Erin Easlon

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells.

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.

Tissue-specific regulation of SIRT1 by calorie restriction

Calorie restriction (CR) has been reported to increase SIRT1 protein levels in mice, rats, and humans, and elevated activity of SIRT1 orthologs extends life span in yeast, worms, and flies. In this study, we challenge the paradigm that CR induces SIRT1 activity in all tissues by showing that activity of this sirtuin in the liver is, in fact, reduced by CR and activated by a high-caloric diet. We demonstrate this change both by assaying levels of SIRT1 and its small molecule regulators, NAD and NADH, as well as assessing phenotypes of a liver-specific SIRT1 knockout mouse on various diets. Our findings suggest that designing CR mimetics that target SIRT1 to provide uniform systemic benefits may be more complex than currently imagined.

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate-aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1Deltaaat1Delta double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate-aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.

The role of calorie restriction and SIRT1 in prion-mediated neurodegeneration

A central focus of aging research is to determine how calorie restriction (CR) extends lifespan and delays diseases of aging. SIRT1, the mammalian ortholog of Sir2 in yeast, is a longevity factor which mediates dietary restriction in diverse species. In addition, SIRT1 plays a protective role in several models of neurodegenerative disease. We tested the role of SIRT1 in mediating the effects of CR in a mouse model of prion disease. Prion diseases are protein misfolding disorders of the central nervous system with many similarities to other neurodegenerative diseases, including deposition of aggregated protein, gliosis, and loss of synapses and neurons. We report that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum. CR exerts no further effect on the SIRT1 KO strain, suggesting the effects of CR and SIRT1 deletion are mechanistically coupled. In conjunction, SIRT1 is downregulated in certain brain regions of CR mice. The expression of PrP mRNA and protein is reduced in the brains of CR mice and in SIRT1 knockout mice, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb to prion disease. Surprisingly, CR greatly shortens the duration of clinical symptoms of prion disease and ultimately shortens lifespan of prion-inoculated mice in a manner that is independent of SIRT1. Taken together, our results suggest a more complex interplay between CR, SIRT1, and neurodegenerative diseases than previously appreciated.

Calorie restriction and the nutrient sensing signaling pathways

Abstract.Calorie restriction (CR) is the most potent regimen known to extend the life span in multiple species. CR has also been shown to ameliorate several age-associated disorders in mammals and perhaps humans. CR induces diverse metabolic changes in organisms, and it is currently unclear whether and how these metabolic changes lead to life span extension. Recent studies in model systems have provided insight into the molecular mechanisms by which CR extends life span. In this review, we summarize and provide recent updates on multiple nutrient signaling pathways that have been connected to CR and longevity regulation. The roles of highly conserved longevity regulators — the Sirtuin family- in CR are also discussed.

The Dihydrolipoamide Acetyltransferase Is a Novel Metabolic Longevity Factor and Is Required for Calorie Restriction-mediated Life Span Extension

Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.

Deleting the 14-3-3 Protein Bmh1 Extends Life Span in Saccharomyces cerevisiae by Increasing Stress Response

Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from age-dependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1Δ-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1Δ mutant also displays a decreased reactive oxygen species level and increased heat-shock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR- and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

Identification of Potential Calorie Restriction-Mimicking Yeast Mutants with Increased Mitochondrial Respiratory Chain and Nitric Oxide Levels

Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Correction: Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells.

The authors would like to clarify that the controls previously depicted in Figs ​Figs4E4E and ​and7A7A were for different experiments and were included in error. Fig 4 UCP2 is Up-Regulated in Sirt1 Knockdown Cells and in Sirt1 KO Mice. Fig 7 UCP2 mRNA or Protein Levels in Fed or Starved Wild-Type Mice. The correct control for Fig 4E was located and used to prepare a corrected figure. The correct control for the original Fig 7A could not be located; this panel has therefore been removed after a careful assessment and investigation determined that the result for which original Fig 7A was cited is supported elsewhere in this article, and that removal of this panel does not affect the conclusions of the paper. We have also taken this opportunity to provide new versions of several figures (Figs ​(Figs4,4, ​,5,5, ​,6,6, ​,7)7) in which gel/blot splices and a non-linear level adjustment were made but were not previously indicated or declared, or to replace incorrectly spliced gels/blots with the un-spliced originals. We also take the opportunity to correct two errors in the legend to Fig 6, first to remove a redundant and incorrect sentence, and second to address incorrect description of p values. Fig 5 Sirt1 Binds at the UCP2 Promoter and Represses the Gene. Fig 6 Knockdown of UCP2 in Sirt1 Knockdown Cells Restores Glucose-Induced Insulin Secretion. The text in the Results section titled “UCP2 Levels Increase in Food-Deprived Mice” has been edited to accommodate the removal of the original Fig 7A and the relabeling of Fig 7B, 7C and 7D as Fig 7A, 7B and 7C, respectively. The corrected text and Figs ​Figs4,4, ​,5,5, ​,66 and ​and77 are provided here.

The Effects of Practice-Based Training on Graduate Teaching Assistants’ Classroom Practices

This article describes implementation of a practice-based graduate teaching assistant (GTA)-training program. GTA implementation of evidence-based teaching practices is measured across 160 hours of videotaped classroom instruction, and longitudinal changes in instructional practices are investigated. The impact of feedback on GTA adoption of evidence-based practices is also assessed.