Long term alcohol ingestion may produce severe oxidant stress and lead to skeletal muscle dysfunction. Emerging evidence has suggested that members of the interleukin-6 (IL-6) family of cytokines play diverse roles in the regulation of skeletal muscle mass. Thus, our goals were (1) to minimize the degree of oxidant stress and attenuate atrophy by supplementing the diets of alcohol-fed rats with the glutathione precursor, procysteine, and (2) to identify the roles of IL-6 family members in alcoholic myopathy.
Age- and gender-matched Sprague-Dawley rats were fed the Lieber-DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 35 wk. Subgroups of alcohol-fed rats received procysteine (0.35%, w/v) for the final 12 wk. Plantaris morphology was assessed by hematoxylin and eosin staining. Major components of glutathione metabolism were determined by assay kits. Real time PCR was used to determine expression levels of several genes.
Plantaris muscles from alcohol-fed rats displayed extensive atrophy, as well as decreased glutathione levels, decreased activities of glutathione reductase and glutathione peroxidase, decreased superoxide dismutase (SOD)-2 (Mn-SOD2), and increased NADPH oxidase-1 gene expression - each indicative of significant oxidant stress. Alcohol also induced gene expression of catabolic factors including IL-6, oncostatin M, atrogin-1, muscle ring finger protein-1, and IGFBP-1. Procysteine treatment attenuated plantaris atrophy, restored glutathione levels, and increased catalase, Cu/Zn-SOD1, and Mn-SOD2 mRNA expression, but did not reduce other markers of oxidant stress or levels of these catabolic factors. Instead, procysteine stimulated gene expression of anabolic factors such as insulin-like growth factor-1, ciliary neurotrophic factor and cardiotrophin-1.
Procysteine significantly attenuated, but did not completely abrogate, alcohol-induced oxidant stress or catabolic factors. Rather, procysteine minimized the extent of plantaris atrophy by inducing components of several anabolic pathways. Therefore, anti-oxidant treatments such as procysteine supplementation may benefit individuals with alcoholic myopathy.
alcoholic myopathy; cardiotrophin-1; ciliary neurotrophic factor; interleukin-6; procysteine
Aims: To assess the effectiveness of procysteine (PRO) supplementation provided during a period of abstinence (ABS) on alcohol-induced skeletal muscle atrophy and oxidant stress. Methods: Age- and gender-matched Sprague–Dawley rats were fed the Lieber–DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 12 week. Next, subgroups of alcohol-fed rats were fed the control diet for 2 week (ABS) supplemented with either PRO (0.35%, w/v) or vehicle. Plantaris morphology was assessed by hematoxylin and eosin staining. Total, reduced and oxidized glutathione (GSH) levels and total antioxidant potential were determined by commercially available assay kits. Antibody arrays were used to determine cytokine levels. Real-time polymerase chain reaction was used to determine gene expressions of two E3 ubiquitin ligases, atrogin-1 and muscle ring finger protein-1 (MuRF-1). Results: Plantaris muscles from alcohol-fed rats displayed extensive atrophy, as well as decreased GSH levels, a trend for decreased total antioxidant potential and elevated atrogin-1 and MuRF-1 mRNA levels. GSH levels and total antioxidant potential continued to decrease during 2 weeks of ABS from alcohol, which were normalized in abstinent rats provided PRO. Gene levels of both E3 ligases returned to baseline during ABS. In parallel, plantaris cross-sectional area increased in both groups during ABS. Conclusions: PRO supplementation during ABS significantly attenuated alcohol-induced redox stress compared with untreated abstinent rats. Thus, our data may suggest that GSH restoration therapy may provide therapeutic benefits to the overall antioxidant state of skeletal muscle when prescribed in conjunction with an established detoxification program for recovering alcoholics.
Alcohol-related chronic myopathy is characterized by severe biochemical and structural changes to skeletal muscle. Our goals were to: (1) identify early regulatory elements that precede the overt manifestation of plantaris atrophy; and (2) circumvent these derangements by supplementing alcohol-fed rats with the glutathione precursor, procysteine. After 6 weeks of daily ingestion, before the development of overt atrophy of the plantaris muscle, alcohol increased several markers of oxidative stress and increased gene expressions of atrogin-1 and transforming growth factor-β1 (TGF-β1) by ~60- and ~65-fold, respectively, which were attenuated by procysteine supplementation. Interestingly, after 28 weeks of alcohol ingestion, when overt plantaris atrophy had developed, atrogin-1 and TGF-β1 gene expression had returned to baseline levels. Together, these findings suggest that alcohol-induced, redox-sensitive alterations drive pro-atrophy signaling pathways that precede muscle atrophy. Therefore, targeted anti-oxidant treatments such as procysteine supplementation may benefit individuals with chronic alcohol abuse, particularly if given prior to the development of clinically significant myopathy.
alcoholic myopathy; atrogin-1; glutathione; oxidative stress; transforming growth factor-β1
Alcohol abuse and HIV-1 infection frequently co-exist and these individuals are at high risk for serious lung infections and respiratory failure. Although alcohol ingestion and HIV-1 transgene expression have been shown to independently cause oxidative stress and disrupt alveolar epithelial barrier function in experimental models, their interactive effects have not been examined.
Methods and Results
In this study we determined that chronic alcohol ingestion (12 wks) exacerbated the already significant defects in alveolar epithelial paracellular permeability and lung liquid clearance in HIV-1 transgenic rats. Further, immunocytochemical analyses of tight junction protein expression in primary alveolar epithelial cells showed that occludin and zonula occludens-1 (ZO-1) localization within the plasma membrane was more disrupted than in either condition alone, consistent with the observed defects in epithelial barrier function. Interestingly, expression of Nrf2, the transcription factor required to activate the antioxidant response element, was decreased in primary alveolar epithelial cells isolated from HIV-1 transgenic rats. In parallel, exposing lung epithelial cells in vitro to either alcohol or the HIV-related protein gp120 also decreased Nrf2 expression. Importantly, treatment with procysteine, which increases thiol antioxidants including glutathione, improved tight junction protein localization in the plasma membrane and restored alveolar epithelial barrier function in alcohol-fed HIV-1 transgenic rats.
These results provide novel evidence that HIV-related proteins and alcohol together causes more barrier dysfunction in the lung epithelium than either stress alone. However, these significant effects on the alveolar barrier can be mitigated by augmenting the thiol antioxidant pool, a strategy with potential clinical applications in subjects who are highly vulnerable to lung disease because of co-existent alcohol abuse and HIV infection.
alveolar barrier function; tight junction proteins; Nrf2; procysteine; glutathione
This study investigated the effects of voluntary wheel running on satellite cells in the rat plantaris muscle. Seventeen 5-week-old male Wistar rats were assigned to a control (n = 5) or training (n = 12) group. Each rat in the training group ran voluntarily in a running-wheel cage for 8 weeks. After the training period, the animals were anesthetized, and the plantaris muscles were removed, weighed, and analyzed immunohistochemically and biochemically. Although there were no significant differences in muscle weight or fiber area between the groups, the numbers of satellite cells and myonuclei per muscle fiber, percentage of satellite cells, and citrate synthase activity were significantly higher in the training group compared with the control group (p < 0.05). The percentage of satellite cells was also positively correlated with distance run in the training group (r = 0.61, p < 0.05). Voluntary running can induce an increase in the number of satellite cells without changing the mean fiber area in the rat plantaris muscle; this increase in satellite cell content is a function of distance run.
Key pointsThere is no study about the effect of voluntary running on satellite cells in the rat plantaris muscle.Voluntary running training causes an increase of citrate synthase activity in the rat plantaris muscle but does not affect muscle weight and mean fiber area in the rat plantaris muscle.Voluntary running can induce an increase in the number of satellite cells without hypertrophy of the rat plantaris muscle.
Endurance training; muscle damage; hypertrophy; myonuclear; Pax7.
Previous studies have demonstrated that endurance exercise training increases the level of heat shock proteins (HSPs) in skeletal muscles. However, little attention has been drawn to the effects of high intensity-short duration exercise, or sprint- interval training (SIT) on HSP72 level in rat skeletal muscles. This study performed to test the hypothesis that the SIT would induce the HSP72 in fast and slow skeletal muscles of rats. Young male Wistar rats (8 weeks old) were randomly assigned to a control (CON) or a SIT group (n = 8/group). Animals in the SIT group were trained (1 min/sprint, 6~10 sets/day and 5~6 days/week) on a treadmill for 9 weeks. After the training period, HSP72 levels in the plantaris (fast) and soleus (slow) muscles were analyzed by Western blotting method. Enzyme activities (hexokinase, phosphofructokinase and citrate synthase) and histochemical properties (muscle fiber type compositions and cross sectional area) in both muscles were also determined. The SIT resulted in significantly (p < 0.05) higher levels of HSP72 in both the plantaris and soleus muscles compared to the CON group, with the plantaris producing a greater HSP72 increase than the soleus (plantaris; 550 ± 116%, soleus; 26 ± 8%, p < 0.05). Further, there were bioenergetic improvements, fast-to-slow shift of muscle fiber composition and hypertrophy in the type IIA fiber only in the plantaris muscle. These findings indicate that the SIT program increases HSP72 level of the rat hindlimb muscles, and the SIT-induced accumulation of HSP72 differs between fast and slow muscles.
Key PointsThere is no study about the effects of high intensity but short duration exercise, or sprint-interval training (SIT) on heat shock protein 72 (HSP72) level in skeletal muscles.The SIT program (≤ 10 min·day-1) accumulated HSP72 in rat skeletal muscles.The SIT-induced accumulation of HSP72 in the plantaris (fast) muscle was drastic compared to the soleus (slow) muscle and accompanied with the improvements of enzyme activities, fast-to-slow shift within fast muscle fiber type and muscle hypertrophy.
Hindlimb; treadmill running; enzyme activity; fiber type shift; hypertrophy
The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-13C]-thiazolidine-4-carboxylate, 13C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by 13C magnetic resonance spectroscopy. Although uptake and clearance of 13C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of 13C-OTZ over 20 h, however, resulted in 13C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.
Magnetic resonance; Metabolism; Cysteine; Glutathione; Taurine
Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.
acquired immunodeficiency syndrome; antioxidants; superoxide
Previously we have shown that chronic alcohol intake causes alcohol-induced ciliary dysfunction (AICD), leading to non-responsive airway cilia. AICD likely occurs through the downregulation of nitric oxide (NO) and cyclic nucleotide-dependent kinases, protein kinase G (PKG) and protein kinase A (PKA). Studies by others have shown that dietary supplementation with the antioxidants N-acetylcysteine (NAC) and procysteine prevent other alcohol-induced lung complications. This led us to hypothesize that dietary supplementation with NAC or procysteine prevents AICD. To test this hypothesis, C57BL/6 mice drank an alcohol/water solution (20% w/v) ad libitum for 6 weeks and were concurrently fed dietary supplements of either NAC or procysteine. Ciliary beat frequency (CBF) was measured in mice tracheas, and PKG/PKA responsiveness to β-agonists and NOx levels were measured from bronchoalveolar lavage (BAL) fluid. Long-term alcohol drinking reduced CBF, PKG and PKA responsiveness to β-agonists, and lung NOx levels in BAL fluid. In contrast, alcohol-drinking mice fed NAC or procysteine sustained ciliary function and PKG and PKA responsiveness to β-agonists. However, BAL NO levels remained low despite antioxidant supplementation. We also determined that removal of alcohol from the drinking water for as little as 1 week restored ciliary function, but not PKG and PKA responsiveness to β-agonists. We conclude that dietary supplementation with NAC or procysteine protects against AICD. In addition, alcohol removal for 1 week restores cilia function independent of PKG and PKA activity. Our findings provide a rationale for the use of antioxidants to prevent damage to airway mucociliary functions in chronic alcohol-drinking individuals.
alcohol; ciliary dysfunction; nitric oxide; PKA; PKG; N-acetylcysteine; procysteine; ciliary beat frequency
Human immunodeficiency virus type 1 (HIV-1) infection and the consequent acquired immunodeficiency syndrome (AIDS) has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef). Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1) and Transforming Growth Factor-β1 (TGFβ1), three factors associated with muscle catabolism.
Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss), the oxidized form of the anti-oxidant cysteine (Cys), and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold) and TGFβ1 (~5-fold and ~3-fold) in heart and plantaris muscle tissues, respectively.
We provide compelling experimental evidence that HIV-1-related proteins can lead to significant cardiac and skeletal muscle complications independently of viral infection or replication. Our data support the concept that HIV-1-related proteins are not merely disease markers, but rather have significant biological activity that may lead to increased oxidative stress, the stimulation of redox-sensitive pathways, and altered muscle morphologies. If correct, this pathophysiological scheme suggests that the use of dietary thiol supplements could reduce skeletal and cardiac muscle dysfunction in HIV-1-infected individuals.
Late stage cancer malignancies may result in severe skeletal muscle wasting, fatigue and reduced quality of life. Resistance training may attenuate these derangements in cancer patients, but how this hypertrophic response relates to normal muscle adaptations in healthy subjects is unknown. Here, we determined the effect of resistance training on muscle mass and myosin heavy chain (MHC) isoform composition in plantaris muscles from tumor-bearing (TB) rats.
Age- and gender-matched Buffalo rats were used for all studies (n = 6/group). Suspensions of Morris Hepatoma MH7777 cells or normal saline were injected subcutaneously into the dorsum. Six weeks after cell implantation, muscles from TB rats were harvested, weighed and processed for ATP-independent proteasome activity assays. Once tumor-induced atrophy had been established, subgroups of TB rats underwent unilateral, functional overload (FO). Healthy, sham-operated rats served as controls. After six weeks, the extent of plantaris hypertrophy was calculated and MHC isoform compositions were determined by gel electrophoresis.
Six weeks of tumor growth reduced body mass and the relative masses of gastrocnemius, plantaris, tibialis anterior, extensor digitorum longus, and diaphragm muscles (p ≤ 0.05). Percent reductions in body mass had a strong, negative correlation to final tumor size (r = -0.78). ATP-independent proteasome activity was increased in plantaris muscles from TB rats (p ≤ 0.05). In healthy rats, functional overload (FO) increased plantaris mass ~44% compared to the contralateral control muscle, and increased the relative percentage of MHC type I and decreased the relative percentage of MHC type IIb compared to the sham-operated controls (p ≤ 0.05). Importantly, plantaris mass was increased ~24% in TB-FO rats and adaptations to MHC isoform composition were consistent with normal, resistance-trained muscles.
Despite significant skeletal muscle derangements due to cancer, muscle retains the capacity to respond normally to hypertrophic stimuli. Specifically, when challenged with functional overload, plantaris muscles from TB rats displayed greater relative mass, increased percentages of MHC type I and decreased percentages of MHC type IIb. Therefore, resistance training paradigms should provide relative morphological and functional benefits to cancer patients suffering from muscle wasting.
Chronic intermittent alcohol vapor exposure and selective breeding procedures have been used separately for many years to model specific aspects of alcohol dependence. The purpose of the present investigation was to combine these 2 approaches by exposing alcohol-preferring (P) rats to chronic intermittent alcohol vapor for extended periods of time and then testing them for operant alcohol responding in parallel with a group of outbred Wistar rats at multiple time points following the termination of vapor exposure.
P rats (n = 20) and Wistar rats (n = 18) were trained to respond for 10% (w/v) ethanol in an operant situation, then divided into groups matched for intake levels. Animals were then exposed to chronic intermittent alcohol vapor (14 hours ON/10 hours OFF) or air for 8 weeks. Rats were then tested for operant alcohol responding under various conditions and at multiple time points during alcohol withdrawal (6 hours) and protracted abstinence (1 to 15 days).
Chronic alcohol vapor exposure produced similar increases in operant alcohol responding in P rats and Wistar rats during acute withdrawal and protracted abstinence.
These results illustrate the separate and combined effects of genetic selection for high alcohol preference and dependence on alcohol drinking behavior. Furthermore, these results confirm past findings that dependent rats consume more alcohol than nondependent controls well into abstinence following extended periods of alcohol vapor exposure.
Dependence; P Rats; Alcohol Vapor; Withdrawal; Abstinence
Separately, chronic alcohol ingestion and HIV-1 infection are associated with severe skeletal muscle derangements, including atrophy and wasting, weakness, and fatigue. One prospective cohort study reported that 41% of HIV-infected patients met the criteria for alcoholism, however; few reports exist on the co-morbid effects of these two disease processes on skeletal muscle homeostasis. Thus, we analyzed the atrophic effects of chronic alcohol ingestion in HIV-1 transgenic rats and identified alterations to several catabolic and anabolic factors.
Relative plantaris mass, total protein content, and fiber cross-sectional area were reduced in each experimental group compared to healthy, control-fed rats. Alcohol abuse further reduced plantaris fiber area in HIV-1 transgenic rats. Consistent with previous reports, gene levels of myostatin and its receptor activin IIB were not increased in HIV-1 transgenic rat muscle. However, myostatin and activin IIB were induced in healthy and HIV-1 transgenic rats fed alcohol for 12 weeks. Catabolic signaling factors such as TGFβ1, TNFα, and phospho-p38/total-p38 were increased in all groups compared to controls. There was no effect on IL-6, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), or ciliary neurotrophic factor (CNTF) in control-fed, transgenic rats. However, the co-morbidity of chronic alcohol abuse and HIV-1-related protein expression decreased expression of the two anabolic factors, CT-1 and CNTF.
Consistent with previous reports, alcohol abuse accentuated skeletal muscle atrophy in an animal model of HIV/AIDS. While some catabolic pathways known to drive alcoholic or HIV-1-associated myopathies were also elevated in this co-morbid model (e.g., TGFβ1), consistent expression patterns were not apparent. Thus, specific alterations to signaling mechanisms such as the induction of the myostatin/activin IIB system or reductions in growth factor signaling via CT-1- and CNTF-dependent mechanisms may play larger roles in the regulation of muscle mass in alcoholic, HIV-1 models.
This investigation sought to determine if aging affected adaptations of the neuromuscular junction (NMJ) to exercise training. Twenty young adult (8 mo), and 20 aged (24 mo) rats were assigned to either a program of treadmill exercise, or sedentary conditions. Following the 10 week experimental period, rats were euthanized, and soleus and plantaris muscles were removed and frozen. Longitudinal sections of the muscles were fluorescently stained to visualize pre-synaptic nerve terminals, and post-synaptic endplates on both slow- and fast-twitch fibers. Images were collected with confocal microscopy and quantified. Muscle cross-sections were histochemically stained to assess muscle fiber profiles (size and fiber type). Our analysis of NMJs revealed a high degree of specificity and sensitivity to aging, exercise training, and their interaction. In the soleus, slow-twitch NMJs demonstrated significant (P ≤ 0.05) training-induced adaptations in young adult, but not aged rats. In the fast-twitch NMJs of the soleus, aging, but not training was associated with remodeling. In the plantaris, aging but not training, remodeled the predominant fast-twitch NMJs, but only pre-synaptically. In contrast, the slow-twitch NMJs of the plantaris displayed morphologic adaptations to both aging and exercise in pre- and post-synaptic components. Muscle fiber profiles indicated that changes in NMJ size were unrelated to adaptations of their fibers. Our data show that aging interferes with the ability of NMJs to adapt to exercise training. Results also reveal complexity in the coordination of synaptic responses among different muscles, and different fiber types within muscles, in their adaptation to aging and exercise training.
synapse; endplate; nerve terminal; neuromuscular junction; muscle fiber; exercise
Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42°C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non–heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress–elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress–induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to non– heat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.
Acute administration of neuropeptide Y (NPY) modulates alcohol intake in genetic and chemical models of high intake, while leaving intake unaffected during ‘normal’ or baseline conditions. In non-selected, normal rat lines, alcohol consumption can be increased by prolonged exposure to alcohol, and it is unclear what effect a constitutive increase in NPY function will have on alcohol intake. The purpose of the present study was to examine the effects on alcohol intake of an inducible, constitutive overexpression of NPY, one of the most abundant neuropeptides in the central nervous system. A liquid diet was used in combination with repeated alcohol deprivation sessions to increase alcohol intake in normal Wistar rats. We then examined the effect of NPY overexpression in the amygdala on excessive alcohol intake produced by prolonged exposure to alcohol and alcohol deprivation. Repeated withdrawal increased alcohol consumption in a 24-h continuous access two-bottle choice model. Both the number of withdrawals as well as the length of the withdrawal periods affected alcohol consumption with an increased intake resulting from multiple withdrawals and the alcohol deprivation effect being enhanced by longer periods of abstinence. The increase in intake following repeated abstinence was blunted by intra-amygdala administration of a Sindbis viral vector containing NPY cDNA. Amygdala NPY overexpression also was demonstrated to be anxiolytic in the open field test. Repeated withdrawal in combination with a history of alcohol consumption significantly elevated alcohol intake, and the amygdala may mediate the transition to high-drinking states in this model.
alcoholism; animal model; neuropeptide Y; viral vector; amygdala
Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated T3 levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics.
alcoholism; chronic ethanol; abstinence; relapse; microarray; gene expression
AIM: To determine the evolution of transient elastography (TE) in patients with alcoholic liver disease according to alcohol cessation or continuation.
METHODS: We retrospectively selected in our local database all patients who had two TE between June 2005 and November 2010 with chronic alcohol excessive consumption and excluded those with associated cause of liver disease. TE was performed at least one week apart by senior operator. TE examinations with less than ten successful measures or with an interquartile range above 30% were excluded. We retrospectively reviewed file of all patients to include only patient followed up by trained addictologist and for which definite information on alcohol consumption was available. Concomitant biological parameters [aspartate amino transferase (AST), alanine amino transferase and gamma-glutamyl transpeptidase (GGT)] within 4 wk of initial and final TE were recorded. Putative fibrosis score according to initial and final TE were determined with available cut-off for alcoholic liver disease and hepatitis C. Initial and final putative fibrosis score were compared according to alcohol consumption during follow-up.
RESULTS: During the study period 572 patients had TE examination for alcoholic liver disease and 79 of them had at least two examinations. Thirty-seven patients met our criteria with a median follow-up of 32.5 wk. At the end of the study, 13 (35%) were abstinent, and 24 (65%) relapsers. Eight patients had liver biopsy during follow-up. TE decreased significantly during follow-up in 85% of abstinent patients [median (range): -4.9 (-6.1,-1.9)], leading to a modification of the putative fibrosis stage in 28%-71% of patient according to different cut-off value. In relapsers TE increased in 45% and decreased in 54% of patient. There was no statistical difference between initial and final TE in relapsers. In the overall population, using 22.6 kPa as cut-off for cirrhosis, 4 patients had cirrhosis at initial TE and 3 patients had cirrhosis at final TE. Using 19.5 kPa as cut-off for cirrhosis, 7 patients had cirrhosis at initial TE and 5 patients had cirrhosis at final TE. Using 12.5 kPa as cut-off for cirrhosis, 16 patients had cirrhosis at initial TE and 15 patients had cirrhosis at final TE. Evolution of biological data was in accordance with the relapse or abstinent status: abstinence ratio (duration of abstinence/duration follow-up) was correlated with AST ratio (r = -0.465, P = 0.007) and GGT ratio (r = -0.662, P < 0.0001). GGT was correlated with initial (r = 0.488, P = 0.002) and final TE (r = 0.49, P < 0.005). Final TE was correlated with AST (r = 0.362, P < 0.05). Correlation between TE ratio and AST ratio (r = 0.44, P = 0.01) revealed that TE varied proportionally to AST for all patients irrespective of their alcohol status. The same relationship was observed between TE ratio and GGT ratio (r = 0.65, P < 0.0001). Evolution of TE was significantly correlated with the ratio of time of abstinence to observation time (r = -0.387, P = 0.016) and the evolution of liver enzymes.
CONCLUSION: TE significantly decreased with abstinence. Results of TE in alcoholic liver disease cannot be interpreted without taking into account alcohol consumption and liver enzymes.
Alcohol; Transient elastography; Cirrhosis; Fibrosis; Liver biopsy; Liver stiffness
We previously demonstrated that alcohol-fed adolescent rats exhibit reductions in lumbar spine bone mineral density and vertebral body height, suggesting that chronic alcohol consumption has negative consequences for skeletal development during adolescence. Binge alcohol consumption is common in adolescents and young adults, yet little is known about its consequences on skeletal integrity or the attainment of peak bone mass. We used a previously validated binge alcohol exposure model to test the hypothesis that binge alcohol treatment of adolescent rats would be associated with distinct temporal and site-specific bone loss profiles, with incomplete recovery from bone loss following a period of alcohol abstinence. Seventy-two male adolescent Sprague-Dawley rats were assigned to one of 6 treatment groups (n = 12/group) receiving binge alcohol (3g/kg) or saline ip, 3 consecutive days (acute binge), 4 consecutive weekly (3-day) binge cycles (chronic binge), or 4 weekly binge cycles followed by a 30-day abstinence period without alcohol or saline injections (chronic binge with abstinence). Cancellous BMD was determined by pQCT and compressive strength determined by biomechanical testing. Serum testosterone and osteocalcin levels were measured by ELISA. Tibial cancellous BMD was significantly reduced by 25% (p < 0.05) after both acute and chronic binge alcohol treatment and vertebral cancellous BMD was significantly reduced by 15% (p<0.05) after chronic binge exposure. Vertebral compressive strength was also significantly decreased by 31% (p<0.05) after chronic binge alcohol treatment. Tibial cancellous BMD returned to control levels after the 30-day alcohol abstinence period, but vertebral cancellous BMD remained 15% below control values (p <0.05) 30 days after termination of binge alcohol exposures. Serum osteocalcin levels were significantly decreased following acute binge alcohol exposure (p<0.05). These results show that binge alcohol exposure can produce both short and long-term skeletal damage in the adolescent rat. This data may have relevance to peak bone mass attainment and future risk of skeletal disease in adolescents and young adults who engage in repeated binge drinking episodes.
Binge alcohol; adolescent rat; peak bone mass; osteoporosis; fracture
Cocaine-responsive gene expression changes have been described after either no drug abstinence or short periods of abstinence. Little data exist on the persistence of these changes after long-term abstinence. Previously, we reported that after discrete-trial, cocaine self-administration and 10 days of forced abstinence, incubation of cocaine reinforcement was observable by a progressive ratio schedule. The present study used rat discrete-trial cocaine self-administration and long-term forced abstinence to examine: extinction responding, mRNA abundance of known cocaine-responsive genes, and chromatin remodeling. At 30 and 100 days of abstinence, extinction responding increased compared to 3-day abstinent rats. Decreases in both medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) c-fos, Nr4a1, Arc, and EGR1 mRNA were observed, and in most cases persisted, for 100 days of abstinence. The signaling peptides CART and NPY transiently increased in the mPFC, but returned to baseline levels following 10 days of abstinence. To investigate a potential regulatory mechanism for these persistent mRNA changes, levels of histone H3 acetylation at promoters for genes with altered mRNA expression were examined. In the mPFC, histone H3 acetylation decreased after 1 and 10 days of abstinence at the promoter for EGR1. H3 acetylation increased for NPY after 1 day of abstinence and returned to control levels by 10 days of abstinence. Behaviorally, these results demonstrate incubation after discrete-trial cocaine self-administration and prolonged forced abstinence. This incubation is accompanied by changes in gene expression that persist long after cessation of drug administration and may be regulated by chromatin remodeling.
cocaine; abstinence; behavior; medial prefrontal cortex; nucleus accumbens; functional genomics; extinction; incubation; addiction
Heart failure (HF)-induced skeletal muscle atrophy is often associated to exercise intolerance and poor prognosis. Better understanding of the molecular mechanisms underlying HF-induced muscle atrophy may contribute to the development of pharmacological strategies to prevent or treat such condition. It has been shown that autophagy-lysosome system is an important mechanism for maintenance of muscle mass. However, its role in HF-induced myopathy has not been addressed yet. Therefore, the aim of the present study was to evaluate autophagy signaling in myocardial infarction (MI)-induced muscle atrophy in rats.
Wistar rats underwent MI or Sham surgeries, and after 12 weeks were submitted to echocardiography, exercise tolerance and histology evaluations. Cathepsin L activity and expression of autophagy-related genes and proteins were assessed in soleus and plantaris muscles by fluorimetric assay, qRT-PCR and immunoblotting, respectively. MI rats displayed exercise intolerance, left ventricular dysfunction and dilation, thereby suggesting the presence of HF. The key findings of the present study were: a) upregulation of autophagy-related genes (GABARAPL1, ATG7, BNIP3, CTSL1 and LAMP2) was observed only in plantaris while muscle atrophy was observed in both soleus and plantaris muscles, and b) Cathepsin L activity, Bnip3 and Fis1 protein levels, and levels of lipid hydroperoxides were increased specifically in plantaris muscle of MI rats.
Altogether our results provide evidence for autophagy signaling regulation in HF-induced plantaris atrophy but not soleus atrophy. Therefore, autophagy-lysosome system is differentially regulated in atrophic muscles comprising different fiber-types and metabolic characteristics.
Cognitive factors associated with drinking behavior such as positive alcohol expectancies, self-efficacy, perception of impaired control over drinking and perception of drinking problems are considered to have a significant influence on treatment effects and outcome in alcohol-dependent patients. However, the development of a rating scale on lack of perception or denial of drinking problems and impaired control over drinking has not been substantial, even though these are important factors in patients under abstinence-oriented treatment as well as participants in self-help groups such as Alcoholics Anonymous (AA). The Drinking-Related Cognitions Scale (DRCS) is a new self-reported rating scale developed to briefly measure cognitive factors associated with drinking behavior in alcohol-dependent patients under abstinence-oriented treatment, including positive alcohol expectancies, abstinence self-efficacy, perception of impaired control over drinking, and perception of drinking problems. Here, we conducted a prospective cohort study to explore the predictive validity of DRCS.
Participants in this study were 175 middle-aged and elderly Japanese male patients who met the DSM-IV Diagnostic Criteria for Alcohol Dependence. DRCS scores were recorded before and after the inpatient abstinence-oriented treatment program, and treatment outcome was evaluated one year after discharge.
Of the 175 participants, 30 were not available for follow-up; thus the number of subjects for analysis in this study was 145. When the total DRCS score and subscale scores were compared before and after inpatient treatment, a significant increase was seen for both scores. Both the total DRCS score and each subscale score were significantly related to total abstinence, percentage of abstinent days, and the first drinking occasion during the one-year post-treatment period. Therefore, good treatment outcome was significantly predicted by low positive alcohol expectancies, high abstinence self-efficacy, high perception level of impaired control over drinking, and high perception level of drinking problems measured by DRCS.
The DRCS was considered to have satisfactory predictive validity, which further supports our previous findings. It was suggested that DRCS is a promising rating scale for evaluating multidimensional cognitive factors associated with drinking behavior in alcohol-dependent patients under abstinence-oriented treatment.
Alcohol-dependent; Treatment outcome; Predictive validity; Drinking-related cognitions scale; Abstinence-oriented treatment; Positive alcohol expectancies; Abstinence self-efficacy; Perception of impaired control; Perception of drinking problems; Denial
Aims: Dangerous alcohol consumption practices are common in adolescents, yet little is known about their consequences on attainment of peak bone mass and long-term skeletal integrity. We previously demonstrated that binge alcohol-exposed adolescent rats showed site-specific reductions in accruement of bone mineral density and bone strength, which were incompletely recovered following prolonged alcohol abstinence. Currently, we analysed the vertebral transcriptome of adolescent rats following alcohol treatment and abstinence to identify long-term molecular changes in the lumbar spine. Methods: Sixty male adolescent Sprague-Dawley rats were assigned to one of six treatment groups receiving binge alcohol (3 g/kg) or saline i.p., 3 consecutive days (acute binge), 4 consecutive weekly (3-day) binge cycles (chronic binge) or 4 weekly binge cycles followed by a 30-day abstinence period (chronic binge with abstinence). Following treatment, lumbar vertebrae were assayed for global transcriptional changes using gene array technology. Results: Analysis of the adolescent rat vertebral transcriptome identified clusters of binge alcohol-sensitive genes displaying differential expression patterns starting before bone damage was seen and persisting after alcohol treatment was discontinued. Functional grouping of these gene clusters identified candidate cellular pathways affected following acute and chronic binge treatment, as well as pathways remaining modulated following abstinence. Conclusions: These results demonstrate that binge alcohol exposure can produce disruptions of normal bone gene expression patterns in the adolescent rat that persist well beyond the period of active intoxication. This data may have relevance to peak bone mass attainment and future risk of skeletal disease in adolescents engaging in repeated binge-drinking episodes.
The rationale for proposing the “kindling”/stress hypothesis is to provide a conceptual basis for the insidious development and maintenance of alcohol abuse.
Objective and results
An objective of the hypothesis is to emphasize how continued alcohol abuse is linked to progressive neural adaptation. Work has shown that repeated withdrawals from chronic low levels of alcohol sensitize (“kindle”) anxiety-like behavior (“anxiety”) in rats, a finding consistent with multiple withdrawal kindling of seizure activity. Additionally, stress substitutes for initial cycles of the multiple withdrawal protocol to sensitize withdrawal-induced anxiety, which is indicative that stress is capable of facilitating neuroadaptive processes related to withdrawal. The persistence of adaptation caused by stress and multiple withdrawals is revealed by the appearance of withdrawal-induced anxiety following a future re-exposure to a single 5-day period of alcohol. This persisting adaptation also permits stress to induce anxiety during a period of abstinence—a response not observed in animals without previous exposure to alcohol. Furthermore, stress interacts with repeated withdrawals to enhance voluntary alcohol drinking. Results of other preclinical and clinical studies reported in the literature are integrated with these investigations in support of the proposed hypothesis.
The “kindling”/stress hypothesis is based on the premise that repeated withdrawals from cycles of chronic alcohol exposure contribute to a progressive development of persisting adaptive change that sensitizes withdrawal-induced anxiety and allows stress to evoke symptoms associated with negative affect during abstinence. Thus, these consequences of repeated withdrawals account for the evolution of major characteristics of alcoholism, which include worsened acute withdrawal symptoms and increased stress-induced negative affect during abstinence, both of which enhance the likelihood of relapse—and with relapse an inability to limit an abusive pattern of alcohol intake. The “kindling”/stress hypothesis provides a clear strategy for future studies to explore the advancing neural adaptation proposed to contribute to the pathogenesis of alcoholism.
Multiple withdrawals; Restraint stress; Anxiety; Alcohol drinking; Neural adaptation; Alcohol deprivation effect; Alcoholism; Kindling; Kindling/stress hypothesis
Alcoholism is a chronic relapsing disorder. Major characteristics observed in alcoholics during an initial period of alcohol abstinence are altered physiological functions and a negative emotional state. Evidence suggests that a persistent, cumulative adaptation involving a kindling/allostasis-like process occurs during the course of repeated chronic alcohol exposures that is critical for the negative symptoms observed during alcohol withdrawal. Basic studies have provided evidence for specific neurotransmitters within identified brain sites being responsible for the negative emotion induced by the persistent cumulative adaptation following intermittent-alcohol exposures. After an extended period of abstinence, the cumulative alcohol adaptation increases susceptibility to stress- and alcohol cue-induced negative symptoms and alcohol seeking, both of which can facilitate excessive ingestion of alcohol. In the alcoholic, stressful imagery and alcohol cues alter physiological responses, enhance negative emotion, and induce craving. Brain fMRI imaging following stress and alcohol cues has documented neural changes in specific brain regions of alcoholics not observed in social drinkers. Such altered activity in brain of abstinent alcoholics to stress and alcohol cues is consistent with a continuing ethanol adaptation being responsible. Therapies in alcoholics found to block responses to stress and alcohol cues would presumably be potential treatments by which susceptibility for continued alcohol abuse can be reduced. By continuing to define the neurobiological basis of the sustained alcohol adaptation critical for the increased susceptibility of alcoholics to stress and alcohol cues that facilitate craving, a new era is expected to evolve in which the high rate of relapse in alcoholism is minimized. 250
alcoholism; alcohol; adaptation; stress; corticotropin releasing factor; cytokines; brain sites; substance P; relapse; brain imaging