Phosphate-activated cyclin-dependent kinase stabilizes G1 cyclin to trigger cell cycle entry

G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability. © 2013, American Society for Microbiology.S. Hernández received a postgraduate Junior Faculty fellowship from the UIC and l'Obra Social la Caixa. This work was supported by grants from Ministerio de Ciencia e Innovación of the Spanish government (BFU 2009-09278)Peer Reviewe

Reduction of Obesity and Insulin Resistance through Dual Targeting of VAT and BAT by a Novel Combination of Metabolic Cofactors

Obesity is an epidemic disease worldwide, characterized by excessive fat accumulation associated with several metabolic perturbations, such as metabolic syndrome, insulin resistance, hypertension, and dyslipidemia. To improve this situation, a specific combination of metabolic cofactors (MC) (betaine, N-acetylcysteine, L-carnitine, and nicotinamide riboside) was assessed as a promising treatment in a high-fat diet (HFD) mouse model. Obese animals were distributed into two groups, orally treated with the vehicle (obese + vehicle) or with the combination of metabolic cofactors (obese + MC) for 4 weeks. Body and adipose depots weights; insulin and glucose tolerance tests; indirect calorimetry; and thermography assays were performed at the end of the intervention. Histological analysis of epidydimal white adipose tissue (EWAT) and brown adipose tissue (BAT) was carried out, and the expression of key genes involved in both fat depots was characterized by qPCR. We demonstrated that MC supplementation conferred a moderate reduction of obesity and adiposity, an improvement in serum glucose and lipid metabolic parameters, an important improvement in lipid oxidation, and a decrease in adipocyte hypertrophy. Moreover, MC-treated animals presented increased adipose gene expression in EWAT related to lipolysis and fatty acid oxidation. Furthermore, MC supplementation reduced glucose intolerance and insulin resistance, with an increased expression of the glucose transporter Glut4; and decreased fat accumulation in BAT, raising non-shivering thermogenesis. This treatment based on a specific combination of metabolic cofactors mitigates important pathophysiological characteristics of obesity, representing a promising clinical approach to this metabolic disease. Keywords: obesity; adipose tissue; insulin resistance; thermogenesis; metabolic cofactor

Regulation of p27 and cdk2 expression in different adipose tissue depots in aging and obesity

Aging usually comes associated with increased visceral fat accumulation, reaching even an obesity state, and favoring its associated comorbidities. One of the processes involved in aging is cellular senescence, which is highly dependent on the activity of the regulators of the cell cycle. The aim of this study was to analyze the changes in the expression of p27 and cdk2 in different adipose tissue depots during aging, as well as their regulation by obesity in mice. Changes in the expression of p27 and CDK2 in visceral and subcutaneous white adipose tissue (WAT) biopsies were also analyzed in a human cohort of obesity and type 2 diabetes. p27, but not cdk2, exhibits a lower expression in subcutaneous than in visceral WAT in mice and humans. p27 is drastically downregulated by aging in subcutaneous WAT (scWAT), but not in gonadal WAT, of female mice. Obesity upregulates p27 and cdk2 expression in scWAT, but not in other fat depots of aged mice. In humans, a significant upregulation of p27 was observed in visceral WAT of subjects with obesity. Taken together, these results show a differential adipose depot-dependent regulation of p27 and cdk2 in aging and obesity, suggesting that p27 and cdk2 could contribute to the adipose-tissue depot’s metabolic differences. Further studies are necessary to fully corroborate this hypothesis

Enhanced fatty acid oxidation in adipocytes and macrophages reduces lipid-induced triglyceride accumulation and inflammation

Lipid overload in obesity and type 2 diabetes is associated with adipocyte dysfunction, inflammation, macrophage infiltration, and decreased fatty acid oxidation (FAO). Here, we report that the expression of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme in mitochondrial FAO, is higher in human adipose tissue macrophages than in adipocytes and that it is differentially expressed in visceral vs. subcutaneous adipose tissue in both an obese and a type 2 diabetes cohort. These observations led us to further investigate the potential role of CPT1A in adipocytes and macrophages. We expressed CPT1AM, a permanently active mutant form of CPT1A, in 3T3-L1 CARΔ1 adipocytes and RAW 264.7 macrophages through adenoviral infection. Enhanced FAO in palmitate-incubated adipocytes and macrophages reduced triglyceride content and inflammation, improved insulin sensitivity in adipocytes, and reduced endoplasmic reticulum stress and ROS damage in macrophages. We conclude that increasing FAO in adipocytes and macrophages improves palmitate-induced derangements. This indicates that enhancing FAO in metabolically relevant cells such as adipocytes and macrophages may be a promising strategy for the treatment of chronic inflammatory pathologies such as obesity and type 2 diabetes

Maresin 1 activates brown adipose tissue and promotes browning of white adipose tissue in mice

Objective Maresin 1 (MaR1) is a docosahexaenoic acid-derived proresolving lipid mediator with insulin-sensitizing and anti-steatosis properties. Here, we aim to unravel MaR1 actions on brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning. Methods MaR1 actions were tested in cultured murine brown adipocytes and in human mesenchymal stem cells (hMSC)-derived adipocytes. In vivo effects of MaR1 were tested in diet-induced obese (DIO) mice and lean WT and Il6 knockout (Il6−/−) mice. Results In cultured differentiated murine brown adipocytes, MaR1 reduces the expression of inflammatory genes, while stimulates glucose uptake, fatty acid utilization and oxygen consumption rate, along with the upregulation of mitochondrial mass and genes involved in mitochondrial biogenesis and function and the thermogenic program. In Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6)-depleted brown adipocytes using siRNA, the stimulatory effect of MaR1 on thermogenic genes was abrogated. In DIO mice, MaR1 promotes BAT remodeling, characterized by higher expression of genes encoding for master regulators of mitochondrial biogenesis and function and iBAT thermogenic activation, together with increased M2 macrophage markers. In addition, MaR1-treated DIO mice exhibit a better response to cold-induced BAT activation. Moreover, MaR1 induces a beige adipocyte signature in inguinal WAT of DIO mice and in hMSC-derived adipocytes. MaR1 potentiates Il6 expression in brown adipocytes and BAT of cold exposed lean WT mice. Interestingly, the thermogenic properties of MaR1 were abrogated in Il6−/− mice. Conclusions These data reveal MaR1 as a novel agent that promotes BAT activation and WAT browning by regulating thermogenic program in adipocytes and M2 polarization of macrophages. Moreover, our data suggest that LGR6 receptor is mediating MaR1 actions on brown adipocytes, and that IL-6 is required for the thermogenic effects of MaR1

Maresin 1 activates brown adipose tissue and promotes browning of white adipose tissue in mice

[Objective]: Maresin 1 (MaR1) is a docosahexaenoic acid-derived proresolving lipid mediator with insulin-sensitizing and anti-steatosis properties. Here, we aim to unravel MaR1 actions on brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning. [Methods]: MaR1 actions were tested in cultured murine brown adipocytes and in human mesenchymal stem cells (hMSC)-derived adipocytes. In vivo effects of MaR1 were tested in diet-induced obese (DIO) mice and lean WT and Il6 knockout (Il6−/−) mice. [Results]: In cultured differentiated murine brown adipocytes, MaR1 reduces the expression of inflammatory genes, while stimulates glucose uptake, fatty acid utilization and oxygen consumption rate, along with the upregulation of mitochondrial mass and genes involved in mitochondrial biogenesis and function and the thermogenic program. In Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6)-depleted brown adipocytes using siRNA, the stimulatory effect of MaR1 on thermogenic genes was abrogated. In DIO mice, MaR1 promotes BAT remodeling, characterized by higher expression of genes encoding for master regulators of mitochondrial biogenesis and function and iBAT thermogenic activation, together with increased M2 macrophage markers. In addition, MaR1-treated DIO mice exhibit a better response to cold-induced BAT activation. Moreover, MaR1 induces a beige adipocyte signature in inguinal WAT of DIO mice and in hMSC-derived adipocytes. MaR1 potentiates Il6 expression in brown adipocytes and BAT of cold exposed lean WT mice. Interestingly, the thermogenic properties of MaR1 were abrogated in Il6−/− mice. [Conclusions]: These data reveal MaR1 as a novel agent that promotes BAT activation and WAT browning by regulating thermogenic program in adipocytes and M2 polarization of macrophages. Moreover, our data suggest that LGR6 receptor is mediating MaR1 actions on brown adipocytes, and that IL-6 is required for the thermogenic effects of MaR1.The authors received support for the current study from Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, Spain, MCIN/AEI/10.13039/501100011033 (grants BFU2012-36089 to MJM-A; BFU2015-65937-R to MJM-A, SL-C; PID2019-106982RB-I00 to MJM-A; SAF2017-83813-C3-1-R to LH and PID2021-122766OB-I00 to AMV), cofinanced by the European Regional Development Fund (ERDF); Dept. of Health, Navarra Government (67–2015) to MJM-A; Merck Health Foundation to LH; CIBEROBN (CB12/03/30002; CB06/03/0001; CB06/03/0025) and CIBERDEM (CB07/08/0033) from ISCIII (Spain). “Juan de la Cierva” Grant to MF-G (IJCI-2016-30025) funded by MCIN/AEI/10.13039/501100011033. Predoctoral grant to LML (Asociación de Amigos, Universidad de Navarra/“la Caixa” Banking Foundation) and to LM-F (FPI, BES-2013-064970). S.Q.-V. is supported by a fellowship from the Vicente Lopez Program (Eurecat).Peer reviewe

Zinc-Alpha 2-Glycoprotein Gene Expression in Adipose Tissue Is Related with Insulin Resistance and Lipolytic Genes in Morbidly Obese Patients

10.1371/journal.pone.003326

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.The authors wish to thank the staff from all the WWTPs monitored for their help and technical support during the sampling campaigns. The authors acknowledge the funding received from the ACA and the ASPCAT from the Catalan Government (Generalitat de Catalunya). ICRA authors acknowledge the funding provided by the Generalitat de Catalunya through the Consolidated Research Group grants ICRA-ENV 2017 SGR 1124 and ICRA-TiA 2017 SGR 1318. ICRA researchers also thank the funding from the CERCA program of the Catalan Government.Peer reviewe

Control of cell cycle progression by the last MAPK Hog1

Exposure of yeast to increases in extracellular osmolarity activates the stress-activated Hog1 MAP kinase, which is essential for cell survival upon osmotic stress. Activation of the Hog1 MAPK results in cell growth arrest, suggesting a possible role of the MAP kinase in the control of the cell cycle. Our results have shown that Hog1 activation resulted in accumulation of cells in the G1/S and G2/M transitions. At G1, Hog1 regulates the cell cycle progression by a dual mechanism that involves downregulation of G1 cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. The MAPK interacts with Sic1, and phosphorylates a single residue of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell cycle progression. Consistently, sic1_ cells, or cells containing a SIC1 allele mutated in the Hog1 phosphorylation site, are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that Sic1 is the molecular target for Hog1 that is required to modulate cell cycle progression in response to stress at G1. On the other hand, activation of the Hog1 MAPK also results in an increase of cells in the G2 phase. Arrested cells displayed down regulation of the Clb2-Cdc28 kinase activity and consequently enlarged buds, defects in spindle formation and orientation. These effects were prevented by deletion of the SWE1 gene. Thus, swe1Ä cells failed to arrest at G2, which resulted in a premature entry into mitosis and mislocalization of nuclei. Consistently, swe1Ä cells were osmosensitive. Swe1 degradation was reduced in response to activation of Hog1. Swe1 accumulation is mediated by the activity of the complex Hsl1-Hsl7. Hog1 phosphorylates a single residue at the regulatory domain of Hsl1, which leads to the mislocalization of Hsl7 from the bud neck, and consequent Swe1 accumulation. In addition, Hog1 downregulates G2 cyclin expression, reinforcing the inhibition of cell cycle progression at G2/M. These results indicate that Hog1 imposes a delay in critical phases of cell cycle progression necessary for proper cellular adaptation to new extracellular conditions