Adipocyte Differentiation-related Protein Research Articles

1α,25(OH)2D3 AND ITS 3 EPIMER PROMOTE PULMONARY ALVEOLAR EPITHELIAL-MESENCHYMAL INTERACTIONS AND INHIBIT INTERSTITIAL FIBROBLAST APOPTOSIS.

Background Alveolar epithelial-mesenchymal interactions play a critical role in perinatal pulmonary transition, which is characterized by surfactant maturation and alveolar wall thinning. This alveolar wall thinning has been attributed to apoptosis of interstitial lung fibroblasts (LFs), normally in abundance perinatally. The mechanism underlying this is unclear. Although 1α,25(OH)2D3 (1,25D) has been suggested as a local paracrine/autocrine effector for fetal lung maturation, its effect on alveolar intersitial LFs survival/apoptosis is unknown. We have previously shown that 1,25D is metabolized to its C-3 epimer (3-epi 1,25D) in rat alveolar type II cells (ATII). Relatively high circulating concentrations of 3-epi vitamin D metabolites have recently been reported in neonates. Objective To investigate the role of 3-epi 1,25D in lung development during the perinatal period. Methods Embryonic (e) day 19 and day 21 fetal rat lung ATII and LFs were isolated and cultured using standard methods. At near-confluence, cells were treated with 1,25D or 3-epi 1,25D (1 × 10−11 to 1 × 10−8M) for up to 48 hours. Cell proliferation (thymidine incorporation assay) and apoptosis (TUNEL assay and Western blotting for Bcl and Bax expression in the mitochondrial and cytoplasmic fractions) were studied. Markers of alveolar epithelial-mesenchymal interactions (parathyroid hormone-related protein receptor, peroxisome proliferator-activated receptorγ, adipocyte differentiation-related protein, leptin, leptin receptor, and surfactant protein B) were assessed by Western blotting. Results Both 1,25D and its 3 epimer exhibited dose-dependent increases in expression of the key homeostatic epithelial-mesenchymal markers, an increase in interstitial fibroblast proliferation, and decreased in interstitial fibroblast apoptosis, with 3-epi 1,25D having a more robust antiapoptotic effect compared with its parent compound. Furthermore, both 1,25D and its 3 epimer inhibited the actinomycin D-induced apoptosis of LFs-again more robustly by 3-epi 1,25D. Conclusions Vitamin D plays a significant role in fetal lung maturation by stimulating the key alveolar epithelial-mesenchymal interactions and LFs9 survival. We speculate that perinatal alterations in vitamin D metabolism, which result in significant decreases in C-3 epimer levels, may be a key factor in alveolar interstitial LFs9 apoptosis and alveolar wall thinning. Supported by grants from TRDRP ((14RT-0073 and 15IT-0250), Philip Morris USA Inc. and Philip Morris International, and NIH (HL55268 and HL075405).

THE SAFETY AND EFFICACY OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AGONIST ROSIGLITAZONE FOR LUNG DIFFERENTIATION IN A NEONATAL RAT MODEL.

Background Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the retinoid X-receptor heterodimer family of ligand-activated nuclear receptors, plays a critical role in modulating lung injury/repair. Recently, using both in vitro and in vivo models of acute neonatal lung injury, our laboratory has shown that PPAR-γ agonists can prevent nicotine- and short-term hyperoxia-induced lung injury. These studies clearly suggest the potential therapeutic usefulness of PPAR-γ agonists in preventing neonatal lung injury. However, there is no information on the effects of systemically administered PPAR-γ agonists on lung differentiation and metabolic profile in the developing system. Objectives To determine the effects of systemically administered PPAR-γ agonist rosiglitazone (RGZ) on lung differentiation, serum electrolytes, blood glucose, and blood gases in a developing neonatal rat model. Methods Newborn Sprague-Dawley rat pups were divided into the following six groups: control and RGZ 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 8 mg/kg groups. The diluent (saline) or RGZ was administered IP in 100 μL volumes once daily for either 1 day or 7 days. The pups were sacrificed and the lungs were collected and processed either immediately for choline incorporation and triolein uptake or later for protein (Western hybridization) and mRNA (RT-PCR) analysis for markers of lung maturation. Results Systemically administered RGZ significantly increased the expression of surfactant proteins B and C, cholinephosphate cytidyltransferase, PPAR-γ, and adipocyte differentiation-related protein in a dose-dependent manner, both at day 1 and day 7. This was also accompanied by significantly increased choline incorporation and triolein uptake, the functional markers of surfactant phospholipid synthesis. There were no significant effects on serum electrolytes, blood glucose, and blood gases except at the highest dose of RGZ (8 mg/kg), which resulted in significantly lower blood glucose values. Conclusions Systemically administered RGZ significantly enhances lung differentiation without significantly affecting the serum metabolic profile. We speculate that PPAR-γ agonists may be safe and effective agents for enhancing repair of lung injury in neonates. Supported by grants from NIH (HL55268 and HL075405), TRDRP (14RT-0073 and 15IT-0250), and Philip Morris USA Inc. and Philip Morris International.

Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine

A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.

Role of adipose differentiation-related protein in lung surfactant production: a reassessment

Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.

Up-Regulation of Fetal Rat Lung Parathyroid Hormone-Related Protein Gene Regulatory Network Down-Regulates the Sonic Hedgehog/Wnt/βcatenin Gene Regulatory Network

Lung development depends on endodermal Sonic Hedgehog (Shh) signaling to mesodermal Wingless/int/beta catenin (Wnt/betacatenin), followed by parathyroid hormone-related protein (PTHrP) signaling from endoderm to mesoderm. Fluid distension of fetal rat lung explants up-regulates PTHrP signaling and down-regulates Shh/Wnt/betacatenin signaling, marked by decreases in Patched, Gli, Frizzled, and Dishevelled, inducing fibroblast triglyceride uptake, type II cell saturated phosphatidylcholine, and surfactant protein-B expression. Bumetanide, which inhibits fluid distension, blocked down-regulation of the Shh/Wnt/betacatenin pathway and up-regulation of the PTHrP pathway, whereas PTHrP (1-34, 5 x 10(-7) M) treatment overcame bumetanide inhibition, and the PTHrP receptor antagonist PTHrP (7-34) amide (5 x 10(-6) M) mimicked bumetanide, indicating that PTHrP signaling mediates fluid distension-induced alveolar differentiation. Fetal rat lung explant automaturation was characterized by decreased Wnt/betacatenin signaling and increased PTHrP/PTHrP receptor signaling, up-regulating fibroblast-specific adipocyte differentiation related protein (ADRP) and peroxisome proliferator-activated receptor gamma. Wnt/betacatenin agonists (LiCl or SB415268) maintained Shh/Wnt/betacatenin signaling, blocking spontaneous up-regulation of the PTHrP pathway, whereas PTHrP or cAMP down-regulated Shh/Wnt/betacatenin signaling and stimulated PTHrP signaling for fibroblast and type II cell differentiation. This is the first evidence that alveolar fluid distension is an organizing principle for PTHrP signaling down-regulation of the Shh/Wnt/betacatenin pathway.

Structural Determinants That Target the Hepatitis C Virus Core Protein to Lipid Droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.

Hypoxia Converts Human Macrophages Into Triglyceride-Loaded Foam Cells

Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells. Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in beta-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl-coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of beta-oxidation, whereas the accumulation of triglycerides increased. The results demonstrate that exposure of human macrophages to hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets. This indicates that the hypoxia present in atherosclerotic lesions can contribute to the formation of the lipid-loaded macrophages that characterize the lesion and to the accumulation of triglycerides in such lesions.

Adipocyte Differentiation-Related Protein Promotes Fatty Acid Storage in Cytosolic Triglycerides and Inhibits Secretion of Very Low–Density Lipoproteins

We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into beta-oxidation. ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to beta-oxidation and promotes the secretion of apoB-48 VLDL1.

Ligand Activation of Peroxisome Proliferator–Activated Receptor β Inhibits Colon Carcinogenesis

There is considerable debate whether peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) ligands potentiate or suppress colon carcinogenesis. Whereas administration of a PPARbeta ligand causes increased small intestinal tumorigenesis in Apc(min/+) mice, PPARbeta-null (Pparb-/-) mice exhibit increased colon polyp multiplicity in colon cancer bioassays, suggesting that ligand activation of this receptor will inhibit colon carcinogenesis. This hypothesis was examined by treating wild-type (Pparb+/+) and Pparb-/- with azoxymethane, coupled with a highly specific PPARbeta ligand, GW0742. Ligand activation of PPARbeta in Pparb+/+ mice caused an increase in the expression of mRNA encoding adipocyte differentiation-related protein, fatty acid-binding protein, and cathepsin E. These findings are indicative of colonocyte differentiation, which was confirmed by immunohistochemical analysis. No PPARbeta-dependent differences in replicative DNA synthesis or expression of phosphatase and tensin homologue, phosphoinositide-dependent kinase, integrin-linked kinase, or phospho-Akt were detected in ligand-treated mouse colonic epithelial cells although increased apoptosis was found in GW0742-treated Pparb+/+ mice. Consistent with increased colonocyte differentiation and apoptosis, inhibition of colon polyp multiplicity was also found in ligand-treated Pparb+/+ mice, and all of these effects were not found in Pparb-/- mice. In contrast to previous reports suggesting that activation of PPARbeta potentiates intestinal tumorigenesis, here we show that ligand activation of PPARbeta attenuates chemically induced colon carcinogenesis and that PPARbeta-dependent induction of cathepsin E could explain the reported disparity in the literature about the effect of ligand activation of PPARbeta in the intestine.

Liver‐directed overexpression of mitochondrial glycerol‐3‐phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step in triacylglycerol (TAG) and phospholipid biosynthesis. GPAT activity has been identified in both ER and mitochondrial subcellular fractions. The ER activity dominates in most tissues except in liver, where the mitochondrial isoform (mtGPAT) can constitute up to 50% of the total activity. To study the in vivo effects of hepatic mtGPAT overexpression, mice were transduced with adenoviruses expressing either murine mtGPAT or a catalytically inactive variant of the enzyme. Overexpressing mtGPAT resulted in massive 12- and 7-fold accumulation of liver TAG and diacylglycerol, respectively but had no effect on phospholipid or cholesterol ester content. Histological analysis showed extensive lipid accumulation in hepatocytes. Furthermore, mtGPAT transduction markedly increased adipocyte differentiation-related protein and stearoyl-CoA desaturase-1 (SCD-1) in the liver. In line with increased SCD-1 expression, 18:1 and 16:1 in the hepatic TAG fraction increased. In addition, mtGPAT overexpression decreased ex vivo fatty acid oxidation, increased liver TAG secretion rate 2-fold, and increased plasma TAG and cholesterol levels. These results support the hypothesis that increased hepatic mtGPAT activity associated with obesity and insulin resistance contributes to increased TAG biosynthesis and inhibition of fatty acid oxidation, responses that would promote hepatic steatosis and dyslipidemia.

Detection of differential gene expression in brown adipose tissue of hibernating arctic ground squirrels with mouse microarrays

Hibernation is an energy-saving strategy adopted by a wide range of mammals to survive highly seasonal or unpredictable environments. Arctic ground squirrels living in Alaska provide an extreme example, with 6- to 9-mo-long hibernation seasons when body temperature alternates between levels near 0 degrees C during torpor and 37 degrees C during arousal episodes. Heat production during hibernation is provided, in part, by nonshivering thermogenesis that occurs in large deposits of brown adipose tissue (BAT). BAT is active at tissue temperatures from 0 to 37 degrees C during rewarming and continuously at near 0 degrees C during torpor in subfreezing conditions. Despite its crucial role in hibernation, the global gene expression patterns in BAT during hibernation compared with the nonhibernation season remain largely unknown. We report a large-scale study of differential gene expression in BAT between winter hibernating and summer active arctic ground squirrels using mouse microarrays. Selected differentially expressed genes identified on the arrays were validated by quantitative real-time PCR using ground squirrel specific primers. Our results show that the mRNA levels of the genes involved in nearly every step of the biochemical pathway leading to nonshivering thermogenesis are significantly increased in BAT during hibernation, whereas those of genes involved in protein biosynthesis are significantly decreased compared with summer active animals in August. Surprisingly, the differentially expressed genes also include adipocyte differentiation-related protein or adipophilin (Adfp), gap junction protein 1 (Gja1), and secreted protein acidic and cysteine-rich (Sparc), which may play a role in enhancing thermogenesis at low tissue temperatures in BAT.

Induction of G1 arrest and differentiation in MDA-MB-231 breast cancer cell by boehmeriasin A, a novel compound from plant

Boehmeriasin A is a new phenanthroquinolizidine alkaloid recently isolated from the Boehmeria siamensis Craib (Urticaceae). In vitro biological activity assay demonstrated that this novel compound has wide-range, strong antitumor activity. This study is aimed to determine the effects of boehmeriasin A on breast cancer cell (MDA-MB-231 cell line). Proliferation assay and fluorescence activated cell sorter (FACS) showed that cell growth inhibition and G1 phase arrest of cell cycle were caused by boehmeriasin A. The concentrations resulting in total and 50% growth inhibition are 0.007 and 0.0035 microg/mL, respectively. Exposed in 0.007 microg/mL boehmeriasin A for 12 h, the G1 phase cell percent increased from 44.8% pre-drug treatment to 66.3%. Consistent with G1 arrest and cell growth inhibition, cyclin E2 and cyclin D1 messenger RNA expression in the cell was down-regulated with drug treatment. Then, few apoptotic cells were detected, and most other cells underwent differentiation, which is characterized by specific changes in cell morphology, lots of lipid droplet accumulation, and increasing expression of adipocyte differentiation-related protein. The result first demonstrates that boehmeriasin A potently inhibits the proliferation of breast cancer cell MDA-MB-231 via the G1 phase cell cycle arrest and differentiation induction, and as such, may be considered as candidate chemotherapeutic and/or chemopreventive agent for breast cancer.

Perilipin Targets a Novel Pool of Lipid Droplets for Lipolytic Attack by Hormone-sensitive Lipase

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.

Dynamics of Lipid Droplet-Associated Proteins during Hormonally Stimulated Lipolysis in Engineered Adipocytes: Stabilization and Lipid Droplet Binding of Adipocyte Differentiation-Related Protein/Adipophilin

In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure.

Binding and Functions of ADP-ribosylation Factor on Mammalian and Yeast Peroxisomes

We have analyzed in vitro the binding characteristics of members of the ADP-ribosylation factor (ARF) family of proteins to a highly purified rat liver peroxisome preparation void of Golgi membranes and studied in vivo a role these proteins play in the proliferation of yeast peroxisomes. Although both ARF1 and ARF6 were found on peroxisomes, coatomer recruitment only depended on ARF1-GTP. Recruitment of ARF1 and coatomer to peroxisomes was significantly affected both by pretreating the animals with peroxisome proliferators and by ATP and a cytosolic fraction designated the intermediate pool fraction depleted of ARF and coatomer. In the presence of ATP, the concentrations of ARF1 and coatomer on peroxisomes were reduced, whereas intermediate pool fraction led to a concentration-dependent decrease in ARF and increase in coatomer. Brefeldin A, a fungal toxin that is known to reduce ARF1 binding to Golgi membranes, did not affect ARF1 binding to peroxisomes. In Saccharomyces cerevisiae, both ScARF1 and ScARF3, the yeast orthologs of mammalian ARF1 and ARF6, were implicated in the control of peroxisome proliferation. ScARF1 regulated this process in a positive manner, and ScARF3 regulated it in a negative manner.

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

Adipocyte Differentiation‐related Protein in Human Skeletal Muscle: Relationship to Insulin Sensitivity

To determine whether adipocyte differentiation-related protein (ADRP), a lipid droplet-associated protein that binds to and sequesters intracellular fatty acids, is 1) expressed in human skeletal muscle and 2) differentially regulated in human skeletal muscle obtained from obese non-diabetic (OND) and obese diabetic (OD) subjects. Ten OND subjects and 15 OD subjects underwent a weight loss or pharmacological intervention program to improve insulin sensitivity. Anthropometric data, hemoglobin A(1C), fasting glucose, lipids, and glucose disposal rate were determined at baseline and at completion of studies. Biopsies of the vastus lateralis muscle (SkM) were obtained in the fasting state from OND and OD subjects. Protein expression was determined by Western blotting. ADRP was highly expressed in SkM from OND (4.4 +/- 1.54 AU/10 microg, protein, n = 10) and OD (5.02 +/- 1.33 AU/10 microg, n = 12) subjects. OND subjects undergoing weight loss had decreased triglyceride levels and improved insulin action. SkM ADRP content increased with weight loss from 5.14 +/- 2.15 AU/10 microg to 9.92 +/- 1.57 AU/10 microg (p < 0.025). OD subjects were treated with either troglitazone or metformin, together with glyburide, for 3 to 4 months. Both treatments attained similar levels of glycemic control. OD subjects with lower baseline ADRP content (2.85 +/- 1.07 AU/10 microg, n = 6) displayed up-regulation of ADRP expression (to 9.27 +/- 2.76 AU/10 microg, p < 0.025). ADRP is the predominant lipid droplet-associated protein in SkM, and low ADRP expression is up-regulated in circumstances of improved glucose tolerance. Up-regulation of ADRP may act to sequester fatty acids as triglycerides in discrete lipid droplets that could protect muscle from the detrimental effects of fatty acids on insulin action and glucose tolerance.

Regulation of Renal Lipid Metabolism, Lipid Accumulation, and Glomerulosclerosis in FVBdb/db Mice With Type 2 Diabetes

Diabetic kidney disease has been associated with the presence of lipid deposits, but the mechanisms for the lipid accumulation have not been fully determined. In the present study, we found that db/db mice on the FVB genetic background with loss-of-function mutation of the leptin receptor (FVB-Lepr(db) mice or FVBdb/db) develop severe diabetic nephropathy, including glomerulosclerosis, tubulointerstitial fibrosis, increased expression of type IV collagen and fibronectin, and proteinuria, which is associated with increased renal mRNA abundance of transforming growth factor-beta, plasminogen activator inhibitor-1, and vascular endothelial growth factor. Electron microscopy demonstrates increases in glomerular basement membrane thickness and foot process (podocyte) length. We found that there is a marked increase in neutral lipid deposits in glomeruli and tubules by oil red O staining and biochemical analysis for cholesterol and triglycerides. We also detected a significant increase in the renal expression of adipocyte differentiation-related protein (adipophilin), a marker of cytoplasmic lipid droplets. We examined the expression of sterol regulatory element-binding protein (SREBP)-1 and -2, transcriptional factors that play an important role in the regulation of fatty acid, triglyceride, and cholesterol synthesis. We found significant increases in SREBP-1 and -2 protein levels in nuclear extracts from the kidneys of FVBdb/db mice, with increases in the mRNA abundance of acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-CoA reductase, which mediates the increase in renal triglyceride and cholesterol content. Our results indicate that in FVBdb/db mice, renal triglyceride and cholesterol accumulation is mediated by increased activity of SREBP-1 and -2. Based on our previous results with transgenic mice overexpressing SREBP-1 in the kidney, we propose that increased expression of SREBPs plays an important role in causing renal lipid accumulation, glomerulosclerosis, tubulointerstitial fibrosis, and proteinuria in mice with type 2 diabetes.

Cytosolic Lipid Droplets Increase in Size by Microtubule-Dependent Complex Formation

Adipocyte differentiation-related protein (ADRP)-containing lipid droplets have an essential role in the development of insulin resistance and atherosclerosis. Such droplets form in a cell-free system with a diameter of 0.1 to 0.4 microm, while the droplets present in cells vary in size, from small to very large, suggesting that the droplets can increase in size after being assembled. We have addressed this possibility. Experiments in NIH 3T3 cells demonstrated that the lipid droplets could increase in size independently of triglyceride biosynthesis. NIH 3T3 cells were either microinjected with ADRP-GFP (green fluorescent protein) or stained with Nile Red and followed by confocal microscopy and time-lapse recordings. The results showed that lipid droplets formed complexes with each other, with a volume equal to the sum of the merging particles. The formation of complexes could be inhibited by the nocodazole-induced depolymerization of the microtubules; thus, the process is dependent on microtubules. The presence of dynein on ADRP-containing droplets supports a role for this motor protein. Lipid droplets can grow after they have been assembled. This increase in size is independent of triglyceride biosynthesis and involves formation of complexes, which requires intact microtubules.

Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane

Lipid droplets (LDs) are organelles that store neutral lipids, but their regulatory mechanism is not well understood. In the present study, we identified Rab18 as an LD component of HepG2 cells by proteomic analysis, and confirmed its localization by immunohistochemistry and western blotting. Wild-type and dominant-active Rab18 localized to LDs but the dominant-negative form did not. Endogenous Rab18 coexisted with adipocyte differentiation-related protein (ADRP) in LDs, but the labeling intensity of the two proteins showed clear reciprocity. Consistent with this observation, overexpression of Rab18 induced a decrease in the amounts of ADRP in LDs in HepG2 and BALB/c 3T3 cells. Furthermore, Rab18 overexpression caused close apposition of LDs to membrane cisternae connected to the rough ER. Two other procedures that decrease ADRP, i.e. RNA interference and brefeldin A treatment, induced the same morphological change, indicating that decrease in ADRP was the cause of the LD-ER apposition. In accordance with similar structures found between ER and other organelles, we propose that the ER membrane apposed to LDs should be named the LD-associated membrane, or LAM. The present results suggested that Rab18 regulates LAM formation, which is likely to be involved in mobilizing lipid esters stored in LDs.