Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

Background: The typical western diet contains foods with modest amounts of lipid oxidation products. Previous work by us and others have demonstrated that mildly oxidized lipids promote a gain in fat mass while highly oxidized lipids decrease fat mass in rodents and triglyceride (TAG) accumulation in 3T3-L1 cells. Adipocyte differentiation is regulated by a key nuclear transcription factor known as PPARγ. Objective: To investigate if the alterations in triglyceride accumulation in 3T3-L1 cells pretreated with oxidized soy oil are due to 1) a change in PPARg DNA interactions 2) changes in the expression of SREBP-1c, PPARg, and/or its target genes. Main Methods: Confluent 3T3-L1 cells were pretreated for 24hours with 0.01% soy oil (SO) which was either unheated (unheated SO) or heated for 3, (3h-SO), 6 (6h-SO), or 9hours (9h-SO). The effect of 24hour soy oil exposure was assessed at several time points throughout the differentiation process. Alterations in PPARg DNA interaction was assessed using a PPARγ transcription factor assay kit while alterations in the expression of genes upstream and downstream of PPARγ was determined by RT-PCR. Primary and secondary products of oxidation within the SO were determined by spectrophotometry. Results: The 6hr-SO contained the greatest concentration of peroxides whereas both the 6hr-SO and 9hr-SO contained a significantly higher concentration of conjugated dienes and aldehydes.Nuclear extracts from 3T3-L1 cells pretreated with 6h-SO demonstrated the greatest reduction in PPARγ DNA binding. Compared to the unheated SO and mildly oxidized 3h-SO, cells treated with the 6h-SO had a significant reduction in SREBP-1c, PPARg, LPL, and GLUT4 expression occurring early in the differentiation process. Variations in the gene expression of 6hr-SO pretreated cells persisted within partially differentiated and mature adipocytes. Conclusions: Pre-treatment of preadipocytes with soy oil heated for ³ 6h greatly decreases the activity of PPARγ in the nucleus and adipogenic gene expression . These changes seen in early differentiation seem to correlate the best with the phenotype of reduced triglyceride accumulation seen in mature adipocytes.

The overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line.Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally.Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

\(PGC1\alpha\) is a key transcriptional coregulator of mitochondrial biogenesis, oxidative metabolism and thermogenesis. We developed a quantitative high throughput screen to identify small molecules that can induce \(PGC1\alpha\) expression in adipocytes. Small molecules antagonizing the TRPVs (Transient Receptor Potential Vanilloid), a family of ion channels, induced \(PGC1\alpha\) expression in adipocytes. In particular, inhibition of TRPV4 increased expression of \(PGC1\alpha\), UCP1 and cellular respiration; conversely, chemical activation of TRPV4 repressed this pathway. Blocking TRPV4 in cultured adipocytes also reduced the expression of multiple proinflammatory genes that are involved in the development of insulin resistance. These effects of TRPV4 were mediated by the activation of ERK1/2. Finally, mice with a null mutation for TRPV4 showed higher energy expenditure with no change in movement or food intake, and were protected from diet-induced obesity, adipose inflammation and insulin resistance. This study links TRPV4 to robust pathways that offer therapeutic potential in obesity and related metabolic diseases.

The overall goal of this research is to understand roles of gut microbiota metabolites and adipocyte transcription factor (TF) network in health and disease by developing systematic analysis methods. As microbiota can perform diverse biotransformation reactions, the spectrum of metabolites present in the gastrointestinal (GI) tract is extremely complex but only a handful of bioactive microbiota metabolites have been identified. We developed a metabolomics workflow that integrates in silico discovery with targeted mass spectrometry. A computational pathway analysis where microbiota metabolisms are modeled as a single metabolic network is utilized to predict a focused set of targets for multiple reaction monitoring (MRM) analysis. We validated our methodology by predicting, quantifying in murine cecum and feces and characterizing tryptophan (TRP)-derived metabolites as ligands for the aryl hydrocarbon receptor. The adipocyte process of lipid droplet accumulation and differentiation is regulated by multiple TFs that function together in a network. Although individual TF activation is previously reported, construction of an integrated network has been limited due to different measurement conditions. We developed an integrated network model of key TFs - PPAR, C/EBP, CREB, NFAT, FoxO1, and SREBP-1c - underlying adipocyte differentiation. A hypothetic model was determined based on literature, and stochastic simulation algorithm (SSA) was applied to simulate TF dynamics. TF activation profiles at different stages of differentiation were measured using 3T3-L1 reporter cell lines where binding of a TF to its DNA binding element drives expression of the Gaussia luciferase gene. Reaction trajectories calculated by SSA showed good agreement with experimental measurement. The TF model was further validated by perturbing dynamics of CREB using forskolin, and comparing the predicted response with experimental data. We studied the molecular recognition mechanism underlying anti-inflammatory function of a bacterial metabolite, indole in DC2.4 cells. The indole treatment attenuated the fraction of cells that were producing the pro-inflammatory cytokine, TNFα and knockdown of nuclear receptor related 1 (Nurr1; NR4A2) resulted in less indole-derived suppression of TNFα production. The first discovery of NR4A2 as a molecular mediator of the endogenous metabolite, indole is expected to provide a new strategy for treatment of inflammatory disorders.