Controlling a master switch of adipocyte development and insulin sensitivity: Covalent modifications of PPARγ

Adipocytes are highly specialized cells that play a central role in lipid homeostasis and the maintenance of energy balance. Obesity, an excessive accumulation of adipose tissue, is a major risk factor for the development of Type 2 diabetes mellitus (T2DM), cardiovascular disease, and hypertension. A variety of studies suggest that obesity and T2DM can be linked to a breakdown in the regulatory mechanisms that control the expression and transcriptional activity of PPARγ. PPARγ is a nuclear hormone receptor that functions as a master switch in controlling adipocyte differentiation and development. Also important in controlling glucose homeostasis and insulin sensitivity, PPARγ is a ligand-dependent transcription factor that is the functional receptor for the anti-diabetic thiazolidinediones (TZDs). In the last fifteen years, a variety of covalent modifications of PPARγ activity have been identified and studied. These covalent modifications include phosphorylation, ubiquitylation, O-GlcNAcylation and SUMOylation. Covalent modifications of PPARγ represent key regulatory mechanisms that control both PPARγ protein stability and transcriptional activity. A variety of PPARγ transgenic models, including mice heterozygous for PPARγ, have demonstrated the importance of PPARγ expression in glucose homeostasis and insulin resistance. In the following review, we have highlighted the regulation of PPARγ by covalent modifications, the interplay between these interactions and how these post-translational modifications impact metabolic disease states. © 2012 Elsevier B.V

A New Colorimetric Assay of Apurinic/Apyrimidinic (Abasic) Sites of Deoxyribonucleic acid (DNA) Using Bicinchoninic Acid [

Apurinic/apyrimidinic (AP or abasic) sites are DNA lesions that result from the loss of a nucleobase by hydrolysis of the N-glycosyl bond. It is estimated that AP sites are the most frequent lesions in cells with about 10,000-50,000 times per day per cell under typical aerobic conditions [2]. The formation of AP sites is caused by environmental and cancer therapeutic genotoxins such as alkylating agents, oxidizing agents, ionizing radiation, and ultraviolet radiation.https://scholarworks.moreheadstate.edu/celebration_posters_2022/1024/thumbnail.jp

Interferon-γ-mediated activation and ubiquitin-proteasome-dependent degradation of PPARγ in adipocytes

Interferon-γ (IFNγ) treatment of adipocytes results in a down-regulation of the peroxisome proliferator-activated receptor γ (PPARγ). The decrease in PPARγ expression is mediated by inhibition of PPARγ synthesis and increased degradation of PPARγ. In this study, we demonstrate that both PPARγ1 and PPARγ2 are targeted to the proteasome under basal conditions and that PPARγ1 is more labile than PPARγ2. The IFNγ-induced increase in PPARγ turnover is blocked by proteasome inhibition and is accompanied by an increase in PPARγ-polyubiquitin conjugates. In addition, IFNγ treatment results in the transcriptional activation of PPARγ. Similar to ligand-dependent activation of PPARγ, IFNγ-induced activation was greater in the phosphorylation-deficient S112A form of PPARγ when compared with wild-type PPARγ. Moreover, the inhibition of ERKs 1 and 2 with a MEK inhibitor, U1026, lead to an inhibition in the decay of PPARγ proteins, indicating that serine phosphorylation influences the degradation of PPARγ in fat cells. Our results also demonstrate that the proteasome-dependent degradation of PPARγ does not require nuclear export. Taken together, these results indicate that PPARγ is targeted to the ubiquitin-proteasome pathway for degradation under basal conditions and that IFNγ leads to an increased targeting of PPARγ to the ubiquitin-proteasome system in a process that is affected by ERK-regulated serine phosphorylation of PPARγ proteins

Control of peroxisome proliferator-activated receptor γ2 stability and activity by SUMOylation

Objective: To determine whether small ubiquitin-related modifier (SUMO)ylation of lysine 107 plays a role in regulating the activity of peroxisome proliferator-activated receptor γ (PPARγ). Research Methods and Procedures: Transient expression of wild-type and K107R-PPARγ2 in the NIH 3T3 fibroblast cell line was carried out in conjunction with half-life studies, luciferase activity assays, and indirect immunofluorescence localization studies. Additional in vitro analysis was carried out using recombinant SUMOylation pathway proteins along with in vitro transcribed and translated wild-type or K107R-PPARγ2 to examine the SUMO-1 modification state of wild-type and SUMO-deficient K107R-PPARγ2. Results: While examining PPARγ2 for potential ubiquitylation sites, we identified a strong consensus site for SUMO modification that contains lysine 107. In vitro, SUMOylation studies showed that lysine 107 of PPARγ2 is a major SUMOylation site and that at least one other SUMOylation site is present in PPARγ. In addition, our results demonstrated that SUMO-1 affects PPARγ stability and transcriptional activity but not the nuclear localization of PPARγ. Discussion: These results indicated that SUMOylation plays a role in regulating PPARγ, both indirectly and directly by modification of lysine 107. Because PPARγ is regulated in numerous animal models of obesity, understanding the covalent modifications of PPARγ may enhance our understanding of the metabolic syndrome. Copyright © 2004 NAASO

STAT5A promotes adipogenesis in nonprecursor cells and associates with the glucocorticoid receptor during adipocyte differentiation

The differentiation of adipocytes is regulated by the activity of a variety of transcription factors, including peroxidase proliferator-activated receptor (PPAR)-γ and C/EBPα. Our current study demonstrates that ectopic expression of STAT5A, such as that of PPAR-γ and C/EBPα, promotes adipogenesis in two nonprecursor fibroblast cell lines. Using morphologic and biochemical criteria, we have demonstrated that STAT5A and the combination of STAT5A and STAT5B are sufficient to induce the expression of early and late adipogenic markers in BALB/c and NIH-3T3 cells. Yet, the ectopic expression of STAT5B alone does not induce the expression of adipocyte genes, but enhances the induction of these genes in cells also expressing STAT5A. This finding suggests that STAT5A and STAT5B do not function identically in adipocytes. In addition, these studies demonstrate that the phosphorylation of STAT5 proteins may play a role in adipogenesis. Moreover, we have shown that STAT5A is associated with the glucocorticoid receptor during adipogenesis in a highly regulated manner

The Met Office weather game: investigating how different methods for presenting probabilistic weather forecasts influence decision-making

To inform the way probabilistic forecasts would be displayed on their website, the UK Met Office ran an online game as a mass participation experiment to highlight the best methods of communicating uncertainty in rainfall and temperature forecasts, and to widen public engagement in uncertainty in weather forecasting. The game used a hypothetical “ice-cream seller” scenario and a randomized structure to test decision-making ability using different methods of representing uncertainty and to enable participants to experience being “lucky” or “unlucky” when the most likely forecast scenario did not occur. Data were collected on participant age, gender, educational attainment, and previous experience of environmental modelling. The large number of participants (n > 8000) that played the game has led to the collation of a unique large dataset with which to compare the impact on the decision-making ability of different weather forecast presentation ormats. This analysis demonstrates that within the game the provision of information regarding forecast uncertainty greatly improved decision-making ability and did not cause confusion in situations where providing the uncertainty added no further information

Interferon-γ-induced Regulation of Peroxisome Proliferator-activated Receptor γ and STATs in Adipocytes

Interferon-γ (IFN-γ) is known primarily for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. To further investigate the effects of IFN-γ on fat cells, we examined the effects of this cytokine on the expression of adipocyte transcription factors in 3T3-L1 adipocytes. Although IFN-γ regulated the expression of several adipocyte transcription factors, IFN-γ treatment resulted in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-γ also resulted in a decrease of both CCAAT/enhancer-binding α and sterol regulatory element binding protein (SREBP-1) expression. The short half-life of both the PPARγ mRNA and protein likely contributed to the rapid decline of both cytosolic and nuclear PPARγ in the presence of IFN-γ. Our studies clearly demonstrated that the IFN-γ-induced loss of PPARγ protein is partially inhibited in the presence of two distinct proteasome inhibitors. Moreover, IFN-γ also inhibited the transcription of PPARγ, which was accompanied by a decrease in PPARγ mRNA accumulation. In addition, exposure to IFN-γ resulted in a substantial increase in STAT I expression and a small increase in STAT 3 expression. IFN-γ treatment of 3T3-L1 adipocytes (48-96 h) resulted in a substantial inhibition of insulin-sensitive glucose uptake. These data clearly demonstrate that IFN-γ treatment results in the development of insulin resistance, which is accompanied by the regulation of various adipocyte transcription factors, in particular the synthesis and degradation of PPARγ