Adipose Tissue Protein Research Articles

Effects of insulin on pigment epithelium-derived factor expression in adipocytes of type 2 diabetic mellitus in rats

To explore the effects of insulin therapy on the expression of pigment epithelium-derived factor (PEDF) in adipocytes of type 2 diabetic mellitus (T2DM) in rats. A total of 22 newly diagnosed type 2 diabetics received a 2-week intensive insulin therapy. The levels of fasting plasma glucose (FPG), serum triglyceride and PEDF were measured before and after therapy. T2DM was induced by a high-fat diet and a low-dose streptozotocin (STZ). The Spraque-Dawley rats were divided randomly into diabetic, insulin treatment and gliclazide treatment groups. Another group with a chow diet was designated as normal controls. Differentiated 3T3-L1 adipocytes were then incubated with tumor necrosis factor-alpha (TNF-α) and (or) insulin for 24 h. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of PEDF in adipose tissue or adipocytes. The PEDF levels in both sera and cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Glucose uptake was detected after treatment of PEDF or anti-PEDF antibody simultaneously together with insulin in mature 3T3-L1 adipocytes. Insulin therapy decreased the serum levels of FPG and triglyceride of T2DM patients ((12.9 ± 2.8) vs (5.9 ± 1.4) mmol/L, (3.1 ± 1.8) vs (1.7 ± 0.8) mmol/L, P < 0.05) while the serum level of PEDF decreased significantly after therapy ((22.85 ± 5.73) vs (18.38 ± 5.28) µg/L, P < 0.05). Consistently the serum level of PEDF of diabetic rats was remarkably higher than that of normal controls and insulin-treated group ((28.6 ± 0.5) vs (25.4 ± 0.6) and (25.3 ± 0.6) µg/L, P < 0.05). And the elevated levels of PEDF, TNF-α mRNA and protein in adipose tissue (P < 0.05) could be reduced by insulin treatment (P < 0.05). However, no obvious change was detected in gliclazide treatment group. Further evidences suggested that TNF-α could induce more secretion and expression of PEDF in 3T3-L1 adipocyte while this effect became ameliorated by insulin treatment. Furthermore, decreased capacity of glucose uptake by PEDF might be reversed by anti-PEDF antibody in 3T3-L1 adipocytes (P < 0.05). Insulin can down-regulate the expression of PEDF in adipocytes of T2DM and improve the glucose uptake of adipocytes. It may be one of the mechanisms through which insulin therapy improves peripheral insulin resistance.

Inorganic nitrate promotes the browning of white adipose tissue through the nitrate-nitrite-nitric oxide pathway.

Inorganic nitrate was once considered an oxidation end product of nitric oxide metabolism with little biological activity. However, recent studies have demonstrated that dietary nitrate can modulate mitochondrial function in man and is effective in reversing features of the metabolic syndrome in mice. Using a combined histological, metabolomics, and transcriptional and protein analysis approach, we mechanistically defined that nitrate not only increases the expression of thermogenic genes in brown adipose tissue but also induces the expression of brown adipocyte-specific genes and proteins in white adipose tissue, substantially increasing oxygen consumption and fatty acid β-oxidation in adipocytes. Nitrate induces these phenotypic changes through a mechanism distinct from known physiological small molecule activators of browning, the recently identified nitrate-nitrite-nitric oxide pathway. The nitrate-induced browning effect was enhanced in hypoxia, a serious comorbidity affecting white adipose tissue in obese individuals, and corrected impaired brown adipocyte-specific gene expression in white adipose tissue in a murine model of obesity. Because resulting beige/brite cells exhibit antiobesity and antidiabetic effects, nitrate may be an effective means of inducing the browning response in adipose tissue to treat the metabolic syndrome.

Abstract 256: AMP-activated Protein Kinase Activator AICAR Attenuates TNFα-induced Inflammation In Murine Adipocytes.

Adipose tissue, a metabolically active tissue and important regulator of vascular function, contributes to obesity-related cardiovascular diseases such as hypertension. Molecular mechanisms are still elusive, but inflammation and activation of a local renin-angiotensin-aldosterone system (RAAS) seem to play an important role in adipocyte dysfunction leading to vascular damage. AMP-activated protein kinase (AMPK), a key regulator of cell metabolism, may play an important role in adipose tissue biology/inflammation. In this study, we evaluated whether AMPK activation regulates TNFα-induced inflammation and RAAS status in adipocytes. Murine adipocytes (3T3-L1) were used and treated with 10ng/ml TNFα in the absence/presence of AICAR (1mM - AMPK activator). Pro-inflammatory markers were assessed by qPCR or ELISA and protein expression by immunoblotting. TNFα stimulation increased IL-6 (113.4 fold) and MCP-1 (449.6 fold) mRNA levels, as well as IL-6 release (17.67 fold) into culture medium (p&lt;0.05, vs vehicle); an effect abrogated by AICAR (75% reduction MCP-1 mRNA, 60% reduction IL-6 mRNA and 30% reduction IL-6 release). Adiponectin mRNA levels were decreased by TNFα (60%, p&lt;0.05 vs vehicle), yet preincubation with AICAR had no effect. The effects of TNFα were also evaluated on components of the RAAS. TNFα induced a decrease in angiotensinogen (60%), mineralocorticoid receptor (70%) and AT2R (90%) mRNA levels in adipocytes (p&lt;0.05), which was unaffected by AICAR. Activation of AMPK also suppressed TNFα-stimulated phosphorylation of MAP kinases and NFκB assessed by immunoblotting. In conclusion, AMPK activation seems to play a role in the control of inflammation and RAAS in adipocytes. Our data highlights the importance of AMPK as an important anti-inflammatory signaling protein in adipose tissue and may be an interesting target for treatment of obesity-related vascular injury leading to hypertension. This is especially significant given that the commonly used drug metformin is an AMPK inhibitor.

Regulation of CD163 mRNA and soluble CD163 protein in human adipose tissue in vitro.

CD163-positive macrophages are highly expressed in the human adipose tissue (AT) particularly from obese individuals. However, little is known about the regulation of CD163 mRNA and the protein level of sCD163 in human AT. We aimed to examine the regulation of CD163 and sCD163 in AT. Human s.c. AT samples (n=5) were stimulated with dexamethasone (DEX; 200 nmol/l), lipopolysaccharide (LPS; 100 ng/ml), or DEX+LPS for various time periods up to 24 h. Gene expressions of CD163, ADAM17, IL10, and TNFA (TNF) were measured by RT-PCR. Protein levels of sCD163, IL10, and TNFα (TNF) were measured by ELISA. Furthermore, AT was separated into stromal and adipocyte fraction. We found that CD163 mRNA was strongly expressed in the stromal vascular fraction but hardly detectable in the isolated adipocytes. Incubating whole AT with DEX significantly up-regulated CD163 (P<0.001), whereas incubation with LPS had no effects on CD163 (P>0.05). By contrast, the protein level of sCD163 was not affected by DEX (P>0.05), but LPS significantly increased the level of sCD163 and TNFα (P<0.05). This might be due to the concomitant LPS stimulation of ADAM17, which is known to mediate shedding of the extracellular domains of sCD163 and TNFα. Finally, DEX significantly reduced the LPS-induced TNFα release to the incubation medium but had no effects on sCD163. We conclude that the expression of CD163 and the release of sCD163 are differentially regulated in human AT. Moreover, similar to studies on differentiated blood monocytes, TNFα and sCD163 are concomitantly released in human AT by LPS, which also up-regulate ADAM17.

Circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in metabolic syndrome.

Progranulin (PGRN) is a secreted protein that has recently emerged as an important regulatory adipokine of glucose metabolism and insulin sensitivity. We report here that serum PGRN concentrations were significantly higher in patients with metabolic syndrome (MS) than in subjects without MS and correlated positively with body mass index, waist circumference, fasting insulin, fasting plasma glucose, glycated hemoglobin A1c, triglyceride, and homeostasis model assessment of insulin resistance, and were inversely related to high-density lipoprotein cholesterol and homeostasis model assessment of β cell function. Subgroup analysis in 32 subjects showed that elevated expression levels of PGRN were positively correlated with increased autophagy markers LC3 and Atg7 proteins in omental adipose tissue of subjects with MS. Consistent with these findings, the enhanced PGRN levels were also observed in multiple insulin-resistant cellular models, whereas PGRN-deficient adipocytes were more susceptible to insulin action and refractory to tunicamycin-induced autophagic disorders. PGRN remarkably attenuated insulin sensitivity, increased autophagic activity, and triggered endoplasmic reticulum (ER) stress in cultured human adipocytes, whereas these effects were nullified by reduction of ER stress with phenylbutyric acid chemical chaperone treatment. In addition, PGRN-induced ER stress and impaired insulin sensitivity were improved in TNFR1(-/-) cells, indicating a causative role of TNF receptor in the action of PGRN. Collectively, our findings suggest that circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in adipose tissue and support the notion that PGRN functions as a potential link between chronic inflammation and insulin resistance.

Artemisia scoparia enhances adipocyte development and endocrine function in vitro and enhances insulin action in vivo.

BackgroundFailure of adipocytes to expand during periods of energy excess can result in undesirable metabolic consequences such as ectopic fat accumulation and insulin resistance. Blinded screening studies have indicated that Artemisia scoparia (SCO) extracts can enhance adipocyte differentiation and lipid accumulation in cultured adipocytes. The present study tested the hypothesis that SCO treatment modulates fat cell development and function in vitro and insulin sensitivity in adipose tissue in vivo.Methods In vitro experiments utilized a Gal4-PPARγ ligand binding domain (LBD) fusion protein-luciferase reporter assay to examine PPARγ activation. To investigate the ability of SCO to modulate adipogenesis and mature fat cell function in 3T3-L1 cells, neutral lipid accumulation, gene expression, and protein secretion were measured by Oil Red O staining, qRT-PCR, and immunoblotting, respectively. For the in vivo experiments, diet-induced obese (DIO) C57BL/6J mice were fed a high-fat diet (HFD) or HFD containing 1% w/w SCO for four weeks. Body weight and composition, food intake, and fasting glucose and insulin levels were measured. Phospho-activation and expression of insulin-sensitizing proteins in epididymal adipose tissue (eWAT) were measured by immunoblotting.ResultsEthanolic extracts of A. scoparia significantly activated the PPARγ LBD and enhanced lipid accumulation in differentiating 3T3-L1 cells. SCO increased the transcription of several PPARγ target genes in differentiating 3T3-L1 cells and rescued the negative effects of tumor necrosis factor α on production and secretion of adiponectin and monocyte chemoattractant protein-1 in fully differentiated fat cells. DIO mice treated with SCO had elevated adiponectin levels and increased phosphorylation of AMPKα in eWAT when compared to control mice. In SCO-treated mice, these changes were also associated with decreased fasting insulin and glucose levels.ConclusionSCO has metabolically beneficial effects on adipocytes in vitro and adipose tissue in vivo, highlighting its potential as a metabolically favorable botanical supplement.

Increased body weight induced by perinatal exposure to bisphenol A was associated with down-regulation zinc-alpha2-glycoprotein expression in offspring female rats

We reported that perinatal exposure to bisphenol A (BPA) resulted in obesity in offspring female rats, and down-regulated zinc-alpha2-glycoprotein (ZAG) mRNA expression in subcutaneous adipose tissue maybe was associated with increased body weight in previous study. Considering that the mechanism of ZAG actions has not been clear, and ZAG gene expression levels are different with adipose tissues, we investigate the levels of ZAG mRNA and protein expression in visceral (perigonadal and perirenal) adipose tissue, and the mechanism of ZAG increasing body weight in BPA exposed rats. Pregnant rats were exposed to BPA in water at levels of either 1 μg/mL(low dose of BPA, LBPA) or 10 μg/mL (high dose of BPA, HBPA) from gestation day 6 to the end of lactation. ZAG mRNA and protein expression from visceral adipose tissue of offspring female rats on postnatal day 50 (PND50) were measured. The levels of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) gene expression, and peroxisome proliferator-activated receptors-γ (PPARγ) mRNA and protein in visceral adipose tissue were also determined. There was large size of adipocytes in perigonadal adipose tissues in BPA treated offspring female rats. Perinatal exposure to BPA could decrease the levels of ZAG and PPARγ mRNA and protein expression in visceral adipose tissues, and increase FAS gene expression, but decrease HSL gene expression. These findings indicated that BPA exposure during early development results in large size of adipocytes, which was associated with down-regulated ZAG gene. Low expression of ZAG gene increased lipogenesis and decreased lipolysis, which was associated with down-regulated PPARγ gene induced by BPA.

Quantitative secretome and glycome of primary human adipocytes during insulin resistance.

Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.

Decreased expression of genes encoding thermogenesis-related proteins in adipose tissues of obese patients is not associated with their methylation status

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Silencing of ClC‐3 protects against preadipocyte apoptosis in type 2 diabetes (1142.6)

OBJECTIVE: ClC‐3 chloride channel, plays an important role in the regulation of cell volume,proliferation and apoptosis. Preadipocytes of type 2 diabetes show a tendency to enhanced apoptosis. We hypothesized that there is a relation between ClC‐3 and insulin resistance, and one underlying mechanism may be associated with the modulation of free fatty acid (FFA) ‐induced preadipocyte apoptosis by ClC‐3.METHODS: Type 2 diabetes mellitus (T2DM) models in ClC‐3 KO mice and rats were induced by fed with high‐sucrose‐high‐fat diet combined with a low dose of streptozocin. The cellular apoptosis was detected by Annexin V‐PI staining.RESULTS: Compared with the wild‐type T2DM models, ClC‐3 knockout T2DM mice displayed an improvement in insulin resistance, a decrease in serum plasma free fatty acid (FFA) levels and preadipocyte apoptosis in epididymal fat pad, as evidenced by significant decrease in homeostatic model assessment of insulin resistance (HOMA‐IR) and flow cytometry. In T2DM rat models, we further found that ClC‐3 protein in adipose tissue is increased, and was positively correlated to HOMA‐IR, but was not the case in liver and skeletal muscle. We further found that palmitate‐induced preadipocyte apoptosis in vitro was parallel to a significant increase in the endogenous expression of ClC‐3 protein. Transfection with ClC‐3 siRNA decreased apoptosis and activation of caspase‐3 and 9, whereas increased ratio of Bcl‐2/Bax in palmitate‐treated preadipocytes. ClC‐3 affected apoptosis mainly through regulating ER stress signaling proteins such as Grp78, CHOP and ATF4.CONCLUSION: We concluded that chloride channel ClC‐3 modulates palmitate‐induced apoptosis in preadipocytes via the ER stress pathway. ClC‐3 in adipocyte tissue may be involved in the development of type 2 diabetes.Grant Funding Source: Supported by National Natural Science Foundation of China (No. 81370897,No. 813111115 and No. 812300

Artemisia scoparia enhances adipocyte development and endocrine function in vitro and enhances insulin action in vivo (LB771)

Background: Failure of adipocytes to expand during periods of energy excess can result in undesirable metabolic consequences. Blinded screening studies have indicated that Artemisia scoparia (SCO) extracts can enhance adipocyte differentiation and lipid accumulation in cultured adipocytes. The present study tested the hypothesis that SCO treatment modulates fat cell development and function in vitro and insulin sensitivity in adipose tissue in vivo.Methods: In vitro experiments utilized a Gal4‐PPAR[[Unsupported Character ‐ Symbol Font □]] ligand binding domain (LBD) fusion protein‐luciferase reporter assay to examine PPAR[[Unsupported Character ‐ Symbol Font □]] activation. Neutral lipid accumulation, gene expression, and protein secretion were measured by Oil Red O staining, qRT‐PCR, and immunoblotting, respectively. For the in vivo experiments, diet‐induced obese (DIO) C57BL/6J mice were fed a high‐fat diet (HFD) or HFD containing 1% w/w SCO for four weeks. Body weight and composition, food intake, and fasting glucose and insulin levels were measured. Phospho‐activation and expression of insulin‐sensitizing proteins in epididymal adipose tissue (eWAT) were measured by immunoblotting.Results: Ethanolic extracts of A. scoparia significantly activated the PPAR[[Unsupported Character ‐ Symbol Font □]] LBD and enhanced lipid accumulation in differentiating 3T3‐L1 cells. SCO increased the transcription of several PPAR[[Unsupported Character ‐ Symbol Font □]] target genes in differentiating 3T3‐L1 cells and rescued the negative effects of tumor necrosis factor [[Unsupported Character ‐ Symbol Font □]] on production and secretion of adiponectin and monocyte chemoattractant protein‐1 in fully differentiated fat cells. DIO mice treated with SCO had elevated adiponectin levels and increased phosphorylation of AMPK[[Unsupported Character ‐ Symbol Font □]] in eWAT when compared to control mice. In SCO‐treated mice, these changes were also associated with decreased fasting insulin and glucose levels.Conclusion: SCO has metabolically beneficial effects on adipocytes in vitro and adipose tissue in vivo, highlighting its potential as a metabolically favorable botanical supplement.Grant Funding Source: Supported by NIH 2P30DK072476

Effect of short‐term dietary curcumin supplementation on mitochondrial regulatory proteins in muscle and brown adipose tissue of aged mice (1159.4)

Curcumin, a polyphenol found in the spice turmeric, is shown to have antioxidant and anti‐inflammatory properties in multiple tissues, but whether it alters mitochondrial biogenesis/apoptotic pathways is unknown. The aging process is associated with impaired mitochondrial function and elevated mitochondrial apoptotic susceptibility. Potential pharmacological and/or neutraceutical therapeutic interventions capable of improving mitochondrial function, like curcumin, have been postulated to delay this process. Thus, we investigated whether short‐term (21d) dietary curcumin supplementation (5% diet) altered mitochondrial biogenesis in muscle and brown adipose tissue (BAT) of aged mice (24 month; C57BL/6) compared to control diet mice (n=4‐6/group). While curcumin supplementation increased the mitochondrial content markers cytochrome c (14%) and COX Vb (26%) and enhanced the mitochondrial regulators Tfam (25%) and NRF‐1 (14%) in BAT, it suppressed and/or caused no change in these mitochondrial indices in muscle. In contrast, curcumin treatment evoked significant decreases (P&lt;0.05) in the pro‐apoptotic BAX protein in both BAT (20%) and muscle (55%). Our data indicate short‐term curcumin treatment in aged mice causes tissue‐specific mitochondrial biogenesis adaptations in BAT and muscle while potentially suppressing mitochondrial apoptotic susceptibility in both tissues.

Resistin in Dairy Cows: Plasma Concentrations during Early Lactation, Expression and Potential Role in Adipose Tissue

Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

Proteome Analysis of Mouse Adipose Tissue and Colon Tissue using a Novel Integrated Data Processing Pipeline

Abstract: Liquid chromatography based mass spectrometry (LC-MS) is a key technology for analyzing highly complex anddynamic proteome samples. With highly accurate and sensitive LC-MS analysis of complex proteome samples, efficient dataprocessing is another critical issue to obtain more information from LC-MS data. A typical proteomic data processing starts wit hprotein database search engine which assigns peptide sequences to MS/MS spectra and finds proteins. Although several searchengines, such as SEQUEST and MASCOT, have been widely used, there is no unique standard way to interpret MS/MS spectraof peptides. Each search engine has pros and cons depending on types of mass spectrometers and physicochemical properties ofpeptides. In this study, we describe a novel data process pipeline which identifies more peptides and proteins by correcting pre-cursor ion mass numbers and unifying multi search engines results. The pipeline utilizes two open-source software, i PE-MMRfor mass number correction, and iProphet to combine several search results. The integrated pipeline identified 25% more pro-teins in mouse epididymal adipose tissue compared with the conventional method. Also the pipeline was validated using controland colitis induced colon tissue. The results of the present study shows that the integrated pipeline can efficiently identifyincreased number of proteins compared to the conventional method which can be a breakthrough in identification of a potentialbiomarker candidate.Key words: iPE-MMR, iProphet, Q-TOF, TPP

Integrative analysis reveals novel pathways mediating the interaction between adipose tissue and pancreatic islets in obesity in rats

Comprehensive characterisation of the interrelation between the peripancreatic adipose tissue and the pancreatic islets promises novel insights into the mechanisms that regulate beta cell adaptation to obesity. Here, we sought to determine the main pathways and key molecules mediating the crosstalk between these two tissues during adaptation to obesity by the way of an integrated inter-tissue, multi-platform analysis. Wistar rats were fed a standard or cafeteria diet for 30 days. Transcriptomic variations by diet in islets and peripancreatic adipose tissue were examined through microarray analysis. The secretome from peripancreatic adipose tissue was subjected to a non-targeted metabolomic and proteomic analysis. Gene expression variations in islets were integrated with changes in peripancreatic adipose tissue gene expression and protein and metabolite secretion using an integrated inter-tissue pathway and network analysis. The highest level of data integration, linking genes differentially expressed in both tissues with secretome variations, allowed the identification of significantly enriched canonical pathways, such as the activation of liver/retinoid X receptors, triacylglycerol degradation, and regulation of inflammatory and immune responses, and underscored interaction network hubs, such as cholesterol and the fatty acid binding protein 4, which were unpredicted through single-tissue analysis and have not been previously implicated in the peripancreatic adipose tissue crosstalk with beta cells. The integrated analysis reported here allowed the identification of novel mechanisms and key molecules involved in peripancreatic adipose tissue interrelation with beta cells during the development of obesity; this might help the development of novel strategies to prevent type 2 diabetes.

Insulin Resistance Modulates Iron-Related Proteins in Adipose Tissue

OBJECTIVE Circulating markers of iron overload are associated with insulin resistance. Less is known about the impact of iron overload on adipose tissue (AT). We hypothesized that gene expression markers of iron metabolism in AT could be associated with insulin action. RESEARCH DESIGN AND METHODS The AT expression of ferroportin (SLC40A1), transferrin (TF), TF receptor (TFRC), ferritin (FT) heavy polypeptide 1 (FTH1), and FT light polypeptide (FTL) was analyzed cross-sectionally in three independent cohorts and also after weight loss-induced changes in insulin sensitivity (clamp M value) in an independent fourth cohort. RESULTS In human AT, TF mRNA and protein levels were decreased with obesity and insulin resistance in the three cohorts and were positively associated with adipogenic mRNAs and insulin action. Otherwise, FTL mRNA and protein and SLC40A1 transcripts were positively associated with BMI and negatively linked to adipogenic genes and insulin action. Bariatric surgery-induced weight loss led to increased TF and decreased TFRC, FTH1, FTL, and SLC40A1 in subcutaneous AT in parallel to improved insulin action. CONCLUSIONS These results suggest that iron overload impacts on AT in association with insulin resistance.

Sex-Dependent Expression of Caveolin 1 in Response to Sex Steroid Hormones Is Closely Associated with Development of Obesity in Rats

Caveolin-1 (CAV1) is a conserved group of structural membrane proteins that form special cholesterol and sphingolipid-rich compartments, especially in adipocytes. Recently, it has been reported that CAV1 is an important target protein in sex hormone-dependent regulation of various metabolic pathways, particularly in cancer and diabetes. To clarify distinct roles of CAV1 in sex-dependent obesity development, we investigated the effects of high fat diet (HFD) and sex steroid hormones on CAV1 expression in adipose tissues of male and female rats. Results of animal experiments revealed that estrogen (17-β-estradiol, E2) and androgen (dihydrotestosterone, DHT) had opposite effects on body weight gain as well as on the regulation of CAV1, hormone sensitive lipase (HSL) and uncoupling protein 1 (UCP1) in adipose tissues. Furthermore, sex hormone receptors and aromatase were differentially expressed in a sex-dependent manner in response to E2 and DHT treatments. In vivo data were confirmed using 3T3-L1 and HIB1B cell lines, where Cav1 knock down stimulated lipogenesis but suppressed sex hormone receptor signaling proteins. Most importantly, co-immunoprecipitation enabled the identification of previously unrecognized CAV1-interacting mitochondrial or lipid oxidative pathway proteins in adipose tissues. Taken together, current data showed that CAV1 may play important preventive role in the development of obesity, with more prominent effects in females, and proved to be an important target protein for the hormonal regulation of adipose tissue metabolism by manipulating sex hormone receptors and mitochondrial oxidative pathways. Therefore, we can report, for the first time, the molecular mechanism underlying the effects of sex steroid hormones in the sex-dimorphic regulation of CAV1.

Fibrous Hamartoma of Infancy

Fibrous hamartoma of infancy is a benign soft tissue tumor with a characteristic triphasic organoid histologic appearance. It typically occurs within the first 2 years of life. The usual anatomic locations include the upper extremities, axilla, and upper back. Diagnostic challenges occur when this tumor arises in older children, outside of the usual anatomic sites, or when unusual histologic features are encountered. This study reports 60 cases of fibrous hamartoma of infancy from institutional and consultation files. All had a triphasic organoid histologic pattern, but half also displayed an unusual pseudoangiomatous histologic pattern. The male to female ratio was 2.0 (40 boys, 20 girls), with a mean age of 1.5 years (range, 16 d to 8 y) at diagnosis. Tumor size ranged from 0.5 to 9 cm, with a mean of 3.7 cm. Sites included the trunk (40 cases), extremities (17 cases), and head and neck (3 cases). All cases had triphasic elements of mature fibrous tissue, mature adipose tissue, and immature mesenchymal tissue in varying proportions, with the additional pseudoangiomatous pattern in 32 cases. Immunohistochemical analysis demonstrated reactivity for smooth muscle actin and CD34 in the mature fibrous tissue, S100 protein in the mature adipose tissue, and variable CD34 reactivity in immature mesenchymal and pseudoangiomatous foci. Ki-67 proliferative activity was noted in the immature mesenchymal and pseudoangiomatous foci, and Bcl-2 reactivity was restricted to mesenchymal and pseudoangiomatous foci. Follow-up information in 12 cases revealed no evidence of recurrence in 10 patients and local recurrence in 2 patients, each at 3.5 years after primary excision. This study demonstrates an expanded age range (up to 8 y) and anatomic distribution (30 cases outside of the classic locations of the upper extremities, axilla, and upper back) of fibrous hamartoma of infancy. The pseudoangiomatous morphologic variation can lead to challenges in diagnosis and may reflect a maturational phenomenon from the immature mesenchymal component.

Downregulation of IRS-1 in adipose tissue of offspring of obese mice is programmed cell-autonomously through post-transcriptional mechanisms.

We determined the effects of maternal diet-induced obesity on offspring adipose tissue insulin signalling and miRNA expression in the aetiology of insulin resistance in later life. Although body composition and glucose tolerance of 8-week-old male offspring of obese dams were not dysregulated, serum insulin was significantly (p<0.05) elevated. Key insulin signalling proteins in adipose tissue were down-regulated, including the insulin receptor, catalytic (p110β) and regulatory (p85α) subunits of PI3K as well as AKT1 and 2 (all p<0.05). The largest reduction observed was in IRS-1 protein (p<0.001), which was regulated post-transcriptionally. Concurrently, miR-126, which targets IRS-1, was up-regulated (p<0.05). These two features were maintained in isolated primary pre-adipocytes and differentiated adipocytes in-vitro. We have therefore established that maternal diet-induced obesity programs adipose tissue insulin resistance. We hypothesise that maintenance of the phenotype in-vitro strongly suggests that this mechanism is cell autonomous and may drive insulin resistance in later life.

Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells

Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor β (TGF-β) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-β or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-β. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome.