Bringing metabolic networks to life: convenience rate law and thermodynamic constraints

BACKGROUND: Translating a known metabolic network into a dynamic model requires rate laws for all chemical reactions. The mathematical expressions depend on the underlying enzymatic mechanism; they can become quite involved and may contain a large number of parameters. Rate laws and enzyme parameters are still unknown for most enzymes. RESULTS: We introduce a simple and general rate law called "convenience kinetics". It can be derived from a simple random-order enzyme mechanism. Thermodynamic laws can impose dependencies on the kinetic parameters. Hence, to facilitate model fitting and parameter optimisation for large networks, we introduce thermodynamically independent system parameters: their values can be varied independently, without violating thermodynamical constraints. We achieve this by expressing the equilibrium constants either by Gibbs free energies of formation or by a set of independent equilibrium constants. The remaining system parameters are mean turnover rates, generalised Michaelis-Menten constants, and constants for inhibition and activation. All parameters correspond to molecular energies, for instance, binding energies between reactants and enzyme. CONCLUSION: Convenience kinetics can be used to translate a biochemical network – manually or automatically - into a dynamical model with plausible biological properties. It implements enzyme saturation and regulation by activators and inhibitors, covers all possible reaction stoichiometries, and can be specified by a small number of parameters. Its mathematical form makes it especially suitable for parameter estimation and optimisation. Parameter estimates can be easily computed from a least-squares fit to Michaelis-Menten values, turnover rates, equilibrium constants, and other quantities that are routinely measured in enzyme assays and stored in kinetic databases

Delinquent Behavior of Dutch Rural Adolescents

This article compares Dutch rural and non-rural adolescents’ delinquent behavior and examines two social correlates of rural delinquency: communal social control and traditional rural culture. The analyses are based on cross-sectional data, containing 3,797 participants aged 13–18 (48.7% females). The analyses show that rural adolescents are only slightly less likely to engage in delinquent behavior. Furthermore, while rural adolescents are exposed more often to communal social control, this does not substantially reduce the likelihood that they engage in delinquent behavior. Concerning rural culture, marked differences appeared between rural and non-rural adolescents. First, alcohol use and the frequency of visiting pubs were more related to rural adolescents’ engagement in delinquent behavior. Second, the gender gap in delinquency is larger among rural adolescents: whereas rural boys did not differ significantly from non-rural boys, rural girls were significantly less likely to engage in delinquent behavior than non-rural girls. However, the magnitude of the effects of most indicators was rather low. To better account for the variety of rural spaces and cultures, it is recommended that future research into antisocial and criminal behavior of rural adolescents should adopt alternative measurements of rurality, instead of using an indicator of population density only

EFFECTS OF PGC1-a OVEREXPRESSION AT THE ONSET OF MUSCLE REGENERATION

Wesley S. Haynie, Richard, A. Perry, Lemuel A. Brown, David E. Lee, Jacob L. Brown, Megan E. Rosa, Nicholas P. Greene, Tyrone A. Washington. University of Arkansas, Fayetteville, Arkansas Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a transcriptional activator shown to stimulate mitochondrial biogenesis. Down-regulation of PGC-1α is observed during muscular dystrophy, a model of impaired regeneration. However, in healthy muscle PGC-1α expression is unaltered during muscle regeneration. Therefore, the role of PGC-1α at the onset of skeletal muscle regeneration is unclear and needs further elucidation. PURPOSE: To examine the effects of overexpression of PGC-1α on gene expression of lactate dehydrogenase (LDH), TNF-a, and myogenesis markers MyoD and Myogenin at the onset of muscle regeneration. METHODS: 23 C57BL/6 (WT) and 24 Transgenic (A1) mice were used for this study, with A1 mice genetically modified to overexpress the protein PGC-1a. Mice were injected with either PBS or Bupivacaine (MAR) at 12 weeks of age. Tibialis anterior (TA) muscle and tibias were excised 3-days post injection. Tissue was immediately frozen for gene expression analysis using RT-qPCR. RESULTS: There were no differences in body weight, TA weight, tibia length (TL), or TA to TL ratio in any mice 3-days post injection. PGC-1α gene expression was 13-fold greater in the A1-PBS group compared to the WT-PBS group (p\u3c0.05). The A1-MAR group however, expressed approximately 4-fold less PGC-1a compared to the A1-PBS group 3-days post injection (p\u3c0.05). In WT mice, MyoD gene expression was 1.5 fold greater in the MAR group compared to the PBS group (p\u3c0.05). In A1 mice, there was no difference in MyoD expression between the MAR and PBS groups. There was a main effect of MAR to increase Myogenin gene expression in both WT and A1 mice. There was a main effect of genotype to decrease LDH-A expression in both A1 groups (p\u3c0.05). There was a 4-fold increase in LDH-B expression in the A1-PBS group compared to the WT-PBS group (p\u3c0.05). In WT mice, there was no effect of MAR on LDH-B gene expression. However, in A1 mice there was a 50% decrease in the A1-MAR group compared to the A1-PBS group (p\u3c0.05). The inflammation marker TNF-a increased approximately 2-fold as a main effect of genotype in both A1 mice groups (p\u3c0.05). CONCLUSION: A surplus of mitochondria may result in more ROS production and higher levels of TNF-α. Lowered expression of MyoD 3-days post-injection in A1 mice could be attributed to the increased levels in TNF-α. With TNF-α possibly activating NF-κB, a nuclear factor shown to negatively regulate myogenesis

EFFECTS OF AGE AND OBESITY ON MARKERS OF FIBROSIS IN HEARTS OF C57BL/6J MICE

Richard A. Perry, Jr., Lemuel A. Brown, Wesley S. Haynie David E. Lee, Jacob L. Brown, Megan E. Rosa, Nicholas P. Greene, Tyrone A. Washington, University of Arkansas, Fayetteville, Arkansas In 1990, 23.6% of the elderly population (60+ years old) was obese (≥30 BMI). By 2010, it increased to 39.5%. Aging is typically associated with a reduction in lean mass. Loss of muscle mass with advanced age coupled with increased fat mass is referred to as sarcopenic obesity. Cardiac dysfunction is highly associated with both age and obesity. A contributing, underlying mechanism of cardiac dysfunction is cardiac fibrosis. As the prevalence of obesity in the elderly population continues to rise, a firmer understanding of how age and obesity interact to affect cardiac fibrosis is needed. PURPOSE: To examine how sarcopenic obesity affects markers of fibrosis in cardiac tissue. METHODS: Twenty-four C57BL/6J mice were evenly distributed into either a normal chow (17% kcals from fat) or high-fat (60% kcals from fat) diet after weaning. Twelve mice from each diet were euthanized at 12-16 weeks of age (young). The remaining 12 were sacrificed at 22-24 months of age (aged). The classification of the 4 groups is as follows: young-lean, young-obese, aged-lean, aged-obese (n=6 per group). Whole hearts were excised at time of euthanasia, snap-frozen, and processed for use in gene expression assays. Gene expression was measured using RT-qPCR and normalized to 18s. RESULTS: The aged obese group had a 50% decrease (p\u3c0.05) in the collagen III to I ratio compared to all other groups. The aged obese mice had a two-fold higher TIMP-1 gene content compared to the young obese and aged lean groups (p\u3c0.05). There was a main effect of obesity to increase MMP-2 gene content (p\u3c0.05). There was a main effect of obesity to decrease MMP-9 gene content (p\u3c0.05). CONCLUSION: Elevated collagen III to I ratio has been observed in some forms of end-stage cardiomyopathy. Increased mRNA abundance of TIMP-1 has been shown to be related to cardiac fibrosis and dysfunction. Downregulation of MMP-9 mRNA in conjunction with upregulation of MMP-2 mRNA have been correlated with cardiac pathology and fibrosis, respectively. The tendency for the aged, obese group to display all four of these expressions leads to the conclusion that the aged, obese population is at higher risk of developing cardiac fibrosis, a leading cause of cardiac dysfunction

EXTRACELLULAR MATRIX REMODELING AND FIBROSIS DURING THE DEVELOPMENT OF CANCER CACHEXIA

Wesley S. Haynie1, Jacob L. Brown1, David E. Lee1, Megan E. Rosa-Caldwell1, Richard, A. Perry1, Lemuel A. Brown1, Nicholas P. Greene1, Tyrone A. Washington1. 1University of Arkansas, Fayetteville, Arkansas. Cancer-cachexia is the largely irreversible wasting of lean body mass as a result of cancer progression, affecting ~80% of all cancer patients with as much as ~40% of cancer-related deaths being attributed directly to cachexia. Cachexia has been associated with increased fibrosis and reduced physiological function in cardiac muscle, but the possible role and development of fibrosis and associated extracellular matrix (ECM) remodeling in skeletal muscle has lacked evaluation. PURPOSE: To examine the effects of cancer cachexia on ECM remodeling and the development of fibrosis in skeletal muscle. METHODS: 40 C57BL6/J mice were injected with either Lewis Lung Carcinoma cells or a PBS control into their hind-flank at 8 wks of age. The tumor was allowed to develop for 1, 2, 3, or 4 wks (n=8 per group). Tibialis anterior (TA) muscle was extracted and immediately frozen for morphology and mRNA abundance analysis using RT-qPCR. RESULTS: There were no changes in TA muscle weight until 4 wks post-tumor implantation which resulted in a ~22% lower muscle wet weight compared to PBS control (p\u3c0.05). PicroSirius Red staining of TA muscle sections resulted in no change in collagen abundance in all groups with the exception of a 2-3-fold increase at 4 wks relative to all other groups (p\u3c0.05). Collagen 1 mRNA abundance was ~50 % and ~60 % lower than control at 3 and 4 wks post tumor injection, respectively (p\u3c0.05). Collagen 1 mRNA abundance was ~2-fold higher at 1 and 2 wks but there was no difference at 3 or 4wks, all relative to control (p\u3c0.05). The ratio of Collagen 3:1 mRNA abundance decreased ~30-50% from 1-3wks compared to control (p\u3c0.05), but there was no difference at 4-wks. MMP-2 mRNA abundance was ~50% higher at 1-wk compared to control (p\u3c0.05), but was not different 2-4wks from control (p\u3c0.05). MMP-9 mRNA abundance was 3 and 6-fold greater than control at 3 and 4-wks post-injection, respectively (p\u3c0.05). There was a main effect of tumor implantation to reduce TIMP-1 mRNA abundance ~20-70% (p\u3c0.05). CONCLUSION: The development of cancer cachexia results in dysregulation of ECM remodeling and increased collagen deposition within skeletal muscle. This dysregulation could negatively affect skeletal muscle’s ability to maintain muscle mass and respond to other environmental stressors. ACKNOWLEDGEMENTS: This study was funded by the National Institutes of Health, Award number: R15 AR069913/AR/NIAMS

PRELIMINARY STUDY: MUSCLE WASTING IS EXACERBATED IN APCMin/+ MICE WITH LEUCINE SUPPLEMENTATION

Katarina A. Bejarano1, Wesley S. Haynie1, Richard, A. Perry1, Lemuel A. Brown1, David E. Lee1, Jacob L. Brown1, Megan E. Rosa-Caldwell1, Nicholas P. Greene1, Tyrone A. Washington1. 1University of Arkansas, Fayetteville, Arkansas. Cancer Cachexia is a devastating syndrome that affects around 50-80% of cancer patients and is characterized by a rapid, drastic fat and muscle mass loss. The APCMin/+ mouse strain is a well-studied mouse model of human colorectal cancer and cancer cachexia. The branched-chain amino acid leucine is known to stimulate muscle growth/maintenance through activation of the protein mTOR and protein synthesis. PURPOSE: To examine the effects of chronic leucine supplementation on the development of cancer cachexia in APCMin/+ mice. METHODS: 7 APCMin/+ mice (APC) and 11 wild-type (WT) were used for this study. The animals were assigned to the following groups: WT no leucine (WTNL, n=5), WT leucine (WTL, n=5), APCMin/+ no leucine (APCNL, n=5) and APCMin/+ leucine (APCL, n=2). Mice were given ad libitum access to food and water. Mice in the leucine groups received leucine in their water at a concentration of 1.5%. Plantaris muscles and tibias were excised at 20 weeks of age. Tissue was immediately frozen for morphology and gene expression analysis using RT-qPCR. RESULTS: The number of polyps increased in APCMin/+ compared to WT (46.57 ± 2.44 vs 0.00 ± 0.00). The number of polyps \u3c 1 mm was increased (7.75 ± 2.05 vs. 14.33 ± 1.45) in APCL compared to APCNL (p\u3c.05). There was a main effect for APCMin/+ to have lower body mass in than WT mice (p\u3c.0001). There was a main effect of genotype to decrease plantaris weight/tibia length in APCMin/+ mice (p\u3c.0001) and a main effect of leucine to decrease plantaris weight/tibia length in APCMin/+ mice compared to WT mice (p\u3c.05). There was an ~8-fold increase in atrogin-1 gene expression in APCNL compared to WTNL mice (p\u3c.05). Atrogin-1 gene expression was ~7-fold lower in APCL compared to APCNL (p\u3c.05). There was a main effect of genotype to increase MuRF1 expression in APCMin/+ mice compared to WT mice (p\u3c.05). There was a main effect of leucine to decrease MuRF1 expression in APCMin/+ mice compared to WT mice (p\u3c.05). CONCLUSION: Contrary to our initial hypothesis, leucine supplementation seemed to exacerbate muscle mass loss in cachectic mice. Based on gene expression of the E3 ubiquitin ligases, this loss in muscle mass may be independent of protein degradation

CHANGES IN LIVER FIBROSIS DURING THE PROGRESSION OF CANCER-CACHEXIA IN MICE

Kyle W. Turner*1, Megan E. Rosa-CaldwellƗ1, Jacob L. BrownƗ1, David E. LeeƗ1, Richard A. PerryƗ1, Wesley A. HaynieƗ1, Tyrone A. Washingtonǂ1, Michael P. Wiggsǂ2, Nicholas P. Greeneǂ1 1University of Arkansas, Fayetteville, Arkansas; 2Univeristy of Texas at Tyler, Tyler, Texas Cancer is one of the most widespread and deadly diseases in recent history. Cancer-cachexia is a systematic, metabolic disorder that greatly disrupts the patient’s energy balance, causing uncontrollable weight and skeletal muscle loss. This cancer-induced cachexia is one of the major causes of cancer-related death. As the primary metabolic organ controlling energy balance, the liver is likely involved with the progression of cancer-cachexia. However, very little research has investigated the liver’s involvement in the progression of cancer-cachexia. PURPOSE: The purpose of this study was to observe changes in liver fibrosis during the progression of cancer-cachexia. METHODS: C57BL/6J mice were injected with 1X106 lewis lung carcinoma cells in the hind flank and cancer was allowed to progress for 1,2, 3, or 4 wks. Control animals were injected with sterile phosphate buffered saline solution (PBS) and harvested with 4 wks animals, creating five groups (CON, 1, 2, 3 or 4wks cancer progression, n=10-16/group). Livers were harvested and ~8 samples/group were analyzed for collagen deposition and measures of mitochondrial content. Results were analyzed by one-way ANOVA as well as regression analysis. When differences were found, a Tukey-Kramer post hoc was used to determine differences among means, significance was determined at p\u3c0.05. RESULTS: Liver weights normalized to tibia length were ~30% larger in 4wks animals compared to all other groups (p\u3c0.05). Sirius red staining for collagen indicated that collagen area increased over time in an exponential fashion (r2=0.78), with 4wks having statistically greater collagen deposition than all other groups. Preliminary analysis (n=4/group) of COX-IV content in the liver suggested no change in mitochondrial content. CONCLUSION: The liver is clearly affected by the progression of cancer-cachexia, becoming much larger and more fibrotic. It can be inferred that this fibrotic damage may also exacerbate the energy balance problems caused by cancer-cachexia. Therefore, further research needs to be conducted in this area to more thoroughly understand the liver’s role in this pathology. This study was supported by The Arkansas Bioscience Institute and National Institutes of Health R15AR069913