Source: UNIVERSITY OF CALIFORNIA, BERKELEY submitted to NRP
PROTEOME DYNAMICS AS MARKERS AND MECHANISMS OF HEALTH BENEFITS FROM CALORIE RESTRICTION AND RELATED LONGEVITY-PROMOTING INTERVENTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1007814
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 20, 2015
Project End Date
Sep 30, 2020
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720
Performing Department
Nutritional Sciences
Non Technical Summary
Caloric restriction (CR) extends life-span and delays diseases in animal models. This remarkable observation has been difficult, however, to translate into human health benefits. A life-time of food deprivation and reduced body weight is not practical for most people. Moreover, it has not been easy to identify the underlying metabolic and molecular signals responsible for health benefits of CR in rodent models, when each experiment takes up to 3 years. To address these limitations, we have applied a biomarker-based strategy, using highly snsitive, stable isotope-mass spectrometric measurements of fluxes through metabolic pathways in vivo as outcome measures, and looked for dietary and pharmacologic interventions that may mimic the benefits of CR without requiring long term weight loss. Our objective in this project is to explore interventions and identify protein kinetic biomarkers of processes related to interventions that delay or prevent age-related morbidities and mortality. Specifically, we will look for protein kinetic biomarkers related to extension of health-span and maximal lifespan in mice and related these measurements to specific metabolic pathways and molecular signals. Our goal is also to translate these studies into humans by measurement of protein kinetic biomarkers in blood.
Animal Health Component
60%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7027299101045%
7027299102035%
7027299106020%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
7299 - Research equipment and methods, general/other;

Field Of Science
1060 - Biology (whole systems); 1010 - Nutrition and metabolism; 1020 - Physiology;
Goals / Objectives
1. Establish the effects of different candidate dietary and pharmacologic interventions on hepatic and plasma proteome dynamics, including modified alternate day fasting (mADF) and drugs including rapamycin that have been proposed to extend health-span and maximum life-span in mice.2. Identify molecular signals mediating changes in biomarker outcomes, including sirtuin pathway activity, acetylation state of SIRT1 targets, mTORC pathway activity, PGC-1 and PPAR levels, activity of the GH/IGF axis, and insulin levels. We will look for activity of candidate molecular signaling pathways for correlations with biomarker outcome measures.3. Identify molecular signals mediating changes in biomarker outcomes, by use of genetically altered mice, specifically by using gene knock-out mice to evaluate the role of specific pathways proposed to extend health-span and maximum life-span.4. Carry out preliminary studies in healthy human subjects measuring plasma proteome dynamics relevant to calorie restriction and its mimetics proposed to extend health-span and maximum life-span.
Project Methods
Animal studies: Briefly, 12-week-old male C57BL/6 mice from Charles River (Wilmington, MA) will be used. All mice are housed individually. Following ~1 week of acclimation to the animal facility and the AIN-93M diet (Bio-Serv, Frenchtown, NJ), mice are randomly assigned to one of the following two groups: ad libitum-fed (AL) or CR. Mice in the AL group are provided unrestricted access to the AIN-93M diet. Food intake is recorded for each AL mouse every other day. Mice in the CR group are provided with 75% of the average daily food intake of the AL group (from the previous 3 to 4 days) and, therefore, will be 25% calorie restricted. CR mice are provided with food daily at 1200h. All mice in this study are kept on their diets for a total of 5 weeks. The body weight of each mouse will be measured one to three times per week. Mice in this study will be labeled with heavy water for the last 21 days of the study. Mice in the AL group are provided unrestricted access to the NIH-31 diet. Mice in the CR group are provided with 3.0g of the NIH-31/NIA fortified diet daily at 1700hr. The body weight of each mouse will be measured once per week. Mice in this study will be labeled with heavy water for the last 1, 2, 4, 8, 15 or 32 days of the study.Heavy water labeling protocols-In order to measure rates of in vivo protein replacement, mice will be labeled with an intraperitoneal injection of 100% 2H2O (0.35ml/ 10g body weight) at the abovementioned specified times prior to the end of the study. Mice are then provided free access to 8% heavy water as drinking water for the remainder of the studies, as previously described (48). Human studies: Heavy water labeling protocols in humans for plasma protein turnover rates have been described previously (51). Subjects will be enrolled at the UCSF SF General Hospital General Clinical Research Center for all studies. Written informed consent will be provided prior to all procedures, by the UCSF Committee on Human Research and Declaration of Helsinki protocols will be followed. Subjects will drink a loading dose of 8 doses of 60 mL 70% 2H2O over a 48-hour period followed by 60mL 2H2O daily until day 21. Blood, saliva and urine and saliva samples will be collected. Analyses will be as described below.Blood, plasma and tissue collection and cell isolation-Upon completion of each study, blood will collected via cardiac puncture (in mice) or percutaneous blood draw (in humans).Measurement of 2H2O enrichment in body water-Enrichment of 2H2O in body water (blood) are measured via chemical conversion to tetrabromoethane as previously described (47, 49, 50).In vivo hepatic proteome dynamics and quantitative analyses-Sample preparation: Frozen livers from AL and CR mice labeled with heavy water for 1, 4, 8, 15, or 32 days will be prepared and treated as previously described (47). Frozen livers from AL and CR mice labeled with heavy water for 2 days as well as livers from mice in all Snell dwarf and rapamycin studies are homogenized in ~500ul lysis buffer (10mM Tris-base, 150mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1mM dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (PMSF), 7.5ug/mL leupeptin, 1.0ug/mL pepstatin, 2.0ug/mL aprotinin and 1 Phosphatase Inhibitor Cocktail 6t (Roche Applied Science, Indianapolis, IN) per 10mL buffer, pH ~7.5) using a stainless steel bead and a TissueLyserII (Retsch, Newtown, PA) set at 30hz for 1min. Tissue homogenates are sonicated in a sonication water bath for 1min and then centrifuged at 10,000 rcf at 4°C for 10min followed by supernatant collection. Protein concentrations are determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). The reduced samples are then alkylated via incubation in 14.3mM iodoacetamide for 1hr in the dark at room temperature. Tryptic peptides from all protein homogenates are prepared as previously described (47)The relative concentration of proteins in the liver are measured by comparison to exogenously labeled standards mixed into tissue homogenates (Stable Isotope LAbeling in Mammals, SILAM). The exogenously labeled standard is derived from the liver of a C57BL/6 mouse metabolically labeled with 15N (MouseExpress Liver, Cambridge Isotope). Briefly, equal ug amounts of protein derived from the liver homogenates of mice in our studies and protein derived from the SILAM liver are combined prior to protein treatment, fractionation and LC-MS/MS analysis.Human plasma proteome kinetic measurements. Blood (10 ml) is collected in Vacutainer tubes with EDTA and cells are separated from plasma by centrifugation (10 min, 1500g) then frozen.The most abundant proteins are removed from 0.5 ml plasma using a mulitaffinity spin cartridge, denatured with ura (6mM in 100 mM Tris, pH 8.0), reduced with TCEP and heated to ensure cleavage of disulfide bonds and incubated with iodoacetimide (10 mM) in the dark. Digestion with trypsin overnight at 37 degrees C followed, and peptides are concentrated and desalted prior to LC-MS/MS analyses as described (51).

Progress 10/20/15 to 09/30/20

Outputs
Target Audience:Scientists Nutritionists Physicians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students (2) and research scientists have been trained and undergone professional development through these research studies. How have the results been disseminated to communities of interest?Abstracts and presentations to scientific meetings have been presented. Manuscripts have been submitted for review or arein progress toward submission. What do you plan to do during the next reporting period to accomplish the goals?This is the final report for the present proposal. For the new proposal,we plan tocontinue using the same strategies and models. Studies in human subjects are still in the plans.

Impacts
What was accomplished under these goals? We continue to makegood progress in goals #1, #2 and #3. Goal #1: We have completed studies and submitted manuscripts about he effects on proteome dynamics in liver of caloric restriction of different periods of time, and of metformin, and modulators ofitric oxide synthase (pharmacolgic and gene-knockout),and finally of the unfollded protein response in combination with aging or high-fatdiet, and rapamycin have (in mice and rats). Goal #2: The role of inducible nitric oxide synthase in the proteome-wide reduction in protein synthesis induced by caloric restrictionhas been established Goal #3: The role of molecular signals including insulin and nitric oxide synthase on the changes in hepatic proteome dynamics with caloric restriction have been advanced into publishable form and submitted for publicationThe finding of a discrete transition time after 20 - 30 days of caloric restricttion, for inducing the changes in hepatic proteome dynamics, is another important result. that has been advanced to publication readiness.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Powell DR, Zambrowicz B, Morrow L, et al. Sotagliflozin Decreases Postprandial Glucose and Insulin Concentrations by Delaying Intestinal Glucose Absorption 2019 Dec 14.�J Clin Endocrinol Metab. 2019;dgz258
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Smith GI, Mahalakshmi Shankaran, Mihoko Yoshino, George G. Schweitzer, Maria Chondronikola, Joseph W. Beals, Adewole L. Okunade, Bruce W. Patterson, Edna Nyangau, Tyler Field, Claude B. Sirlin, Saswata Talukdar, Marc K. Hellerstein, and Samuel Klein. Insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease.�J Clin Invest. 2020;134165. doi:10.1172/JCI134165
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: cite Cawthon PM, Orwoll ES, Peters KE, Ensrud KE, Cauley JA, Kado DM, Stefanick ML, Shikany JM, Strotmeyer ES, Glynn NW, Caserotti P, Shankaran M, Hellerstein M, Cummings SR, Evans WJ; Osteoporotic Fractures in Men (MrOS) Study Research Group. Strong Relation Between Muscle Mass Determined by D3-creatine Dilution, Physical Performance, and Incidence of Falls and Mobility Limitations in a Prospective Cohort of Older Men. J Gerontol A Biol Sci Med Sci. 2019 May 16;74(6):844-852. doi: 10.1093/gerona/gly129. PMID: 29897420; PMCID: PMC6521914.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Orwoll ES, Peters KE, Hellerstein M, Cummings SR, Evans WJ, Cawthon PM. The Importance of Muscle Versus Fat Mass in Sarcopenic Obesity: A Re-evaluation Using D3-Creatine Muscle Mass Versus DXA Lean Mass Measurements. J Gerontol A Biol Sci Med Sci. 2020 Jun 18;75(7):1362-1368. doi: 10.1093/gerona/glaa064. PMID: 32436565; PMCID: PMC7302180.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hellerstein M. What are the roles of antibodies versus a durable, high quality T-cell response in protective immunity against SARS-CoV2? Vaccine: X (2020), doi: https://doi.org/10.1016/j.jvacx. 2020.100076
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Evans WJ, Scottoline B, Imam F, Hellerstein M, Garton K, Czerwieniec G, Nyangau E, Shankaran M. D3-creatine dilution for the noninvasive measurement of skeletal muscle mass in premature infants. Pediatr Res. 2020 Sep 12. doi: 10.1038/s41390-020-01122-w. Epub ahead of print. PMID: 32919390.
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Ahmed R, Miners KL, Lahoz-Beneytez J, Jones RE, Roger L, Baboonian C, Zhang Y, Wang ECY, Hellerstein MK, McCune JM, Baird DM, Price DA, Macallan DC, Asquith B, Ladell K. CD57+ Memory T Cells Proliferate In Vivo. Cell Rep. 2020 Dec 15;33(11):108501. doi: 10.1016/j.celrep.2020.108501. PMID: 33326780; PMCID: PMC7758161.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hellerstein M. Surprising findings in a 'well-understood' nutrient-assimilation pathway. Nat Metab. 2020 Jul;2(7):561-563. doi: 10.1038/s42255-020-0242-5. PMID: 32694796.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kelly KL, Reagan WJ, Sonnenberg GE, Clasquin M, Hales K, Asano S, Amor PA, Carvajal-Gonzalez S, Shirai N, Matthews MD, Li KW, Hellerstein MK, Vera NB, Ross TT, Cappon G, Bergman A, Buckeridge C, Sun Z, Qejvanaj EZ, Schmahai T, Beebe D, Pfefferkorn JA, Esler WP. De novo lipogenesis is essential for platelet production in humans. Nat Metab. 2020 Oct;2(10):1163-1178. doi: 10.1038/s42255-020-00272-9. Epub 2020 Sep 14. PMID: 32929234.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Scientists Nutritionists Physicians Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students (2) and research scientists have been trained and undergone professional development throughthese researchstudies. How have the results been disseminated to communities of interest?Abstracts and presentations to scientific meetings have been presented. Manuscripts are in progress orunder process of submission. What do you plan to do during the next reporting period to accomplish the goals?We will continue using the same startegies and models. Studies in human subjects are still in the plans.

Impacts
What was accomplished under these goals? We have made goodprogress in goals #1 and #3. Goal #1: The effects on proteome dynamics in liverof caloric restriction of different periods of time, metformin, modulators of nitric oxide synthase (pharmacolgic and gene-knockout), the unfollded protein response in combination with aging or high-fat diet,andrapamycin have all been charatcterized inmice and rats. Goal #3: The role of molecular signals including insulin and nitric oxide synthase on the changes in hepatic proteome dynamics with caloric restrictionhave been advanced. The finding of a discrete transition time after 20 - 30 days of caloric restricttion, forinducingthe changes in hepatic proteome dynamics, is an important result.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Murphy CH, Shankaran M&, Hellerstein MK, Phillips S� Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction. J Physiol 11:2091-212018. 2018 PMID:29532476
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mazzarello AN, Fitch M, Hellerstein MK, Chiorazzi N. Methods Mol Biol. Measurement of Leukemic B-Cell Growth Kinetics in Patients with Chronic Lymphocytic Leukemia. 2019;1881:129-151. PMID: 30350203
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Esler WP, Tesz GJ, Hellerstein MK, et al. Human sebum requires de novo lipogenesis, which is increased in acne vulgaris and suppressed by acetyl-CoA carboxylase inhibition.�Sci Transl Med. 2019;11(492): PMID:�31092695
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Dall'Era M, Pauli ML, Remedios K, Taravati K, Sandova PM, Putnam AL, Lares A, Haemel A, Tang Q, Hellerstein M, Fitch M, McNamara J, Welch B, Bluestone JA, Wofsy D, Rosenblum MD; Autoimmunity Centers of Excellence. Adoptive Treg Cell Therapy in a Patient With Systemic Lupus Erythematosus. Arthritis Rheumatol. 2019 Mar;71(3):431-440. PMID: 30277008
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: White UA, Fitch MD, Beyl RA, Hellerstein MK, Ravussin E. Racial differences in in vivo adipose lipid kinetics in humans. J Lipid Res. 2018 Sep;59(9):1738-1744. PMID: 29910190
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Beysen C, Deines K, Tsang E, Riiff T, Protasio J, Chan M, Matthews M, Czerwieniec G, Hellerstein MK and Turner SM. The effect of Bile Acid Sequestration in Combination with Dipeptidyl Peptidase-4 Inhibition on Glucose Metabolism in People with Type 2 Diabetes: A Randomized Open-Label Diabetes Care 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Loomba R, Decaris M, Li KW, Shankaran M, Mohammed H, Matthews M, Richards LM, Nguyen P, Rizo E, Andrews B, Soto R, Angel T, Suri V, Kitrinos KM, Barnes D, Czerwieniec G, Brendza K, Subramanian GM, Gaggar A, Hellerstein MK. Discovery of half-life of circulating HBsAg in patients with chronic hepatitis B infection using heavy water labeling. Clin Infect Dis. Dec 24. doi: 10.1093/cid/ciy1100. [Epub ahead of print] 2018 PMID: 30590481
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Chen TC, Lee RA, Tsai SL, Kanamaluru D, Gray NE, Yiv N, Cheang RT, Tan JH, Lee JY, Fitch MD, Hellerstein MK, Wang JC. An ANGPTL4-ceramide-protein kinase C? axis mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia in mice. J Biol Chem. 2019 Jun 7;294(23):9213-9224. 2019 May 3. PMID: 31053639
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Krois CR, Vuckovic MG, Huang P, Zaversnik C, Liu CS, Gibson CE, Wheeler MR, Obrochta KM, Min JH, Herber CB, Thompson AC, Shah ID, Gordon SP, Hellerstein MK, Napoli JL. RDH1 suppresses adiposity by promoting brown adipose adaptation to fasting and re-feeding. Cell Mol Life Sci. 2019 Jun;76(12):2425-2447. PMID: 30788515
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Hellerstein M. Measure for measure?Am J Clin Nutr. 2019 Feb 1;109(2):247-248. No abstract available. PMID: 30721927
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: NouwsJ, Fitch M, Mata M, Santoro N, Galuppo B, Kursawe R, Narayan D, Vash-Margita A, Pierpont B, Shulman G, Hellerstein M, Caprio S. Altered in vivo Lipid Fluxes and Cell Dynamics in Subcutaneous Adipose Tissues are Associated with the Unfavorable Pattern of Fat Distribution in Obese Adolescent Girls. Diabetes. 2019 Jun;68(6):1168-1177. PMID: 30936147
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Evans WJ,�Hellerstein M, Orwoll E, Cummings S, Cawthon PM. D3 -Creatine dilution and the importance of accuracy in the assessment of skeletal muscle mass. J Cachexia Sarcopenia Muscle. 2019 Feb;10(1):14-21. Review. PMID: 30900400
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Cawthon PM, Orwoll ES, Peters KE, Ensrud KE, Cauley JA, Kado DM, Stefanick ML, Shikany JM, Strotmeyer ES, Glynn NW, Caserotti P, Shankaran M,�Hellerstein M, Cummings SR, Evans WJ; Osteoporotic Fractures in Men (MrOS) Study Research Group. Strong Relation Between Muscle Mass Determined by D3-creatine Dilution, Physical Performance, and Incidence of Falls and Mobility Limitations in a Prospective Cohort of Older Men. J Gerontol A Biol Sci Med Sci. 2019 May 16;74(6):844-852. PMID: 29897420
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lawitz EJ, Coste A, Poordad F, Alkourhi N, Loo N, McColgan B, Tarrant JM, Nguyen T, Han L, Chung C, Ray A, McHutchison JG, Subramanian GM, Myers RP, Middleton MS, Sirlin C, Loomba R, Nyangau E, Fitch M, Li K, Hellerstein MK. Acetyl-CoA Carboxylase Inhibitor GS-0976 for 12 Weeks Reduces Hepatic De Novo Lipogenesis and Improves Hepatic Steatosis in Patients with Nonalcoholic Steatohepatitis Clin Gastroenterol Hepatol. 2018 Dec;16(12):1983-1991. PMID: 29705265
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gonz�lez-Granillo M, Savva L, FitchM, Pedrelli M, Hellerstein M, Parini P, Korach-Andr� M and Gustafsson J-A. ER beta Activation in Obesity Improves Whole Body Metabolism Via Restoration of Adipose Tissue Function and Enhanced Mitochondria Biogenesis. Mol Cell Endocrinol. 2019 Jan 5;479:147-158. PMID: 30342056
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Beysen C, Ruddy M, Stoch A, et al. Dose-dependent quantitative effects of acute fructose administration on hepatic de novo lipogenesis in healthy humans. Am J Physiol Endocrinol Metab. 2018;315(1):E126E132. doi:10.1152/ajpendo.00470.2017
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miranda DA, Krause WC, Cazenave-Gassiot A, et al. LRH-1 regulates hepatic lipid homeostasis and maintains arachidonoyl phospholipid pools critical for phospholipid diversity.�JCI Insight. 2018;3(5):e96151.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Shankaran M, Czerwieniec G, Fessler C, et al. Dilution of oral D3 -Creatine to measure creatine pool size and estimate skeletal muscle mass: development of a correction algorithm. J Cachexia Sarcopenia Muscle. 2018;9(3):540546.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientists Nutritionists Physicians Policymakers Changes/Problems:No major problems What opportunities for training and professional development has the project provided?Training of 2 graduate students How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Continue the present strategy in animal models. Also try to begin testing of plasma proteome synthesis rates in plasma in human subjects in response to dietary interventions

Impacts
What was accomplished under these goals? We have made progress in several areas: 1.Identifying signals involved in reduced global protein synthesis observed withcalorie restriction (CR) and the unfolded protein response (UPR). Results to date include identifying arole of nitric oxide synthesis in CR, based on mouse knock-out models and pharamacologic interventions; 2. The role of changes in synthesis rates of chaperones and other proteinsin the golobal reduction of UPR after tunicamycin exposure; and the manifestationof UPR onbrain proteins synthesis. 3. We have also identified the time-frame required for induction of the CR effectson global protein synthesis rates in the liver of mice. Surprisingly, the onset is not gradual but shows a thresh-hold effect or "switch" aftera discrete oeriod of CR exposure. This result may allow idenfification of the specific signals that are induced and mediate lower global protein synthesis rates. These findingshave not yet been published.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Magkos F, Sullivan S, Fitch M, Smith G, Fabbrini E, Mittendorfer B, Hellerstein M, Klein S. Effect of Weight Gain and Weight Loss on In Vivo Colonocyte Proliferation Rate in People with Obesity. Obesity (Silver Spring). 2017 Nov;25 Suppl 2:S81-S86. PMID: 29086514 Buehring B, Siglinsky E, Krueger D, Evans W,�Hellerstein M, Yamada Y, Binkley N. Comparison of muscle/lean mass measurement methods: correlation with functional and biochemical testing. Osteoporos Int. 2017 PMID: 29116169 Akondy RA, Fitch M, Edupuganti S, Yang S, Kissick HT, Li KW, Youngblood BA,Abdelsamed HA, McGuire DJ, Cohen KW, Alexe G, Nagar S, McCausland MM, Gupta S, Tata P,Haining NW, McElrath MJ, Zhang D, Hu B, Greenleaf WJ, Goronzy JJ, Mulligan MJ, Hellerstein M# and Ahmed R# (# corresponding authors). Tracking the origin and differentiation of human memory CD8 T cells after vaccination. Nature. 2017 Dec 21;552(7685): 362-367; PMID:29236685 Shankaran M, Czerwieniec G, Fessler C, Wong PA, Killion S, Turner SM, Hellerstein MK, Evans WJ. Dilution of oral D3-Creatine to measure creatine pool size and estimate skeletal muscle mass: development of a correction algorithm Journal of Cachexia, Sarcopenia and Muscle, 2018 Cawthon PG, Orwoll E, Peters K, Ensrud KE, Cauley JA, Kado D, Stefanick M, Shikany J, Strotmeyer E, Caserotti P, Shaunkaran M, Hellerstein M, Cummings SR, Evans WJ.. Functional muscle mass assessed by D3-creatine dilution and its relation to physical performance, incident falls and mobility problems in community dwelling older men, J Gerontol 2018


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Research scientists Nutritionists/Dieticians Physicians; public health workers Biopharmaceutical companies; FDA General public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several graduate students and undergraduates received tarining and laboratory experience How have the results been disseminated to communities of interest?Several publications in refereed journals Review articles YouTube videos What do you plan to do during the next reporting period to accomplish the goals?Continue to work in animal models and in human subjects

Impacts
What was accomplished under these goals? Identified proteome dynamic signature of integrated stress response and its inhibition in tissues of animal models (liver, kidney,brain). Discovered blood proteincandidates as markers of calorie restriction and calorie restrictionmimetics and as potential targets for gene knock-out or knock-down. Confirmed muscle proteome dynamic responses to different types of exercise. Discovered and validatedplasma protein biomarkers for which kinetics reveal synthesis of liver collagen inhepatitis C infection (in response to curative treatment) and in non-alcoholic fatty liver disease (in response to experimetal drug treatments).

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Decaris ML, Li KW, Emson CL, Gatmaitan M, Liu S, Wang Y, Nyangau E, Colangelo M, Angel TE, Beysen C, Cui J, Hernandez C, Lazaro L, Brenner DA, Turner SM, Hellerstein MK, Loomba R. Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood. Hepatology. 2017;65(1):78-88. PMID: 27706836 Hellerstein M, Evans W. Recent advances for measurement of protein synthesis rates, use of the 'Virtual Biopsy' approach, and measurement of muscle mass. Curr Opin Clin Nutr Metab Care. 2017 May;20(3):191-200. PMID: 28257333 Cockerham LR, Yukl SA, Harvill K, Somsouk M, Joshi SK, Sinclair E, Liegler T, Hoh R, Lyons S, Hunt PW, Rupert A, Sereti I, Morcock DR, Rhodes A, Emson C, Hellerstein MK, Estes JD, Lewin S, Deeks SG, Hatano H. A Randomized Controlled Trial of Lisinopril to Decrease Lymphoid Fibrosis in Antiretroviral-Treated, HIV-infected Individuals. Pathog Immun. 2017;2(3):310-334. PMID: 28936485 Murphy CH, Shankaran M, Churchward-Venne TA, Mitchell CJ, Burke LM, Hawley JA, Kassis A, Karagounis LG, Li K, King C, Hellerstein MK, and Phillips S� Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction. J Physiol (In Press), 2018


Progress 10/20/15 to 09/30/16

Outputs
Target Audience:Other scientists, physicians, dieticians, policy makers, pharmaceutical researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of a graduate student and an undergraduate, particularly on the protein elongation rate project. Graduate student: Hector Pallacios. Learnt lab techniques, animal investiagtion, mass spectrometric kinetic analysis. Teaching and supervision was by lab pesonnel. Undergraduate: Chase Garcia. Learnt lab techniques, animal investiagtion, mass spectrometric kinetic analysis. Teaching and supervision was by lab personnel. How have the results been disseminated to communities of interest?No special dissemination, other than publications and scientific meetings (e.g., Experimental Biology, American Society for Liver Disease, International Society for Sarcopenia and Cachexia) What do you plan to do during the next reporting period to accomplish the goals?We plan to continue with the current projects and progress toward translational studies in human subjects. These studies will include: Studies to advance the proteome dynamics method. In particular, our novel proposed method for measuring protein elongation rates in vivo will be worked on and human plasma proteome dynamic methods will be advanced (for instruments, protocols and analyses). Studies of pathways potentially involved as signals. The roles of protein initiation factors, structural elements and signaling pathways altering protein elongation rates, the influence of the unfolded protein response, and various intermediate signaling processes will be explored. Genetic models. As noted, genetically altered mice can be helpful in these studies. Human translation. Opportunities to explore in humans the effects of relevant interventions or conditions (dietary, exercise, diseases) using these tools will be explored.

Impacts
What was accomplished under these goals? Impact of the research over the past year will be discussed across several dimensions: methodologic advances, physiologic insights and clinical translation. 1. Establish the effects of different candidate dietary and pharmacologic interventions on hepatic and plasma proteome dynamics. We published in Methods in Enzymology a comprehensive review of the proteome dynamics method that we have deveoped (Holmes et al., 2015). We published our work demonstrating a quantitative correlation between hepatic proteome dynamics and maximum life-span in various longevity-promoting interventions: calorie restriction [CR], Snell Dwarf mice, and rapamycin treatment (Thompson et al., Ageing Cell, 2015). We also confirmed similar effects of CR on general slowing of muscle proteome dynamics and showed that this was an opposite directional signature as occurs with endurance exercise in rats (Khambatta et al., manuscript in preparation). For translation into humans, plamsa proteome dynamics have been explored in various settings, including sprint exercise (Shankaran eta l, 2016), power exercise ( Murphy et al. manuscript in preparation), hepatitis C infection and obesity/fatty liver disease (Decaris et al., 2015 and 2017), to extend the method and establish normative data as well as to test and validate our concept of the "virtual biospy" for measuring intracellular protein synthesis rates from stable isotope-mass spectrometric measurements on proteins that spilled into the bloodstream. The impact of these studies includes creation of a powerful new investigative technology (proteome dynamics), new insights into the potential role of protein synthesis rates as a general cellular stressor and promoter of senescence, surprising findings on the effects of life-span extending inerventions on proteome dynamics, and a translatable biomarker tool for use in human studies. 2. Identify molecular signals mediating changes in biomarker outcomes. The molecular flux mechanism underlying slowed proteome turnover iwith CR and other life-span extending interventions is being actively investigated through development and validation of a novel method for measuring in vivo the elongation rate of peptides during translation on ribosomes (Pallacios et al., work in progress). This method could provide fundamental information for investigation of the molecular flux basis of improved proteostasis and the impact of many signaling factors on one of the most basic processes in biology (elongation rate of nascent proteins on ribosomes). This method is in principle translational directly into humans. In addition, we have characterized the effects of the unfolded protein response on global proteome tunover in liver and kidney (the finding being slowing of turnover for a large subset of proteins) in rodents, and whether these effects can be reversed by pharamacologic means. These studies complemet and can be combined with effects of CR and CR-mimetics. 3. Identify molecular signals mediating changes in biomarker outcomes, by use of genetically altered mice, specifically by using gene knock-out mice to evaluate the role of specific pathways proposed to extend health-span and maximum life-span. Studies with genetically altered mice are being started this current year, including mice with genetically altered protein initiation or elongation. 4. Carry out preliminary studies in healthy human subjects measuring plasma proteome dynamics relevant to calorie restriction and its mimetics proposed to extend health-span and maximum life-span. Human studies of CR or other interventions have not yet been started.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Holmes W., Angel T., Li K., & Hellerstein MK. Dynamic Proteomics: In vivo proteome-wide measurement of protein kinetics using metabolic labeling. Methods Enz. 2015;561:219-76. PMID: 263589072015 Shankaran M, Shearer TW, Stimpson SA, Turner SM, King C, Wong PA, Shen Y, Turnbull PS, Kramer F, Clifton L, Russell AJ, Hellerstein MK, Evans WJ. Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats. Am J Physiol Endocrinol Metab. 2016 PMID: 26714847 Thompson ACS, Bruss M, Price JC, KhambattaCF, HolmesWE, Colangelo M, DaliddM, Roberts LS, Astle CM, Harrison DE and Hellerstein MK. Reduced in vivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice. Ageing Cell 2016,15(1):118-27; PMID: 26541492 Shankaran M, King CL, Angel TE, Holmes WE, Li KW, Colangelo M, Price JC, Turner SM, Bell C, Hamilton KL, Miller BF, Hellerstein MK. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics. J Clin Invest. 2016; 126(1):288-302. PMID: 26657858