α-gustducin. Taken together, our data strongly suggest that male germ cells, like taste bud cells in the oral cavity and solitary chemosensory cells in the airway, utilize T2r receptors to sense chemicals in the milieu that may affect sperm behavior and fertilization.

Functional interchangeability of rod and cone transducin alpha-subunits.

Rod and cone photoreceptors use similar but distinct sets of phototransduction proteins to achieve different functional properties, suitable for their role as dim and bright light receptors, respectively. For example, rod and cone visual pigments couple to distinct variants of the heterotrimeric G protein transducin. However, the role of the structural differences between rod and cone transducin alpha subunits (Talpha) in determining the functional differences between rods and cones is unknown. To address this question, we studied the translocation and signaling properties of rod Talpha expressed in cones and cone Talpha expressed in rods in three mouse strains: rod Talpha knockout, cone Talpha GNAT2(cpfl3) mutant, and rod and cone Talpha double mutant rd17 mouse. Surprisingly, although the rod/cone Talpha are only 79% identical, exogenously expressed rod or cone Talpha localized and translocated identically to endogenous Talpha in each photoreceptor type. Moreover, exogenously expressed rod or cone Talpha rescued electroretinogram responses (ERGs) in mice lacking functional cone or rod Talpha, respectively. Ex vivo transretinal ERG and single-cell recordings from rd17 retinas treated with rod or cone Talpha showed comparable rod sensitivity and response kinetics. These results demonstrate that cone Talpha forms a functional heterotrimeric G protein complex in rods and that rod and cone Talpha couple equally well to the rod phototransduction cascade. Thus, rod and cone transducin alpha-subunits are functionally interchangeable and their signaling properties do not contribute to the intrinsic light sensitivity differences between rods and cones. Additionally, the technology used here could be adapted for any such homologue swap desired.

Short-Wavelength (Violet) Light Protects Mice From Myopia Through Cone Signaling.

Purpose: Exposure to short-wavelength light influences refractive development and inhibits myopic development in many animal models. Retinal mechanisms underlying this response remain unknown. This study used a mouse model of lens-induced myopia to evaluate the effect of different wavelength light on refractive development and dopamine levels in the retina. A possible retinal pathway is tested using a mutant mouse with dysfunctional cones. Methods: Wild-type C57BL/6J (WT) and ALS/LtJ/Gnat2cpfl3 (Gnat2-/-) mice were exposed to one of three different light conditions beginning at postnatal day 28: broad-spectrum "white" (420-680 nm), medium wavelength "green" (525 ± 40 nm), and short wavelength "violet" (400 ± 20 nm). One-half of the mice received hyperopic lens defocus. All mice were exposed to the light for 4 weeks; animals were measured weekly for refractive error and axial parameters. Retinal dopamine and the dopamine metabolite 3,4-dihydroxyphenylacetic acid were measured by HPLC. Results: In WT mice, short-wavelength violet light induced hyperopia and violet light inhibited lens-induced myopia when compared with mice exposed to white light. Hyperopia could be attributed to shallower vitreous chambers in WT animals. There were no changes in the levels of dopamine or its metabolite. In Gnat2-/- mice, violet light did not induce hyperopia or inhibit lens-induced myopia. Conclusions: These findings show that short-wavelength light slows refractive eye growth, producing hyperopic responses in mice and inhibiting lens-induced myopia. The lack of inhibition in mice with dysfunctional cones suggests that cone signaling plays a role in the hyperopic response to short-wavelength (violet) light.

Speed, spatial, and temporal tuning of rod and cone vision in mouse.

Rods and cones subserve mouse vision over a 100 million-fold range of light intensity (-6 to 2 log cd m(-2)). Rod pathways tune vision to the temporal frequency of stimuli (peak, 0.75 Hz) and cone pathways to their speed (peak, approximately 12 degrees/s). Both pathways tune vision to the spatial components of stimuli (0.064-0.128 cycles/degree). The specific photoreceptor contributions were determined by two-alternative, forced-choice measures of contrast thresholds for optomotor responses of C57BL/6J mice with normal vision, Gnat2(cpfl3) mice without functional cones, and Gnat1-/- mice without functional rods. Gnat2(cpfl3) mice (threshold, -6.0 log cd m(-2)) cannot see rotating gratings above -2.0 log cd m(-2) (photopic vision), and Gnat1-/- mice (threshold, -4.0 log cd m(-2)) are blind below -4.0 log cd m(-2) (scotopic vision). Both genotypes can see in the transitional mesopic range (-4.0 to -2.0 log cd m(-2)). Mouse rod and cone sensitivities are similar to those of human. This parametric study characterizes the functional properties of the mouse visual system, revealing the rod and cone contributions to contrast sensitivity and to the temporal processing of visual stimuli.

Female adipose tissue-specific Bscl2 knockout mice develop only moderate metabolic dysfunction when housed at thermoneutrality and fed a high-fat diet.

Mutations affecting the BSCL2 gene cause the most severe form of congenital generalised lipodystrophy. Affected individuals almost completely lack adipose tissue and suffer from severe diabetes and metabolic complications. Likewise, mice lacking Bscl2 in all tissues have dramatically reduced adipose mass, glucose intolerance and hyperinsulinaemia. However, male adipose tissue-specific Bscl2 knockout mice fail to develop the metabolic dysfunction observed in Bscl2 null mice and BSCL2 deficient patients, despite a similar generalised lack of adipose tissues. Clinical reports indicate gender differences frequently exist in cases of lipodystrophy, with female patients more adversely affected than male patients. We therefore generated and characterised female mice lacking Bscl2 specifically in adipose tissue (Ad-B2(-/-)). We show that female Ad-B2(-/-) mice also develop early-onset lipodystrophy when fed a chow diet and are maintained under standard housing conditions (21 °C) or thermoneutrality (30 °C). Despite this, female Ad-B2(-/-) mice fail to develop severe metabolic dysfunction. Only when female Ad-B2(-/-) mice are maintained at thermoneutrality and fed a high-fat diet do subtle alterations to metabolic homeostasis manifest. This is despite a striking inability to expand adipose mass. Our findings provide further evidence that loss of Bscl2 in non-adipose tissues may contribute to the severity of metabolic dysfunction in this condition.

Dyslipidemia, steatohepatitis and atherogenesis in lipodystrophic apoE deficient mice with Seipin deletion.

SEIPIN is an integral membrane protein located in the endoplasmic reticulum, regulating adipocytes differentiation and lipolysis. Deficiency of Seipin in mice causes severe general lipodystrophy, accompanied by insulin resistance, postprandial hypertriglyceridemia and steatohepatitis. In atherosclerosis-prone Ldlr null (Ldlr-/-) mice, lipodystrophy caused by Seipin deletion even led to severe hypercholesteremia and accelerated atherogenesis, when challenged with an atherogenic diet. However, whether the phenotypes observed in Seipin-/-Ldlr-/- mice were a common consequence due to lipodystrophy, rather than genetic background restricted or diet dependent, was unknown. Herein we explored the lipodystrophy-related dyslipidemia, steatohepatitis and atherogenesis in another atherosclerosis-prone murine model, apolipoprotein E null (apoE-/-) mice. Besides, we also compared phenotypes between sexes in apoE-/- mice with Seipin deletion (Seipin-/-apoE-/-). We found that compared with apoE-/- controls, Seipin-/-apoE-/- mice also developed severe general lipodystrophy with hyperlipidemia, steatohepatitis and increased atherogenesis. Although the severity of adipose loss in female and male Seipin-/-apoE-/- mice were similar, hyperlipidemia, steatohepatitis and atherosclerosis were less severe in females than in males. Therefore, we demonstrated that lipodystrophy-related metabolic disorders, caused by Seipin deletion, were independent of genetic background and experimental diet, as seen in Ldlr-/- and apoE-/- mice. However, gender factor affected the disease progression, with females more resistant to developing lipodystrophy-related metabolic consequences.

Seipin deficiency in mice causes loss of dopaminergic neurons via aggregation and phosphorylation of α-synuclein and neuroinflammation.

Seipin gene is originally found in type 2 congenital generalized lipodystrophy (CGL2) to involve lipid droplet formation. Recently, decrease of seipin expression is reported in substantia nigra of Parkinson's disease patients. Dopaminergic neurons in substantia nigra pars compacta expressed the seipin protein. The objective of this study is to investigate influence of the seipin deficiency on dopaminergic neurons and motor behaviors. Neuronal seipin knockout (seipin-nKO) mice (3-12 months of age) displayed an age-related deficit in motor coordination. The number of dopaminergic neurons in seipin-nKO mice was age dependently reduced with increase in cleaved caspase-3. The levels of αSyn oligomers and oligomer phosphorylation (S129), but not αSyn monomers, were elevated in dopaminergic neurons and substantia nigra of seipin-nKO mice. The PPARγ expression in seipin-nKO mice was reduced. In seipin-nKO mice, the phosphorylation of GSK3ß was increased at Tyr216 and was reduced at Ser9, which was corrected by the PPARγ agonist rosiglitazone. The increased IL-6 level in seipin-nKO mice was sensitive to rosiglitazone and GSK3ß inhibitor AR-A014418. The enhanced phosphorylation of αSyn was prevented by rosiglitazone and AR-A014418, while the increase in αSyn oligomers was corrected only by rosiglitazone. The treatment of seipin-nKO mice with rosiglitazone and AR-A014418 rescued the death of dopaminergic neurons, which was accompanied by the improvement of motor coordination. Therefore, the results indicate that seipin deficiency causes an age-related loss of dopaminergic neurons and impairment of motor coordination through reducing PPARγ to enhance aggregation and phosphorylation of αSyn and neuroinflammation.

Normal hematopoiesis and inflammatory responses despite discrete signaling defects in Galpha15 knockout mice.

Galpha15 activates phospholipase Cbeta in response to the greatest variety of agonist-stimulated heptahelical receptors among the four Gq class G-protein alpha subunits expressed in mammals. Galpha15 is primarily expressed in hematopoietic cells in fetal and adult mice. We disrupted the Galpha15 gene by homologous recombination in embryonic stem cells to identify its biological functions. Surprisingly, hematopoiesis was normal in Galpha15(-/-) mice, Galpha15(-/-) Galphaq(-/-) double-knockout mice (which express only Galpha11 in most hematopoietic cells), and Galpha11(-/-) mice, suggesting functional redundancy in Gq class signaling. Inflammatory challenges, including thioglycolate-induced peritonitis and infection with Trichinella spiralis, stimulated similar responses in Galpha15(-/-) adults and wild-type siblings. Agonist-stimulated Ca(2+) release from intracellular stores was assayed to identify signaling defects in primary cultures of thioglycolate-elicited macrophages isolated from Galpha15(-/-) mice. C5a-stimulated phosphoinositide accumulation and Ca(2+) release was significantly reduced in Galpha15(-/-) macrophages. Ca(2+) signaling was abolished only in mutant cells pretreated with pertussis toxin, suggesting that the C5a receptor couples to both Galpha15 and Galphai in vivo. Signaling evoked by other receptors coupled by Gq class alpha subunits appeared normal in Galpha15(-/-) macrophages. Despite discrete signaling defects, compensation by coexpressed Gq and/or Gi class alpha subunits may suppress abnormalities in Galpha15-deficient mice.

Seipin deficiency alters brown adipose tissue thermogenesis and insulin sensitivity in a non-cell autonomous mode.

Loss-of-function mutations in BSCL2 are responsible for Berardinelli-Seip congenital lipodystrophy, a rare disorder characterized by near absence of adipose tissue associated with insulin resistance. Seipin-deficient (Bscl2-/-) mice display an almost total loss of white adipose tissue (WAT) with residual brown adipose tissue (BAT). Previous cellular studies have shown that seipin deficiency alters white adipocyte differentiation. In this study, we aimed to decipher the consequences of seipin deficiency in BAT. Using a brown adipocyte cell-line, we show that seipin knockdown had very little effect on adipocyte differentiation without affecting insulin sensitivity and oxygen consumption. However, when submitted to cold acclimation or chronic ß3 agonist treatment, Bscl2-/- mice displayed altered thermogenic capacity, despite several signs of BAT remodeling. Under cold activation, Bscl2-/- mice were able to maintain their body temperature when fed ad libitum, but not under short fasting. At control temperature (i.e. 21 °C), fasting worsened Bscl2-/- BAT properties. Finally, Bscl2-/- BAT displayed obvious signs of insulin resistance. Our results in these lipodystrophic mice strongly suggest that BAT activity relies on WAT as an energetic substrate provider and adipokine-producing organ. Therefore, the WAT/BAT dialogue is a key component of BAT integrity in guaranteeing its response to insulin and cold-activated adrenergic signals.

SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase.

Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT) in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Overexpression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin-/- mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients.