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.

Involvement of Lipophagy and Chaperone-Mediated Autophagy in the Pathogenesis of Non-Alcoholic Fatty Liver Disease by Regulation of Lipid Droplets.

Non-alcoholic fatty liver disease (NAFLD) is defined as the accumulation of lipids in the form of lipid droplets in more than 5% of hepatocytes. It is regarded as a range of diverse pathologies, including simple steatosis and steatohepatitis. The structural characteristics of lipid droplets, along with their protein composition, mainly including perilipins, have been implicated in the etiology of the disease. These proteins have garnered increasing attention as a pivotal regulator since their levels and distinct expression appear to be associated with the progression from simple steatosis to steatohepatitis. Perilipins are target proteins of chaperone-mediated autophagy, and their degradation is a prerequisite for lipolysis and lipophagy to access the lipid core. Both lipophagy and chaperone-mediated autophagy have significant implications on the development of the disease, as evidenced by their upregulation during the initial phases of simple steatosis and their subsequent downregulation once steatosis is established. On the contrary, during steatohepatitis, the process of chaperone-mediated autophagy is enhanced, although lipophagy remains suppressed. Evidently, the reduced levels of autophagic pathways observed in simple steatosis serve as a defensive mechanism against lipotoxicity. Conversely, in steatohepatitis, chaperone-mediated autophagy fails to compensate for the continuous generation of small lipid droplets and thus cannot protect hepatocytes from lipotoxicity.

The Receptor Tyrosine Kinase c-Met Promotes Lipid Accumulation in 3T3-L1 Adipocytes.

The receptor tyrosine kinase c-Met is elaborated in embryogenesis, morphogenesis, metabolism, cell growth, and differentiation. JNJ38877605 (JNJ) is an inhibitor of c-Met with anti-tumor activity. The c-Met expression and its role in adipocyte differentiation are unknown. Here, we investigated the c-Met expression and phosphorylation, knockdown (KD) effects, and pharmacological inhibition of c-Met by JNJ on fat accumulation in murine preadipocyte 3T3-L1 cells. During 3T3-L1 preadipocyte differentiation, strikingly, c-Met expression at the protein and mRNA levels and the protein phosphorylation on Y1234/1235 and Y1349 is crucial for inducing its kinase catalytic activity and activating a docking site for signal transducers were increased in a time-dependent manner. Of note, JNJ treatment at 20 µM that strongly inhibits c-Met phosphorylation without altering its total expression resulted in less lipid accumulation and triglyceride (TG) content with no cytotoxicity. JNJ further reduced the expression of adipogenic regulators, including CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A. Moreover, JNJ treatment increased cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) phosphorylation but decreased ATP levels. Significantly, KD of c-Met suppressed fat accumulation and triglyceride (TG) quantity and reduced the expression of C/EBP-α, PPAR-γ, FAS, ACC, and perilipin A. Collectively, the present results demonstrate that c-Met is a novel, highly conserved mediator of adipogenesis regulating lipid accumulation in murine adipocytes.

Effects of Long-Term Physical Activity and BCAA Availability on the Subcellular Associations between Intramyocellular Lipids, Perilipins and PGC-1α.

Cellular skeletal muscle lipid metabolism is of paramount importance for metabolic health, specifically through its connection to branched-chain amino acids (BCAA) metabolism and through its modulation by exercise. In this study, we aimed at better understanding intramyocellular lipids (IMCL) and their related key proteins in response to physical activity and BCAA deprivation. By means of confocal microscopy, we examined IMCL and the lipid droplet coating proteins PLIN2 and PLIN5 in human twin pairs discordant for physical activity. Additionally, in order to study IMCLs, PLINs and their association to peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in cytosolic and nuclear pools, we mimicked exercise-induced contractions in C2C12 myotubes by electrical pulse stimulation (EPS), with or without BCAA deprivation. The life-long physically active twins displayed an increased IMCL signal in type I fibers when compared to their inactive twin pair. Moreover, the inactive twins showed a decreased association between PLIN2 and IMCL. Similarly, in the C2C12 cell line, PLIN2 dissociated from IMCL when myotubes were deprived of BCAA, especially when contracting. In addition, in myotubes, EPS led to an increase in nuclear PLIN5 signal and its associations with IMCL and PGC-1α. This study demonstrates how physical activity and BCAA availability affects IMCL and their associated proteins, providing further and novel evidence for the link between the BCAA, energy and lipid metabolisms.

Evaluation of the Effect of Metoprolol Dosage on Fat Graft Survival.

AIM: The aim of this study was to evaluate the effect of different metoprolol doses on fat graft survival. MATERIAL AND METHOD: A total of 10 Sprague-Dawley rats were used in the study. The dorsal regions of the rats were separated into four quadrants: right and left cranial, and right and left caudal. Each quadrant was determined as a separate group. Fat grafts were harvested from the groin areas and incubated in 5 ml solutions containing 0.9% sodium chloride (control group), 1 mg/mL metoprolol (Group 1), 2 mg/mL metoprolol (Group 2), and 3 mg/mL metoprolol (Group 3), respectively. The fat grafts were then placed in pockets dissected in each of the 4 dorsal quadrants. After 3 months all the rats were euthanized. The fat grafts were removed together with the surrounding area to which they had passed. Histopathological examination was made with hematoxylin and eosin (HE) and Masson Trichrome staining, and immunohistochemical examination with fibroblast growth factor-2 and perilipin staining. RESULTS: In the examinations made with HE and Masson Trichrome staining, the scores of Group 2 and Group 3 were determined to be significantly higher than those of the control group (p < 0.05). The Group 3 scores were significantly higher than those of Group 1 (p < 0.05). In the examinations made with fibroblast growth factor-2 staining, the scores of Group 2 and Group 3 were determined to be significantly higher than those of the control group (p < 0.05). The Group 3 scores were significantly higher than those of Group 1 and Group 2 (p < 0.05). In the examinations made with perilipin staining, the scores in Groups 1, 2, and 3 were higher than those of the control group (p < 0.05). CONCLUSION: Although metoprolol has previously been shown to prolong the survival of fat grafts, the results of this study demonstrated immunohistochemically that as the metoprolol dose increased, so the quality and vitality of fat graft also increased. 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 .

Differential glycosylation of tissue non-specific alkaline phosphatase in mesenchymal stromal cells differentiated into either an osteoblastic or adipocytic phenotype.

It has long been known that tissue non-specific alkaline phosphatase (TNAP) is essential for the correct formation of bone, as altered expression or function of this enzyme results in hypophosphatasia, a disease characterised by compromised bone structure, density and strength. However, recent evidence strongly suggests that the enzyme also has a role in lipid accrual and adipogenesis, a function that seems far removed from bone formation. Given that mesenchymal stromal cells (MSCs) are progenitors of both osteoblasts and adipocytes, the question arises of how TNAP is regulated to potentially have a different function when MSCs undergo either osteogenesis or adipogenesis. As the primary protein sequence is unchanged for the enzyme during both types of differentiation, any differences in function must be attributed to post-translational modification and/or localisation. We therefore examined the location of TNAP in bone- or adipose-derived MSCs differentiated into an adipocytic phenotype and compared the glycosylation state of the enzyme in MSCs differentiated into either osteoblasts or adipocytes. TNAP was found to co-locate with perilipin around lipid droplets in MSCs from bone, subcutaneous- and visceral adipose tissue during adipocytic differentiation. Treatment of TNAP with wheat germ lectin followed by electrophoresis showed minor differences in glycosylation between the phosphatase isolated from cells from these tissues, whereas electrophoresis after neuraminidase digestion highlighted differential glycosylation between cell types and during adipogenesis and osteoblastogenesis. This infers that post-translational modification of TNAP is altered during differentiation and is dependent on the eventual phenotype of the cells.

Development of skeletal muscle atrophy and intermuscular adipose tissue in patients with early breast cancer treated with chemotherapy.

Chemotherapy is a common therapy to treat patients with breast cancer but also leads to skeletal muscle deconditioning. Skeletal muscle deconditioning is multifactorial and intermuscular adipose tissue (IMAT) accumulation is closely linked to muscle dysfunction. To date, there is no clinical study available investigating IMAT development through a longitudinal protocol and the underlying mechanisms remain unknown. Our study was dedicated to investigating IMAT content in patients with early breast cancer who were treated with chemotherapy and exploring the subsequent cellular mechanisms involved in its development. We included 13 women undergoing chemotherapy. Muscle biopsies and ultrasonography assessment were performed before and after chemotherapy completion. Histological and Western blotting analyses were conducted. We found a substantial increase in protein levels of three mature adipocyte markers (perilipin, +901%; adiponectin, +135%; FABP4, +321%; P < 0.05). These results were supported by an increase in oil red O-positive staining (+358%; P < 0.05). A substantial increase in PDGFRα protein levels was observed (+476%; P < 0.05) highlighting an increase in fibro-adipogenic progenitors (FAPs) content. The cross-sectional area of the vastus lateralis muscle fibers substantially decreased (-21%; P < 0.01), and muscle architecture was altered, as shown by a decrease in fascicle length (-15%; P < 0.05) and a decreasing trend in muscle thickness (-8%; P = 0.08). We demonstrated both IMAT development and muscle atrophy in patients with breast cancer who were treated with chemotherapy. FAPs, critical stem cells inducing both IMAT development and skeletal muscle atrophy, also increased, suggesting that FAPs likely play a critical role in the skeletal muscle deconditioning observed in patients with breast cancer who were treated with chemotherapy.

Growth hormone/IGF-I-dependent signaling restores decreased expression of the myokine SPARC in aged skeletal muscle.

Skeletal muscle exerts many beneficial effects on the human body including the contraction-dependent secretion of peptides termed myokines. We have recently connected the myokine secreted protein acidic and rich in cysteine (SPARC) to the formation of intramuscular adipose tissue (IMAT) in skeletal muscle from aged mice and humans. Here, we searched for inducers of SPARC in order to uncover novel treatment approaches for IMAT. Endurance exercise in mice as well as forskolin treatment in vitro only modestly activated SPARC levels. However, through pharmacological treatments in vitro, we identified IGF-I as a potent inducer of SPARC expression in muscle cells, likely through a direct activation of its promoter via phosphatidylinositol 4,5-bisphospate 3-kinase (PI3K)-dependent signaling. We employed two different mouse models of growth hormone (GH)/IGF-I deficiency to solidify our understanding of the relationship between IGF-I and SPARC in vivo. GH administration robustly increased intramuscular SPARC levels (3.5-fold) in GH releasing hormone receptor-deficient mice and restored low intramuscular SPARC expression in skeletal muscle from aged mice. Intramuscular glycerol injections induced higher levels of adipocyte markers (adiponectin, perilipin) in aged compared to young mice, which was not prevented by GH treatment. Our study provides a roadmap for the study of myokine regulation during aging and demonstrates that the GH/IGF-I axis is critical for SPARC expression in skeletal muscle. Although GH treatment did not prevent IMAT formation in the glycerol model, targeting SPARC by exercise or by activation of IGF-I signaling might offer a novel therapeutic strategy against IMAT formation during aging. KEY MESSAGES : IGF-I regulates the myokine SPARC in muscle cells directly at the promoter level. GH/IGF-I is able to restore the decreased SPARC levels in aged skeletal muscle. The glycerol model induces higher adipocyte markers in aged compared to young muscle. GH treatment does not prevent IMAT formation in the glycerol model.

[Lipid Droplets and Perilipins in Cardiovascular Diseases].

Lipid droplet (LD) are multifunctional organelles which take part into intracellular lipid metabolism,mainly consisting of a neutral lipid core,a single-layer phospholipid shell,and LD-related protein (LRP).LRP on the shell can regulate the storage,transport,and metabolism of lipid.The imbalance of LD regulation could cause the abnormality of lipid metabolism,leading to the blood lipid changes and immune disorders.The available studies have demonstrated that LRP are capable of influencing the lipid metabolism in heart and atherosclerosis (AS) by regulating the physical processes in myocardial and vascular cells.This article reviewed the recent studies about LD in heart and vessels,and illustrated the effects of LD on myocardial cells,the formation of foam cells,and the development of atherosclerosis.Furthermore,we summarized the relationship between LD and cardiovascular diseases,providing new insights for the treatment of such diseases based on LD.

Lipid droplets and perilipins in canine osteosarcoma. Investigations on tumor tissue, 2D and 3D cell culture models.

Lipid droplets were identified as important players in biological processes of various tumor types. With emphasis on lipid droplet-coating proteins (perilipins, PLINs), this study intended to shed light on the presence and formation of lipid droplets in canine osteosarcoma. For this purpose, canine osteosarcoma tissue samples (n = 11) were analyzed via immunohistochemistry and electron microscopy for lipid droplets and lipid droplet-coating proteins (PLINs). Additionally, we used the canine osteosarcoma cell lines D-17 and COS4288 in 2D monolayer and 3D spheroid (cultivated for 7, 14, and 21 days) in vitro models, and further analyzed the samples by means of histochemistry, immunofluorescence, molecular biological techniques (RT-qPCR, Western Blot) and electron microscopical imaging. Lipid droplets, PLIN2, and PLIN3 were detected in osteosarcoma tissue samples as well as in 2D and 3D cultivated D-17 and COS4288 cells. In spheroids, specific distribution patterns of lipid droplets and perilipins were identified, taking into consideration cell line specific zonal apportionment. Upon external lipid supplementation (oleic acid), a rise of lipid droplet amount accompanied with an increase of PLIN2 expression was observed. Detailed electron microscopical analyzes revealed that lipid droplet sizes in tumor tissue were comparable to that of 3D spheroid models. Moreover, the biggest lipid droplets were found in the central zone of the spheroids at all sampling time-points, reaching their maximum size at 21 days. Thus, the 3D spheroids can be considered as a relevant in vitro model for further studies focusing on lipid droplets biology and function in osteosarcoma.

Perilipins at a glance.

Lipid droplets (LDs) are ubiquitous organelles that store and supply lipids for energy metabolism, membrane synthesis and production of lipid-derived signaling molecules. While compositional differences in the phospholipid monolayer or neutral lipid core of LDs impact their metabolism and function, the proteome of LDs has emerged as a major influencer in all aspects of LD biology. The perilipins (PLINs) are the most studied and abundant proteins residing on the LD surface. This Cell Science at a Glance and the accompanying poster summarize our current knowledge of the common and unique features of the mammalian PLIN family of proteins, the mechanisms through which they affect cell metabolism and signaling, and their links to disease.

Oral administration of megestrol acetate can increase fat graft survival in a rat model.

Insufficient and inconsistent survival is a significant shortcoming of fat grafts. Reportedly, megestrol acetate (MA) could induce proliferation, migration, and adipogenic differentiation of adipose-derived stem cells in vitro. Thus, we tested whether MA could promote fat graft survival in a rat model. Twenty-eight Sprague-Dawley rats (8 weeks old, male) were divided into two groups: experimental (MA group, n = 14) and control (n = 14). The inguinal fat pad (1 g) was extracted en bloc and re-implanted under the scalp in both groups. MA (100 mg/kg/day) was administered orally for 14 postoperative days in the experimental group. After 6 weeks, the volume and weight of the grafted fat were measured. Histologic examination with hematoxylin and eosin (HE) and real-time polymerase chain reaction (PCR) for vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and CCAAT/enhancer-binding protein alpha (C/EBP-α) were performed. Perilipin staining was performed to check the viability of grafted fat. Graft fat volume was greater in the MA group, compared with that in the control (P = 0.023). The MA group also had more viable cells, including more adipocytes, and less fibrosis or vacuoles than the control on HE and perilipin staining. MA upregulated the expression of FGF2 (P<0.001), VEGF (P = 0.008), and C/EBP-α (P = 0.002) at the second postoperative week. MA increased survival of grafted fat in an animal model. Increased vascularization and adipogenesis were related to these results. Further human clinical trials are necessary to evaluate adjunctive oral administration of MA after fat grafting to promote graft survival.

Adding collagen to adipose tissue transplant increases engraftment by promoting cell proliferation, neovascularisation and macrophage activity in a rat model.

To clarify the effect of collagen addition to transplanted adipose tissue on angiogenesis, cell proliferation and tissue remodelling process and reveal whether collagen addition contributes to improving transplanted adipose tissue engraftment in rats. Adipose tissue was harvested from the inguinal and injected into the back of the rat, in addition to collagen. Engraftment tissue was harvested, semi-quantitatively evaluated and underwent haematoxylin and eosin or Perilipin staining. Moreover, we evaluated viable adipocyte counts and neovascularisation. Macrophages were evaluated using flow cytometry, and the adiponectin or vascular endothelial growth factor (VEGF) mRNA was detected using real-time polymerase chain reaction. By collagen addition to transplanted adipose tissue, higher engraftment rate semi-quantitatively and a greater number of new blood vessels histologically were identified. Perilipin staining revealed a higher adipocyte number. The total cell, M1 macrophage and M2 macrophage count were higher. There was increased adiponectin mRNA significantly at week 4 compared to that at week 1 after transplantation. Note that the expression levels of VEGF mRNA increased. In rats, adding collagen enhanced cell proliferation, induced M2 macrophages, which are involved in wound healing, and promoted adipocytes and neovascularisation. Therefore, collagen addition to transplanted adipose tissue could increase the engraftment rate of adipose tissue.

Expression pattern of perilipins in human brain during aging and in Alzheimer's disease.

AIMS: Perilipins are conserved proteins that decorate intracellular lipid droplets and are essential for lipid metabolism. To date, there is limited knowledge on their expression in human brain or their involvement in brain aging and neurodegeneration. The aim of this study was to characterise the expression levels of perilipins (Plin1-Plin5) in different cerebral areas from subjects of different age, with or without signs of neurodegeneration. METHODS: We performed real-time RT-PCR, western blotting, immunohistochemistry and confocal microscopy analyses in autoptic brain samples of frontal and temporal cortex, cerebellum and hippocampus from subjects ranging from 33 to 104 years of age, with or without histological signs of neurodegeneration. To test the possible relationship between Plins and inflammation, correlation analysis with IL-6 expression was also performed. RESULTS: Plin2, Plin3 and Plin5, but not Plin1 and Plin4, are expressed in the considered brain areas with different intensities. Plin2 appears to be expressed more in grey matter, particularly in neurons in all the areas analysed, whereas Plin3 and Plin5 appear to be expressed more in white matter. Plin3 seems to be expressed more in astrocytes. Only Plin2 expression is higher in old subjects and patients with early tauopathy or Alzheimer's disease and is associated with IL-6 expression. CONCLUSIONS: Perilipins are expressed in human brain but only Plin2 appears to be modulated with age and neurodegeneration and linked to an inflammatory state. We propose that the accumulation of lipid droplets decorated with Plin2 occurs during brain aging and that this accumulation may be an early marker and initial step of inflammation and neurodegeneration.

THRAP3 depletion reduces PPARγ mRNA and anti-inflammatory action in 3T3-L1 adipocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator of adipocytes and the cellular target of thiazolidinedione (TZD) drugs. Suppression of pro-inflammatory actions, including pro-inflammatory gene expression and lipolysis in adipocytes, contributes to PPARγ-mediated anti-diabetic effects of TZDs. However, adverse side effects largely limited the clinical use of TZDs, despite their potent insulin-sensitizing effects. Therefore, it is important to understand how PPARγ is regulated. Thyroid hormone receptor-associated protein 3 (THRAP3) was previously reported to promote diabetic gene expression by acting as a transcriptional coregulator of PPARγ in adipocytes. Therefore, we tested if THRAP3 modulated anti-inflammatory functions of PPARγ in 3T3-L1 adipocytes. THRAP3 depletion increased basal and tumor necrosis factor α (TNFα)-induced lipolysis, pro-inflammatory gene expression, and phosphorylation of extracellular signal-regulated kinases (ERKs), suggesting elevated pro-inflammatory response after THRAP3 depletion in adipocytes. Moreover, TZD-mediated suppression of TNFα-induced lipolysis, pro-inflammatory gene expression, and ERK phosphorylation was attenuated or alleviated after THRAP3 depletion. Interestingly, the mRNA and protein levels of PPARγ were greatly reduced in THRAP3-depleted adipocytes. Actinomycin D treatment revealed that the stability of PPARγ mRNA was greatly reduced by THRAP3 depletion in adipocytes. Thus, in addition to modulating PPARγ function, THRAP3 may directly regulate the transcript of PPARγ in differentiated adipocytes.

Effects of (-)-epicatechin on the time course of the expression of perilipins in a diet-induced model of nonalcoholic steatohepatitis.

The existing treatments for nonalcoholic steatohepatitis (NASH) are not completely effective. The need for new alternatives without adverse effects and low cost, such as the flavonoid (-)-epicatechin (EC), which has beneficial effects on lipid metabolism and cardiovascular diseases, arises. The objective of this work was to analyze EC effects in the NASH induced by a Paigen-type diet (PD). Mice were administered with (1) normal chow and water, (2) PD + fructose 30% and (3) PD + fructose 30% + EC (1 mg/kg) per gavage during 9 weeks. At the end of each treatment, serum was collected for analysis of the biochemical profile and liver enzymes. The liver was collected for microscopic analysis and for the evaluation of the relative expression of Plin2, Plin3, CD36, adiponectin and UCP2. Results showed that EC reduced weight gain and decreased triglyceride (TG), low-density lipoprotein cholesterol, TG/high-density lipoprotein and the activity of liver enzymes (alanine aminotransferase and alkaline phosphatase), suggesting lower liver damage. The microscopic analysis showed less "balloonization" of the hepatocyte, small drops of lipids, less accumulation of collagen and infiltration of inflammatory cells as compared to nontreated group. Finally, a decrease in the expression of Plin 2 was observed. While CD36 decreased, adiponectin and UCP2 increased. In conclusion, EC improves the biochemical profile, the microscopic characteristics and protein expression. Therefore, it may be a possible therapeutic approach for NASH since it prevents the progression of the hepatic and metabolic damage induced by high-fat diets.

A 7-day high-fat, high-calorie diet induces fibre-specific increases in intramuscular triglyceride and perilipin protein expression in human skeletal muscle.

KEY POINTS: We have recently shown that a high-fat, high-calorie (HFHC) diet decreases whole body glucose clearance without impairing skeletal muscle insulin signalling, in healthy lean individuals. These diets are also known to increase skeletal muscle IMTG stores, but the effect on lipid metabolites leading to skeletal muscle insulin resistance has not been investigated. This study measured the effect of 7 days' HFHC diet on (1) skeletal muscle concentration of lipid metabolites, and (2) potential changes in the perilipin (PLIN) content of the lipid droplets storing intramuscular triglyceride (IMTG). The HFHC diet increased PLIN3 protein expression and redistributed PLIN2 to lipid droplet stores in type I fibres. The HFHC diet increased IMTG content in type I fibres, while lipid metabolite concentrations remained the same. The data suggest that the increases in IMTG stores assists in reducing the accumulation of lipid metabolites known to contribute to skeletal muscle insulin resistance. ABSTRACT: A high-fat, high-calorie (HFHC) diet reduces whole body glucose clearance without impairing skeletal muscle insulin signalling in healthy lean individuals. HFHC diets also increase skeletal muscle lipid stores. However, unlike certain lipid metabolites, intramuscular triglyceride (IMTG) stored within lipid droplets (LDs) does not directly contribute to skeletal muscle insulin resistance. Increased expression of perilipin (PLIN) proteins and colocalisation to LDs has been shown to assist in IMTG storage. We aimed to test the hypothesis that 7 days on a HFHC diet increases IMTG content while minimising accumulation of lipid metabolites known to disrupt skeletal muscle insulin signalling in sedentary and obese individuals. We also aimed to identify changes in expression and subcellular distribution of proteins involved in IMTG storage. Muscle biopsies were obtained from the m. vastus lateralis of 13 (11 males, 2 females) healthy lean individuals (age: 23 ± 2.5 years; body mass index: 24.5 ± 2.4 kg m-2 ), following an overnight fast, before and after consuming a high-fat (64% energy), high-calorie (+47% kcal) diet for 7 days. After the HFHC diet, IMTG content increased in type I fibres only (+101%; P < 0.001), whereas there was no change in the concentration of either total diacylglycerol (P = 0.123) or total ceramides (P = 0.150). Of the PLINs investigated, only PLIN3 content increased (+50%; P < 0.01) solely in type I fibres. LDs labelled with PLIN2 increased (+80%; P < 0.01), also in type I fibres only. We propose that these adaptations of LDs support IMTG storage and minimise accumulation of lipid metabolites to protect skeletal muscle insulin signalling following 7 days' HFHC diet.

Skeletal muscle lipid droplets are resynthesized before being coated with perilipin proteins following prolonged exercise in elite male triathletes.

Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg-1·min-1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (-53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers (P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.

Formation and degradation of lipid droplets in human adipocytes and the expression of aldehyde oxidase (AOX).

Lipid droplet (LD) binding proteins in mammary glands and in adipocytes were previously compared and striking similar sets of these specific proteins demonstrated. Xanthine oxidoreductase (XOR) together with perilipins and the lactating mammary gland protein butyrophilin play an important role in the secretion process of LDs into milk ducts. In contrast, in adipose tissue and in adipocytes, mainly perilipins have been described. Moreover, XOR was reported in mouse adipose tissue and adipocyte culture cells as "novel regulator of adipogenesis". This obvious coincidence of protein sets prompted us to revisit the formation of LDs in human-cultured adipocytes in more detail with special emphasis on the possibility of a LD association of XOR. We demonstrate by electron and immunoelectron microscopy new structural details on LD formation in adipocytes. Surprisingly, by immunological and proteomic analysis, we identify in contrast to previous data showing the enzyme XOR, predominantly the expression of aldehyde oxidase (AOX). AOX could be detected tightly linked to LDs when adipocytes were treated with starvation medium. In addition, the majority of cells show an enormous interconnected, tubulated mitochondria network. Here, we discuss that (1) XOR is involved-together with perilipins-in the secretion of LDs in alveolar epithelial cells of the lactating mammary gland and is important in the transcytosis pathway of capillary endothelial cells. (2) In cells, where LDs are not secreted, XOR cannot be detected at the protein level, whereas in contrast in these cases, AOX is often present. We detect AOX in adipocytes together with perilipins and find evidence that these proteins might direct LDs to mitochondria. Finally, we here report for the first time the exclusive and complementary localization of XOR and AOX in diverse cell types.

Forskolin induced remodeling of lipid droplets in rat adipocytes.

Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 µmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.