The lipid droplet assembly complex consists of seipin and four accessory factors in budding yeast.

Seipin, a crucial protein for cellular lipid droplet (LD) assembly, oligomerizes at the interface between the endoplasmic reticulum (ER) and LDs to facilitate neutral lipid packaging. Using proximity labeling, we identify four proteins-Ldo45, Ldo16, Tgl4, and Pln1-that are recruited to the vicinity of yeast seipin, the Sei1-Ldb16 complex, exclusively when seipin function is intact, hence termed seipin accessory factors. Localization studies identify Tgl4 at the ER-LD contact site, in contrast to Ldo45, Ldo16 and Pln1 at the LD surface. Cells with compromised seipin function resulted in uneven distribution of these proteins with aberrant LDs, supporting a central role of seipin in orchestrating their association with the LD. Overexpression of any seipin accessory factor causes LD aggregation and affects a subset of LD protein distribution, highlighting the importance of their stoichiometry. Although single factor mutations show minor LD morphology changes, combined mutations have additive effects. Lastly, we present evidence that seipin accessory factors assemble and interact with seipin in the absence of neutral lipids and undergo dynamically rearrangements during LD formation induction, with Ldo45 acting as a central hub recruiting other factors to interact with the seipin complex.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

Hypertriglyceridemia Results From an Impaired Catabolism of Triglyceride-Rich Lipoproteins in PLIN1-Related Lipodystrophy.

BACKGROUND: Pathogenic variants in PLIN1-encoding PLIN1 (perilipin-1) are responsible for an autosomal dominant form of familial partial lipodystrophy (FPL) associated with severe insulin resistance, hepatic steatosis, and important hypertriglyceridemia. This study aims to decipher the mechanisms of hypertriglyceridemia associated with PLIN1-related FPL. METHODS: We performed an in vivo lipoprotein kinetic study in 6 affected patients compared with 13 healthy controls and 8 patients with type 2 diabetes. Glucose and lipid parameters, including plasma LPL (lipoprotein lipase) mass, were measured. LPL mRNA and protein expression were evaluated in abdominal subcutaneous adipose tissue from patients with 5 PLIN1-mutated FPL and 3 controls. RESULTS: Patients with PLIN1-mutated FPL presented with decreased fat mass, insulin resistance, and diabetes (glycated hemoglobin A1c, 6.68±0.70% versus 7.48±1.63% in patients with type 2 diabetes; mean±SD; P=0.27). Their plasma triglycerides were higher (5.96±3.08 mmol/L) than in controls (0.76±0.27 mmol/L; P<0.0001) and patients with type 2 diabetes (2.94±1.46 mmol/L, P=0.006). Compared with controls, patients with PLIN1-related FPL had a significant reduction of the indirect fractional catabolic rate of VLDL (very-low-density lipoprotein)-apoB100 toward IDL (intermediate-density lipoprotein)/LDL (low-density lipoprotein; 1.79±1.38 versus 5.34±2.45 pool/d; P=0.003) and the indirect fractional catabolic rate of IDL-apoB100 toward LDL (2.14±1.44 versus 7.51±4.07 pool/d; P=0.005). VLDL-apoB100 production was not different between patients with PLIN1-related FPL and controls. Compared with patients with type 2 diabetes, patients with PLIN1-related FPL also showed a significant reduction of the catabolism of both VLDL-apoB100 (P=0.031) and IDL-apoB100 (P=0.031). Plasma LPL mass was significantly lower in patients with PLIN1-related FPL than in controls (21.03±10.08 versus 55.76±13.10 ng/mL; P<0.0001), although the LPL protein expression in adipose tissue was similar. VLDL-apoB100 and IDL-apoB100 indirect fractional catabolic rates were negatively correlated with plasma triglycerides and positively correlated with LPL mass. CONCLUSIONS: We show that hypertriglyceridemia associated with PLIN1-related FPL results from a marked decrease in the catabolism of triglyceride-rich lipoproteins (VLDL and IDL). This could be due to a pronounced reduction in LPL availability, related to the decreased adipose tissue mass.

Peridroplet mitochondria are associated with the severity of MASLD and the prevention of MASLD by diethyldithiocarbamate.

Mitochondria can contact lipid droplets (LDs) to form peridroplet mitochondria (PDM) which trap fatty acids in LDs by providing ATP for triglyceride synthesis, and prevent lipotoxicity. However, the role of PDM in metabolic dysfunction associated steatotic liver disease (MASLD) is not clear. Here, the features of PDM in dietary MASLD models with different severity in mice were explored. Electron microscope photographs show that LDs and mitochondria rarely come into contact with each other in normal liver. In mice fed with high-fat diet, PDM can be observed in the liver as early as the beginning of steatosis in hepatocytes. For the first time, we show that PDM in mouse liver varies with the severity of MASLD. PDM and cytosolic mitochondria (CM) were isolated from the liver tissue of MASLD and analyzed by quantitative proteomics. Compared with CM, PDM have enhanced mitochondrial respiration and ATP synthesis. Diethyldithiocarbamate (DDC) alleviates choline-deficient, L-amino acid-defined diet-induced MASLD, while increases PDM in the liver. Similarly, DDC promotes the contact of mitochondria-LDs in steatotic C3A cells in vitro. Meanwhile, DDC promotes triglyceride synthesis and improves mitochondrial dysfunction in MASLD. In addition, DDC upregulates perilipin 5 both in vivo and in vitro, which is considered as a key regulator in PDM formation. Knockout of Plin5 inhibits the contact of mitochondria-LDs induced by DDC in C3A cells. These results demonstrate that PDM might be associated with the progression of MASLD and the prevention of MASLD by DDC. The regulation of PDM might be a new pharmacological strategy for MASLD.

Adverse cardiac remodeling augments adipose tissue ß-adrenergic signaling and lipolysis counteracting diet-induced obesity.

Cardiac triacylglycerol accumulation is a common characteristic of obesity and type 2 diabetes and strongly correlates with heart morbidity and mortality. We have previously shown that cardiomyocyte-specific perilipin 5 overexpression (Plin5-Tg) provokes significant cardiac steatosis via lowering cardiac lipolysis and fatty acid (FA) oxidation. In strong contrast to cardiac steatosis and lethal heart dysfunction in adipose triglyceride lipase deficiency, Plin5-Tg mice do not develop heart dysfunction and show a normal life span on chow diet. This finding prompted us to study heart function and energy metabolism in Plin5-Tg mice fed high-fat diet (HFD). Plin5-Tg mice showed adverse cardiac remodeling on HFD with heart function only being compromised in one-year-old mice, likely due to reduced cardiac FA uptake, thereby delaying deleterious cardiac lipotoxicity. Notably, Plin5-Tg mice were less obese and protected from glucose intolerance on HFD. Changes in cardiac energy catabolism in Plin5-Tg mice increased ß-adrenergic signaling, lipolytic, and thermogenic protein expression in adipose tissue ultimately counteracting HFD-induced obesity. Acute cold exposure further augmented ß-adrenergic signaling in Plin5-Tg mice, whereas housing at thermoneutrality did not protect Plin5-Tg mice from HFD-induced obesity albeit blood glucose and insulin levels remained low in transgenic mice. Overall, our data suggest that the limited capacity for myocardial FA oxidation on HFD increases cardiac stress in Plin5-Tg mice, thereby stimulating adipose tissue ß-adrenergic signaling, triacylglycerol catabolism, and thermogenesis. However, long-term HFD-mediated metabolic stress causes contractile dysfunction in Plin5-Tg mice, which emphasizes the importance of a carefully controlled dietary regime in patients with cardiac steatosis and hypertrophy.

Binding of perilipin 3 to membranes containing diacylglycerol is mediated by conserved residues within its PAT domain.

Perilipins (PLINs) constitute an evolutionarily conserved family of proteins that specifically associate with the surface of lipid droplets (LDs). These proteins function in LD biogenesis and lipolysis and help to stabilize the surface of LDs. PLINs are typically composed of three different protein domains. They share an N-terminal PAT domain of unknown structure and function, a central region containing 11-mer repeats that form amphipathic helices, and a C-terminal domain that adopts a 4-helix bundle structure. How exactly these three distinct domains contribute to PLIN function remains to be determined. Here, we show that the N-terminal PAT domain of PLIN3 binds diacylglycerol (DAG), the precursor to triacylglycerol, a major storage lipid of LDs. PLIN3 and its PAT domain alone bind liposomes with micromolar affinity and PLIN3 binds artificial LDs containing low concentrations of DAG with nanomolar affinity. The PAT domain of PLIN3 is predicted to adopt an amphipathic triangular shaped structure. In silico ligand docking indicates that DAG binds to one of the highly curved regions within this domain. A conserved aspartic acid residue in the PAT domain, E86, is predicted to interact with DAG, and we found that its substitution abrogates high affinity binding of DAG as well as DAG-stimulated association with liposome and artificial LDs. These results indicate that the PAT domain of PLINs harbor specific lipid-binding properties that are important for targeting these proteins to the surface of LDs and to ER membrane domains enriched in DAG to promote LD formation.

Lipid droplets-related Perilipin-3: potential immune checkpoint and oncogene in oral squamous cell carcinoma.

BACKGROUND: Lipid droplets (LDs) as major lipid storage organelles are recently reported to be innate immune hubs. Perilipin-3 (PLIN3) is indispensable for the formation and accumulation of LDs. Since cancer patients show dysregulated lipid metabolism, we aimed to elaborate the role of LDs-related PLIN3 in oral squamous cell carcinoma (OSCC). METHODS: PLIN3 expression patterns (n = 87), its immune-related landscape (n = 74) and association with B7-H2 (n = 51) were assessed by immunohistochemistry and flow cytometry. Real-time PCR, Western blot, Oil Red O assay, immunofluorescence, migration assay, spheroid-forming assay and flow cytometry were performed for function analysis. RESULTS: Spotted LDs-like PLIN3 staining was dominantly enriched in tumor cells than other cell types. PLIN3high tumor showed high proliferation index with metastasis potential, accompanied with less CD3+CD8+ T cells in peripheral blood and in situ tissue, conferring immunosuppressive microenvironment and shorter postoperative survival. Consistently, PLIN3 knockdown in tumor cells not only reduced LD deposits and tumor migration, but benefited for CD8+ T cells activation in co-culture system with decreased B7-H2. An OSCC subpopulation harbored PLIN3highB7-H2high tumor showed more T cells exhaustion, rendering higher risk of cancer-related death (95% CI 1.285-6.851). CONCLUSIONS: LDs marker PLIN3 may be a novel immunotherapeutic target in OSCC.

Perilipin 5 protects the mitochondrial oxidative functions and improves the alcoholic liver injury in mice.

BACKGROUND AND AIMS: Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated. METHODS: The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444-464 aa) were used for in vitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes. RESULTS: Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions. CONCLUSION: Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues.

Behavior of intracellular lipid droplets during cell division in HuH7 hepatoma cells.

Intracellular lipid droplets (LDs) are ubiquitous organelles found in many cell types. During mitosis, membranous organelles, including mitochondria, are divided into small pieces and transferred to daughter cells; however, the process of LD transfer to daughter cells is not fully elucidated. Herein, we investigated the behavior of LDs during mitosis in HuH7 human hepatoma cells. While fragments of the Golgi apparatus were scattered in the cytosol during mitosis, intracellular LDs retained their size and spherical morphology as they translocated to the two daughter cells. LDs were initially distributed throughout the cell during prophase but positioned outside the spindle in metaphase, aligning at the far sides of the centrioles. A similar distribution of LDs during mitosis was observed in another hepatocarcinoma HepG2 cells. When the spindle was disrupted by nocodazole treatment or never in mitosis gene A-related kinase 2A knockdown, LDs were localized in the area outside the chromosomes, suggesting that spindle formation is not necessary for LD localization at metaphase. The amount of major LD protein perilipin 2 reduced while LDs were enriched in perilipin 3 during mitosis, indicating the potential alteration of LD protein composition. Conclusively, the behavior of LDs during mitosis is distinct from that of other organelles in hepatocytes.

Depletion of lipid storage droplet-1 delays endoreplication progression and induces cell death in Drosophila salivary gland.

Perilipins are evolutionarily conserved from insects to mammals. Drosophila lipid storage droplet-1 (LSD-1) is a lipid storage droplet membrane surface-binding protein family member and a counterpart to mammalian perilipin 1 and is known to play a role in lipolysis. However, the function of LSD-1 during specific tissue development remains under investigation. This study demonstrated the role of LSD-1 in salivary gland development. Knockdown of Lsd-1 in the salivary gland was established using the GAL4/UAS system. The third-instar larvae of knockdown flies had small salivary glands containing cells with smaller nuclei. The null mutant Drosophila also showed the same phenotype. The depletion of LSD-1 expression induced a delay of endoreplication due to decreasing CycE expression and increasing DNA damage. Lsd-1 genetically interacted with Myc in the third-instar larvae. These results demonstrate that LSD-1 is involved in cell cycle and cell death programs in the salivary gland, providing novel insight into the effects of LSD-1 in regulating salivary gland development and the interaction between LSD-1 and Myc.

Endocrine modulation of brain-skeleton axis driven by neural stem cell-derived perilipin 5 in the lipid metabolism homeostasis for bone regeneration.

Factors released from the nervous system always play crucial roles in modulating bone metabolism and regeneration. How the brain-driven endocrine axes maintain bone homeostasis, especially under metabolic disorders, remains obscure. Here, we found that neural stem cells (NSCs) residing in the subventricular zone participated in lipid metabolism homeostasis of regenerative bone through exosomal perilipin 5 (PLIN5). Fluorescence-labeled exosomes tracing and histological detection identified that NSC-derived exosomes (NSC-Exo) could travel from the lateral ventricle into bone injury sites. Homocysteine (Hcy) led to osteogenic and angiogenic impairment, whereas the NSC-Exo were confirmed to restore it. Mecobalamin, a clinically used neurotrophic drug, further enhanced the protective effects of NSC-Exo through increased PLIN5 expression. Mechanistically, NSC-derived PLIN5 reversed excessive Hcy-induced lipid metabolic imbalance and aberrant lipid droplet accumulation through lipophagy-dependent intracellular lipolysis. Intracerebroventricular administration of mecobalamin and/or AAV-shPlin5 confirmed the effects of PLIN5-driven endocrine modulations on new bone formation and vascular reconstruction in hyperhomocysteinemic and high-fat diet models. This study uncovered a novel brain-skeleton axis that NSCs in the mammalian brain modulated bone regeneration through PLIN5-driven lipid metabolism modulation, providing evidence for lipid- or bone-targeted medicine development.

Perilipin2 depletion causes lipid droplet enlargement in the ovarian corpus luteum in mice.

Lipid droplets (LDs) are endoplasmic reticulum-derived organelles that store neutral lipids (mostly triglycerides and cholesterol esters) within a phospholipid monolayer and appear in most eukaryotic cells. Perilipins (PLINs, comprising PLIN1-5) are abundant LD-associated proteins with highly variable expression levels among tissues. Although PLINs are expressed in the mammalian ovaries, little is known about their subcellular localization and physiological functions. In this study, we investigated the localization of PLIN1-3 and their relationship with LD synthesis using mCherry-HPos reporter mice, thereby enabling the visualization of LD biogenesis in vivo. PLIN2 and PLIN3 were localized as puncta in granulosa cells with low levels of LD synthesis in developing follicles. This localization pattern was quite different from that of PLIN1, which was mainly localized in the theca and interstitial cells with high levels of LD synthesis. In the corpus luteum, where LD synthesis is highly induced, PLIN2 and PLIN3 are abundant in the particulate structures, whereas PLIN1 is poorly distributed. We also generated global Plin2-deficient mice using the CRSPR/Cas9 system and demonstrated that the lack of PLIN2 did not alter the distribution of PLIN1 and PLIN3 but unexpectedly induced LD enlargement in the corpus luteum. Collectively, our results suggest that the localization of PLIN1-3 is spatiotemporally regulated and that PLIN2 deficiency influences LD mobilization in the corpus luteum within the ovaries.

Sex-Specific Effects of Long-Term Antipsychotic Drug Treatment on Adipocyte Tissue and the Crosstalk to Liver and Brain in Rats.

Antipsychotic drug (APD) medication can lead to metabolic dysfunctions and weight gain, which together increase morbidity and mortality. Metabolically active visceral adipose tissue (VAT) in particular plays a crucial role in the etiopathology of these metabolic dysregulations. Here, we studied the effect of 12 weeks of drug medication by daily oral feeding of clozapine and haloperidol on the perirenal fat tissue as part of VAT of male and female Sprague Dawley rats in the context of complex former investigations on brain, liver, and blood. Adipocyte area values were determined, as well as triglycerides, non-esterified fatty acids (NEFAs), glucose, glycogen, lactate, malondialdehyde equivalents, ferric iron and protein levels of Perilipin-A, hormone-sensitive-lipase (HSL), hepcidin, glucose transporter-4 (Glut-4) and insulin receptor-ß (IR-ß). We found increased adipocyte mass in males, with slightly higher adipocyte area values in both males and females under clozapine treatment. Triglycerides, NEFAs, glucose and oxidative stress in the medicated groups were unchanged or slightly decreased. In contrast to controls and haloperidol-medicated rats, perirenal adipocyte mass and serum leptin levels were not correlated under clozapine. Protein expressions of perilipin-A, Glut-4 and HSL were decreased under clozapine treatment. IR-ß expression changed sex-specifically in the clozapine-medicated groups associated with higher hepcidin levels in the perirenal adipose tissue of clozapine-treated females. Taken together, clozapine and haloperidol had a smaller effect than expected on perirenal adipose tissue. The perirenal adipose tissue shows only weak changes in lipid and glucose metabolism. The main changes can be seen in the proteins examined, and probably in their effect on liver metabolism.

In silico modeling human VPS13 proteins associated with donor and target membranes suggests lipid transfer mechanisms.

The VPS13 protein family constitutes a novel class of bridge-like lipid transferases. Autosomal recessive inheritance of mutations in VPS13 genes is associated with the development of neurodegenerative diseases in humans. Bioinformatic approaches previously recognized the domain architecture of these proteins. In this study, we model the first ever full-length structures of the four human homologs VPS13A, VPS13B, VPS13C, and VPS13D in association with model membranes, to investigate their lipid transfer ability and potential structural association with membrane leaflets. We analyze the evolutionary conservation and physicochemical properties of these proteins, focusing on conserved C-terminal amphipathic helices that disturb organelle surfaces and that, adjoined, resemble a traditional Venetian gondola. The gondola domains share significant structural homology with lipid droplet surface-binding proteins. We introduce in silico protein-membrane models displaying the mode of association of VPS13A, VPS13B, VPS13C, and VPS13D to donor and target membranes, and present potential models of action for protein-mediated lipid transfer.

A novel HIF2A mutation causes dyslipidemia and promotes hepatic lipid accumulation.

Hypoxia-inducible factor-2α (HIF-2α) is a transcription factor responsible for regulating genes related to angiogenesis and metabolism. This study aims to explore the effect of a previously unreported mutation c.C2473T (p.R825S) in the C-terminal transactivation domain (CTAD) of HIF-2α that we detected in tissue of patients with liver disease. We sequenced available liver and matched blood samples obtained during partial liver resection or liver transplantation performed for clinical indications including hepatocellular carcinoma and liver failure. In tandem, we constructed cell lines and a transgenic mouse model bearing the corresponding identified mutation in HIF-2α from which we extracted primary hepatocytes. Lipid accumulation was evaluated in these cells and liver tissue from the mouse model using Oil Red O staining and biochemical measurements. We identified a mutation in the CTAD of HIF-2α (c.C2473T; p.R825S) in 5 of 356 liver samples obtained from patients with hepatopathy and dyslipidemia. We found that introduction of this mutation into the mouse model led to an elevated triglyceride level, lipid droplet accumulation in liver of the mutant mice and in their extracted primary hepatocytes, and increased transcription of genes related to hepatic fatty acid transport and synthesis in the mutant compared to the control groups. In mutant mice and cells, the protein levels of nuclear HIF-2α and its target perilipin-2 (PLIN2), a lipid droplet-related gene, were also elevated. Decreased lipophagy was observed in mutant groups. Our study defines a subpopulation of dyslipidemia that is caused by this HIF-2α mutation. This may have implications for personalized treatment.

Lipid-Independent Regulation of PLIN5 via IL-6 through the JAK/STAT3 Axis in Hep3B Cells.

Perilipin 5 (PLIN5) is a lipid droplet coat protein that is highly expressed in oxidative tissues such as those of muscles, the heart and the liver. PLIN5 expression is regulated by a family of peroxisome proliferator-activated receptors (PPARs) and modulated by the cellular lipid status. So far, research has focused on the role of PLIN5 in the context of non-alcoholic fatty liver disease (NAFLD) and specifically in lipid droplet formation and lipolysis, where PLIN5 serves as a regulator of lipid metabolism. In addition, there are only limited studies connecting PLIN5 to hepatocellular carcinoma (HCC), where PLIN5 expression is proven to be upregulated in hepatic tissue. Considering that HCC development is highly driven by cytokines present throughout NAFLD development and in the tumor microenvironment, we here explore the possible regulation of PLIN5 by cytokines known to be involved in HCC and NAFLD progression. We demonstrate that PLIN5 expression is strongly induced by interleukin-6 (IL-6) in a dose- and time-dependent manner in Hep3B cells. Moreover, IL-6-dependent PLIN5 upregulation is mediated by the JAK/STAT3 signaling pathway, which can be blocked by transforming growth factor-ß (TGF-ß) and tumor necrosis factor-α (TNF-α). Furthermore, IL-6-mediated PLIN5 upregulation changes when IL-6 trans-signaling is stimulated through the addition of soluble IL-6R. In sum, this study sheds light on lipid-independent regulation of PLIN5 expression in the liver, making PLIN5 a crucial target for NAFLD-induced HCC.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice.

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

Endoplasmic reticulum stress induces hepatic steatosis by transcriptional upregulating lipid droplet protein perilipin2.

Previous studies have shown that endoplasmic reticulum (ER) stress contributes to hepatic steatosis in several manners. However, how lipid droplet (LD) proteins participate in this process has rarely been reported. In the present study, ER stress was induced at both in vitro and in vivo levels with tunicamycin in large yellow croaker (Larimichthys crocea). Effects of LD protein perilipin2 (PLIN2) on hepatic lipid accumulation and lipoprotein transport under normal physiological condition and ER stress were then explored using dsRNA mediated knockdown. Subsequently, the transcriptional regulation of plin2 expression by transcription factors generated in the unfolded protein response (UPR) was determined by dual-luciferase reporter assays, chromatin immunoprecipitation and electrophoretic mobility-shift assay. We demonstrated that ER stress could promote LDs accumulation and inhibit lipoprotein transport by transcriptionally upregulating PLIN2 in liver. Among the transcription factors generated by UPR, spliced X-box binding protein1 can directly upregulated the expression of plin2, whereas C/EBP homologous protein can upregulate the expression of plin2 through peroxisome proliferator activated-receptor α. These results revealed that the LD protein PLIN2 played an important role in ER stress-induced hepatic steatosis, which might be a novel mechanism explaining hepatic steatosis triggered by ER stress.

The Effect of Ultrasonic Liposuction Energy Levels on Fat Graft Viability.

BACKGROUND: The use of fat obtained from ultrasound-assisted liposuction is popular. However, no study has considered the effect of different energy levels on fat grafts. OBJECTIVES: We hypothesized that different ultrasonic energy levels could change the fat graft viability. METHODS: Both flanks of 15 CD1 nude mice (30 experimental areas) were used, with experimental areas randomly distributed into five groups. Using different energy settings, fat grafts were obtained from a patient's abdominoplasty material and applied to the mouse flank regions. Device settings were intermittent mode with 50% vibration amplitude in group 1, continuous mode with 50% vibration amplitude in group 2, intermittent mode with 90% vibration amplitude in group 3, and continuous mode with 90% vibration amplitude in group 4. The control group was grafted with fat obtained via the conventional method. After 6 weeks, all mice were sacrificed, and fat grafts were excised. Sections were stained with hematoxylin-eosin, Masson's trichrome, and anti-perilipin A antibody. RESULTS: The perilipin A immunostaining result was lowest in group 4, indicating the lowest viable cell count (p < 0.01). There was no significant difference between groups for the other parameters (p > 0.05). CONCLUSION: High ultrasonic energy may affect fat graft survival. If fat injection is planned, avoiding high energy settings (our recommendation is not to exceed 16 Watts.) should be considered. We also recommend increasing the vibration amplitude rather than switching from intermittent to continuous mode in body parts that are relatively resistant to liposuction. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Metformin Inhibits Lipid Droplets Fusion and Growth via Reduction in Cidec and Its Regulatory Factors in Rat Adipose-Derived Stem Cells.

Metformin is still being investigated due to its potential use as a therapeutic agent for managing overweight or obesity. However, the underlying mechanisms are not fully understood. Inhibiting the adipogenesis of adipocyte precursors may be a new therapeutic opportunity for obesity treatments. It is still not fully elucidated whether adipogenesis is also involved in the weight loss mechanisms by metformin. We therefore used adipose-derived stem cells (ADSCs) from inguinal and epididymal fat pads to investigate the effects and mechanisms of metformin on adipogenesis in vitro. Our results demonstrate the similar effect of metformin inhibition on lipid accumulation, lipid droplets fusion, and growth in adipose-derived stem cells from epididymal fat pads (Epi-ADSCs) and adipose-derived stem cells from inguinal fat pads (Ing-ADSCs) cultures. We identified that cell death-inducing DFFA-like effector c (Cidec), Perilipin1, and ras-related protein 8a (Rab8a) expression increased ADSCs differentiation. In addition, we found that metformin inhibits lipid droplets fusion and growth by decreasing the expression of Cidec, Perilipin1, and Rab8a. Activation of AMPK pathway signaling in part involves metformin inhibition on Cidec, Perilipin1, and Rab8a expression. Collectively, our study reveals that metformin inhibits lipid storage, fusion, and growth of lipid droplets via reduction in Cidec and its regulatory factors in ADSCs cultures. Our study supports the development of clinical trials on metformin-based therapy for patients with overweight and obesity.