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• Research Paper Volume 12, Issue 19 pp 19677-19700

  Reduced mitochondrial translation prevents diet-induced metabolic dysfunction but not inflammation

  Relevance score: 7.168065

  Kara L. Perks, Nicola Ferreira, Judith A. Ermer, Danielle L. Rudler, Tara R. Richman, Giulia Rossetti, Vance B. Matthews, Natalie C. Ward, Oliver Rackham, Aleksandra Filipovska

  Keywords: mitochondria, stress response, mTOR and insulin signaling pathways, obesity, metabolic syndrome

  Published in Aging on October 6, 2020

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  The contribution of dysregulated mitochondrial gene expression and consequent imbalance in biogenesis is not well understood in metabolic disorders such as insulin resistance and obesity. The ribosomal RNA maturation protein PTCD1 is essential for mitochondrial protein synthesis and its reduction causes adult-onset obesity and liver steatosis. We used haploinsufficient Ptcd1 mice fed normal or high fat diets to understand how changes in mitochondrial biogenesis can lead to metabolic dysfunction. We show that Akt-stimulated reduction in lipid content and upregulation of mitochondrial biogenesis effectively protected mice with reduced mitochondrial protein synthesis from excessive weight gain on a high fat diet, resulting in improved glucose and insulin tolerance and reduced lipid accumulation in the liver. However, inflammation of the white adipose tissue and early signs of fibrosis in skeletal muscle, as a consequence of reduced protein synthesis, were exacerbated with the high fat diet. We identify that reduced mitochondrial protein synthesis and OXPHOS biogenesis can be recovered in a tissue-specific manner via Akt-mediated increase in insulin sensitivity and transcriptional activation of the mitochondrial stress response.

• Research Paper Volume 12, Issue 18 pp 18033-18051

  Insulin sensitivity in long-lived growth hormone-releasing hormone knockout mice

  Relevance score: 7.1157327

  Fang Zhang, Mert Icyuz, Zhenghui Liu, Michael Fitch, Liou Y. Sun

  Keywords: GHRH^-/- mice, tissues-specific insulin signaling, hyperinsulinemic-euglycemic clamp, AKT/S6, ERK1/2

  Published in Aging on July 8, 2020

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  Our previous studies showed that loss-of-function mutation of growth hormone releasing hormone (GHRH) results in increased longevity and enhanced insulin sensitivity in mice. However, the details of improved insulin action and tissue-specific insulin signaling are largely unknown in this healthy-aging mouse model. We conducted hyperinsulinemic-euglycemic clamp to investigate mechanisms underlying enhanced insulin sensitivity in growth hormone (GH) deficient mice. Further, we assessed in vivo tissue-specific insulin activity via activation of PI3K-AKT and MAPK-ERK1/2 cascades using western blot. Clamp results showed that the glucose infusion rate required for maintaining euglycemia was much higher in GHRH^-/- mice compared to WT controls. Insulin-mediated glucose production was largely suppressed, whereas glucose uptake in skeletal muscle and brown adipose tissue were significant enhanced in GHRH^-/- mice compared to WT controls. Enhanced capacity of insulin-induced activation of the PI3K-AKT and MAPK-ERK1/2 signaling were observed in a tissue-specific manner in GHRH^-/- mice. Enhanced systemic insulin sensitivity in long-lived GHRH^-/- mice is associated with differential activation of insulin signaling cascades among various organs. Improved action of insulin in the insulin sensitive tissues is likely to mediate the prolonged longevity and healthy-aging effects of GH deficiency in mice.

• Research Paper Volume 7, Issue 4 pp 223-232

  Liver-specific deletion of Ppp2cα enhances glucose metabolism and insulin sensitivity

  Relevance score: 9.049522

  Li Xian, Siyuan Hou, Zan Huang, An Tang, Peiliang Shi, Qinghua Wang, Anying Song, Shujun Jiang, Zhaoyu Lin, Shiying Guo, Xiang Gao

  Keywords: PP2A, glucose, insulin signaling, liver, disorder

  Published in Aging on February 21, 2015

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  Protein phosphatase 2A (PP2A) is a key negative regulator of phosphatidylinositol 3-kinase/Akt pathway. Previous study showed that, in the liver, the catalytic subunit of PP2A (PP2Ac) is closely associated with insulin resistance syndrome, which is characterized by glucose intolerance and dyslipidemia. Here we studied the role of liver PP2Ac in glucose metabolism and evaluated whether PP2Ac is a suitable therapeutic target for treating insulin resistance syndrome. Liver-specific Ppp2cα knockout mice (Ppp2cα^loxp/loxp: Alb) exhibited improved glucose homeostasis compared with littermate controls in both normal and high-fat diet conditions, despite no significant changes in body weight and liver weight under chow diet. Ppp2cα^loxp/loxp: Alb mice showed enhanced glycogen deposition, serum triglyceride, cholesterol, low density lipoprotein and high density lipoprotein, activated insulin signaling, decreased expressions of gluconeogenic genes G₆P and PEPCK, and lower liver triglyceride. Liver-specific Ppp2cα knockout mice showed enhanced glucose homeostasis and increased insulin sensitivity by activation of insulin signaling through Akt. These findings suggest that inhibition of hepatic Ppp2cα may be a useful strategy for the treatment of insulin resistance syndrome

  

  Ppp2cα is highly expressed in two models of insulin resistant and liver-specific deletion of Ppp2cα. (a) Q-PCR was performed to measure Ppp2cα mRNA levels in liver from ob/ob of 8weeks and 16 weeks mice fed HFD beginning at 8 weeks of age compared to appropriate controls (n=5). (b) Deletion efficiency of the Ppp2cα allele was analyzed using Q-PCR and western blotting. (c) Body weight (BW) of mice on a normal chow diet (n=8). BW was monitored every week from 6 weeks of age for 34 weeks. (d) Liver weight (normalized to BW) of 8-week-old mice fed ad libitum (n=8).

  
  
  

  

  Deletion of Ppp2cα in the liver improves glucose tolerance (a,b) GTT on male Ppp2cα^loxp/loxp: Alb and control mice (n=7-8) on chow diet (a) at 8 weeks of age and on HFD (b) for 8 weeks (16 weeks of age). Bar graphs to the right show the respective area under the curve (AUC) of glucose. (c) Fed and fasting serum glucose levels. (d) Fed and fasting serum insulin levels. (e) ALT activity. Ppp2cα^loxp/loxp: Alb and WT: Alb control groups are indicated in the figures. Data are represented as mean ± SEM. Data were analyzed using two-tailed Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001).

  
  
  

  

  Loss of Ppp2cα significantly alters metabolism. (a) Accumulation of glycogen was detected by PAS staining and (b) decrease of lipid droplets was detected by Oil Red staining (c) Hepatic glycogen content measured in liver and (d) Hepatic TG content of the liver of 10-week-old Ppp2cα^loxp/loxp: Alb and littermate controls following fasting overnight or random fed. Data were analyzed using two-tailed Student's t test (*p < 0.05)

  
  
  

  

  Enhanced insulin sensitivity in Ppp2cα^loxp/loxp: Alb mice. (a, b) Western blot of insulin signaling involved Akt pathway in the liver (a) and muscle (b). Livers and skeletal muscle of overnight fasted mice on chow were isolated 5mins after 1U/kg insulin treatment. (c) Relative expression of G₆P, PEPCK, and PGC1α mRNAs normalized against 36B4 mRNA levels, measured by Q-PCR in livers from fasted overnight mice. Data were analyzed by two-tailed Student's t test (*p < 0.05, ***p < 0.001).

  
  
  

  

  Effects of wortmannin on glucose of Ppp2cα^loxp/loxp: Alb and WT: Alb mice. (a) GTT and AUC of over fasted Ppp2cα^loxp/loxp: Alb and WT: Alb mice treated with wortmannin lasted 4weeks. (b) Immunoblot analysis with antibodies to Akt, phosphor-Akt (pSer⁴⁷³ and pThr³⁰⁸) was examined in Ppp2cα^loxp/loxp: Alb and WT: Alb mice after injection with saline or insulin after 4 weeks wortmannin treatment. Data were analysed by two-tailed Student's t test (*p < 0.05).

  
  
  

  

  Proposed model for the role of PP2A on insulin signaling. Red and purple arrows illustrate the direct and indirect actions.

  
  
  

• Research Paper Volume 6, Issue 12 pp 1019-1032

  The suppression of ghrelin signaling mitigates age-associated thermogenic impairment

  Relevance score: 5.578198

  Ligen Lin, Jong Han Lee, Odelia Y. N. Bongmba, Xiaojun Ma, Xiongwei Zhu, David Sheikh-Hamad, Yuxiang Sun

  Keywords: ghrelin, growth hormone secretagogue receptor (GHSR), brown adipose tissue (BAT), thermogenesis, insulin signaling, mitochondrial biogenesis and dynamics

  Published in Aging on December 15, 2014

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  Aging is associated with severe thermogenic impairment, which contributes to obesity and diabetes in aging. We previously reported that ablation of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), attenuates age-associated obesity and insulin resistance. Ghrelin and obestatin are derived from the same preproghrelin gene. Here we showed that in brown adipocytes, ghrelin decreases the expression of thermogenic regulator but obestatin increases it, thus showing the opposite effects. We also found that during aging, plasma ghrelin and GHS-R expression in brown adipose tissue (BAT) are increased, but plasma obestatin is unchanged. Increased plasma ghrelin and unchanged obestatin during aging may lead to an imbalance of thermogenic regulation, which may in turn exacerbate thermogenic impairment in aging. Moreover, we found that GHS-R ablation activates thermogenic signaling, enhances insulin activation, increases mitochondrial biogenesis, and improves mitochondrial dynamics of BAT. In addition, we detected increased norepinephrine in the circulation, and observed that GHS-R knockdown in brown adipocytes directly stimulates thermogenic activity, suggesting that GHS-R regulates thermogenesis via both central and peripheral mechanisms. Collectively, our studies demonstrate that ghrelin signaling is an important thermogenic regulator in aging. Antagonists of GHS-R may serve as unique anti-obesity agents, combating obesity by activating thermogenesis.

  

  UCP1 expression in HIB1B cells treated with saline or different concentrations of ghrelin (A), des-acyl ghrelin, DAG (B), and obestatin (C). Summary of UCP1 expression in HIB1B cells treated with saline, 1 nM ghrelin, 10 nM DAG, or 10 nM obestatin (D). N = 9, and each assay was measured in triplicate. *p<0.05, Treatments vs. Controls.

  
  
  

  

  Active ghrelin (A), des-acyl ghrelin, DAG (B), obestatin (C), and insulin (D) of young, middle-aged and old WT mice under fed or fasting conditions. Young (4-months), middle-aged (10-months) and old (22-months) mice were used. The mice were fasted overnight. N = 10-12. *p<0.05, fed vs. fasting. #p<0.05, ## P<0.001, young vs. middle-aged or old.

  
  
  

  

  (A) Energy expenditure of young, middle-aged and old WT and Ghsr^−/− mice normalized by lean body mass. (B) Ghsr expression in BAT of young, middle-aged and old WT mice. (C) BAT mass and percentage of BAT weight over body weight in young, middle-aged and old WT and Ghsr^−/− mice. (D) UCP1 expression in BAT of young, middle-aged and old WT and Ghsr^−/− mice. IR (E) and IRS-1 (F) expression in BAT of young, middle-aged and old WT and Ghsr^−/− mice. Young (4-months), middle-aged (10-months) and old (22-months) mice were used. N = 6-14. *p<0.05, **p<0.001, WT vs. Ghsr^−/−; #p<0.05, ##p<0.001, old or middle-aged vs. young.

  
  
  

  

  The representative data presented here were from 22 month-old WT and Ghsr^−/− mice. (A) Representative Western blots of key thermogenic regulators in BAT of aged WT and Ghsr^−/− mice. (B) Ex vivo lipolysis of BAT of aged WT and Ghsr^−/− mice, treated with or without 10 μM CL316243. (C) Representative Western blots show phosphorylated AMPK α (p-AMPK-α), total AMPK (t-AMPK-α) and UCP1 protein expression in BAT of old WT and Ghsr^−/− mice after 4 hours of cold exposure. (D) Expression of mitochondrial fission genes in BAT of aged WT and Ghsr^−/− mice. (E) Expression of mitochondrial fusion genes in BAT of aged WT and Ghsr^−/− mice. (F) Expression of mitochondrial complex biogenesis markers in BAT of aged WT and Ghsr^−/− mice. N = 6. *p<0.05, **p<0.001, WT vs. Ghsr^−/−; #p<0.05, basal vs. CL316243 treatment.

  
  
  

  

  NE concentrations in the urine (A) and NE levels in BAT (B) of young, middle-aged and old WT and Ghsr^−/− mice. Young (4-months), middle-aged (10-months) and old (22-months) mice were used. (C) β3-AR mRNA expression in BAT of 22 month-old WT and Ghsr^−/− mice. N = 6-10. *p<0.05, WT vs. Ghsr^−/−; #p<0.05, ##p<0.001, old or middle-aged vs. young.

  
  
  

  

  (A) Oxygen consumption of old WT and Ghsr^−/− mice after 1.5 mg/Kg subcutaneous injection of NE. N = 10-12. *p<0.05, WT vs. Ghsr^−/−. (B) Expression of Ghsr, UCP1 and PPARγ in shGHS-R knockdown HIB1B cells. shScr represents scramble shRNA; shGHS-R represents shRNA specific for GHS-R. N = 9. **p<0.001, shScr vs. shGHS-R.

  
  
  

  

  Our data suggest that ghrelin signaling may regulate thermogenesis in BAT via the following 4 independent and interconnected signaling pathways: 1) Ablation of GHS-R stimulates SNS-mediated NE release, which in turn induces β3-AR expression, subsequently activating thermogenic signaling cascades in BAT. This involves activation of thermogenic signaling pathway PKA-CREB-UCP1 and lipolytic pathway PKA-HSL-UCP1. 2) Ablation of GHS-R enhances insulin signaling in BAT, which improves insulin sensitivity of BAT and activates key thermogenic regulator PKA. 3) Ablation of GHS-R enhances AMPK activity in BAT, which increases DNA and protein synthesis of mitochondria, thus increasing mitochondrial biogenesis. 4) Ablation of GHS-R augments mitochondrial dynamics, enhancing both mitochondrial fission and fussion; this restores mitochondrial architecture and improves mitochondrial homeostasis. Improved mitochondrial homeostasis enhances the sensitivity of mitochondria to FFA to further promote mitochondrial uncoupling. Collectively, GHS-R ablation increases thermogenesis in BAT by activating thermogenic signaling, sensitizing insulin signaling, increasing mitochondrial biogenesis, and enhancing mitochondrial dynamics.

  
  
  

• Research Paper Volume 4, Issue 3 pp 206-223

  dSir2 deficiency in the fatbody, but not muscles, affects systemic insulin signaling, fat mobilization and starvation survival in flies

  Relevance score: 7.8115664

  Kushal Kr. Banerjee, Champakali Ayyub, Samudra Sengupta, Ullas Kolthur-Seetharam

  Keywords: Sir2, fat metabolism, starvation, survival, fatbody, muscles, insulin signaling

  Published in Aging on March 10, 2012

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  Sir2 is an evolutionarily conserved NAD⁺ dependent protein. Although, SIRT1 has been implicated to be a key regulator of fat and glucose metabolism in mammals, the role of Sir2 in regulating organismal physiology, in invertebrates, is unclear. Drosophila has been used to study evolutionarily conserved nutrient sensing mechanisms, however, the molecular and metabolic pathways downstream to Sir2 (dSir2) are poorly understood. Here, we have knocked down endogenous dSir2 in a tissue specific manner using gene-switch gal4 drivers. Knockdown of dSir2 in the adult fatbody leads to deregulated fat metabolism involving altered expression of key metabolic genes. Our results highlight the role of dSir2 in mobilizing fat reserves and demonstrate that its functions in the adult fatbody are crucial for starvation survival. Further, dSir2 knockdown in the fatbody affects dilp5 (insulin-like-peptide) expression, and mediates systemic effects of insulin signaling. This report delineates the functions of dSir2 in the fatbody and muscles with systemic consequences on fat metabolism and insulin signaling. In conclusion, these findings highlight the central role that fatbody dSir2 plays in linking metabolism to organismal physiology and its importance for survival.

  

  (A) Starvation survival of dSir2 mutants (Sir2^2A.7.11) (p < 0.001) and (B) whole body dSir2^RNAi (+ RU486) (p < 0.001), with respective controls (n = 60). (C)dSir2 transcript levels increase in response to 48-hours starvation in control (- RU486) but not in whole body dSir2^RNAi(+ RU486) flies (n = 8/24). (D) Increase in dSir2 protein levels in control flies in response to 48-hours starvation. (E) Increase in NAD⁺ levels in response to 48-hours starvation in control flies (n = 36). 200 μM RU486 was used to knockdown dSir2 expression inpSw-tub-gal4>dSir2^RNAi flies. Log Rank was used to plot survival curves and Mantel-Cox test was used for statistical analysis. Student's t-test and ANOVA were used to analyze statistical significance of the data (*, p < 0.05; **, p < 0.01; ***, p < 0.001 or mentioned otherwise).

  
  
  

  

  (A) Total body triglyceride (TAG) in dSir2 mutants (Sir2^2A.7.11) and whole body dSir2^RNAi (+ RU486) flies, with respective controls (n = 36). (B) Oil-Red O staining of fatbodies from control and Sir2^2A.7.11. (C) Relative expression of fat metabolism genes lipase-3 (lip3), brummer (brmm), mitochondrial acyl carrier protein (mtACP), medium chain acyl CoA dehydrogenase (mcad), aceto acetyl CoA thiolase (ACoT), long chain acyl CoA dehydrogenase (lcad), acetyl CoA carboxylase (ACC), fatty acid synthase (fas) and diacyl glycerol synthase (dDAG) (n = 24). 200 μM RU486 was used to knockdown dSir2 expression in pSw-tub-gal4>dSir2^RNAi flies. Student's t-test was used to analyze statistical significance of the data (*, p < 0.05; **, p < 0.01; ***, p < 0.001 or mentioned otherwise).

  
  
  

  

  (A) RT-PCR to show knockdown of dSir2 in the fatbody (FB) of fatbody dSir2^RNAiand body wall with muscles (BWM) of muscle dSir2^RNAi, with respective controls (n = 24). (B-C) Total body triglyceride levels in (B) fatbody dSir2^RNAi flies and (C) muscles dSir2^RNAi flies (n = 36). (D) Triglyceride levels in the isolated fatbody of fatbody dSir2^RNAi flies (n = 60). (E) Relative expression of fat metabolism genes lipase-3 (lip3), brummer (brmm), medium chain acyl CoA dehydrogenase (mcad), long chain acyl CoA dehydrogenase (lcad) and fatty acid synthase (fas) in fatbody isolated from fatbody dSir2^RNAi flies (n = 24). 200 μM RU486 was used to knockdown dSir2 expression pSw-S₁106-gal4>dSir2^RNAi and pSw-MHC-gal4> dSir2^RNAi flies. Student's t-test was used to analyze statistical significance of the data (*, p < 0.05; **, p < 0.01; ***, p < 0.001 or mentioned otherwise).

  
  
  

  

  (A and B) Starvation survival in (A) fatbody dSir2 knockdown flies (p < 0.001) and (B) muscle dSir2 knockdown flies (non-significant, ns)(n = 60). 200 μM RU486 was used to knockdown dSir2 expression pSw-S₁106-gal4>dSir2^RNAi and pSw-MHC-gal4> dSir2^RNAi flies. Log Rank was used to plot survival curves and Mantel-Cox test was used for statistical analysis. (C-D) Total body triglyceride levels in fed and starved conditions in (C) whole bodydSir2^RNAiflies, with respective controls (n = 36) and (D) fatbody dSir2^RNAi (n = 36). Relative expression of fat metabolism genes (E) lipase-3 (lip3), (F) brummer (brmm), (G) medium chain acyl CoA dehydrogenase (mcad), (H) fatty acid synthase (fas) in fatbody dSir2 knockdown flies under fed and starved conditions (n = 24). 200 μM RU486 was used to knockdown dSir2 expression pSw-S₁106-gal4>dSir2^RNAi and pSw-MHC-gal4> dSir2^RNAi flies. ANOVA was used to analyze statistical significance of the data (*, p < 0.05; **, p < 0.01; ***, p < 0.001 or mentioned otherwise).

  
  
  

  

  (A-C) Relative dilp5 levels in the heads of (A) whole body dSir2^RNAi (B) fatbody dSir2^RNAi and (C) muscle dSir2^RNAi flies, with respective controls, under fed and starved conditions (n = 24). Relative expression of (D) dInR and (E) d4eBP in fatbody dSir2^RNAi flies under fed and starved conditions (n = 24). (F) Starvation survival of fatbody dSir2 knockdown (pSw-S₁106-gal4>+/+;dSir2^RNAi + RU486) and control (pSw-S₁106-gal4>+/+;dSir2^RNAi - RU486) flies, fatbody dSir2 knockdown in chico heterozygote flies (pSw-S₁106-gal4>ch^+/-; dSir2^RNAi + RU486) and (pSw-S₁106-gal4>ch^+/-; dSir2^RNAi- RU486) (statistical significance indicated in supplementary figure 9) (n = 60).200 μM RU486 was used to knockdown dSir2 expression. Log Rank was used to plot survival curves and Mantel-Cox test was used for statistical analysis. Student's t-test and ANOVA were used to analyze statistical significance of the data (*, p < 0.05; **, p < 0.01; ***, p < 0.001 or mentioned otherwise).

  
  
  

• Research Paper Volume 3, Issue 2 pp 108-124

  The microRNA cluster miR-106b~25 regulates adult neural stem/progenitor cell proliferation and neuronal differentiation

  Relevance score: 6.871448

  Jamie O. Brett, Valérie M. Renault, Victoria A. Rafalski, Ashley E. Webb, Anne Brunet

  Keywords: aging, neural stem cells, microRNAs, FoxO transcription factors, insulin signaling, neuronal differentiation

  Published in Aging on February 20, 2011

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  In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93, and miR-25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice. We find that knocking down miR-25 decreases NSPC proliferation, whereas ectopically expressing miR-25 promotes NSPC proliferation. Expressing the entire miR-106b~25 cluster in NSPCs also increases their ability to generate new neurons. Interestingly, miR-25 has a number of potential target mRNAs involved in insulin/insulin-like growth factor-1 (IGF) signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR-106b~25 is bound by FoxO3, a member of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF signaling. These results suggest that miR-106b~25 regulates NSPC function and is part of a network involving the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.

  

  (A) Genomic locus of the mouse miR-106b~25 cluster and its host gene, Mcm7. (B) NSPCs (age 12 weeks, passage 2) were grown in multi-lineage differentiation conditions (no EGF or bFGF, with 1% FBS) for 7 days and then stained for Tuj1 (a marker of neurons), GFAP (a marker of astrocytes), or O4 (a marker of oligodendrocytes). Scale bar: 100 μm. (C) miRNA expression was determined by RT-qPCR in NSPCs in self-renewal conditions (with EGF and bFGF, no FBS) or differentiation conditions (no EGF or bFGF, with 1% FBS) for 4 days. Mean and SEM of gene expression relative to self-renewal conditions for 3 independent NSPC cultures (age 12 weeks, passage 2) are shown. One-sample two-tailed t-test, *: p<0.05.

  
  
  

  

  NSPCs were transfected to knock down miR-106b, miR-93, or miR-25 or were transfected with a scrambled control oligonucleotide. Two days after transfection, NSPCs were incubated with EdU for 1 hour and then immediately fixed for analysis. (A) Representative photos for control knockdown and miR-25 knockdown. Scale bar: 100 μm. (B) Mean and SEM of the proportion of EdU+ cells for each condition, for experiments on 5 independent NSPC cultures (age 8-14 weeks, passage 3-7). Paired two-tailed t-test, **: p<0.01.

  
  
  

  

  NSPCs were infected with an empty control retrovirus (expressing a GFP marker only) or a retrovirus expressing miR-25. NSPCs were grown to full neurospheres for about 1 week after infection before miRNA expression and proliferation were analyzed. (A) miR-25 expression was assessed with RT-qPCR in control versus miR-25-overexpressing NSPCs. Mean and SEM of 2 independent NSPC cultures (age 12 weeks, passage 2-5) are shown. (B) Representative photos for each condition. Scale bar: 100 μm. (C) Control and miR-25-overexpressing NSPCs were dissociated and incubated with EdU for 1 hour. Mean and SEM of the proportion of EdU+ cells for each condition, for experiments on 4 independent NSPC cultures (age 12 weeks, passage 3-6), are shown. Paired two-tailed t-test, *: p<0.05.

  
  
  

  

  NSPCs were infected with an empty control retrovirus (expressing a GFP marker only) or a retrovirus expressing miR-106b, miR-93, and miR-25 simultaneously (miR-106b~25). NSPCs were grown to full neurospheres for about 1 week after infection before miRNA expression and proliferation were analyzed. (A) miR-106b, miR-93, and miR-25 expression was assessed with RT-qPCR in control versus miR-106b~25-overexpressing NSPCs. Mean and SEM of 4 independent NSPC cultures (age 12-14 weeks, passage 5-14) are shown. (B) Representative photos for each condition. Scale bar: 100 μm. (C) Control and miR-106b~25-overexpressing NSPCs were dissociated and incubated with EdU or BrdU for 1 hour. Mean and SEM of the proportion of EdU+ or BrdU+ cells for each condition, for 6 experiments on independent NSPC cultures (age 12-14 weeks, passage 3-14), are shown. Paired two-tailed t-test, *: p<0.05.

  
  
  

  

  NSPCs were infected with an empty control virus or virus to overexpress miR-106b_~25. Three days after infection, NSPCs were placed in differentiation conditions for 7 days, and then stained for Tuj1, a marker of neurons. (A) Representative photos for each condition. Scale bar: 50 μm. (B) Mean and SEM of the proportion of Tuj1+ cells (total Tuj1+ cells/total DAPI-stained nuclei) normalized to control infection, for experiments on 4 independent NSPC cultures (age 12 weeks, passage 2), are shown. Paired two-tailed t-test, **: p<0.01.

  
  
  

  

  (A) The PANTHER gene classification program was used to analyze TargetScan-predicted conserved targets for mouse miR-25 (~600 targets total). Shown are the top 5 biological pathways (ordered by Bonferroni-corrected binomial test p-values). (B) The GSEA program was used to analyze the same TargetScan-predicted target list as in (A), using the Canonical Pathways and GO Gene Sets categories. Shown are the top 5 categories (ordered by hypergeometric distribution-generated p-values). (C) The DIANA-microT program was used to generate a stringent list of mouse miR-25 targets. Shown are the top KEGG categories (ordered by Pearson's chi-square test p-values). (D) Pathway diagrams based on those in PANTHER Pathways for TGFβ and insulin/IGF-Akt signaling pathways, modified for simplicity and with select miR-25 predicted targets listed.

  
  
  

  

  (A) Location of the FoxO binding site (FHRE) within the first intron of the miR-106b~25/Mcm7 gene, and the sequence locations used for EMSA, ChIP, and luciferase experiments. (B) EMSA with recombinant FoxO3-GST and a radioactively-labeled (hot) probe corresponding to the FoxO binding site in miR-106b~25/Mcm7 (FHRE WT). + Ctrl: FoxO3-GST incubated with a probe for a known FoxO binding site. The specificity of the interaction was tested by increasing amounts of unlabeled (cold) probe or cold probe with mutations in the FoxO consensus binding sequence (FHRE Mut). (C) Wild-type and FoxO3-null NSPCs were dissociated and the next day treated with 4 hours growth factor removal followed by addition of LY294002 for 1 hour. Antibodies to FoxO3 or control IgG antibodies were used for ChIP. qPCR was used to assess the enrichment of FHRE and of a negative control site (- Ctrl). Shown is the relative enrichment for 1 experiment (age 12 weeks, passage 10). These results were confirmed in ChIP-Seq studies (Webb et al. submitted). (D) HEK 293T cells were co-transfected with a plasmid to express FoxO3 (empty control, wild-type FoxO3, FoxO3 lacking the DNA binding domain, or constitutively nuclear FoxO3), a firefly luciferase reporter containing FHRE with or without the FoxO consensus sequence mutated, and a Renilla luciferase reporter to normalize for transfection efficiency. As a positive control, a luciferase reporter containing a known FoxO3-activated site was used (+ Ctrl); as a negative control, a luciferase reporter without an enhancer site was used (- Ctrl). Luciferase activity was assessed two days after transfection. Mean and SEM for 4 independent experiments (- Ctrl, + Ctrl, and FHRE WT) or 2 independent experiments (FHRE Mut) are shown. Unpaired two-tailed t-test, **: p<0.01. (E) NSPCs from wild-type and FoxO3-null mice were isolated and cultured. Total RNA was collected, and the levels of mature miR-106b~25 members (relative to 5S RNA) and Mcm7 mRNA (relative to β-actin mRNA) were assessed by RT-qPCR. Mean and SEM of the FoxO3-null/wild-type fold change for 5-6 independent cultures (age 10-13 weeks, passage 2-5) are shown. One-sample two-tailed t-test, *: p<0.05.

  
  
  

• Research Paper Volume 2, Issue 11 pp 843-853

  Activation of mitochondrial energy metabolism protects against cardiac failure

  Relevance score: 8.598157

  Tim J. Schulz, Dirk Westermann, Frank Isken, Anja Voigt, Beate Laube, René Thierbach, Doreen Kuhlow, Kim Zarse, Lutz Schomburg, Andreas F. H. Pfeiffer, Carsten Tschöpe, Michael Ristow

  Keywords: OXPHOS, cardiac failure, cardiomyopathy, insulin signaling, mitohormesis

  Published in Aging on November 16, 2010

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  Cardiac failure is the most prevalent cause of death at higher age, and is commonly associated with impaired energy homeostasis in the heart. Mitochondrial metabolism appears critical to sustain cardiac function to counteract aging. In this study, we generated mice transgenically over-expressing the mitochondrial protein frataxin, which promotes mitochondrial energy conversion by controlling iron-sulfur-cluster biogenesis and hereby mitochondrial electron flux. Hearts of transgenic mice displayed increased mitochondrial energy metabolism and induced stress defense mechanisms, while overall oxidative stress was decreased. Following standardized exposure to doxorubicin to induce experimental cardiomyopathy, cardiac function and survival was significantly improved in the transgenic mice. The insulin/IGF-1 signaling cascade is an important pathway that regulates survival following cytotoxic stress through the downstream targets protein kinase B, Akt, and glycogen synthase kinase 3. Activation of this cascade is markedly inhibited in the hearts of wild-type mice following induction of cardiomyopathy. By contrast, transgenic overexpression of frataxin rescues impaired insulin/IGF-1 signaling and provides a mechanism to explain enhanced cardiac stress resistance in transgenic mice. Taken together, these findings suggest that increased mitochondrial metabolism elicits an adaptive response due to mildly increased oxidative stress as a consequence of increased oxidative energy conversion, previously named mitohormesis. This in turn activates protective mechanisms which counteract cardiotoxic stress and promote survival in states of experimental cardiomyopathy. Thus, induction of mitochondrial metabolism may be considered part of a generally protective mechanism to prevent cardiomyopathy and cardiac failure.

  

  (A) Representative anti-hemagglutinin immunoblot showing several tissues from a transgene-negative littermate (“-”) and a transgenic (“+”) animal each. “Control” is a previously published cell line over-expressing frataxin [20]. (B) Aconitase activity measured in murine heart samples. Grey bars indicate wild-type (WT) and white bars indicate frataxin-transgenic (FX) animals (also applies to subsequent figures). (C) ATP, (D) NADH, (E)) NADPH, (F) reduced glutathione (GSH) and (G) thiobarbituric acid reactive substances (TBARS) contents in the hearts of wild-type and frataxin-transgenic animals. Error bars represent S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001 (applies to this and all subsequent figures) (n=4).

  
  
  

  

  (A) Maximum rate of pressure development in the left ventricle (dP/dtmax), (B) left-ventricular maximum rate of pressure decrease (dP/dtmin), (C) end-systolic pressure (Pes), (D) end-systolic volume (Ves), (E) end-diastolic pressure (Ped), (F) end-diastolic volume (Ved), (G) time constant of isovolumetric left ventricular pressure decline (tau, τ). (H) stroke work, (I) stroke volume, (J) cardiac output, (K) ejection fraction, and (L) heart rate in wild-type vs. frataxin-transgenic animals following administration of doxorubicin. Grey bars depict wild-type litter mates, white bars frataxin-transgenic mice (n=6 each).

  
  
  

  

  Survival plot of animals exposed to doxorubicin. Triangles depict frataxin-transgenic (FX) animals; squares depict wild-type (WT) animals (n=24 per genotype).

  
  
  

  

  Three animals were analyzed by western blotting for each group. Lanes 1-3: WT untreated; lanes 4-6: FX untreated; lanes 7-9: WT doxorubicin-treated; lanes 10-12: FX doxorubicin-treated. Membranes were probed with the indicated antibody, then stripped and re-probed with α-tubulin as loading control. Approximate protein band size to the left as indicated by arrows.

  
  
  

• Research Perspective Volume 2, Issue 10 pp 742-747

  EAK proteins: novel conserved regulators of C. elegans lifespan

  Relevance score: 7.6582413

  Travis W. Williams, Kathleen J. Dumas, Patrick J. Hu

  Keywords: C. elegans, aging, lifespan, longevity, insulin signaling, Akt, FoxO, eak, diabetes, cancer, osteoporosis

  Published in Aging on October 25, 2010

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  FoxO transcription factors (TFs) extend lifespan in invertebrates and may participate in the control of human longevity. The role of FoxO TFs in lifespan regulation has been studied most extensively in C. elegans, where a conserved insulin/insulin-like growth factor signaling (IIS) pathway and the germline both control lifespan by regulating the subcellular localization of the FoxO transcription factor DAF-16. Although the control of FoxO activity through modulation of its subcellular localization is well established, nuclear translocation of FoxO is not sufficient for full FoxO activation, suggesting that undiscovered inputs regulate FoxO activity after its translocation to the nucleus. We have recently discovered a new conserved pathway, the EAK (enhancer-of-akt-1) pathway, which acts in parallel to the Akt/PKB family of serine-threonine kinases to regulate DAF-16/FoxO activity. Whereas mutation of Akt/PKB promotes the nuclear accumulation of DAF-16/FoxO, mutation of eak genes increases nuclear DAF-16/FoxO activity without influencing DAF-16/FoxO subcellular localization. Thus, EAK proteins regulate the activity of nuclear DAF-16/FoxO. Two EAK proteins, EAK-2/HSD-1 and EAK-7, influence C. elegans lifespan and are conserved in mammals. The discovery of the EAK pathway defines a new conserved FoxO regulatory input and may have implications relevant to aging and the pathogenesis of aging-associated diseases.

  

  The nucleus is denoted by the dashed ellipse. A. In the presence of an intact germline and IIS, most FoxO is sequestered in the cytoplasm, and EAK-7 inhibits the activity of the small fraction of cellular FoxO that resides in the nucleus. B. In animals with reduced IIS and/or germline signaling, EAK-7 has a greater influence on FoxO activity, since relative concentrations of nuclear FoxO are increased.