Image analysis methods have been developed to provide quantitative assessment of microscopy data. In this unit, basic aspects of image analysis are outlined, including software installation, data ...import, image processing functions, and analytical tools that can be used to extract information from microscopy data using ImageJ. Step-by-step protocols for analyzing objects in a fluorescence image and extracting information from two-color tissue images collected by bright-field microscopy are included.
Overnutrition and sedentary activity reinforce the growing trend of worldwide obesity, insulin resistance, and type 2 diabetes. However, we have limited insight into how food intake generates ...sophisticated metabolic perturbations associated with obesity. Accumulation of mitochondrial oxidative stress contributes to the metabolic changes in obesity, but the mechanisms and significance are unclear. In white adipose tissue (WAT), mitochondrial oxidative stress, and the generation of reactive oxygen species (ROS) impact the endocrine and metabolic function of fat cells. The central role of mitochondria in nutrient handling suggests pharmacological targeting of pathological oxidative stress likely improves the metabolic profile of obesity. This review will summarize the critical pathogenic mechanisms of obesity-driven oxidative stress in WAT.
Background and Aims
Obesity‐induced chronic inflammation is a key component in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and insulin resistance. Increased secretion of ...proinflammatory cytokines by macrophages in metabolic tissues promotes disease progression. In the diet‐induced obesity (DIO) mouse model, activation of liver resident macrophages, or Kupffer cells (KCs), drives inflammatory responses, which recruits circulating macrophages and promotes fatty liver development, and ultimately contributes to impaired hepatic insulin sensitivity. Hepatic macrophages express the highest level of vitamin D receptors (VDRs) among nonparenchymal cells, whereas VDR expression is very low in hepatocytes. VDR activation exerts anti‐inflammatory effects in immune cells.
Approach and Results
Here we found that VDR activation exhibits strong anti‐inflammatory effects in mouse hepatic macrophages, including those isolated from DIO livers, and mice with genetic loss of Vdr developed spontaneous hepatic inflammation at 6 months of age. Under the chronic inflammation conditions of the DIO model, VDR activation by the vitamin D analog calcipotriol reduced liver inflammation and hepatic steatosis, significantly improving insulin sensitivity. The hyperinsulinemic euglycemic clamp revealed that VDR activation greatly increased the glucose infusion rate, while hepatic glucose production was remarkably decreased. Glucose uptake in muscle and adipose did not show similar effects, suggesting that improved hepatic insulin sensitivity is the primary contributor to the beneficial effects of VDR activation. Finally, specifically ablating liver macrophages by treatment with clodronate liposomes largely abolished the beneficial metabolic effects of calcipotriol, confirming that VDR activation in liver macrophages is required for the antidiabetic effect.
Conclusions
Activation of liver macrophage VDRs by vitamin D ligands ameliorates liver inflammation, steatosis and insulin resistance. Our results suggest therapeutic paradigms for treatment of NAFLD and type 2 diabetes mellitus.
The delta cell is uniquely positioned in the islet to integrate local signals and circulating nutrient cues to regulate alpha and beta cell function through somatostatin (SST) secretion. In type 2 ...diabetes (T2D), delta cell responses to ambient glucose are disproportionate and consequently alter insulin and glucagon secretion leading to dysglycemia. However, we know relatively little about the mechanisms influencing delta cells. Thus, understanding factors that regulate delta cell SST secretion may reveal pathogenic mechanisms contributing to T2D and direct new therapies to achieve glucose homeostasis. Leptin is a circulating hormone that potently inhibits insulin and glucagon secretion, but notably, the leptin receptor (LepR) is exclusively expressed on delta cells of human islets. Our preliminary studies demonstrate for the first time that leptin stimulates SST secretion from human islets. Leptin-induced SST corresponded with decreased glucagon and insulin secretion. Leptin effects were conserved in mouse islets and required delta cell specific LepR expression. Our single-cell RNA-seq studies revealed Stat3 target genes become depleted in delta cells that lack LepR, suggesting canonical leptin signals regulate transcriptional activity in delta cells. Along these lines, delta cell specific Stat3 KO islets (Sst-Cre+/wt;Stat3fl/fl) do not respond to leptin. On-going studies in human islets employ pharmacologic and genetic interventions with live functional imaging and unbiased approaches that will reveal the mechanisms of leptin action in human delta cells. These studies are the first to describe leptin effects on delta cells and uncover a unifying mechanism whereby leptin acts indirectly on alpha and beta cells through paracrine SST signaling. As such, our findings are expected to reveal new signaling mechanisms governing islet function that potentially exert therapeutic benefits for T2D.
Disclosure
S.M.Hartig: None. A.Cox: None.
Progesterone Receptor Signaling Mechanisms Grimm, Sandra L.; Hartig, Sean M.; Edwards, Dean P.
Journal of molecular biology,
09/2016, Letnik:
428, Številka:
19
Journal Article
Recenzirano
Progesterone receptor (PR) is a master regulator in female reproductive tissues that controls developmental processes and proliferation and differentiation during the reproductive cycle and ...pregnancy. PR also plays a role in progression of endocrine-dependent breast cancer. As a member of the nuclear receptor family of ligand-dependent transcription factors, the main action of PR is to regulate networks of target gene expression in response to binding its cognate steroid hormone, progesterone. This paper summarizes recent advances in understanding the structure–function properties of the receptor protein and the tissue/cell-type-specific PR signaling pathways that contribute to the biological actions of progesterone in the normal breast and in breast cancer.
Dynamic structures of the steroid hormone receptor (SHR). The conformational flexibility of SHR is mediated by the intrinsically disordered amino-terminal domain (NTD) and by allosteric interactions between domains ligand-binding domain (LBD) and DNA-binding domain (DBD). The apo SHR (left side of diagram) in response to binding co-regulatory proteins (CoRs), DNA, and steroidal ligands undergoes conformational changes to a transcriptionally active receptor (right side of diagram). Changes include folding and stabilization of the NTD, allosteric interdomain interactions, and repositioning of helix 12 in the LBD, enabling the binding of CoRs containing LXXLL motifs. Shadows around LBD and DBD are potential avenues of interdomain interactions. Binding proteins that induce folding and stabilization of NTD may function directly as a CoR or by reorganizing structure of the NTD enabling binding of other CoRs. Display omitted
•PR mediates variable responses to progesterone in a context-dependent manner.•Conformational flexibility of PR structure contributes to functional diversity.•Unique signaling mechanisms contribute to cell-specific actions of progesterone.•New tools are needed to advance structure studies of intact PR–protein complexes.•New models are advancing the understanding of the role of PR in breast cancer progression.
Summary
White adipose tissue (WAT) is a vital endocrine organ that regulates energy balance and metabolic homeostasis. In addition to fat cells, WAT harbors macrophages with distinct phenotypes that ...play crucial roles in immunity and metabolism. Nutrient demands cause macrophages to accumulate in WAT niches, where they remodel the microenvironment and produce beneficial or detrimental effects on systemic metabolism. Given the abundance of macrophages in WAT, this review summarizes the heterogeneity of WAT macrophages in physiological and pathological conditions, including their alterations in quantity, phenotypes, characteristics, and functions during WAT growth and development, as well as healthy or unhealthy expansion. We will discuss the interactions of macrophages with other cell partners in WAT including adipose stem cells, adipocytes, and T cells in the context of various microenvironment niches in lean or obese condition. Finally, we highlight how adipose tissue macrophages merge immunity and metabolic changes to govern energy balance for the organism.
The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the ...molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.
Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors ...indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.
The functional conversion of white adipose tissue (WAT) into a tissue with brown adipose tissue (BAT)-like activity, often referred to as “browning,” represents an intriguing strategy for combating ...obesity and metabolic disease. We demonstrate that thyroid hormone receptor (TR) activation by a synthetic agonist markedly induces a program of adaptive thermogenesis in subcutaneous WAT that coincides with a restoration of cold tolerance to cold-intolerant mice. Distinct from most other browning agents, pharmacological TR activation dissociates the browning of WAT from activation of classical BAT. TR agonism also induces the browning of white adipocytes in vitro, indicating that TR-mediated browning is cell autonomous. These data establish TR agonists as a class of browning agents, implicate the TRs in the browning of WAT, and suggest a profound pharmacological potential of this action.
Display omitted
•TR activation elicits a program of thermogenesis in subcutaneous white adipocytes•TR-mediated browning coincides with anti-obesogenic and anti-diabetic effects•TR-agonist-induced browning of white adipocytes is cell autonomous•TR-mediated browning dissociates activation of WAT from classical BAT
Lin et al. demonstrate that activation of the thyroid hormone receptors by a synthetic agonist strongly induces a brown-fat-like program of adaptive thermogenesis and increased metabolism in white adipocytes both in vivo and in vitro.
The pancreatic islet is a dense cellular network comprised of several cell types with endocrine function vital in the control of glucose homeostasis, metabolism, and feeding behavior. Within the ...islet, endocrine hormones also form an intricate paracrine network with supportive cells (endothelial, neuronal, immune) and secondary signaling molecules regulating cellular function and survival. Modulation of these signals has potential consequences for diabetes development, progression, and therapeutic intervention. Beta cell loss, reduced endogenous insulin secretion, and dysregulated glucagon secretion are hallmark features of both type 1 and 2 diabetes that not only impact systemic regulation of glucose, but also contribute to the function and survival of cells within the islet. Advancing research and technology have revealed new islet biology (cellular identity and transcriptomes) and identified previously unrecognized paracrine signals and mechanisms (somatostatin and ghrelin paracrine actions), while shifting prior views of intraislet communication. This review will summarize the paracrine signals regulating islet endocrine function and survival, the disruption and dysfunction that occur in diabetes, and potential therapeutic targets to preserve beta cell mass and function.
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