Mitochondria Bound to Lipid Droplets Have Unique Bioenergetics,Composition, and Dynamics that Support Lipid Droplet Expansion

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Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b

Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1. These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.     

Cerebral Cortex Synaptic Heavy Mitochondria May Represent the Oldest Synaptic Mitochondrial Population: Biochemical Heterogeneity and Effects of L-Acetylcarnitine

The microheterogeneous nature of intrasynaptic mitochondria has been demonstrated and iswidely accepted. However, evidence is still lacking about the role played by the differentintrasynaptic mitochondrial subpopulations. The data obtained support the hypothesis thatheavy mitochondria could represent old mitochondrial populations: in fact, in addition tothe well known impairment of typical mitochondrial functions, they possess the highest levelsof hydroperoxides and their fatty acids pattern is completely modified. The qualitative andquantitative fatty acid modifications suffered by these organelles deeply altered theirprotein/lipid ratio, thus modifying their mode of action. The present work also collects a large bodyof evidence that a subchronic L-acetylcarnitine treatment in 28 days does not structurallyaffect both nonsynaptic and intrasynaptic mitochondria of normal rat in asteady-statemetabolic condition.     

Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.     

Dietary lipids inhibit mitochondria transfer to macrophages to divert adipocyte-derived mitochondria into the blood

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A Mixture of Paraphenylenediamine and Imidazole: Its Effect on the Extraction of Lipid Droplets during Electron Microscopy Staining

To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.     

Pex11a deficiency causes dyslipidaemia and obesity in mice

Peroxisomes play a central role in lipid metabolism. We previously demonstrated that Pex11a deficiency impairs peroxisome abundance and fatty acid β‐oxidation and results in hepatic triglyceride accumulation. The role of Pex11a in dyslipidaemia and obesity is investigated here with Pex11a knockout mice (Pex11a^?/?). Metabolic phenotypes including tissue weight, glucose tolerance, insulin sensitivity, cholesterol levels, fatty acid profile, oxygen consumption, physical activity were assessed in wild‐type (WT) and Pex11a^?/? fed with a high‐fat diet. Molecular changes and peroxisome abundance in adipose tissue were evaluated through qRT‐PCR, Western blotting, and Immunofluorescence. Pex11a^?/? showed increased fat mass, decreased skeletal muscle, higher cholesterol levels, and more severely impaired glucose and insulin tolerance. Pex11a^?/? consumed less oxygen, indicating a decrease in fatty acid oxidation, which is consistent with the accumulation of very long‐ and long‐chain fatty acids. Adipose palmitic acid (C16:0) levels were elevated in Pex11a^?/?, which may be because of dramatically increased fatty acid synthase mRNA and protein levels. Furthermore, Pex11a deficiency increased ventricle size and macrophage infiltration, which are related to the reduced physical activity. These data demonstrate that Pex11a deficiency impairs physical activity and energy expenditure, decreases fatty acid β‐oxidation, increases de novo lipogenesis and results in dyslipidaemia and obesity.     

Kynurenic Acid and Gpr35 Regulate Adipose Tissue Energy Homeostasis and Inflammation

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Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.     

Disruption of Acetyl-Lysine Turnover in Muscle Mitochondria Promotes Insulin Resistance and Redox Stress without Overt Respiratory Dysfunction

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Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates

Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.