Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in ß-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

Calcium-Sensing Receptor in Adipose Tissue: Possible Association with Obesity-Related Elevated Autophagy.

Autophagy is upregulated in adipose tissue (AT) from people with obesity. We showed that activation of the calcium-sensing receptor (CaSR) elevates proinflammatory cytokines through autophagy in preadipocytes. Our aim is to understand the role of CaSR on autophagy in AT from humans with obesity. We determined mRNA and protein levels of CaSR and markers of autophagy by qPCR and western blot in human visceral AT explants or isolated primary preadipocytes (60 donors: 72% female, 23-56% body fat). We also investigated their association with donors' anthropometric variables. Donors' % body fat and CaSR mRNA expression in AT were correlated (r = 0.44, p < 0.01). CaSR expression was associated with mRNA levels of the autophagy markers atg5 (r = 0.37, p < 0.01), atg7 (r = 0.29, p < 0.05) and lc3b (r = 0.40, p < 0.01). CaSR activation increased becn and atg7 mRNA expression in AT. CaSR activation also upregulated LC3II by ~50%, an effect abolished by the CaSR inhibitor. Spermine (CaSR agonist) regulates LC3II through the ERK1/2 pathway. Structural equation model analysis suggests a link between donors' AT CaSR expression, AT autophagy and expression of Tumor Necrosis Factor alpha TNF-α. CaSR expression in visceral AT is directly associated with % body fat, and CaSR activation may contribute to obesity-related disruption in AT autophagy.

Calcium sensing receptor effects in adipocytes and liver cells: Implications for an adipose-hepatic crosstalk.

The calcium sensing receptor (CaSR) is expressed in human adipose cells, and its activation may associate with adipose tissue (AT) dysfunction. We evaluated whether CaSR stimulation influences adipocyte triglyceride (TG) and fatty acid binding protein 4 (aP2) content, and hepatocyte TGs and proinflammatory cytokine expression. The effect of the calcimimetic cinacalcet on TGs (fluorimetry), lipogenic genes (qPCR) and aP2 (immunoblot) was evaluated in LS14 adipocytes or AT. In the human HepG2 hepatic cell line, we assessed CaSR expression and cinacalcet effect on TGs and lipogenic and proinflammatory genes. CaSR activation decreased adipocyte TG content by 20% and the expression of GPD and LPL by 34% and 20%, respectively. Cinacalcet increased aP2 protein expression by 60%. CaSR expression was shown in HepG2 cells and human liver samples. Cinacalcet-treated HepG2 cells in the presence of oleic acid exhibited a19% increased TG content. No changes were observed in the expression of lipogenic genes in HepG2 cells, however there was a 50%-300% elevation in the expression of proinflammatory cytokines. CaSR activation in adipocytes may associate with decreased TG storage ability and increased aP2. Hepatic CaSR stimulation may elevate steatosis and proinflammatory factors. We propose that CaSR may contribute to obesity-associated hepatic metabolic consequences.

Insulin and leptin oscillations license food-entrained browning and metabolic flexibility.

Timed feeding drives adipose browning, although the integrative mechanisms for the same remain unclear. Here, we show that twice-a-night (TAN) feeding generates biphasic oscillations of circulating insulin and leptin, representing their entrainment by timed feeding. Insulin and leptin surges lead to marked cellular, functional, and metabolic remodeling of subcutaneous white adipose tissue (sWAT), resulting in increased energy expenditure. Single-cell RNA-sequencing (scRNA-seq) analyses and flow cytometry demonstrate a role for insulin and leptin surges in innate lymphoid type 2 (ILC2) cell recruitment and sWAT browning, since sWAT depot denervation or loss of leptin or insulin receptor signaling or ILC2 recruitment each dampens TAN feeding-induced sWAT remodeling and energy expenditure. Consistently, recreating insulin and leptin oscillations via once-a-day timed co-injections is sufficient to favorably remodel innervated sWAT. Innervation is necessary for sWAT remodeling, since denervation of sWAT, but not brown adipose tissue (BAT), blocks TAN-induced sWAT remodeling and resolution of inflammation. In sum, reorganization of nutrient-sensitive pathways remodels sWAT and drives the metabolic benefits of timed feeding.

Corrigendum: Impact of short and long exposure to cafeteria diet on food intake and white adipose tissue lipolysis mediated by glucagon-like peptide 1 receptor.

[This corrects the article DOI: 10.3389/fendo.2023.1164047.].

Impact of short and long exposure to cafeteria diet on food intake and white adipose tissue lipolysis mediated by glucagon-like peptide 1 receptor.

Introduction: The modern food environment facilitates excessive calorie intake, a major driver of obesity. Glucagon-like peptide 1 (GLP1) is a neuroendocrine peptide that has been the basis for developing new pharmacotherapies against obesity. The GLP1 receptor (GLP1R) is expressed in central and peripheral tissues, and activation of GLP1R reduces food intake, increases the expression of thermogenic proteins in brown adipose tissue (BAT), and enhances lipolysis in white adipose tissue (WAT). Obesity decreases the efficiency of GLP1R agonists in reducing food intake and body weight. Still, whether palatable food intake before or during the early development of obesity reduces the effects of GLP1R agonists on food intake and adipose tissue metabolism remains undetermined. Further, whether GLP1R expressed in WAT contributes to these effects is unclear. Methods: Food intake, expression of thermogenic BAT proteins, and WAT lipolysis were measured after central or peripheral administration of Exendin-4 (EX4), a GLP1R agonist, to mice under intermittent-short exposure to CAF diet (3 h/d for 8 days) or a longer-continuous exposure to CAF diet (24 h/d for 15 days). Ex-vivo lipolysis was measured after EX4 exposure to WAT samples from mice fed CAF or control diet for 12 weeks. . Results: During intermittent-short exposure to CAF diet (3 h/d for 8 days), third ventricle injection (ICV) and intra-peritoneal administration of EX4 reduced palatable food intake. Yet, during a longer-continuous exposure to CAF diet (24 h/d for 15 days), only ICV EX4 administration reduced food intake and body weight. However, this exposure to CAF diet blocked the increase in uncoupling protein 1 (UCP1) caused by ICV EX4 administration in mice fed control diet. Finally, GLP1R expression in WAT was minimal, and EX4 failed to increase lipolysis ex-vivo in WAT tissue samples from mice fed CAF or control diet for 12 weeks. . Discussion: Exposure to a CAF diet during the early stages of obesity reduces the effects of peripheral and central GLP1R agonists, and WAT does not express a functional GLP1 receptor. These data support that exposure to the obesogenic food environment, without the development or manifestation of obesity, can alter the response to GLP1R agonists. .

mTORC2-NDRG1-CDC42 axis couples fasting to mitochondrial fission.

Fasting triggers diverse physiological adaptations including increases in circulating fatty acids and mitochondrial respiration to facilitate organismal survival. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during fasting remain incompletely understood. Here we show that fasting or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its downstream target NDRG1 at serine 336 sustains mitochondrial fission and respiratory sufficiency. Time-lapse imaging shows that NDRG1, but not the phosphorylation-deficient NDRG1Ser336Ala mutant, engages with mitochondria to facilitate fission in control cells, as well as in those lacking DRP1. Using proteomics, a small interfering RNA screen, and epistasis experiments, we show that mTORC2-phosphorylated NDRG1 cooperates with small GTPase CDC42 and effectors and regulators of CDC42 to orchestrate fission. Accordingly, RictorKO, NDRG1Ser336Ala mutants and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions; however, paradoxical reactivation of mTORC2 during fasting unexpectedly drives mitochondrial fission and respiration.

Integrating the effects of sucrose intake on the brain and white adipose tissue: Could autophagy be a possible link?

Excess dietary sucrose is associated with obesity and metabolic diseases. This relationship is driven by the malfunction of several cell types and tissues critical for the regulation of energy balance, including hypothalamic neurons and white adipose tissue (WAT). However, the mechanisms behind these effects of dietary sucrose are still unclear and might be independent of increased adiposity. Accumulating evidence has indicated that dysregulation of autophagy, a fundamental process for maintenance of cellular homeostasis, alters energy metabolism in hypothalamic neurons and WAT, but whether autophagy could mediate the detrimental effects of dietary sucrose on hypothalamic neurons and WAT that contribute to weight gain is a matter of debate. In this review, we examine the hypothesis that dysregulated autophagy in hypothalamic neurons and WAT is an adiposity-independent effect of sucrose that contributes to increased body weight gain. We propose that excess dietary sucrose leads to autophagy unbalance in hypothalamic neurons and WAT, which increases caloric intake and body weight, favoring the emergence of obesity and metabolic diseases.

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line.

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

Leptin stimulates autophagy/lysosome-related degradation of long-lived proteins in adipocytes.

Obesity, a condition most commonly associated with hyper-leptinemia, is also characterized by increased expression of autophagy genes and likely autophagic activity in human adipose tissue (AT). Indeed, circulating leptin levels were previously shown to positively associate with the expression levels of autophagy genes such as Autophagy related gene-5 (ATG5). Here we hypothesized that leptin acts in an autocrine-paracrine manner to increase autophagy in two major AT cell populations, adipocytes and macrophages. We followed the dynamics of autophagosomes following acute leptin administration with or without a leptin receptor antagonist (SMLA) using high-throughput live-cell imaging in murine epididymal adipocyte and macrophage (RAW264.7) cell-lines. In macrophages leptin exerted only a mild effect on autophagy dynamics, tending to attenuate autophagosomes growth rate. In contrast, leptin-treated adipocytes exhibited a moderate, ~20% increase in the rate of autophagosome growth, an effect that was blocked by SMLA. This finding corresponded to mild increases in mRNA and protein expression of key autophagy genes. Interestingly, a long-lived proteins degradation assay uncovered a robust, >2-fold leptin-mediated stimulation of the autophagy/lysosome-related (bafilomycin-inhibited) activity, which was entirely blocked by SMLA. Collectively, leptin regulates autophagy in a cell-type specific manner. In adipocytes, autophagosome dynamics is moderately enhanced, but even more pronounced stimulation is seen in autophagy-related long-lived protein degradation. These findings suggest a causal link between obesity-associated hyperleptinemia and elevated adipocyte and AT autophagy-related processes.

Autophagy mediates calcium-sensing receptor-induced TNFα production in human preadipocytes.

Obesity is a major current public health problem worldwide due to the severe co-morbid conditions that this disease entails. The development of obesity-related cardiometabolic disorders is in direct association with adipose tissue inflammation that leads to its functional impairment. Activation of the Calcium-Sensing Receptor (CaSR) in adipose tissue contributes to inflammation and adipose dysfunction. Autophagy, a process of cell component degradation, is closely related to inflammation in many diseases, however, whether autophagy is associated with CaSR-induced inflammation remains unknown. Using LS14 and SW872 preadipose cell lines as well as primary human preadipocytes, we show that CaSR activation with the allosteric activator cinacalcet induces autophagosome formation. Cinacalcet-induced LC3II content elevation was precluded by knockdown of the CaSR and enhanced by CaSR overexpression, indicating a specific effect. Autophagy inhibition using 3-methyladenine prevented CaSR-induced TNFα production, indicating that autophagy contributes to CaSR-induced inflammation in human preadipocytes. Our results suggest that modulation of CaSR-induced autophagy is an attractive target in obese inflamed adipose tissue, to prevent the development of diseases triggered by adipose dysfunction. We describe a novel mechanism and possible new target to modulate and prevent adipose inflammation and hence the resulting disease-generating adipose tissue dysfunction.

Calcium Sensing Receptor as a Novel Mediator of Adipose Tissue Dysfunction: Mechanisms and Potential Clinical Implications.

Obesity is currently a serious worldwide public health problem, reaching pandemic levels. For decades, dietary and behavioral approaches have failed to prevent this disease from expanding, and health authorities are challenged by the elevated prevalence of co-morbid conditions. Understanding how obesity-associated diseases develop from a basic science approach is recognized as an urgent task to face this growing problem. White adipose tissue (WAT) is an active endocrine organ, with a crucial influence on whole-body homeostasis. WAT dysfunction plays a key role linking obesity with its associated diseases such as type 2 diabetes mellitus, cardiovascular disease, and some cancers. Among the regulators of WAT physiology, the calcium-sensing receptor (CaSR) has arisen as a potential mediator of WAT dysfunction. Expression of the receptor has been described in human preadipocytes, adipocytes, and the human adipose cell lines LS14 and SW872. The evidence suggests that CaSR activation in the visceral (i.e., unhealthy) WAT is associated with an increased proliferation of adipose progenitor cells and elevated adipocyte differentiation. In addition, exposure of adipose cells to CaSR activators in vitro elevates proinflammatory cytokine expression and secretion. An increased proinflammatory environment in WAT plays a key role in the development of WAT dysfunction that leads to peripheral organ fat deposition and insulin resistance, among other consequences. We propose that CaSR may be one relevant therapeutic target in the struggle to confront the health consequences of the current worldwide obesity pandemic.

Preadipocyte proliferation is elevated by calcium sensing receptor activation.

Obesity is a major worldwide problem, despite considerable efforts against it. While excess body fat defines obesity, adipose tissue quality and functionality are key to whether cardiovascular and metabolic comorbidities develop. Adipose tissue cellular composition can vary considerably, and excess adipocyte progenitors (preadipocytes) is associated with obesity. We have proposed that calcium sensing receptor (CaSR) activation in adipose tissue leads to dysfunction. This study evaluated whether CaSR activation elevates preadipocyte proliferation. Human LS14 preadipocytes were exposed to CaSR activators cinacalcet (2 µM), GdCl3 (5 µM) and spermine (1 µM), and cell viability was evaluated after 72h. CaSR activators elevated proliferation by 19-24%, and CaSR silencing (siRNA) abolished the effect. Cinacalcet elevated phospho-ERK1/2 content, and upstream inhibition of ERK1/2 phosphorylation reverted cinacalcet-induced proliferation. Cinacalcet also elevated expression of the proinflammatory factors IL1ß, IL6 and CCL2. The results suggest that CaSR induces preadipocyte proliferation, partly through ERK1/2 activation. Considering reported proinflammatory and adipogenic CaSR effects, excess preadipocyte proliferation further supports the dysfunctional effect of CaSR in obesity.

Antilipolytic effect of calcimimetics depends on the allelic variant of calcium-sensing receptor gene polymorphism rs1042636 (Arg990Gly).

Calcium-sensing receptor polymorphism rs1042636 (Arg990Gly) affects the response to the calcimimetic cinacalcet, used to treat hypercalcemia in secondary hyperparathyroidism (sHPT) or parathyroid carcinoma. Carriers of the Arg allelle, show less parathyroid hormone secretion suppression in response to the drug. This effect was reproducible in transfected cultured human embryonic kidney cells, supporting a causal relationship on the protein level. We previously established that cinacalcet has an antilipolytic effect in isolated human adipocytes; however, there were a number of samples that did not respond to the treatment. The present work aimed to investigate whether the variable antilipolytic response to cinacalcet in adipocytes was consistent with the effect reported for the rs1042636 polymorphism. Lipolysis was assessed by measuring glycerol release after exposure to cinacalcet (10 µM) or vehicle in adipocytes isolated from 38 donors. Responsiveness was defined as lipolysis suppression (cinacalcet vs vehicle control) greater than 20%. Genotype analysis showed that 23 adipocyte donors were homozygous for Arg at position 990, 14 heterozygous and 1 homozygous Gly-Gly. Among the Arg homozygotes, one was responsive to cinacalcet, whereas five Gly carriers responded to the calcimimetic. In all, 83% of adipocytes showing response to cinacalcet carried the glycine allele, whereas in 96% of Arg-Arg individuals adipocytes did not respond to the calcimimetic (P=0.027, Fisher's exact test). Confirming sHPT observations, adipocytes from rs1042636 Gly-allele carriers show higher sensitivity to the antilipolytic action of cinacalcet. The potential benefit of cinacalcet as a suppressor of basal lipolysis and free fatty acid release in uremic patients needs to consider the rs1042636 single-nucleotide polymorphism.