Sirtuin 6 regulates glucose-stimulated insulin secretion in mouse pancreatic beta cells

AIMS/HYPOTHESIS: Sirtuin 6 (SIRT6) has been implicated in ageing, DNA repair and metabolism; however, its function in pancreatic beta cells is unclear. The aim of this study is to elucidate the role of SIRT6 in pancreatic beta cells. METHODS: To investigate the function of SIRT6 in pancreatic beta cells, we performed Sirt6 gene knockdown in MIN6 cells and generated pancreatic- and beta cell-specific Sirt6 knockout mice. Islet morphology and glucose-stimulated insulin secretion (GSIS) were analysed. Glycolysis and oxygen consumption rates in SIRT6-deficient beta cells were measured. Cytosolic calcium was monitored using the Fura-2-AM fluorescent probe (Invitrogen, Grand Island, NY, USA). Mitochondria were analysed by immunoblots and electron microscopy. RESULTS: Sirt6 knockdown in MIN6 beta cells led to a significant decrease in GSIS. Pancreatic beta cell Sirt6 knockout mice showed a ~50% decrease in GSIS. The knockout mouse islets had lower ATP levels compared with the wild-type controls. Mitochondrial oxygen consumption rates were significantly decreased in the SIRT6-deficient beta cells. Cytosolic calcium dynamics in response to glucose or potassium chloride were attenuated in the Sirt6 knockout islets. Numbers of damaged mitochondria were increased and mitochondrial complex levels were decreased in the SIRT6-deficient islets. CONCLUSIONS/INTERPRETATION: These data suggest that SIRT6 is important for GSIS from pancreatic beta cells and activation of SIRT6 may be useful to improve insulin secretion in diabetes

Sirt6 Regulates Insulin Secretion from the Pancreatic Beta Cells

poster abstractSirt6 is an NAD-dependent histone deacetylase, which is involved in multiple biological processes, including aging, DNA repair, and metabolism; however, it is unclear what its functions in pancreatic beta-cells are. The beta cells play an essential role in metabolic regulation by secreting insulin in response to an elevated glucose concentration in the circulation. To examine the role of Sirt6 in beta cells, we initially used adenovirus-mediated shRNA to knock down the Sirt6 gene expression in a mouse pancreatic beta cell line - MIN6. Knockdown of the Sirt6 gene significantly reduced glucose-stimulated insulin secretion. To further validate this phenotype in vivo, we generated pancreatic beta-cell-specific Sirt6 knockout mice (bKO) using mouse genetic approach. Indeed, the bKO mice showed remarkable impairment in both first and second phases of insulin secretion in response to a glucose load. While morphometric analyses did not reveal significant difference in islet area between wild-type and bKO mice, biochemical analysis of ATP concentrations showed a 22% decrease in bKO mouse islets relative to control wild-type islets after glucose stimulation. To assess mitochondrial function in Sirt6-deficient beta cells, we also performed Seahorse bioenergetics assays in MIN6 cells after the Sirt6 gene was knocked down. Glucose oxidation in mitochondria was decreased 20-30% in Sirt6- knockdown MIN6 cells as compared to the control cells. Since calcium signaling is critical to insulin secretion, we also measured intracellular calcium concentrations using a fluorescent imaging approach. The results showed a significant decrease in cytoplasmic calcium in the bKO islets as compared to the wild-type controls. Overall, our data demonstrate that Sirt6 plays a critical role in the regulation of pancreatic insulin secretion. This work was supported in part by the NIDDK grant R01DK091592

The epigenetic regulator SIRT6 protects the liver from alcohol-induced tissue injury by reducing oxidative stress in mice

BACKGROUND & AIMS: As a nicotinamide adenine dinucleotide-dependent deacetylase and a key epigenetic regulator, sirtuin 6 (SIRT6) has been implicated in the regulation of metabolism, DNA repair, and inflammation. However, the role of SIRT6 in alcohol-related liver disease (ALD) remains unclear. The aim of this study was to investigate the function and mechanism of SIRT6 in ALD pathogenesis. METHODS: We developed and characterized Sirt6 knockout (KO) and transgenic mouse models that were treated with either control or ethanol diet. Hepatic steatosis, inflammation, and oxidative stress were analyzed using biochemical and histological methods. Gene regulation was analyzed by luciferase reporter and chromatin immunoprecipitation assays. RESULTS: The Sirt6 KO mice developed severe liver injury characterized by a remarkable increase of oxidative stress and inflammation, whereas the Sirt6 transgenic mice were protected from ALD via normalization of hepatic lipids, inflammatory response, and oxidative stress. Our molecular analysis has identified a number of novel Sirt6-regulated genes that are involved in antioxidative stress, including metallothionein 1 and 2 (Mt1 and Mt2). Mt1/2 genes were downregulated in the livers of Sirt6 KO mice and patients with alcoholic hepatitis. Overexpression of Mt1 in the liver of Sirt6 KO mice improved ALD by reducing hepatic oxidative stress and inflammation. We also identified a critical link between SIRT6 and metal regulatory transcription factor 1 (Mtf1) via a physical interaction and functional coactivation. Mt1/2 promoter reporter assays showed a strong synergistic effect of SIRT6 on the transcriptional activity of Mtf1. CONCLUSIONS: Our data suggest that SIRT6 plays a critical protective role against ALD and it may serve as a potential therapeutic target for ALD. LAY SUMMARY: The liver, the primary organ for ethanol metabolism, can be damaged by the byproducts of ethanol metabolism, including reactive oxygen species. In this study, we have identified a key epigenetic regulator SIRT6 that plays a critical role in protecting the liver from oxidative stress-induced liver injury. Thus, our data suggest that SIRT6 may be a potential therapeutic target for alcohol-related liver disease

Neurospora COP9 Signalosome Integrity Plays Major Roles for Hyphal Growth, Conidial Development, and Circadian Function

The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EXnHXHX10D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCFFWD-1 complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa

Food Safety Practices of Food Handlers in China and their Correlation with Self-reported Foodborne Illness

Food service facilities are important sites where foodborne diseases have been reported to occur frequently. This study aims to determine the correlation between self-reported foodborne diseases and food-safety practices followed by food handlers of various food service facilities. A cross-sectional survey was conducted from March 1, 2022 to December 30, 2022 in Wuhu City, Anhui Province, China. Data were collected through face-to-face interviews and having the selected food handlers fill in a self-compiled questionnaire. Of the 1072 food handlers included in the study, 88 (8.2%) reported having experienced symptoms of foodborne diseases in the past 4 weeks. The following food-safety practices correlated with self-reported foodborne diseases: (1) infrequently using 3-compartment sinks to separately clean different types of raw food materials (P < 0.05, OR = 2.312); (2) infrequently removing non-edible parts of aquatic products outside a specific room for food processing (P < 0.001, OR = 3.916); (3) infrequently immediately refrigerating cold dishes prepared in advance to be consumed later (P < 0.001, OR = 4.048); (4) often store perishable foods at 8–60°C in the indoor environment after cooking and before eating (P < 0.05, OR = 2.068); (5) infrequently reheating cooked perishable food stored at 8–60°C for more than 2 h before eating (P < 0.05, OR = 1.934); and (6) often storing raw and cooked food in the same container (P < 0.001, OR = 3.818). Hence, a better supervision of food-safety practices of catering workers may reduce the frequency of foodborne-disease outbreaks in food service facilities

Mutations within the JAMM motif of CSN-5 abolish CSN–mediated deneddylation activity for Cul1, Cul3, and Cul4.

<p>(A) Amino acid alignment of conserved JAMM motifs of CSN-5 homologs from <i>Neurospora crassa</i> (Ncr), <i>Homo sapiens</i> (Hsa), <i>Arabidopsis thaliana</i> (Ath), <i>Drosophila melanogaster</i> (Dme), <i>Caenorhabditis elegans</i> (Cel), and <i>Schizosaccharomyces pombe</i> (Spb). (B) Predicted structure of the <i>N. crassa</i> CSN-5 JAMM domain. The structure was generated by the SWISS-MODEL using the structure of the pre-mRNA splicing factor Prp8 as template (PDB accession number 2P8R), and the functional sites (His127, His129, and Asp140) were mapped according to the structure alignment with the AfJAMM structure (PDB accession number 1R5X). (C–E) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 (C), Myc-Cul3 (D), and Myc-Cul4 (E) in the wild-type strain, <i>csn-5^KO</i>, and CSN-5 complementation strains. (F) Western blot analysis showing the expression of Cul4 in the wild-type, <i>cul4^KO</i>, and <i>csn-5^KO</i> strains. (G) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 in the wild-type strain, <i>csn-5^KO</i>, and <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5 or <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5tri complementation strains. (H) Western blot analyses showing the expression of endogenous Cul4 in the wild-type strain, <i>csn-5^KO</i>, <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5 or <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5tri complementation strains. The asterisk indicates a nonspecific cross-reacted protein band recognized by our Cul4 antiserum (in F and H).</p

Mutations in the JAMM motif of CSN-5 partially restore SCF-mediated FRQ degradation in the <i>csn-5^KO</i> strain.

<p>(A) Western blot analyses showing degradation of FRQ protein in <i>csn-5</i> mutant and the different CSN-5 complementation strains after addition of cycloheximide (10 mg/mL). Asterisks indicate nonspecific bands detected by FRQ antibody. (B) Densitometric analyses from four independent experiments showing the degradation of FRQ in different strains.</p

CSN-5–mutated CSN efficiently prevents degradation of components of the SCF^FWD-1 complex.

<p>(A–C) Western blot analyses with labeled antibodies showing degradation of Myc-Cul1 (A), Myc-SKP-1 (B), and FWD-1 (C) after addition of cycloheximide (10 mg/mL) in the wild-type, <i>csn-5^KO</i>, and different CSN-5 complementation strains. Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FWD-1 antibody. (D–F) Densitometric analyses from four independent experiments showing the degradation of Myc-Cul1 (D), Myc-SKP-1 (E), and FWD-1 (F).</p

Stakeholders' willingness to pay for the new construction and demolition waste landfill charge scheme in Shenzhen : a contingent valuation approach

To promote the sorting and recycling rate of construction and demolition waste (CDW), economically incentive policy has been widely adopted by developed economies. Referring to the successful landfill charge scheme in Hong Kong, a renewal CDW landfill charge scheme was assumed to be implemented in Shenzhen. Instead of thumb rules, this study employed contingent valuation method (CVM) to evaluate the local stakeholders’ willingness to pay (WTP) for disposal of CDW. This study aims to set a reasonable landfill charge level instead of a theoretically ideal one in order to facilitate industry's behavioral change. In essence, this study attempts to find the tipping point that the stakeholders will change their waste handling behavior. The construction industry of Shenzhen was surveyed and a total of 106 valid questionnaires were collected. Results show the mean WTPs for disposal of Inert CDW, Half mixed CDW and Mixed CDW were RMBÂ¥6.1, 24.0 and 33.6 (about US$ 0.89, 3.53 and 4.94) per ton respectively. Through regression analysis, firm size, ownership, position, environmental concern, perceived benefit and effectiveness were found to have significant effect on stakeholders’ WTP for the disposal of CDW. These results provide useful references for improving the current CDW landfill charge scheme in Shenzhen

CAND1 is not required for regulation of the circadian rhythm.

<p>(A–C) Normal conidiation rhythms of <i>cand1</i> mutants in dark–dark (A), light–dark (B), and temperature cycles (C) measured by race tube assays. At least four replicates were tested under each condition. Black lines indicate the growth fronts every 24 h.</p