Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA replication and repair and development. Several recent proteomic studies uncovered a great variety of nuclear envelope transmembrane proteins (NETs). Diseases associated with several nuclear envelope proteins, mostly NETs, affect many tissues e.g. muscle, adipose tissue, skin, bones. Many NETs of the inner nuclear membrane have been shown to interact with chromatin, suggesting that their influencing gene expression might explain NET roles in disease. This work is focused on finding novel interactions of NETs with chromatin. First, SUN2 post-translational modifications were analysed and the effect of phosphomimetic and phospho-null mutants on heterochromatin and the cytoskeleton was tested by overexpression. However, no obvious changes were found. Second, several tissue-preferential NETs were tested in an adipocyte differentiation system. NET29 changed chromosome 6 position in pre-adipocytes. This matched changes in chromosome positioning that occur during adipocyte differentiation when NET29 is normally induced. Post-translational modifications of NET29 are likely to play a vital role in this process because a phospho-null mutant dominantly blocked chromosome repositioning. The effect of over-expression and down-regulation of NET29 on transcription was tested and results suggest that NET29 negatively regulates expression of myogenic genes during adipogenesis. This thesis is split into six chapters. Chapter I is an overview of the nuclear envelope, adipogenesis and chromatin remodelling, Chapter II is a detailed description of methods used in this study. Chapter III focuses on post-translational modifications of SUN2, as well as trials to identify novel partners of SUN2. Chapter IV and V deal with a novel nuclear envelope transmembrane protein and its role in adipogenesis. Finally, the last chapter includes a discussion and recommended future directions.

No description available.

In addition to providing heat insulation and mechanical cushioning, adipose tissue regulates overall metabolic homeostasis and serves as an essential energy storage site. Excess adipose tissue, or obesity, is on the rise in the US among all demographics. The expansion of adipose tissue results from both adipocyte hypertrophy and hyperplasia but the mechanisms that regulate these processes are not fully understood. Destabilizing actin has been shown to promote adipogenesis while actin stabilization inhibits this process. In addition, decreased actin synthesis is known to occur. However, these studies examined total actin and did not consider that actin is in fact a family of functionally diverse isoforms and that individual isoforms may have different functions in adipogenesis. I hypothesized that actin isoforms contribute differently to adipogenic actin reorganization. To measure this, I developed a novel fractionation method that allowed for the reliable quantification of actin polymerization. I used this actin fractionation method to identify an early loss in polymerized α-smooth muscle actin (α-SMA) relative to polymerized β-actin and γ-actin and to also rule out a role for the actin severing protein gelsolin in the loss of polymerized actin. Furthermore, I showed that the loss of α-SMA expression precedes the loss of β-actin and γ-actin expression. A known regulator of actin cytoskeleton genes is the transcription factor serum response factor (SRF) and its co-activator, myocardin related transcription factor (MRTF). I identified a role for MRTF/SRF in the downregulation of actin expression during adipogenesis, particularly α-SMA. There was an additional cAMP-responsive decrease in α-SMA expression during the initiation of adipogenesis by exposure to established inducers. Overall, my findings are consistent with growing evidence suggesting that genetic markers of smooth muscle cells, including α-SMA, help control adipogenic commitment. Understanding these early stages of adipogenesis could open new therapeutic avenues for obesity and its co-morbidities.

The differentiation of osteoblasts and bone marrow adipocytes are closely associated yet mutually exclusive processes that are essential for maintaining bone homeostasis. Various diseases have been shown to develop once the delicate balance between adipogenesis and osteoblastogenesis is disrupted. Investigating the underlying molecular mechanisms of the osteoblasto-adipogenic switch under osteoporotic conditions will facilitate our understanding of the pathogenesis of osteoporosis and may eventually lead to the development of clinical therapeutic approaches for this life-threatening disease. While changes in cell morphology and cytoskeletal integrity can alter pre-committed mesenchymal stem cell (MSC) differentiation of certain lineages, previous studies have shown that cellular morphological changes can affect the early commitment of pluripotent MSCs via modulation of Ras homolog gene family, member A (RhoA) activity. The RhoA pathway regulates actin polymerization to promote the incorporation of globular-actin (G-actin) into filamentous-actin (F-actin). Actin polymerization releases G-actin bound myocardin-related transcription factors (MRTFs), which translocate to the nucleus and co-activate serum response factor (SRF) target gene expression. Exactly how the RhoA-actin-MRTF-SRF circuit is involved in the regulation of early commitment of MSCs remains poorly understood. Here we show that global MRTFA knockout mice (MRTFA KO) exhibited lower body weight, shorter femur and tibia lengths, and decreased trabecular bone volume. Furthermore, bone marrow MSCs isolated from MRTFA KO mice showed increased adipogenesis and brown fat gene expression as well as compromised osteoblastogenic differentiation as compared to WT controls. Treatment of WT bone marrow MSCs with the SRF inhibitor, CCG1423, mimicked these effects in that the compound inhibited osteoblastogenesis and promoted adipogenesis. Over-expression of MRTFA or SRF inhibited adipogenesis and enhanced osteoblastogenesis in C3H/10T1/2 cell lines, whereas over-expression of dominant-negative MRTFA or SRF variants had the opposite effects. In conclusion, our study identified MRTFA as a crucial regulator of skeletal homeostasis via regulating the balance between adipogenic and osteoblastogenic differentiation of the MSCs. Furthering our understanding of how the RhoA-actin-MRTFA-SRF circuit is involved in regulating the fate commitment of MSCs may ultimately lead to novel therapeutic strategies for treating osteoporosis and obesity.

This study was conducted to assess the effects of specific oxysterols on the adipogenic differentiation and expression of adipogenic transcripts in C3H10T1/2 mouse stem cells. In the first study, four oxysterols namely; 20S, 22R, 22S and 25 hydroxycholesterol (25-HC) were tested to determine which one best inhibits adipogenesis in C3H10T1/2 mouse stem cells. Adipogenic differentiation was induced using an adipogenic media (DMITro) consisting of dexamethasone (DEX), 3-isobutyl-1-methyl-xanthine (IBMX), insulin and troglitazone (Tro). Inhibition of adipogenesis was assessed by treatment of cells with DMITro+20S, 22R, 22S or 25-HC for six days. Oil red O pictures and gene expression analysis showed that 25-HC was more effective in inhibiting the expression of adipogenic genes compared to the other oxysterols. Further investigation of the mechanisms of action of 25-HC showed that the inhibitory effects of 25-HC on adipogenesis are not mediated by hedgehog signalling. / October 2015

No description available.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Doctor of Philosophy. Johannesburg, 2004 / Alkaline phosphatase (ALP) is a ubiquitously expressed enzyme, that has been shown to play a role in cell differentiation and organogenesis. One study has also demonstrated ALP activity in rat adipocytes. The purpose of the present study was therefore to determine whether ALP is expressed in preadipocytes and what role it may have in adipogenesis. ALP activity was detected in the murine preadipocyte cell line, 3T3-L1, and in human preadipocytes isolated from mammary tissue, and from subcutaneous abdominal fat depots. In all the cell types studied ALP activity increased in parallel with adipogenesis. In the 3T3 -L1 cell line the tissue- non -specific ALP inhibitors, levamisole and histidine inhibited ALP activity, and adipogenesis, whereas the tissue specific ALP inhibitor Phe- Gly-Gly did not inhibit ALP or adipogenesis. In human preadipocytes, histidine inhibited adipogenesis and ALP activity, whereas levamisole inhibited adipogenesis, but did not block ALP activity in intact cells. However, levamisole did inhibit ALP activity by 50% in cell extracts. Levamisole was able to inhibit adipogenesis in human preadipocytes. The tissue specific ALP inhibitor, Phe Gly Gly, did not inhibit ALP activity or adipogenesis in human preadipocytes. ALP activity and adipogenesis, were compared in preadipocytes isolated from mammary tissue taken from black (13) and white (15) female subjects. Both ALP activity and adipogenesis, were lower in white compared to black female subjects. iii Immunocytochemical, analysis of the 3T3-L1 cell line and human preadipocytes demonstrated that ALP activity was restricted to the lipid droplets of these cells. ALP activity was also measured in serum samples obtained from 100 African subjects (74 females and 26 males) of varying BMI. ALP activity was found to be higher in obese than lean subjects, whereas, the other liver enzymes or products measured in serum were not. In fact these variables correlated to varying degrees with waist-hip ratio, whereas ALP levels did not. This suggest that liver function is predominantly influenced by abdominal obesity whereas serum ALP levels are more influenced by overall body adiposity. In conclusion, ALP, may be involved in the control of adipogenesis, in the 3T3- L1 preadipocyte cell line and in human preadipocytes isolated from mammary adipose tissue and subcutaneous abdominal adipose tisssue. The presence of ALP activity in lipid droplets in 3T3-L1 cells and human preadipocytes, and the ability of ALP inhibitors to block adipogenesis strongly suggest that ALP plays a role in the control of adipogenesis. / IT2017

Endocytosis is the process in which a cell engulfs extracellular cargo by creating invaginations within its plasma membrane. The cargo that has entered the cell enters an endosome and then is delivered to either the trans-Golgi network for recycling to the plasma membrane or to the lysosome for its degradation (Trousdale & Kim, 2015). Retromer is a peripheral membrane protein complex that plays a key role in sorting of these cargo molecules (Collins, 2008). More specifically, retromer deliver cargo from the endosome to the trans-Golgi Network, the process which is called retrograde transport of cargo molecules. Retromer dysfunction is strongly linked to neurodegenerative diseases such as Alzheimer’s and Parkinson’s Disease. However, recent Genome Wide Association Studies suggest that a mutation in retromer subunit VPS26a, has been linked to Type II Diabetes (Trousdale & Kim, 2015). A 2016 study published in The Faseb Journal attempts to characterize the role of retromer in adipocyte differentiation and insulin-stimulated uptake of glucose through transporter GLUT4 (Yang et al., 2016). The aim of this study is to further investigate the role of retromer in adipogenesis and to determine whether retromer plays a role at the transcriptional level or translational level. In this study, retromer’s VPS35 subunit was knocked down in four mouse 3T3-L1 fibroblast cell lines using the CRISPR-Cas9 approach. These cell lines were differentiated into mature adipocytes and analyzed by Oil-Red O staining, Western Blotting and quantitative PCR. The knockdown of retromer produced varying effects on adipocyte differentiation. In two of the knockdown cell lines, adipocyte differentiation was downregulated whereas adipocyte differentiation was upregulated in the other two cell lines. Although the results from Oil-Red O staining and Western Blot analyses complemented each other, results obtained from qPCR were not as straightforward and further analysis is needed to fully comprehend how retromer acts at the transcriptional level of cell differentiation. Based on the results of this study, retromer is involved in adipogenesis at both the transcriptional and translational level, however it’s mechanism of action remains unclear as both cases of impaired differentiation and upregulated differentiation were observed. Further studies are necessary to determine retromer’s exact role in adipogenesis.

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. By inhibiting HMG-CoA reductase (HMGCR) statins deplete the cellular isoprenoid biosynthetic pathway products farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Current thought in the field is that statins stimulate bone formation through the depletion of GGPP, since exogenous GGPP prevents the effects of statins on osteoblasts in vitro. We hypothesized that direct inhibition of GGPP synthase (GGPPS) would similarly stimulate osteoblast differentiation. Digeranyl bisphosphonate (DGBP), a specific inhibitor of GGPPS, decreased GGPP levels in MC3T3-E1 pre-osteoblasts and calvarial osteoblasts leading to impaired protein geranylgeranylation. In contrast to our hypothesis, DGBP inhibited the matrix mineralization of MC3T3-E1 cells and the expression of osteoblast differentiation markers in calvarial osteoblasts. The effect on mineralization was not prevented by exogenous GGPP. By inhibiting GGPPS, DGBP led to an accumulation of the GGPPS substrate FPP. We show that FPP and GGPP levels decreased during MC3T3-E1 and calvarial osteoblast differentiation, which correlated with decreased expression of HMGCR and FPP synthase. The decrease in FPP during differentiation was prevented by DGBP treatment. The accumulation of FPP following 24 h DGBP treatment correlated with activation of the glucocorticoid receptor, suggesting a potential mechanism by which DGBP-induced FPP accumulation may inhibit osteoblast differentiation. To further investigate whether FPP inhibits osteoblast differentiation, we utilized the squalene synthase (SQS) inhibitor zaragozic acid (ZGA), which causes a greater accumulation of FPP than can be achieved with GGPPS inhibition. ZGA treatment decreased osteoblast proliferation, gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization of calvarial osteoblasts. Prevention of ZGA-induced FPP accumulation with HMGCR inhibition prevented the effects of ZGA on osteoblast differentiation. Treatment of osteoblasts with exogenous FPP similarly inhibited matrix mineralization. These results suggest that the accumulation of FPP negatively regulates osteoblast differentiation. While we did not find that specific depletion of GGPP stimulates osteoblast differentiation, we obtained evidence that GGPP does negatively regulate the differentiation of these cells. Exogenous GGPP treatment inhibited primary calvarial osteoblast gene expression and matrix mineralization. Interestingly, GGPP pre-treatment increased markers of insulin signaling, despite reduced phosphorylation of the insulin receptor (InsR). Inhibition of osteoblast differentiation by GGPP led to the induction of PPARã and enhanced adipogenesis in osteoblastic cultures, suggesting that GGPP may play a role in the osteoblast versus adipocyte fate decision. Adipogenic differentiation of primary bone marrow stromal cell (BMSC) cultures was prevented by DGBP treatment. Altogether these data present novel roles for the isoprenoids FPP and GGPP in the regulation of osteoblast differentiation and have intriguing implications for the isoprenoid biosynthetic pathway in the regulation of skeletal homeostasis.

Obesity-related comorbidities, including type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) have become a major public health concerns. These complications primarily arise in response to cellular changes in white adipose tissue (WAT). In particular, when a majority of fat cells become hypertrophic it promotes a metabolically unhealthy phenotype, which is characterized by chronic low-grade inflammation, insulin resistance, and ectopic lipid accumulation. Research has implicated synthetic (i.e., Bisdehydrodoisynolic acid, BDDA) and natural (i.e., genistein and daidzein) xenoestrogens in the protection against obesity-related pathologies. Bisdehydrodoisynolic acid (BDDA) reduced weight gain and adiposity, as well as improved lipid homeostasis in obese rodents. Alternatively, phytoestrogens, such as genistein and daidzein were reported to induce adipocyte differentiation through potential interactions with PPARγs, canonical WNT proteins, and estrogen receptors (ER) signaling. The current investigation was conducted to test the effects of synthetic and natural estrogenic compounds (BDDA, daidzein, and genistein) for their effects on the induction of adipogenic signaling, which may potentially improve WAT morphology by reducing adipocyte hypertrophy.