Formation Of Cytoplasmic Lipid Droplets Research Articles

C-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling. Maf inactivation perturbed the villus zonation program by increasing carbohydrate-related transcripts while suppressing transcripts linked to amino-acid and lipid absorption. The formation of cytoplasmic lipid droplets, shuttling dietary fat to chylomicrons, was impaired upon Maf loss indicating its role in dietary lipid handling. Maf inactivation under homeostatic conditions expanded tuft cells and led to compensatory gut lengthening, preventing weight loss. However, delayed Maf-/- enterocyte maturation impaired weight recovery after acute intestinal injury, resulting in reduced survival. Our results identify c-MAF as a regulator of the intestinal villus zonation program, while highlighting the importance of coordination between stem/progenitor and differentiation programs for intestinal regeneration.

Larval excretion/secretion of dipters of Lucilia cuprina species induces death in promastigote and amastigote forms of Leishmania amazonensis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Larval excretion/secretion (ES) of the larvae of flies of the Calliphoridae family has microbicidal activity against Gram-positive and Gram-negative bacteria, in addition to some species of Leishmania. Our study aimed at assessing the in vitro efficacy of Lucilia cuprina larval ES against the promastigote and amastigote forms of Leishmania amazonensis, elucidating possible microbicidal mechanisms and routes of death involved. Larval ES was able to inhibit the viability of L. amazonensis at all concentrations, induce morphological and ultrastructural changes in the parasite, retraction of the cell body, roughness of the cytoplasmic membrane, leakage of intracellular content, ROS production increase, induction of membrane depolarization and mitochondrial swelling, the formation of cytoplasmic lipid droplets and phosphatidylserine exposure, thus indicating the possibility of apoptosis-like death. To verify the efficacy of larval ES on amastigote forms, we performed a phagocytic assay, measurement of total ROS and NO. Treatment using larval ES reduced the percentage of infection and the number of amastigotes per macrophage of lineage J774A.1 at all concentrations, increasing the production of ROS and TNF-α, thus indicating possible pro-inflammatory immunomodulation and oxidative damage. Therefore, treatment using larval ES is effective at inducing the death of promastigotes and amastigotes of L. amazonensis even at low concentrations.

The Roles of Cytoplasmic Lipid Droplets in Modulating Intestinal Uptake of Dietary Fat.

Dietary fat absorption is required for health but also contributes to hyperlipidemia and metabolic disease when dysregulated. One step in the process of dietary fat absorption is the formation of cytoplasmic lipid droplets (CLDs) in small intestinal enterocytes; these CLDs serve as dynamic triacylglycerol storage organelles that influence the rate at which dietary fat is absorbed. Recent studies have uncovered novel factors regulating enterocyte CLD metabolism that in turn influence the absorption of dietary fat. These include peroxisome proliferator-activated receptor α activation, compartmentalization of different lipid pools, the gut microbiome, liver X receptor and farnesoid X receptor activation, obesity, and physiological factors stimulating CLD mobilization. Understanding how enterocyte CLD metabolism is regulated is key in modulating the absorption of dietary fat in the prevention of hyperlipidemia and its associated metabolic disorders.

EEF1D facilitates milk lipid synthesis by regulation of PI3K-Akt signaling in mammals.

Mammal's milk is an abundantly foremost source of proteins, lipids, and micronutrients for human nutrition and health. Understanding the molecular mechanisms underlying synthesis of milk components provides practical benefits to improve the milk quality via systematic breeding program in mammals. Through RNAi with EEF1D in primary bovine mammary epithelial cells, we phenotypically observed aberrant formation of cytoplasmic lipid droplets and significantly decreased milk triglyceride level by 37.7%, and exploited the mechanisms by which EEF1D regulated milk lipid synthesis via insulin (PI3K-Akt), AMPK, and PPAR pathways. In the EEF1D CRISPR/Cas9 knockout mice, incompletely developed mammary glands at 9th day postpartum with small or unformed lumens, and significantly decreased triglyceride concentration in milk by 23.4% were observed, as well as the same gene expression alterations in the three pathways. For dairy cattle, we identified a critical regulatory mutation modifying EEF1D transcription activity, which interpreted 7% of the genetic variances of milk lipid yield and percentage. Our findings highlight the significance of EEF1D in mammary gland development and milk lipid synthesis in mammals.

Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB

For many years, scientists have been looking for specific biomarkers associated with cancer cells for diagnosis purposes. These biomarkers mainly consist of proteins located at the cell surface (e.g. the TrkB receptor) whose activation is associated with specific metabolic modifications. Identification of these metabolic changes usually requires cell fixation and specific dye staining. MCARS microspectroscopy is a label-free, non-toxic, and minimally invasive method allowing to perform analyses of live cells and tissues. We used this method to follow the formation of lipid droplets in three colorectal cancer cell lines expressing TrkB. MCARS images of cells generated from signal integration of CH2 stretching modes allow to discriminate between lipid accumulation in the endoplasmic reticulum and the formation of cytoplasmic lipid droplets. We found that the number of the latter was related to the TrkB expression level. This result was confirmed thanks to the creation of a HEK cell line which over-expresses TrkB. We demonstrated that BDNF-induced TrkB activation leads to the formation of cytoplasmic lipid droplets, which can be abolished by K252a, an inhibitor of TrkB. So, MCARS microspectroscopy proved useful in characterizing cancer cells displaying an aberrant lipid metabolism.

HADHA overexpression disrupts lipid metabolism and inhibits tumor growth in clear cell renal cell carcinoma

Hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) is a key lipid metabolic enzyme with a novel role in carcinogenesis. We previously reported that HADHA, a prognostic marker, was downregulated in clear cell renal cell carcinoma (ccRCC). Herein, the tumor inhibitory role of HADHA overexpression in ccRCC was investigated further. The quantitative proteomic analysis displayed that a total of 1293 and 1293 proteins were identified in HADHA overexpressed 786-O-hadha and vector-transfected control 786-O-vc cells, respectively, and 206 proteins were found to be up- or downregulated. PANTHER, OmicsNet, STRING, and DAVID tools were utilized on the dysregulated proteins in order to elucidate multiple metabolic pathways (especial lipid metabolism) and lipid metabolism-related proteins (e.g. ACAT1, ACLY). The dysregulation of the lipid metabolic enzymes, ACAT1, ACLY, CYB5R3 and FASN, were confirmed by Western blotting. Further assays demonstrated that HADHA overexpression significantly inhibited cell growth, induced cell apoptosis, and decreased the formation of cytoplasmic lipid droplets (LDs); moreover, it also inhibited tumor growth and lessened the formation of LDs in xenografted mouse. Collectively, these data revealed that HADHA overexpression disrupted lipid metabolism and inhibited tumor growth, which shed light on HADHA as a potential therapeutic target for clinical intervention of ccRCC.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

Metabolic control of the scaffold protein TKS5 in tissue-invasive, proinflammatory T cells.

Pathogenic T cells in rheumatoid arthritis (RA) infiltrate non-lymphoid tissue sites, maneuver through extra-cellular matrix and form lasting inflammatory microstructures. Here, we found that RA T cells abundantly express the podosome scaffolding protein TKS5, enabling them to form tissue-invasive membrane structures. TKS5 overexpression was regulated by the intracellular metabolic environment of RA T cells; specifically, reduced glycolytic flux resulting in ATP and pyruvate deficiencies. ATPlo, pyruvatelo conditions triggered fatty acid biosynthesis and the formation of cytoplasmic lipid droplets. Restoring pyruvate production or inhibiting fatty acid synthesis corrected the tissue-invasiveness of RA T cells in vivo and reversed their pro-arthritogenic behavior. Thus, metabolic control of T cell locomotion provides new opportunities to interfere with T cell invasion into specific tissue sites.

Relationships between milks differentiated on native milk fat globule characteristics and fat, protein and calcium compositions

Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a threshold but mechanisms are still unknown.

Cardiac mesenchymal progenitors differentiate into adipocytes via Klf4 and c-Myc.

Direct reprogramming of differentiated cells to pluripotent stem cells has great potential to improve our understanding of developmental biology and disorders such as cancers, and has implications for regenerative medicine. In general, the effects of transcription factors (TFs) that are transduced into cells can be influenced by pre-existing transcriptional networks and epigenetic modifications. However, previous work has identified four key TFs, Oct4, Sox2, Klf4 and c-Myc, which can reprogram various differentiated cells to generate induced pluripotent stem cells. Here, we show that in the heart, the transduction of cardiac mesenchymal progenitors (CMPs) with Klf4 and c-Myc (KM) was sufficient to drive the differentiation of these cells into adipocytes without the use of adipogenic stimulation cocktail, that is, insulin, 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone. KM-transduced CMPs exhibited a gradually increased expression of adipogenic-related genes, such as C/Ebpα, Pparγ and Fabp4, activation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway, inactivation of the cell cycle-related pathway and formation of cytoplasmic lipid droplets within 10 days. In contrast, NIH3T3 fibroblasts, 3T3-L1 preadipocytes, and bone marrow-derived mesenchymal stem cells transduced with KM did not differentiate into adipocytes. Both in vitro and in vivo cardiac ischemia reperfusion injury models demonstrated that the expression of KM genes sharply increased following a reperfusion insult. These results suggest that ectopic adipose tissue formation in the heart following myocardial infarction results from CMPs that express KM following a stress response.

Lipogenic Enzymes Complexes and Cytoplasmic Lipid Droplet Formation During Adipogenesis.

Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.

Potential anti-tumor effects of Mugil cephalus processed roe extracts on colon cancer cells

The salted-semidried mullet ovary product, bottarga, is a Mediterranean food rich in n-3 PUFA EPA and DHA. We studied and compared the effects on cell viability, sensitivity to the anti-tumor drug 5-fluorouracil, and lipid composition, in colon cancer Caco-2 cells after 24 h incubation with oils and hydrophilic extracts obtained from two bottarga samples stored at different conditions. The cellular absorption of bottarga lipids was assessed in cancer cells by the evaluation of lipid accumulation in cytoplasmic lipid droplets by fluorescence microscopy. Bottarga oil showed a significant in vitro inhibitory effect on the growth of cancer Caco-2 cells and the ability to potentiate, at non-toxic concentration, the growth inhibitory effect of 5-fluorouracil. Moreover, bottarga oil induced in cancer Caco-2 cells marked changes in fatty acid composition, with a significant accumulation of the n-3 PUFA EPA and DHA, and cytoplasmic lipid droplet formation. Also bottarga hydrophilic extract, characterized by means of ¹H NMR spectroscopy, exhibited a reduction in cancer cell viability, without affecting cell lipid profile. Cell cholesterol levels were unmodified by all treatments. The results showed interesting anti-tumor properties of bottarga lipids, and qualify this fish product as a food with nutraceutical properties and potential benefits in colon cancer prevention.

Lipid droplet formation on opposing sides of the endoplasmic reticulum

In animal cells, the primary repositories of esterified fatty acids and alcohols (neutral lipids) are lipid droplets that form on the lumenal and/or cytoplasmic side of the endoplasmic reticulum (ER) membrane. A monolayer of amphipathic lipids, intermeshed with key proteins, serves to solubilize neutral lipids as they are synthesized and desorbed. In specialized cells, mobilization of the lipid cargo for delivery to other tissues occurs by secretion of lipoproteins into the plasma compartment. Serum lipoprotein assembly requires an obligate structural protein anchor (apolipoprotein B) and a dedicated chaperone, microsomal triglyceride transfer protein. By contrast, lipid droplets that form on the cytoplasmic face of the ER lack an obligate protein scaffold or any required chaperone/lipid transfer protein. Mobilization of neutral lipids from the cytosol requires regulated hydrolysis followed by transfer of the products to different organelles or export from cells. Several proteins play a key role in controlling droplet number, stability, and catabolism; however, it is our premise that their formation initiates spontaneously, solely as a consequence of neutral lipid synthesis. This default pathway directs droplets into the cytoplasm where they accumulate in many lipid disorders.

Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation

A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid β-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species.

Abstract B69: Cellular neutral lipid content is a novel biomarker of cancer preventive activity

Abstract Introduction: Commonly used biomarkers to assess the cancer preventive activity of cancer preventive agents include markers of cell proliferation or apoptosis. However, the expression of proliferation or apoptosis markers is highly variable, and measurement is often done using semi-quantitative assays. In addition, cancer preventive agents may act through mechanisms that do not depend on proliferation or apoptosis. Therefore, identification of new biomarkers indicative of the cancer preventive activity of chemopreventive agents is essential for the development of new cancer preventive drugs. Methods: We applied high-content microscopic analysis to assess the phenotypic changes occurring in normal mammary epithelial cells (HMECs) after treatment with cancer preventive drugs by measuring cell proliferation, DNA and neutral lipid content and localization, as well as cell cycle distribution of the cells. Gene specific knock-down was achieved by transfections with siRNA duplexes. Measurement of transcript levels was performed using quantitative real-time RT-PCR. Results: Multiparametric analysis of high-throughput image based studies have revealed that anti-proliferative activity of cancer preventive rexinoids is associated with increased formation of cytoplasmic lipid droplets. Accumulation of cytoplasmic neutral lipids was restricted to cell types that undergo significant growth suppression by bexarotene (HMECs, SUM225, and ZR75.1), but not observed in retinoid-resistant cells (MCF10A, HCC1937, and HCC1143). Other growth suppressive drugs including the kinase inhibitor lapatinib, vitamin D, and certain natural compounds, including epicatechine monogallate, duartin, actinonin and homoisoflavonoids, were also found to increase cellular neutral lipid content in breast cells. The addition of external fatty acids, such as oleic and palmitoleic acids did not result in dose-dependent growth suppression or accumulation of neutral esterified lipid droplets. Lipid accumulation and growth suppression were associated with the up-regulation of genes involved in regulating the formation of neutral lipids, triglycerides and phospholipids, but not genes involved in de novo fatty acid synthesis. Conversely, knock-down of key proteins controlling cellular triglyceride synthesis and storage, diacylglycerol acyltransferase 1 (DGAT1) and adipophyllin (Adfp) increased cell proliferation whereas their induction was associated with growth suppression in HMECs. Conclusion: These results suggest that increased accumulation of cellular triglycerides may be a useful biomarker to assess the preventive effect of putative cancer preventive agents. These results support the systematic testing of cellular neutral lipid levels in preclinical and clinical models testing novel chemopreventive agents. This work was supported by the grant RO1 CA 78480 (to P.H.B.). Citation Information: Cancer Prev Res 2011;4(10 Suppl):B69.

Adipophilin regulates maturation of cytoplasmic lipid droplets and alveolae in differentiating mammary glands

Milk lipids originate by secretion of triglyceride-rich cytoplasmic lipid droplets (CLDs) from mammary epithelial cells. Adipophilin (ADPH)/Plin2, a member of the perilipin family of CLD binding proteins, is hypothesized to regulate CLD production in these cells during differentiation of the mammary gland into a secretory organ. We tested this hypothesis by comparing CLD accumulation in differentiating mammary glands of wild-type and ADPH-deficient mice. ADPH deficiency did not prevent CLD formation; however, it disrupted the increase in CLD size that normally occurs in differentiating mammary epithelial cells. Failure to form large CLDs in ADPH-deficient mice correlated with localization of adipose triglyceride lipase (ATGL) to the CLD surface, suggesting that ADPH promotes CLD growth by inhibiting lipolytic activity. Significantly, mammary alveoli also failed to mature in ADPH-deficient mice, and pups born to these mice failed to survive. The possibility that CLD accumulation and alveolar maturation defects in ADPH-deficient mice are functionally related was tested by in vivo rescue experiments. Transduction of mammary glands of pregnant ADPH-deficient mice with adenovirus encoding ADPH as an N-terminal GFP fusion protein prevented ATGL from localizing to CLDs and rescued CLD size and alveolar maturation defects. Collectively, these data provide direct in vivo evidence that ADPH inhibition of ATGL-dependent lipolysis is required for normal CLD accumulation and alveolar maturation during mammary gland differentiation. We speculate that impairing CLD accumulation interferes with alveolar maturation and lactation by disrupting triglyceride homeostasis in mammary epithelial cells.

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.

Modulation of lipid droplet size and lipid droplet proteins by trans-10, cis-12 conjugated linoleic acid parallels improvements in hepatic steatosis in obese, insulin-resistant rats

The isomer-specific effects of conjugated linoleic acid (CLA) on hepatic steatosis were assessed in fa/fa Zucker rats, a model for insulin resistance and the metabolic syndrome. Eight weeks of feeding trans-10, cis-12 CLA significantly improved glucose tolerance without changing body weight or visceral adipose mass. The trans-10, cis-12 isomer was also associated with reduced liver lipid content, improved liver function and reduced inflammation; these effects were not observed in rats fed the cis-9, trans-11 CLA isomer. Reduced liver lipid content did not correlate with activation of AMP-activated protein kinase or suppressed activation of sterol-regulatory element binding protein-1, two key regulators of hepatic lipid metabolism. Interestingly, rats fed cis-9, trans-11 CLA had fewer cytoplasmic lipid droplets in hepatocytes compared to rats fed control diet, but these droplets were larger in size. Conversely, fa/fa rats fed the trans-10, cis-12 CLA isomer had greater numbers of hepatic lipid droplets that were smaller in size, resulting in overall lower total lipid within these droplets. Changes in lipid droplets were associated with lower hepatic levels of PERILIPIN-2 (formerly known as adipophilin) in rats fed trans-10, cis-12 CLA, whereas amounts of other members of the PERILIPIN family of lipid droplet proteins were unaffected by dietary CLA. However, CLA isomers differentially affected the subcellular localization of these proteins. Treatment of H4IIE rat hepatoma cells with CLA isomers neither prevented nor reversed, but rather induced cytoplasmic lipid droplet formation, suggesting that the anti-steatotic effects of trans-10, cis-12 CLA are likely indirect and potentially mediated via increased lipid utilization by peripheral tissues.

Albumin expression is required for adipocyte differentiation of 3T3-L1 cells

We have previously demonstrated that albumin is directly involved in the formation of cytoplasmic lipid droplets in pancreatic stellate cells and may act as a downstream effector of adipogenic transcription factors, PPAR-γ and C/EBP-α. Here, we investigated the role of albumin in adipocyte differentiation using 3T3-L1 cells. Albumin expression was significantly increased at later stages of adipocyte differentiation, which was accompanied with increased C/EBP-β binding to albumin promoter. Suppression of albumin expression using short-hairpin RNA (shRNA) during differentiation led to a considerable reduction in lipid droplet formation, whereas albumin overexpression was stimulatory. Furthermore, point mutation in its fatty acid-binding sites inhibited lipid droplet formation. Consistent with these in vitro finding, Nagase analbuminemic rats displayed reduced fat accumulation. Therefore, our findings suggest that albumin may play a distinct role in adipocyte differentiation by promoting lipid accumulation.

Metformin regulates osteoblast and adipocyte differentiation of rat mesenchymal stem cells

Abstract Metformin is widely used for the treatment of type 2 diabetes mellitus. In this study, we evaluated the effects of metformin on the osteoblast and adipocyte differentiation of rat marrow mesenchymal stem cells (MSCs) in culture. Treatment of MSCs in osteoblastic or adipogenic medium with 100 μM metformin for 21 days led to an increased mRNA expression of the osteoblast markers but a decreased mRNA expression of the adipocyte markers in the MSCs. Metformin markedly stimulated deposition of mineralized nodules and blocked the formation of cytoplasmic lipid droplets. In addition, alkaline phosphate activity and Western blot analysis for core binding factor a1 (Cbfa 1) and peroxisome proliferator-activated receptor gamma 2 (PPARγ2) proteins also confirmed that metformin inhibited adipocyte differentiation and promoted osteoblast differentiation. The reciprocal relationship between osteoblastic and adipogenic differentiation suggests that metformin may regulate osteoblastic and adipogenic differentiation through inhibition of PPARγ.