Ventromedial hypothalamic nucleus subset stimulates tissue thermogenesis via preoptic area outputs.

OBJECTIVE: Hypothalamic signals potently stimulate energy expenditure by engaging peripheral mechanisms to restore energy homeostasis. Previous studies have identified several critical hypothalamic sites (e.g. preoptic area (POA) and ventromedial hypothalamic nucleus (VMN)) that could be part of an interconnected neurocircuit that controls tissue thermogenesis and essential for body weight control. However, the key neurocircuit that can stimulate energy expenditure has not yet been established. METHODS: Here, we investigated the downstream mechanisms by which VMN neurons stimulate adipose tissue thermogenesis. We manipulated subsets of VMN neurons acutely as well as chronically and studied its effect on tissue thermogenesis and body weight control, using Sf1Cre and Adcyap1Cre mice and measured physiological parameters under both high-fat diet and standard chow diet conditions. To determine the node efferent to these VMN neurons, that is involved in modulating energy expenditure, we employed electrophysiology and optogenetics experiments combined with measurements using tissue-implantable temperature microchips. RESULTS: Activation of the VMN neurons that express the steroidogenic factor 1 (Sf1; VMNSf1 neurons) reduced body weight, adiposity and increased energy expenditure in diet-induced obese mice. This function is likely mediated, at least in part, by the release of the pituitary adenylate cyclase-activating polypeptide (PACAP; encoded by the Adcyap1 gene) by the VMN neurons, since we previously demonstrated that PACAP, at the VMN, plays a key role in energy expenditure control. Thus, we then shifted focus to the subpopulation of VMNSf1 neurons that contain the neuropeptide PACAP (VMNPACAP neurons). Since the VMN neurons do not directly project to the peripheral tissues, we traced the location of the VMNPACAP neurons' efferents. We identified that VMNPACAP neurons project to and activate neurons in the caudal regions of the POA whereby these projections stimulate tissue thermogenesis in brown and beige adipose tissue. We demonstrated that selective activation of caudal POA projections from VMNPACAP neurons induces tissue thermogenesis, most potently in negative energy balance and activating these projections lead to some similar, but mostly unique, patterns of gene expression in brown and beige tissue. Finally, we demonstrated that the activation of the VMNPACAP neurons' efferents that lie at the caudal POA are necessary for inducing tissue thermogenesis in brown and beige adipose tissue. CONCLUSIONS: These data indicate that VMNPACAP connections with the caudal POA neurons impact adipose tissue function and are important for induction of tissue thermogenesis. Our data suggests that the VMNPACAP → caudal POA neurocircuit and its components are critical for controlling energy balance by activating energy expenditure and body weight control.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

OBJECTIVE: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. METHODS: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. RESULTS: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. CONCLUSIONS: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

Robust single nucleus RNA sequencing reveals depot-specific cell population dynamics in adipose tissue remodeling during obesity.

Single nucleus RNA sequencing (snRNA-seq), an alternative to single cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying adipose tissue remodeling during obesity. By integrating bulk nuclear RNA-seq from adipocyte nuclei of different sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot. Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses. Furthermore, our differential gene expression analysis reveals distinct contributions of adipocyte subpopulations to the overall pathophysiology of adipose tissue. Our study establishes a robust snRNA-seq method, providing novel insights into the mechanisms orchestrating adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

Objective: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. Methods: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice, to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active UCP1 expression. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. Results: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. However, endogenous Ucp1 was not actively expressed as Ucp1-CreERT2 failed to induce the reporter expression in the mammary glands. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. Conclusions: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

Chronic cAMP activation induces adipocyte browning through discordant biphasic remodeling of transcriptome and chromatin accessibility.

OBJECTIVE: Adipose tissue thermogenesis has been suggested as a new therapeutic target to promote energy metabolism for obesity and metabolic disease. Cold-inducible thermogenic adipocytes, called beige adipocytes, have attracted significant attention for their potent anti-obesity activity in adult humans. In this study, we identified the mechanisms underlying beige adipocyte recruitment, so-called adipocyte browning, by different stimuli. METHODS: We generated a new adipocyte cell line with enhanced browning potentials and determined its transcriptomic and epigenomic responses following cAMP (forskolin, FSK) versus PPARγ activation (rosiglitazone). We performed time-course RNA-seq and compared the treatments and in vivo adipocyte browning. We also developed an improved protocol for Assay for Transposase Accessible Chromatin-sequencing (ATAC-seq) and defined changes in chromatin accessibility in a time course. The RNA-seq and ATAC-seq data were integrated to determine the kinetics of their coordinated regulation and to identify a transcription factor that drives these processes. We conducted functional studies using pharmacological and genetic approaches with specific inhibitors and shRNA-mediated knockdown, respectively. RESULTS: FSK, not rosiglitazone, resulted in a biphasic transcriptomic response, resembling the kinetics of in vivo cold-induced browning. FSK promoted tissue remodeling first and subsequently shifted energy metabolism, concluding with a transcriptomic profile similar to that induced by rosiglitazone. The thermogenic effects of FSK were abolished by PPARγ antagonists, indicating PPARγ as a converging point. ATAC-seq uncovered that FSK leads to a significant chromatin remodeling that precedes or persists beyond transcriptomic changes, whereas rosiglitazone induces minimal changes. Motif analysis identified nuclear factor, interleukin 3 regulated (NFIL3) as a transcriptional regulator connecting the biphasic response of FSK-induced browning, as indicated by disrupted thermogenesis with NFIL3 knockdown. CONCLUSIONS: Our findings elucidated unique dynamics of the transcriptomic and epigenomic remodeling in adipocyte browning, providing new mechanistic insights into adipose thermogenesis and molecular targets for obesity treatment.

Adipocytes fail to maintain cellular identity during obesity due to reduced PPARγ activity and elevated TGFß-SMAD signaling.

OBJECTIVE: Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. METHODS: We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), 3) and PPARγ cistrome (ChIP-seq) profiles in mice fed chow or a high-fat diet (HFD) for 10 weeks. We also assessed the impact of the PPARγ agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from the HFD-fed mice. We integrated these data to determine the transcription factors underlying adipocyte responses to HFD and conducted functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. RESULTS: Adipocytes from the HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes and enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARγ binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGFß1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and associated with myofibroblast signature genes. TGFß1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFß1. CONCLUSIONS: Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPARγ activity and elevated TGFß-SMAD signaling. This cellular identity crisis may be a fundamental mechanism that drives functional decline of adipose tissues during obesity.

Warming Induces Significant Reprogramming of Beige, but Not Brown, Adipocyte Cellular Identity.

Beige and brown adipocytes generate heat in response to reductions in ambient temperature. When warmed, both beige and brown adipocytes exhibit morphological "whitening," but it is unknown whether or to what extent this represents a true shift in cellular identity. Using cell-type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a white chromatin state. Despite this profound shift in cellular identity, warm whitened beige adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised enhancers that prime thermogenic genes for rapid response during a second bout of cold exposure. We further show that a transcriptional cascade involving glucocorticoid receptor and Zfp423 can drive warm-induced whitening of beige adipocytes. These studies identify the epigenomic and transcriptional bases of an extraordinary example of cellular plasticity in response to environmental signals.

Adipocyte glucocorticoid receptor is important in lipolysis and insulin resistance due to exogenous steroids, but not insulin resistance caused by high fat feeding.

OBJECTIVE: The critical role of adipose tissue in energy and nutrient homeostasis is influenced by many external factors, including overnutrition, inflammation, and exogenous hormones. Prior studies have suggested that glucocorticoids (GCs) in particular are major drivers of physiological and pathophysiological changes in adipocytes. In order to determine whether these effects directly require the glucocorticoid receptor (GR) within adipocytes, we generated adipocyte-specific GR knockout (AGRKO) mice. METHODS: AGRKO and control mice were fed chow or high fat diet (HFD) for 14 weeks. Alternatively, AGRKO and control mice were injected with dexamethasone for two months. Glucose tolerance, insulin sensitivity, adiposity, lipolysis, thermogenesis, and insulin signaling were assessed. RESULTS: We find that obesity, insulin resistance, and dysglycemia associated with high fat feeding do not require an intact GR in the adipocyte. However, exogenous dexamethasone (Dex) promotes metabolic dysfunction in mice, and this effect is reduced in mice lacking GR in adipocytes. The ability of Dex to promote "whitening" of brown fat is also reduced in these animals. We also show that GR is required for ß-adrenergic and cold stimulation-mediated lipolysis via expression of the key lipolytic enzyme ATGL. CONCLUSIONS: Our data suggest that the GR plays a role in normal adipose physiology via effects on lipolysis and mediates at least some of the adverse effects of exogenous steroids on metabolic function. The data also indicate that intra-adipocyte GR plays less of a role than previously believed in the local and systemic pathology associated with overnutrition.

Vitreous Hyper-Reflective Dots in Optical Coherence Tomography and Retinal Tear in Patients with Acute Posterior Vitreous Detachment.

PURPOSE: To report the presence of hyper-reflective dots in the vitreous cavity using spectral-domain optical coherence tomography (SD-OCT) in patients with acute symptomatic posterior vitreous detachment (PVD) and investigate their association with the presence of retinal tear. METHODS: The medical records of 77 patients with acute symptomatic PVD, who were examined between March 2013 and February 2015, were reviewed. The severity of vitreous hyper-reflective dots (VHDs) was graded using SD-OCT images, and the presence of retinal tear was assessed. RESULTS: Forty-one (53.2%) eyes had mild VHDs, 13 (16.9%) eyes had moderate VHDs, and 14 (18.2%) eyes had severe VHDs. Retinal tear was found in 21 (27.3%) eyes. The presence of severe VHDs was associated with an increased likelihood of retinal tear (positive likelihood ratio, 9.78; 95% confidence interval, 3.02-31.63). In 14 (66.7%) eyes with retinal tear, the mean number of VHDs significantly decreased from 23.2 ± 20.27 to 2.3 ± 2.66 at a mean follow-up interval of 2.8 ± 1.48 weeks (P = 0.002). CONCLUSIONS: The presence of severe VHDs is suggestive of retinal tear in patients with acute symptomatic PVD. However, this SD-OCT finding should be limited to the acute phase of PVD.

Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo.

Epigenomic mechanisms direct distinct gene expression programs for different cell types. Various in vivo tissues have been subjected to epigenomic analysis; however, these studies have been limited by cellular heterogeneity, resulting in composite gene expression and epigenomic profiles. Here, we introduce "NuTRAP," a transgenic mouse that allows simultaneous isolation of cell-type-specific translating mRNA and chromatin from complex tissues. Using NuTRAP, we successfully characterize gene expression and epigenomic states of various adipocyte populations in vivo, revealing significant differences compared to either whole adipose tissue or in vitro adipocyte cell lines. We find that chromatin immunoprecipitation sequencing (ChIP-seq) using NuTRAP is highly efficient, scalable, and robust with even limited cell input. We further demonstrate the general utility of NuTRAP by analyzing hepatocyte-specific epigenomic states. The NuTRAP mouse is a resource that provides a powerful system for cell-type-specific gene expression and epigenomic profiling.

IRF3 promotes adipose inflammation and insulin resistance and represses browning.

The chronic inflammatory state that accompanies obesity is a major contributor to insulin resistance and other dysfunctional adaptations in adipose tissue. Cellular and secreted factors promote the inflammatory milieu of obesity, but the transcriptional pathways that drive these processes are not well described. Although the canonical inflammatory transcription factor NF-κB is considered to be the major driver of adipocyte inflammation, members of the interferon regulatory factor (IRF) family may also play a role in this process. Here, we determined that IRF3 expression is upregulated in the adipocytes of obese mice and humans. Signaling through TLR3 and TLR4, which lie upstream of IRF3, induced insulin resistance in murine adipocytes, while IRF3 knockdown prevented insulin resistance. Furthermore, improved insulin sensitivity in IRF3-deficient mice was associated with reductions in intra-adipose and systemic inflammation in the high fat-fed state, enhanced browning of subcutaneous fat, and increased adipose expression of GLUT4. Taken together, the data indicate that IRF3 is a major transcriptional regulator of adipose inflammation and is involved in maintaining systemic glucose and energy homeostasis.

Comparison of impact of four surgical methods on surgical outcomes in endoscopic dacryocystorhinostomy.

PURPOSE: To evaluate differences in the surgical outcomes of endoscopic dacryocystorhinostomy (DCR) according to four different surgical methods. MATERIAL AND METHODS: This retrospective study included 222 patients who underwent endoscopic DCR from 2011 to 2013. All patients were assigned to one of four groups according to instruments for incision of nasal mucosa and the formation of mucosal flap: group 1, a sickle knife with mucosal flap; group 2, a sickle knife without mucosal flap; group 3, electrocautery with mucosal flap; and group 4, electrocautery without mucosal flap. The follow up period was at least 6 months. RESULTS: There were 33 eyes in group 1, 44 eyes in group 2, 49 eyes in group 3, and 97 eyes in group 4. There were no significant differences in success rate between groups (P = 0.878). Wound healing time was significantly different between groups (P < 0.001). In post hoc analysis, wound healing time was significantly shorter in group 1 and group 2 than in group 3 and group 4. The vertical ostium size and postsurgical complication were not significantly different between groups. CONCLUSIONS: The use of cold instruments such as sickle knife may be more helpful and effective for shortening wound healing time rather than making mucosal flaps in endoscopic DCR.

Changes of the Macular Ganglion Cell-Inner Plexiform Layer Thickness after Cataract Surgery in Glaucoma Patients.

Purpose. To investigate the effect of uneventful cataract surgery on macular ganglion cell-inner plexiform layer (mGC-IPL) thickness in glaucoma patients. Methods. This retrospective study included 65 eyes of 65 subjects who underwent uneventful cataract surgery, including 13 glaucoma eyes and 52 normal eyes. Using spectral domain optical coherence tomography, the mGC-IPL thickness was measured and compared between glaucoma and normal eyes preoperatively as well as 1 month and 3 months postoperatively. Linear regression analysis was used to determine the factors associated with postoperative change in mGC-IPL thickness. Results. The mean mGC-IPL significantly increased in both groups 1 month and 3 months after surgery (all P values equal to or less than 0.001). The postoperative changes between groups were not significantly different (P = 0.171). In the multivariate regression analysis, preoperative mGC-IPL thickness showed a significant association with the change of average mGC-IPL thickness 1 month and 3 months after surgery (all P values < 0.001). Conclusions. The mean mGC-IPL thickness was increased after cataract surgery, and the postoperative mGC-IPL thickness changes were associated with preoperative mGC-IPL thickness in both groups and axial length in normal eye. The effects of cataract surgery on mean mGC-IPL thickness were not different in glaucomatous and normal eyes.

Systemic Comorbidities of Dry Eye Syndrome: The Korean National Health and Nutrition Examination Survey V, 2010 to 2012.

PURPOSE: To identify systemic comorbidities in patients with dry eye syndrome in South Korea. METHODS: From 2010 to 2012, 17,364 participants aged 20 or older were randomly included in the nationwide Korean National Health and Nutrition Examination Survey V. The prevalence of dry eye syndrome and demographics of these patients were investigated. We performed conditional logistic regression analyses based on age, sex, residential area, education level, occupation type, and household income level to obtain the odds ratio for each systemic comorbidity among subjects with and without dry eye syndrome. RESULTS: The prevalence of dry eye syndrome in this study was 10.4%. Age [adjusted odds ratio (AOR): 1.02], female gender (AOR: 3.01), and indoor occupation (AOR: 1.30) were associated with a higher prevalence of dry eye syndrome and found to be less prevalent in those residing in rural areas (AOR: 0.73) and with lower education levels (AOR: 0.66-0.99). With regard to systemic comorbidities, dyslipidemia (AOR: 1.63), degenerative arthritis (AOR: 1.56), rheumatoid arthritis (AOR: 1.44), thyroid disease (AOR: 1.79), and renal failure (AOR: 2.56) were associated with a significantly higher prevalence of dry eye syndrome. CONCLUSIONS: We found that patients with dry eye syndrome have a higher prevalence of several systemic comorbidities. A more comprehensive therapeutic approach considering the effect of systemic medication may be necessary in these patients.

The effect of corneal irregularity on astigmatism measurement by automated versus ray tracing keratometry.

The aim of this study was to compare the effect of corneal irregularity on astigmatism assessment using automated keratometry (AK) (IOLMaster) versus ray tracing keratometry (Pentacam). This is an observational case series approved by the institutional review board of Dongguk University Hospital, Goyang, South Korea. A total of 207 eyes of 207 cataract patients were included. Preoperative corneal astigmatism was measured by both IOLMaster and Pentacam. Corneal irregularity index (IR) was calculated in Fourier analysis map of Pentacam. AK by IOLMaster and total corneal refractive power (TCRP, 3 mm and 4 mm zone analysis with pupil centered) by Pentacam were selected and the difference between the 2 measurements (delta Δ) was calculated using vector analysis. Ocular residual astigmatism (ORA) after cataract surgery was calculated by subtracting 6-month postoperative refractive astigmatism (RA) measurements from corresponding preoperative values (AK, TCRP3, and TCRP4). The mean irregularity index measured was 0.042 ±â€Š0.019 mm (mean ±â€Šstandard deviation) and was positively correlated with age and magnitude of corneal astigmatism (P < 0.001 and P < 0.05). The difference (Δ) between TCRPs and AK (ΔTCRPs-AK) was 0.43 ±â€Š0.37 (TCRP3) and 0.39 ±â€Š0.35 (TCRP4) diopters. Linear regression analysis revealed that age (P < 0.001), IR (P < 0.001), and AK (P < 0.001) were positively correlated with ΔTCRPs-AK. In highly irregular corneas (IR over 0.77 diopters: mean + 2 standard deviation), postoperative ORAs calculated using TCRPs were significantly lower than ORAs calculated using AK. Corneal irregularities significantly impact astigmatism assessment by IOLMaster (AK) and Pentacam (TCRPs). Compared with AK, TCRPs were more accurate in predicting postoperative residual astigmatism in highly irregular corneas.

A modular system of DNA enhancer elements mediates tissue-specific activation of transcription by high dietary zinc in C. elegans.

Zinc is essential for biological systems, and aberrant zinc metabolism is implicated in a broad range of human diseases. To maintain homeostasis in response to fluctuating levels of dietary zinc, animals regulate gene expression; however, mechanisms that mediate the transcriptional response to fluctuating levels of zinc have not been fully defined. Here, we identified DNA enhancer elements that mediate intestine-specific transcriptional activation in response to high levels of dietary zinc in C. elegans. Using bioinformatics, we characterized an evolutionarily conserved enhancer element present in multiple zinc-inducible genes, the high zinc activation (HZA) element. The HZA was consistently adjacent to a GATA element that mediates expression in intestinal cells. Functional studies using transgenic animals demonstrated that this modular system of DNA enhancers mediates tissue-specific transcriptional activation in response to high levels of dietary zinc. We used this information to search the genome and successfully identified novel zinc-inducible genes. To characterize the mechanism of enhancer function, we demonstrated that the GATA transcription factor ELT-2 and the mediator subunit MDT-15 are necessary for zinc-responsive transcriptional activation. These findings define new mechanisms of zinc homeostasis and tissue-specific regulation of transcription.

ttm-1 encodes CDF transporters that excrete zinc from intestinal cells of C. elegans and act in a parallel negative feedback circuit that promotes homeostasis.

Zinc is an essential metal involved in a wide range of biological processes, and aberrant zinc metabolism is implicated in human diseases. The gastrointestinal tract of animals is a critical site of zinc metabolism that is responsible for dietary zinc uptake and distribution to the body. However, the role of the gastrointestinal tract in zinc excretion remains unclear. Zinc transporters are key regulators of zinc metabolism that mediate the movement of zinc ions across membranes. Here, we identified a comprehensive list of 14 predicted Cation Diffusion Facilitator (CDF) family zinc transporters in Caenorhabditis elegans and demonstrated that zinc is excreted from intestinal cells by one of these CDF proteins, TTM-1B. The ttm-1 locus encodes two transcripts, ttm-1a and ttm-1b, that use different transcription start sites. ttm-1b expression was induced by high levels of zinc specifically in intestinal cells, whereas ttm-1a was not induced by zinc. TTM-1B was localized to the apical plasma membrane of intestinal cells, and analyses of loss-of-function mutant animals indicated that TTM-1B promotes zinc excretion into the intestinal lumen. Zinc excretion mediated by TTM-1B contributes to zinc detoxification. These observations indicate that ttm-1 is a component of a negative feedback circuit, since high levels of cytoplasmic zinc increase ttm-1b transcript levels and TTM-1B protein functions to reduce the level of cytoplasmic zinc. We showed that TTM-1 isoforms function in tandem with CDF-2, which is also induced by high levels of cytoplasmic zinc and reduces cytoplasmic zinc levels by sequestering zinc in lysosome-related organelles. These findings define a parallel negative feedback circuit that promotes zinc homeostasis and advance the understanding of the physiological roles of the gastrointestinal tract in zinc metabolism in animals.

Lysosome-related organelles in intestinal cells are a zinc storage site in C. elegans.

Zinc is an essential trace element involved in many biological processes and human diseases. Because zinc deficiency and excess are deleterious, animals require homeostatic mechanisms to maintain zinc levels in response to dietary fluctuations. Here, we demonstrate that lysosome-related organelles in intestinal cells of C. elegans, called gut granules, function as the major site of zinc storage. Zinc storage in gut granules promotes detoxification and subsequent mobilization, linking cellular and organismal zinc metabolism. The cation diffusion facilitator protein CDF-2 plays a critical role in this process by transporting zinc into gut granules. In response to high dietary zinc, gut granules displayed structural changes characterized by a bilobed morphology with asymmetric distributions of zinc and molecular markers. We defined a genetic pathway that mediates the formation of bilobed morphology. These findings elucidate mechanisms of zinc storage, detoxification, and mobilization in C. elegans and may be relevant to other animals.

Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-κB-dependent pathway, leading to monocyte adhesion to endothelial cells.

Enterotoxigenic Bacteroides fragilis (ETBF) produces a ∼ 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although a variety of inflammatory cells is found at ETBF-infected sites, little is known about leukocyte adhesion in response to BFT stimulation. We investigated whether BFT affected the expression of ICAM-1 and monocytic adhesion to endothelial cells (ECs). Stimulation of HUVECs and rat aortic ECs with BFT resulted in the induction of ICAM-1 expression. Upregulation of ICAM-1 was dependent on the activation of IκB kinase (IKK) and NF-κB signaling. In contrast, suppression of AP-1 did not affect ICAM-1 expression in BFT-stimulated cells. Suppression of NF-κB activity in HUVECs significantly reduced monocytic adhesion, indicating that ICAM-1 expression is indispensable for BFT-induced adhesion of monocytes to the endothelium. Inhibition of JNK resulted in a significant attenuation of BFT-induced ICAM-1 expression in ECs. Moreover, inhibition of aldose reductase significantly reduced JNK-dependent IKK/NF-κB activation, ICAM-1 expression, and adhesion of monocytes to HUVECs. These results suggest that a signaling pathway involving aldose reductase, JNK, IKK, and NF-κB is required for ICAM-1 induction in ECs exposed to BFT, and may be involved in the leukocyte-adhesion cascade following infection with ETBF.

The cation diffusion facilitator gene cdf-2 mediates zinc metabolism in Caenorhabditis elegans.

Zinc is essential for many cellular processes. To use Caenorhabditis elegans to study zinc metabolism, we developed culture conditions allowing full control of dietary zinc and methods to measure zinc content of animals. Dietary zinc dramatically affected growth and zinc content; wild-type worms survived from 7 microm to 1.3 mm dietary zinc, and zinc content varied 27-fold. We investigated cdf-2, which encodes a predicted zinc transporter in the cation diffusion facilitator family. cdf-2 mRNA levels were increased by high dietary zinc, suggesting cdf-2 promotes zinc homeostasis. CDF-2 protein was expressed in intestinal cells and localized to cytosolic vesicles. A cdf-2 loss-of-function mutant displayed impaired growth and reduced zinc content, indicating that CDF-2 stores zinc by transport into the lumen of vesicles. The relationships between three cdf genes, cdf-1, cdf-2, and sur-7, were analyzed in double and triple mutant animals. A cdf-1 mutant displayed increased zinc content, whereas a cdf-1 cdf-2 double mutant had intermediate zinc content, suggesting cdf-1 and cdf-2 have antagonistic functions. These studies advance C. elegans as a model of zinc metabolism and identify cdf-2 as a new gene that has a critical role in zinc storage.