Targeted delivery of CRISPR interference system against Fabp4 to white adipocytes ameliorates obesity, inflammation, hepatic steatosis, and insulin resistance.

Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.

Identification of biomarkers, pathways and potential therapeutic agents for white adipocyte insulin resistance using bioinformatics analysis.

For the better understanding of insulin resistance (IR), the molecular biomarkers in IR white adipocytes and its potential mechanism, we downloaded two mRNA expression profiles from Gene Expression Omnibus (GEO). The white adipocyte samples in two databases were collected from the human omental adipose tissue of IR obese (IRO) subjects and insulin-sensitive obese (ISO) subjects, respectively. We identified 86 differentially expressed genes (DEGs) between the IRO and ISO subjects using limma package in R software. Gene Set Enrichment Analysis (GSEA) provided evidence that the most gene sets enriched in kidney mesenchyme development in the ISO subjects, as compared with the IRO subjects. The Gene Ontology (GO) analysis indicated that the most significantly enriched in cellular response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the DEGs were most significantly enriched in cytokine-cytokine receptor interaction. Protein-Protein Interaction (PPI) network was performed with the STRING, and the top 10 hub genes were identified with the Cytohubba. CMap analysis found several small molecular compounds to reverse the altered DEGs, including dropropizine, aceclofenac, melatonin, and so on. Our outputs could empower the novel potential targets to treat omental white adipocyte insulin resistance, diabetes, and diabetes-related diseases.

Fat Tissue Growth and Development in Humans.

Lipid storage and release from fat cells in adipose tissue are key factors in the regulation of the energy balance. During infancy and adolescence, adipose tissue is growing by a combination of increase in fat cell size (to a lesser extent) and (above all) the number of these cells. In adults, fat cell number is constant over time in spite of a large turnover (about 10% of the fat cells per year) when body weight is stable. A decrease in body weight only changes fat cell size (becoming smaller), whereas an increase in body weight causes elevation of both fat cell size and number in adults. An important source of renewal of fat cells during the entire life span is the bone marrow. This is most apparent in obesity when ∼20% of all fat cells are derived from the bone marrow. Fat cell turnover is also important for the size of fat cells. Low turnover may cause large fat cells which, in turn, is linked to cardiovascular disease and type 2 diabetes. There is also a rapid turnover of fat cell lipids, which constitute a single active pool and are renewed about 6 times during the life span of individual fat cells. Overweight and obesity are associated with decreased lipid turnover due to high input in combination with low output of lipids from the fat cells. Low fat cell lipid turnover is associated with insulin resistance and dyslipidemia. Thus, changes in the turnover of fat cells and their lipid content are important for the development of adipose tissue mass and its cellularity (fat cell size and number) and, in turn, for metabolic disturbances.

Proteomic differences between white and brown adipocytes.

Although a series of protein levels from several protein pathways have been shown to differ between white (WA) and brown (BA) adipocytes, proteomic work on this subject with the exception of mitochondrial protein differences is limited. It was, therefore, the aim of the study to compare WA with BA soluble protein levels. Proteins were extracted from WA and BA and the soluble fraction was run on two-dimensional gel electrophoresis. Quantification of spot volume was carried out and protein spots, statistically different between groups (P < 0.01), were in-gel digested with trypsin and peptides were identified using nano-LC-ESI-MS/MS in the CID and ETD mode. Differences between selected proteins were evaluated by immunoblotting. A network was generated using the ingenuity pathway analysis. Five proteins, protein DJ-1, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase subunit alpha, electron transfer flavoprotein subunit alpha and immunoglobulin-binding protein 1, were increased in BA based on a gel-based proteomic method and differential expression was verified by immunoblotting. These individual proteins were represented by one spot each and sequence coverages were between 28 and 65%. A network generated based on these results indicated a link to ubiquitination. Differential protein levels between WA and BA allow interpretation of previous work on adipocyte biochemistry and form the basis for future studies with genetic or pharmacological inhibition of these proteins accompanied by work on phenotype and adipocyte function.

Lipid droplets: size matters.

The lipid droplet (LD), an organelle that exists ubiquitously in various organisms, from bacteria to mammals, has attracted much attention from both medical and cell biology fields. The LD in white adipocytes is often treated as the prototype LD, but is rather a special example, considering that its size, intracellular localization and molecular composition are vastly different from those of non-adipocyte LDs. These differences confer distinct properties on adipocyte and non-adipocyte LDs. In this article, we address the current understanding of LDs by discussing the differences between adipocyte and non-adipocyte LDs.

Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS.

Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to (1) comprehensively characterize the human adipocyte proteome for the first time and (2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and high performance liquid chromatography-electron spray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from approximately 0.5 g of subcutaneous abdominal adipose tissue of three healthy, lean subjects, we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis, and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases.

n-3 PUFA-enriched diet delays the occurrence of cancer cachexia in rat with peritoneal carcinosis.

The aim of this study was to evaluate the effects of a fish oil (FO) diet (rich in long chain, n-3, polyunsaturated fatty acid) on cancer cachexia symptoms in rats. To this end, peritoneal carcinosis (PC) was generated by an intraperitoneal injection of cancer cells in BDIX rats fed FO or standard (Std) diets. Food intake and body weight were recorded throughout the study until sacrifice. PC rats were sacrificed when food intake was significantly and severely reduced. Fat and skeletal muscles masses were weighed and serum inflammatory cytokines concentration measured at sacrifice. Occurrence of anorexia in PC rats was delayed in the FO diet group (median time was multiplied by 2.5) in comparison with Std diet. At the time of sacrifice, PC rats displayed a lower body weight gain as well as lower muscle and fat masses than pair-fed rats, suggesting the presence of a hypermetabolism state. Serum TNF-alpha was significantly increased in PC rats compared with controls rats. There was no effect of FO diet on tissue mass (skeletal muscle and fat) or on TNF-alpha concentration. In conclusion, FO diet delays the appearance of anorexia induced by PC in rats.

Global analysis of triacylglycerols including oxidized molecular species by reverse-phase high resolution LC/ESI-QTOF MS/MS.

Recently, global analysis of triacylglycerols (TAGs) has become increasingly important in studies of abnormality of lipid metabolism in metabolic syndrome. TAGs consist of various molecular species, caused by their three fatty acyl chains with a large variety of carbon chain lengths and degrees of unsaturation. Therefore, most previously reported methods have been insufficient in global detection of TAGs including their structural isomers and TAGs with oxidized or odd number acyl carbon chain. Here we report an effective method for global analysis of TAG molecular species from complex lipid mixtures of mouse liver and white adipose tissue (WAT) using reverse-phased high resolution liquid chromatography (LC) coupled with electrospray ionization (ESI)-quadrapole/time of flight hybrid mass spectrometer (QTOF-MS). For effective profiling of TAG molecular species, sensitive two-dimensional (2D) maps were constructed and individual structures were correctly identified by the elution profile and MS/MS. As a result, TAGs including their structural isomers and TAGs with an odd number acyl carbon chain were separated and detected effectively on the 2D map as compared with conventional high performance LC. It was also found that our 2D profiling method was useful in searching characteristic molecular species globally. In mouse WAT, novel oxidized TAGs, which were mainly formed by hydroperoxidation of one of their linoleic acyl chains, were effectively detected in comparison with TAG molecular species of mouse liver.

Distribution and regulation of protein expression of the free fatty acid receptor GPR120.

GPR120 is a G-protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids. It has been implicated as playing an important role in the control of lipid and glucose metabolism by regulating the secretion of glucagon-like peptide-1 and cholecystokinin. We have developed an antibody against the extracellular domain of GPR120. The specificity of the antibody was demonstrated by immunoprecipitation, Western blotting, flow cytometry, and immunocytochemistry using GPR120-transfected cells. Immunoreactivity for GPR120 was abundant in the mouse large intestine, lung, and adipose tissue. Furthermore, we found that the expression of GPR120 protein was up-regulated during the adipogenic differentiation of 3T3-L1 cells, which corresponded well with changes in mRNA expression. The anti-GPR120 antibody will be of value for the further study of the function of this nutrient-sensing receptor.

Acute and chronic regulation of leptin synthesis, storage, and secretion by insulin and dexamethasone in human adipose tissue.

Serum leptin levels are upregulated in proportion to body fat and also increase over the short term in response to meals or insulin. To understand the mechanisms involved, we assessed leptin synthesis and secretion in samples of adipose tissue from subjects with a wide range of BMI. Tissue leptin content and relative rates of leptin biosynthesis, as determined by metabolic labeling, were highly correlated with each other and with BMI and fat cell size. To understand mechanisms regulating leptin synthesis in obesity, we used biosynthetic labeling to directly assess the effects of insulin and glucocorticoids (dexamethasone) on leptin synthesis and secretion in human adipose tissue. Chronic treatment (1-2 days in organ culture) with insulin increased relative rates of leptin biosynthesis without affecting leptin mRNA levels. In contrast, dexamethasone increased leptin mRNA and biosynthesis in parallel. Acute treatment with insulin or dexamethasone (added during 1-h preincubation and 45-min pulse labeling) did not affect relative rates of leptin biosynthesis, but pulse-chase studies showed that addition of insulin nearly doubled the release of [35S]leptin after a 1-h chase. We conclude that the higher leptin stores in adipose tissue of obese humans are maintained by chronic effects of insulin and glucocorticoids acting at pre- and posttranslational levels and that the ability of insulin to increase the release of preformed leptin may contribute to short-term variations in circulating leptin levels.

A growth hormone-releasing peptide promotes mitochondrial biogenesis and a fat burning-like phenotype through scavenger receptor CD36 in white adipocytes.

Whereas the uptake of oxidized lipoproteins by scavenger receptor CD36 in macrophages has been associated with foam cell formation and atherogenesis, little is known about the role of CD36 in regulating lipid metabolism in adipocytes. Here we report that treatment of 3T3-L1 adipocytes with hexarelin, a GH-releasing peptide that interacts with CD36, resulted in a depletion of intracellular lipid content with no significant change in CD36 expression. Microarray analysis revealed an increased pattern in several genes involved in fatty acid mobilization toward the mitochondrial oxidative phosphorylation process in response to hexarelin. Interestingly, many of these up-regulated genes are known targets of peroxisomal proliferator-activated receptor (PPAR)-gamma, such as FATP, CPT-1, and F(1)-ATPase, suggesting that adipocyte response to hexarelin may involve PPARgamma activation. Expression studies also indicate an increase in thermogenic markers PPARgamma coactivator 1alpha and uncoupling protein-1, which are normally expressed in brown adipocytes. Electron microscopy of hexarelin-treated 3T3-L1 adipocytes showed an intense and highly organized cristae formation that spans the entire width of mitochondria, compared with untreated cells, and cytochrome c oxidase activity was enhanced by hexarelin, two features characteristic of highly oxidative tissues. A similar mitochondrial phenotype was detected in epididymal white fat of mice treated with hexarelin, along with an increased expression of thermogenic markers that was lost in treated CD36-null mice, suggesting that the ability of hexarelin to promote a brown fat-like phenotype also occurs in vivo and is dependent on CD36. These results provide a potential role for CD36 to impact the overall metabolic activity of fat usage and mitochondrial biogenesis in adipocytes.