Re-patterning of skeletal muscle energy metabolism by fat storage-inducing transmembrane protein 2

Triacylglyceride stored in cytosolic lipid droplets (LDs) constitutes a major energy reservoir in most eukaryotes. The regulated turnover of triacylglyceride in LDs provides fatty acids for mitochondrial β-oxidation and ATP generation in physiological states of high demand for energy. The mechanisms for the formation of LDs in conditions of energy excess are not entirely understood. Fat storage-inducing transmembrane protein 2 (FIT2/FITM2) is the anciently conserved member of the fat storage-inducing transmembrane family of proteins implicated to be important in the formation of LDs, but its role in energy metabolism has not been tested. Here, we report that expression of FIT2 in mouse skeletal muscle had profound effects on muscle energy metabolism. Mice with skeletal muscle-specific overexpression of FIT2 (CKF2) had significantly increased intramyocellular triacylglyceride and complete protection from high fat diet-induced weight gain due to increased energy expenditure. Mass spectrometry-based metabolite profiling suggested that CKF2 skeletal muscle had increased oxidation of branched chain amino acids but decreased oxidation of fatty acids. Glucose was primarily utilized in CKF2 muscle for synthesis of the glycerol backbone of triacylglyceride and not for glycogen production. CKF2 muscle was ATP-deficient and had activated AMP kinase. Together, these studies indicate that FIT2 expression in skeletal muscle plays an unexpected function in regulating muscle energy metabolism and indicates an important role for lipid droplet formation in this process.     

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2^−/−) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2^−/− mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2^−/− primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.     

Adipose differentiation related protein induces lipid accumulation and lipid droplet formation in hepatic stellate cells

Summary The function of adipose differentiation-related protein (ADRP) is known to be the uptake of long-chain fatty acids and formation of lipid droplets in lipid-accumulating cells. We hypothesized that ADRP might stimulate activated hepatic stellate cells (HSCs) to accumulate lipids, resulting in their transition to the quiescent state. In this study, cultured HSCs in fifth passages isolated from rat were infected by adenovirus vector expressing ADRP (Ad.GFP-ADRP), and morphologic and functional changes were evaluated in comparison with control HSCs infected by recombinant adenovirus-expressing β-galactosidase (Ad. LacZ). In Ad. GFP-ADRP-infected cells only, many tiny lipid droplets were apparent in the cytoplasm, while the outline of the cells was not changed. The ADRP was detected around the lipid droplets. In HSCs with intracellular actin filaments, the staining pattern of the filaments before and after infection with Ad.GFP-ADRP or Ad.LacZ did not differ. The cell proliferation rate was not influenced by infection with Ad.LacZ or Ad.GFP-ADRP. Type I collagen secretion from cells overexpressing ADRP was not significantly different from that of Ad.LacZ-infected cells. In our in vitro study, ADRP overexpression induced the formation of cytoplasmic lipid droplets in activated HSCs but could not convert other characteristics of the activated form into those of the quiescent form.     

Dynamics and regulation of lipid droplet formation in lipopolysaccharide (LPS)-stimulated microglia

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 α/β (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A(2) (cPLA(2)-α), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 α/β in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.     

Dynamics and regulation of lipid droplet formation in lipopolysaccharide (LPS)-stimulated microglia

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 α/β (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A₂ (cPLA₂-α), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 α/β in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.     

EHD1 regulates cholesterol homeostasis and lipid droplet storage

Endocytic transport is critical for the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its trafficking. The C-terminal EHD protein, EHD1, controls receptor recycling through the ERC and affects free cholesterol distribution in the cell. We utilized embryonic fibroblasts from EHD1 knockout mice (Ehd1(-/-)MEF) and SiRNA in normal MEF cells to assess the role of EHD1 in intracellular transport of cholesterol. Surprisingly, Ehd1(-/-)MEFs displayed reduced levels of esterified and free cholesterol, which returned to normal level upon re-introduction of wild-type, but not dysfunctional EHD1. Moreover, triglyceride and cholesterol storage organelles known as 'lipid droplets' were smaller in size in cells lacking EHD1, indicating that less esterified cholesterol and triglycerides were being stored. Decreased cellular cholesterol and reduced lipid droplet size in Ehd1(-/-)MEFs correlated with ineffectual cholesterol uptake via LDL receptor, suggesting involvement of EHD1 in LDL receptor internalization.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed early during adipose differentiation. The present study was undertaken to reveal the role of ADRP in adipose differentiation. In murine fibroblasts infected with green fluorescent protein (GFP)-ADRP fusion protein expression adenovirus vector, confocal microscopic analysis showed the number and size of lipid droplets apparently increased comparing with those of control cells. Overexpressed GFP-ADRP were mainly located at the surface of lipid droplets and appeared to be "ring-shaped." Triacylglycerol content was also significantly (P < 0.001) increased in GFP-ADRP-overexpressed cells compared with control cells. ADRP-induced lipid accumulation did not depend on adipocyte-specific gene induction, such as peroxisome proliferator-activated receptor-gamma, lipoprotein lipase, or other lipogenic genes, including acyl-CoA synthetase, fatty acid-binding protein, and fatty acid transporter. In conclusion, ADRP stimulated lipid accumulation and lipid droplet formation without induction of other adipocyte-specific genes or other lipogenic genes in murine fibroblasts. The detailed molecular mechanisms of ADRP on lipid accumulation remain to be elucidated.     

Uptake of the Egyptian solar fractions in relation to fat biosynthesis by Candida parapsilosis (Wild type KSh 21)

Summary The metabolic pattern of solar in relation to the biosynthesis of different lipid groups of Candida parapsilosis (Wild type, KSh 21) was studied.The yeast showed high affinity towards hydrocarbons with short chain length. Within 2 days, about 39% of the starting hydrocarbon had been utilized by the yeast, although the addition of caproic acid to the culture medium retarded the rate of uptake of different hydrocarbons until it was itself absorbed.Addition of caproic acid also increased the triglycerides, sterol esters and phospholipid content of the cellular lipid. Cellular sterols, on the other hand, showed inferior results.Tridecanoic acid was one of the main acids obtained after 2 days of incubation.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occassion of his 60th birthday     

PERILIPIN-dependent control of lipid droplet structure and fat storage in Drosophila

Lipid droplets are intracellular organelles enriched in adipose tissue that govern the body fat stores of animals. In mammals, members of the evolutionarily conserved PERILIPIN protein family are associated with the lipid droplet surface and participate in lipid homeostasis. Here, we show that Drosophila mutants lacking the PERILIPIN PLIN1 are hyperphagic and suffer from adult-onset obesity. PLIN1 is a central and Janus-faced component of fat metabolism. It provides barrier function to storage lipid breakdown and acts as a key factor of stimulated lipolysis by modulating the access of proteins to the lipid droplet surface. It also shapes lipid droplet structure, transforming unilocular into multilocular fat cells. We generated flies devoid of all PERILIPIN family members and show that they exhibit impaired yet functional body fat regulation. Our data reveal the existence of a basal and possibly ancient lipid homeostasis system.     

Inhibition of Candida parapsilosis Fatty Acid Synthase (Fas2) Induces Mitochondrial Cell Death in Serum

We have recently observed that a fatty acid auxotrophic mutant (fatty acid synthase, Fas2Δ/Δ) of the emerging human pathogenic yeast Candida parapsilosis dies after incubation in various media including serum. In the present study we describe the mechanism for cell death induced by serum and glucose containing media. We show that Fas2Δ/Δ yeast cells are profoundly susceptible to glucose leading us to propose that yeast cells lacking fatty acids exhibit uncontrolled metabolism in response to glucose. We demonstrate that incubation of Fas2Δ/Δ yeast cells with serum leads to cell death, and this process can be prevented with inhibition of protein or DNA synthesis, indicating that newly synthesized cellular components are detrimental to the mutant cells. Furthermore, we have found that cell death is mediated by mitochondria. Suppression of electron transport enzymes using inhibitors such as cyanide or azide prevents ROS overproduction and Fas2Δ/Δ yeast cell death. Additionally, deletion of mitochondrial DNA, which encodes several subunits for enzymes of the electron transport chain, significantly reduces serum-induced Fas2Δ/Δ yeast cell death. Therefore, our results show that serum and glucose media induce Fas2Δ/Δ yeast cell death by triggering unbalanced metabolism, which is regulated by mitochondria. To our knowledge, this is the first study to critically define a link between cytosolic fatty acid synthesis and mitochondrial function in response to serum stress in C. parapsilosis.     

Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets.     

Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER.     

Ca(2+) transport into an intracellular acidic compartment of Candida parapsilosis.

In this report, we study Ca2+ transport in permeabilized Candida parapsilosis spheroplasts prepared by a new technique using lyticase. An intracellular non-mitochondrial Ca2+ uptake pathway, insensitive to orthovanadate and sensitive to the V-H(+)-ATPase inhibitor bafilomycin A(1), nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone was characterized. Acidification of the compartment in which Ca2+ accumulated was followed using the fluorescent dye acridine orange. Acidification was stimulated by the Ca2+ chelator EGTA and inhibited by Ca2+. These results, when added to the observation that Ca2+ induces alkalization of a cellular compartment, provide evidence for the presence of a Ca2+/nH(+) antiporter in the acid compartment membrane. Interestingly, like in acidocalcisomes of trypanosomatids, the antioxidant 3,5-dibutyl-4-hydroxytoluene inhibits the V-H(+)-ATPase. In addition, the antifungal agent ketoconazole promoted a fast alkalization of the acidic compartment. Ketoconazole effects were dose-dependent and occurred in a concentration range close to that attained in the plasma of patients treated with this drug.     

Discoidin domain receptor 2 (DDR2) regulates body size and fat metabolism in mice

Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens, which act as its endogenous ligand. DDR2 regulates cell proliferation, cell adhesion, migration, extracellular matrix remodeling and reproductive functions. Both DDR2 null allele mice and mice with a recessive, loss-of-function allele for Ddr2 exhibit dwarfing and a reduction in body weight. However, the detailed mechanisms by which DDR2 exerts its positive systemic regulation of whole body size, local skeletal size and fat tissue volume remain to be clarified. To investigate the systemic role of DDR2 in body size regulation, we produced transgenic mice in which the DDR2 protein is overexpressed, then screened the transgenic mice for abnormalities using systematic mouse abnormality screening. The modified-SHIPRA screen revealed that only the parameter of body size was significantly different among the genotypes. We also discovered that the body length was significantly increased, while the body weight was significantly decreased in transgenic mice compared to their littermate controls. We also found that the epididymal fat pads were significantly decreased in transgenic mice compared to normal littermate mice, which may have been the cause of the leptin decrement in the transgenic mice. The new insight that DDR2 might promote metabolism in adipocyte cells is very interesting, but more experiments will be needed to elucidate the direct relation between DDR2 and adipose-derived hormones. Taken together, our data demonstrated that DDR2 might play a systemic role in the regulation of body size thorough skeletal formation and fat metabolism.     

COPI–TRAPPII activates Rab18 and regulates its lipid droplet association

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.     

Trans-Golgi proteins participate in the control of lipid droplet and chylomicron formation

LDs (lipid droplets) carrying TAG (triacylglycerol) and cholesteryl esters are emerging as dynamic cellular organelles that are generated in nearly every cell. They play a key role in lipid and membrane homoeostasis. Abnormal LD dynamics are associated with the pathophysiology of many metabolic diseases, such as obesity, diabetes, atherosclerosis, fatty liver and even cancer. Chylomicrons, stable droplets also consisting of TAG and cholesterol are generated in the intestinal epithelium to transport exogenous (dietary) lipids after meals from the small intestine to tissues for degradation. Defective chylomicron formation is responsible for inherited lipoprotein deficiencies, including abetalipoproteinaemia, hypobetalipoproteinaemia and chylomicron retention disease. These are disorders sharing characteristics such as fat malabsorption, low levels of circulating lipids and fat-soluble vitamins, failure to thrive in early childhood, ataxic neuropathy and visual impairment. Thus understanding the molecular mechanisms governing the dynamics of LDs and chylomicrons, namely, their biogenesis, growth, maintenance and degradation, will not only clarify their molecular role, but might also provide additional indications to treatment of metabolic diseases. In this review, we highlight the role of two small GTPases [ARFRP1 (ADP-ribosylation factor related protein 1) and ARL1 (ADP-ribosylation factor-like 1)] and their downstream targets acting on the trans-Golgi (Golgins and Rab proteins) on LD and chylomicron formation.     

Membrane attachment and structure models of lipid storage droplet protein 1

Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center.     

Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism

Nutrient availability is critical for the physiological functions of all tissues. By contrast, an excess of nutrients such as carbohydrate and fats impair health and shorten life due by stimulating chronic diseases, including diabetes, cancer and neurodegeneration. The control of circulating glucose and lipid levels involve mitochondria in both central and peripheral mechanisms of metabolism regulation. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in physiological and pathological processes related to glucose and lipid metabolism, and in this review we discuss the latest data on the relationships between UCP2 and glucose and lipid sensing from the perspective of specific hypothalamic neuronal circuits and peripheral tissue functions. The goal is to provide a framework for discussion of future therapeutic strategies for metabolism-related chronic diseases.