Featured ArticlesVolume 14 | August 2018
|The lncRNA Blnc1 orchestrates adipose tissue remodeling to preserve metabolic healthBrown fat long noncoding RNA 1 (Blnc1) is a conserved long noncoding RNA (lncRNA) that is enriched in brown adipose tissue and promotes differentiation of cultured brown and beige adipocytes. Zhao et al. investigated the role of Blnc1 in adipose tissue remodeling. They found that fat-specific inactivation of Blnc1 accelerates brown fat whitening and impairs homeostatic fat expansion during a high-fat diet, leading to adipose tissue inflammation, insulin resistance and hepatic steatosis. Adipocyte-specific transgenic expression of Blnc1 elicited the opposite, beneficial metabolic effects. This shows a surprisingly powerful role of lncRNA in orchestrating adipocyte adaptation to obesity and maintaining systemic metabolic health.
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Objective: Long noncoding RNAs (lncRNAs) are emerging as powerful regulators of adipocyte differentiation and gene expression. However, their significance in adipose tissue metabolism and physiology has not been demonstrated in vivo. We previously identified Blnc1 as a conserved lncRNA regulator of brown and beige adipocyte differentiation. In this study, we investigated the physiological role of Blnc1 in thermogenesis, adipose remodeling and systemic metabolism.
Methods: We generated fat-specific Blnc1 transgenic and conditional knockout mouse strains and investigated how adipocyte Blnc1 levels are causally linked to key aspects of metabolic health following diet-induced obesity. We performed studies using cultured adipocytes to establish cell-autonomous role of Blnc1 in regulating adipocyte gene programs.
Results: Blnc1 is highly induced in both brown and white fats from obese mice. Fat-specific inactivation of Blnc1 impairs cold-induced thermogenesis and browning and exacerbates obesity-associated brown fat whitening, adipose tissue inflammation and fibrosis, leading to more severe insulin resistance and hepatic steatosis. On the contrary, transgenic expression of Blnc1 in adipose tissue elicits the opposite and beneficial metabolic effects, supporting a critical role of Blnc1 in driving adipose adaptation and homeostatic remodeling during obesity. Mechanistically, Blnc1 cell-autonomously attenuates proinflammatory cytokine signaling and promotes fuel storage in adipocytes through its protein partner Zbtb7b.
Conclusions: This study illustrates a surprisingly pleiotropic and dominant role of lncRNA in driving adaptive adipose tissue remodeling and preserving metabolic health.[Hide abstract]
|Three-dimensional volume imaging of vascular plasticity in adipose tissuesAdipose tissues are among the most highly vascularized organs of the body, with each adipocyte being in direct contact with capillaries. Intra-adipose vasculatures increase or decrease their density in response to various metabolic stimuli or stresses, a process known as vascular plasticity. Modulation of the vasculatures may exert significant and long-lasting effects on adipose metabolism. Cao, Wang et al. report a 3D volume fluorescence-imaging procedure to visualize the adipose vascular network at single-capillary resolution. To demonstrate the power of this imaging technique, they assess the pathological remodeling of vasculatures in adipose tissues under obese conditions and characterize the physiological changes of vasculatures in response to a cold challenge.|
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Objective: The vascular system is central to sustaining tissue survival and homeostasis. Blood vessels are densely present in adipose tissues and exert essential roles in their metabolism. However, conventional immunohistochemistry methods have intrinsic limitations in examining the 3D vascular network in adipose tissues as well as other organs in general.
Methods: We established a 3D volume fluorescence-imaging technique to visualize the vasculatures in mouse adipose tissues by combining the optimized steps of whole-mount immunolabeling, tissue optical clearing, and lightsheet volume fluorescence-imaging. To demonstrate the strength of this novel imaging procedure, we comprehensively assessed the intra-adipose vasculatures under obese conditions or in response to a cold challenge.
Results: We show the entirety of the vascular network in mouse adipose tissues on the whole-tissue level at a single-capillary resolution for the first time in the field. We accurately quantify the pathological changes of vasculatures in adipose tissues in wild-type or obese mice (ob/ob, db/db, or diet-induced obesity). In addition, we identify significant and reversible changes of the intra-adipose vasculatures in the mice subjected to cold challenge (i.e., 4°). Furthermore, we demonstrate that the cold-induced vascular plasticity depends on the sympathetic-derived catecholamine signal and is involved in the beiging process of white adipose tissues.
Conclusions: We report a 3D volume fluorescence-imaging procedure that is compatible with many areas of vascular research and is poised to serve the field in future investigations of the vascular system in adipose tissues or other research scenarios.[Hide abstract]
|Plasma N-Acylethanolamine levels as a biomarker of obesity and dysmetabolismN-acylethanolamines (NAEs) are bioactive lipids that are involved in a wide spectrum of processes, including inflammation, pain perception, and energy balance. Their exact role in determining, maintaining, and reflecting the obese status has not been clarified. Fanelli and colleagues tested the hypothesis that plasma NAE levels as well as their ratios are influenced by BMI and by menopause in women and ageing in men. Their results support the relevance of the NAE system as a target for interventions aiming at restoring metabolic health in humans.
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Objective: N-acylethanolamines play different roles in energy balance; anandamide (AEA) stimulates energy intake and storage, N-palmitoylethanolamide (PEA) counters inflammation, and N-oleoylethanolamide (OEA) mediates anorectic signals and lipid oxidation. Inconsistencies in the association of plasma N-acylethanolamines with human obesity and cardiometabolic risk have emerged among previous studies, possibly caused by heterogeneous cohorts and designs, and by unstandardized N-acylethanolamine measurements. We aimed to characterize changes in the plasma profile, including N-acylethanolamine levels and ratios associated with obesity, menopause in women, and ageing in men, and to define the significance of such a profile as a biomarker for metabolic imbalance.
Methods: Adult, drug-free women (n = 103 premenopausal and n = 81 menopausal) and men (n = 144) were stratified according to the body mass index (BMI) into normal weight (NW; BMI: 18.5–24.9 kg/m2), overweight (OW; BMI: 25.0–29.9 kg/m2), and obese (OB; BMI ≥30.0 kg/m2). Anthropometric and metabolic parameters were determined. Validated blood processing and analytical procedures for N-acylethanolamine measurements were used. We investigated the effect of BMI and menopause in women, and BMI and age in men, as well as the BMI-independent influence of metabolic parameters on the N-acylethanolamine profile.
Results: BMI and waist circumference directly associated with AEA in women and men, and with PEA in premenopausal women and in men, while BMI directly associated with OEA in premenopausal women and in men. BMI, in both genders, and waist circumference, in women only, inversely associated with PEA/AEA and OEA/AEA. Menopause increased N-acylethanolamine levels, whereas ageing resulted in increasing OEA relative abundance in men. AEA and OEA abundances in premenopausal, and PEA and OEA abundances in lean menopausal women, were directly associated with hypertension. Conversely, PEA and OEA abundances lowered with hypertension in elderly men. Insulin resistance was associated with changes in N-acylethanolamine ratios specific for premenopausal (reduced PEA/AEA and OEA/AEA), menopausal (reduced OEA/AEA) women and men (reduced OEA/AEA and OEA/PEA). PEA and OEA levels increased with total cholesterol, and OEA abundance specifically increased with HDL-cholesterol. Elevated triglyceride levels were associated with increased N-acylethanolamine levels only in menopausal women.
Conclusions: Obesity-related N-acylethanolamine hypertone is characterized by imbalanced N-acylethanolamine ratios. The profile given by a combination of N-acylethanolamine absolute levels and ratios enables imbalances to be identified in relationship with different metabolic parameters, with specific relevance according to gender, menopause and age, representing a useful means for monitoring metabolic health. Finally, N-acylethanolamine system appears a promising target for intervention strategies.[Hide abstract]
|Pancreatic deletion of the IL-1 receptor disrupts glucose homeostasis and promotes β-cell de-differentiationThe interleuking-1 (IL-1) signaling system is associated with both acute and chronic inflammatory events. Inflammation is linked with reductions in β-cell function and mass in both type 1 and type 2 diabetes. Burke and colleagues studied the consequences of pancreatic IL-1 receptor deletion in mice. They found reduced glucose tolerance and insulin secretion, as well as signs of β-cell de-differentiation.|
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Objective: Pancreatic tissue, and islets in particular, are enriched in expression of the interleukin-1 receptor type I (IL-1R). Because of this enrichment, islet β-cells are exquisitely sensitive to the IL-1R ligands IL-1α and IL-1β, suggesting that signaling through this pathway regulates health and function of islet β-cells.
Methods: Herein, we report a targeted deletion of IL-1R in pancreatic tissue (IL-1RPdx1−/−) in C57BL/6J mice and in db/db mice on the C57 genetic background. Islet morphology, β-cell transcription factor abundance, and expression of the de-differentiation marker Aldh1a3 were analyzed by immunofluorescent staining. Glucose and insulin tolerance tests were used to examine metabolic status of these genetic manipulations. Glucose-stimulated insulin secretion was evaluated in vivo and in isolated islets ex vivo by perifusion.
Results: Pancreatic deletion of IL-1R leads to impaired glucose tolerance, a phenotype that is exacerbated by age. Crossing the IL-1RPdx1−/− with db/db mice worsened glucose tolerance without altering body weight. There were no detectable alterations in insulin tolerance between IL-1RPdx1−/− mice and littermate controls. However, glucose-stimulated insulin secretion was reduced in islets isolated from IL-1RPdx1−/− relative to control islets. Insulin output in vivo after a glucose challenge was also markedly reduced in IL-1RPdx1−/− mice when compared with littermate controls. Pancreatic islets from IL-1RPdx1−/− mice displayed elevations in Aldh1a3, a marker of de-differentiation, and reduction in nuclear abundance of the β-cell transcription factor MafA. Nkx6.1 abundance was unaltered.
Conclusions: There is an important physiological role for pancreatic IL-1R to promote glucose homeostasis by suppressing expression of Aldh1a3, sustaining MafA abundance, and supporting glucose-stimulated insulin secretion in vivo.[Hide abstract]
|Secretagogin protects Pdx1 from proteasomal degradation to control β cell specification Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that controls a transcriptional program which ultimately leads to the formation of functional β cells. It is, however, unknown how signal initiation by calcium is linked to the transcriptional effectors. Malenczyk et al. identify the Ca2+ sensor secretagogin as a protective factor for Pdx1 that acts by proteasome inhibition. By its action, a favorable α to β cell ratio is achieved in Langerhans islets.
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Objective: Specification of endocrine cell lineages in the developing pancreas relies on extrinsic signals from non-pancreatic tissues, which initiate a cell-autonomous sequence of transcription factor activation and repression switches. The steps in this pathway share reliance on activity-dependent Ca2+ signals. However, the mechanisms by which phasic Ca2+ surges become converted into a dynamic, cell-state-specific and physiologically meaningful code made up by transcription factors constellations remain essentially unknown.
Methods: We used high-resolution histochemistry to explore the coincident expression of secretagogin and transcription factors driving β cell differentiation. Secretagogin promoter activity was tested in response to genetically manipulating Pax6 and Pax4 expression. Secretagogin null mice were produced with their pancreatic islets morphologically and functionally characterized during fetal development. A proteomic approach was utilized to identify the Ca2+-dependent interaction of secretagogin with subunits of the 26S proteasome and verified in vitro by focusing on Pdx1 retention.
Results: Here, we show that secretagogin, a Ca2+ sensor protein that controls α and β cell turnover in adult, is in fact expressed in endocrine pancreas from the inception of lineage segregation in a Pax4-and Pax6-dependent fashion. By genetically and pharmacologically manipulating secretagogin expression and interactome engagement in vitro, we find secretagogin to gate excitation-driven Ca2+ signals for β cell differentiation and insulin production. Accordingly, secretagogin−/− fetuses retain a non-committed pool of endocrine progenitors that co-express both insulin and glucagon. We identify the Ca2+-dependent interaction of secretagogin with subunits of the 26S proteasome complex to prevent Pdx1 degradation through proteasome inactivation. This coincides with retained Nkx6.1, Pax4 and insulin transcription in prospective β cells.
Conclusions: In sum, secretagogin scales the temporal availability of a Ca2+-dependent transcription factor network to define β cell identity.[Hide abstract]
|Hepatocyte TLR4 deficiency protects against alcohol-induced fatty liver diseaseEvidence suggests that inflammation is an essential factor contributing to the initiation and progression of alcoholic liver disease (ALD). Previous studies have shown that mutant toll-like receptor 4 (TLR4) mice, or mice deficient in TLR4, display attenuated alcohol-induced fatty liver disease, suggesting a direct role for TLR4 in the development of ALD. In this study, Jia et al. showed that selective TLR4 deletion from hepatocytes protects mice from chronic alcohol-induced liver injury and fatty liver.|
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Objective: Recent studies have suggested a critical role for toll-like receptor 4 (TLR4) in the development of alcoholic liver disease. As TLR4 is widely expressed throughout the body, it is unclear which TLR4-expressing cell types contribute to alcohol-induced liver damage.
Methods: We selectively ablated TLR4 in hepatocytes and myeloid cells. Male mice were fed a liquid diet containing either 5% alcohol or pair-fed a control diet for 4 weeks to examine chronic alcohol intake-induced liver damage and inflammation. In addition, mice were administered a single oral gavage of alcohol to investigate acute alcohol drinking-associated liver injury.
Results: We found that selective hepatocyte TLR4 deletion protected mice from chronic alcohol-induced liver injury and fatty liver. This result was in part due to decreased expression of endogenous lipogenic genes and enhanced expression of genes involved in fatty acid oxidation. In addition, mice lacking hepatocyte TLR4 exhibited reduced mRNA expression of inflammatory genes in white adipose tissue. Furthermore, in an acute alcohol binge model, hepatocyte TLR4 deficient mice had significantly decreased plasma alanine transaminase (ALT) levels and attenuated hepatic triglyceride content compared to their alcohol-gavaged control mice. In contrast, deleting TLR4 in myeloid cells did not affect the development of chronic-alcohol induced fatty liver, despite the finding that mice lacking myeloid cell TLR4 had significantly reduced circulating ALT concentrations.
Conclusions: These findings suggest that hepatocyte TLR4 plays an important role in regulating alcohol-induced liver damage and fatty liver disease.[Hide abstract]
|Specific subpopulations of LepR-expressing neurons mediate the effects of early LepR deletion on energy balanceThe hormone leptin, which is produced by adipose tissue to signal the repletion of fat stores, acts via its receptor (LepRb) on hypothalamic neurons to suppress food intake and permit energy expenditure. Leptin- or LepRb-deficient humans and rodent models display dramatic hyperphagia and reduced energy expenditure, leading to severe obesity. However, the specific LepRb neurons that mediate the largest component of this dramatic obesity phenotype remain undefined. Rupp et al. found that the early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance.|
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Objective: To date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance.
Methods: We generated new mouse lines deleted for LepRb in ARC GhrhCre neurons or in Htr2cCre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells.
Results: The early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance.
Conclusions: Although early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH.[Hide abstract]
|PPARγ is dispensable for clear cell renal cell carcinoma progressionClear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, is characterized by robust intracellular lipid and glycogen accumulation. The peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis. Sanchez and colleagues hypothesized that PPARγ promotes lipid storage in ccRCC and contributes to tumorigenesis, which they tested using loss-of-function experiments. PPARγ deletion in two ccRCC cell lines affected neither viability, proliferation, migratory capacity in vitro, nor tumor growth in a subcutaneous xenograft model. Surprisingly, PPARγ also appears to be dispensable for lipid storage and maintenance of total triglyceride levels in ccRCC cells.
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Objective: Clear cell renal cell carcinoma (ccRCC) is a subtype of kidney cancer defined by robust lipid accumulation, which prior studies have indicated plays an important role in tumor progression. We hypothesized that the peroxisome proliferator-activated receptor gamma (PPARγ), detected in both ccRCC tumors and cell lines, promotes lipid storage in ccRCC and contributes to tumorigenesis in this setting. PPARγ transcriptionally regulates a number of genes involved in lipid and glucose metabolism in adipocytes, yet its role in ccRCC has not been described. The objective of this study was to elucidate endogenous PPARγ function in ccRCC cells.
Methods and results: Using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), we found that PPARγ and its heterodimer RXR occupy the canonical DR1 PPAR binding motif at approximately 1000 locations throughout the genome that can be subdivided into adipose-shared and ccRCC-specific sites. CRISPR-Cas9 mediated, loss-of-function studies determined that PPARγ is dispensable for viability, proliferation, and migration of ccRCC cells in vitro and in vivo. Also, surprisingly, PPARγ deletion had little effect on the robust lipid accumulation that typifies the “clear cell” phenotype of kidney cancer.
Conclusion: Our results suggest that PPARγ plays neither a tumor suppressive nor oncogenic role in advanced ccRCC, and thus single-agent therapeutics targeting PPARγ are unlikely to be effective for the treatment of this disease. The unique cistrome of PPARγ in ccRCC cells demonstrates the importance of cell type in determining the functions of PPARγ.[Hide abstract]
|GIP is upregulated in patients with atherosclerosis and stabilizes plaques in ApoE-/- mice Glucose-dependent insulinotropic peptide (GIP) is secreted by enteroendocrine cells following nutrient intake leading to insulin secretion and glucose control. Kahles, Liberman et al. investigated the role of GIP in atherosclerotic cardiovascular disease with a focus on plaque morphology. They found elevated circulating GIP concentrations in patients with atherosclerosis and identified GIP as a vasoprotective peptide stabilizing atherosclerotic lesions in ApoE-/- mice by preventing monocyte migration and blocking proinflammatory activation of macrophages. Given that plaque erosion and rupture are critical steps in the process of myocardial infarction, these findings might open new therapeutic avenues for patients with high cardiovascular risk.|
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Objective: The incretin hormones GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic peptide) are secreted by the gut after food intake leading to pancreatic insulin secretion and glucose lowering. Beyond its role in glucose control, GLP-1 was found in mice and men to beneficially modulate the process of atherosclerosis, which has been linked to improved cardiovascular outcome of patients with diabetes at high cardiovascular risk treated with GLP-1 receptor agonists. However, little is known on the role of the other main incretin in the cardiovascular system. The aim of this study was to characterize GIP in atherosclerotic cardiovascular disease.
Methods and results: Serum concentrations of GIP were assessed in 731 patients who presented for elective coronary angiography at the University Hospital Aachen. While GIP concentrations were not associated with coronary artery disease (CAD), we found 97 patients with PAD (peripheral artery disease) vs. 634 without PAD to have higher circulating GIP levels (413.0 ± 315.3 vs. 332.7 ± 292.5 pg/mL, p = 0.0165). GIP levels were independently related to PAD after multivariable adjustment for CAD, age, sex, BMI, hypertension, diabetes, CRP, WBC, and smoking. To investigate the functional relevance of elevated GIP levels in human atherosclerotic disease, we overexpressed GIP (1–42) in ApoE−/− mice fed a Western diet for 12 weeks using an adeno-associated viral vector system. GIP overexpression led to reduced atherosclerotic plaque macrophage infiltration and increased collagen content compared to control (LacZ) with no change in overall lesion size, suggesting improved plaque stability. Mechanistically, we found GIP treatment to reduce MCP-1-induced monocyte migration under In vitro conditions. Additionally, GIP prevented proinflammatory macrophage activation leading to reduced LPS-induced IL-6 secretion and inhibition of MMP-9 activity, which was attributable to GIP dependent inhibition of NfκB, JNK-, ERK, and p38 in endotoxin activated macrophages.
Conclusion: Elevated concentrations of the incretin hormone GIP are found in patients with atherosclerotic cardiovascular disease, while GIP treatment attenuates atherosclerotic plaque inflammation in mice and abrogates inflammatory macrophage activation in vitro. These observations identified GIP as a counterregulatory vasoprotective peptide, which might open new therapeutic avenues for the treatment of patients with high cardiovascular risk.[Hide abstract]