Methylomic profiling and replication implicates deregulation of PCSK9 in alcohol use disorder.

Alcohol use disorder (AUD) is a common and chronic disorder with substantial effects on personal and public health. The underlying pathophysiology is poorly understood but strong evidence suggests significant roles of both genetic and epigenetic components. Given that alcohol affects many organ systems, we performed a cross-tissue and cross-phenotypic analysis of genome-wide methylomic variation in AUD using samples from 3 discovery, 4 replication, and 2 translational cohorts. We identified a differentially methylated region in the promoter of the proprotein convertase subtilisin/kexin 9 (PCSK9) gene that was associated with disease phenotypes. Biological validation showed that PCSK9 promoter methylation is conserved across tissues and positively correlated with expression. Replication in AUD datasets confirmed PCSK9 hypomethylation and a translational mouse model of AUD showed that alcohol exposure leads to PCSK9 downregulation. PCSK9 is primarily expressed in the liver and regulates low-density lipoprotein cholesterol (LDL-C). Our finding of alcohol-induced epigenetic regulation of PCSK9 represents one of the underlying mechanisms between the well-known effects of alcohol on lipid metabolism and cardiovascular risk, with light alcohol use generally being protective while chronic heavy use has detrimental health outcomes.

Reduced cannabinoid CB1 receptor binding in alcohol dependence measured with positron emission tomography.

Brain cannabinoid CB1 receptors contribute to alcohol-related behaviors in experimental animals, but their potential role in humans with alcohol dependence is poorly understood. We measured CB1 receptors in alcohol dependent patients in early and protracted abstinence, and in comparison with control subjects without alcohol use disorders, using positron emission tomography and [(18)F]FMPEP-d2, a radioligand for CB1 receptors. We scanned 18 male in-patients with alcohol dependence twice, within 3-7 days of admission from ongoing drinking, and after 2-4 weeks of supervised abstinence. Imaging data were compared with those from 19 age-matched healthy male control subjects. Data were also analyzed for potential influence of a common functional variation (rs2023239) in the CB1 receptor gene (CNR1) that may moderate CB1 receptor density. On the first scan, CB1 receptor binding was 20-30% lower in patients with alcohol dependence than in control subjects in all brain regions and was negatively correlated with years of alcohol abuse. After 2-4 weeks of abstinence, CB1 receptor binding remained similarly reduced in these patients. Irrespective of the diagnostic status, C allele carriers at rs2023239 had higher CB1 receptor binding compared with non-carriers. Alcohol dependence is associated with a widespread reduction of cannabinoid CB1 receptor binding in the human brain and this reduction persists at least 2-4 weeks into abstinence. The correlation of reduced binding with years of alcohol abuse suggests an involvement of CB1 receptors in alcohol dependence in humans.

A genetic determinant of the striatal dopamine response to alcohol in men.

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [(11)C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

The relationship between folate and docosahexaenoic acid in men.

OBJECTIVE: Docosahexaenoic acid (DHA, 22:6n-3), an essential omega 3 fatty acid, may protect against disorders of emotional regulation as well as cardiovascular disease. Animal studies demonstrate that dietary folate can increase tissue concentrations of DHA, although the literature, to date, includes no human studies examining the possibility that folate status may affect plasma DHA concentrations. The objective of this study is to determine if the blood concentrations of folate and DHA are correlated in humans. DESIGN: Retrospective study. SETTING: An American research hospital. SUBJECTS: A total of 15 normal and 22 hostile and aggressive subjects, with a mean age of 38 years. METHODS: Concentrations of plasma polyunsaturated essential fatty acids and red blood cell folate (RBC folate) were obtained prior to 1996, before American flour was enriched with folate. RESULTS: RBC folate was significantly correlated with plasma DHA, r=0.57, P=0.005 in the aggressive group. Age, smoking and alcohol consumption did not alter the results. No other essential fatty acids were significantly associated with RBC folate in either group. CONCLUSIONS: The positive relationship between plasma DHA and RBC folate concentrations suggests that these two nutrients should be examined together in order to make the most accurate inferences about their relative contributions to disease pathogenesis. Our findings present one explanation why some conditions associated with hostility and low DHA status, such as cardiovascular disease and emotional disorders, are also associated with low folate status. SPONSORSHIP: National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism.

Serotonin, testosterone and alcohol in the etiology of domestic violence.

In a previous study we administered the panicogenic agent sodium lactate to a select group of perpetrators of domestic violence and comparison groups. Results of that study showed that perpetrators exhibited exaggerated lactate-induced fear, panic and rage. In this current study, we compared the cerebral spinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and testosterone obtained from perpetrators of domestic violence and a group of healthy comparison subjects. All subjects were assessed for DSM-III-R diagnoses. Perpetrators with alcohol dependence (DV-ALC) (n=13), perpetrators without alcohol dependence (DV-NALC) (n=10) and healthy comparison subjects (HCS) (n=20) were clinically assessed using the Spielberger Trait Anxiety, Brown-Goodwin Aggression Scale, Buss Durkee Hostility Inventory and Straus Conflict Tactics. Following an overnight fast and bed rest, subjects received a lumbar puncture to obtain CSF concentrations of 5-HIAA and testosterone. Perpetrators scored significantly higher on measures of aggression than HCS. DV-NALC had significantly lower concentrations of CSF 5-HIAA and higher Straus Conflict Tactics (CT) physical violence scores than DV-ALC and HCS. DV-ALC had significantly higher concentrations of CSF testosterone than DV-NALC. DV-ALC also had significantly higher Straus CT physical violence scores than HCS. DV-NALC and DV-ALC differed on 5-HIAA concentrations, testosterone concentrations, Straus CT physical violence scores and alcohol dependence. These results suggest that DV-NALC and DV-ALC groups could have different biological mechanisms mediating domestic violence.

The time-course of electrocardiographic interbeat interval dynamics in alcoholic subjects after short-term abstinence.

Alcohol dependence has been correlated with decreases in heart rate variability. However, the time course of recovery of heart rate variability after cessation of alcohol consumption is unknown. We used electrocardiogram (ECG) data serially obtained from a population of detoxifying alcoholic subjects to determine the Hurst exponent of the ECG interbeat interval time series. Higher values of the Hurst exponent are associated with decreased heart rate variability when H< or =0.5. We tested a series of response-surface models relating the Hurst exponent (H) thus obtained to the following independent variables: the time interval T (days since last use of alcohol), A (age in years at time of admission), and gender. The best-fit model was: H(T)=(KA+H(m)T+H(f)T)/(1+T), F=5.2, P(F)

Increased coronary artery blood flow with aortomyoplasty in chronic heart failure.

BACKGROUND: We hypothesized that diastolic counter-pulsation using aortomyoplasty will increase coronary blood flow. METHODS: In dogs (n = 6, 20 to 25 kg), the left latissimus dorsi muscle was isolated, wrapped around the descending thoracic aorta, and conditioned by chronic electrical stimulation. Heart failure was induced by rapid ventricular pacing. In a terminal study, left ventricular and aortic pressures, and blood flow in the left anterior descending coronary artery and descending aorta were measured. The endocardial-viability ratio was calculated. RESULTS: Aortomyoplasty increased mean diastolic aortic pressure (70 +/- 5 to 75 +/- 5 mm Hg, p < 0.05) and reduced peak left ventricular pressure (86 +/- 4 to 84 +/- 4 mm Hg, p < 0.05), leading to a 16% increase in endocardial-viability ratio (1.29 +/- 0.05 to 1.49 +/- 0.05, p < 0.05). Coronary blood flow was increased by 15% (8.2 +/- 1.5 to 9.4 +/- 1.6 mL/min, p < 0.05). During muscle contraction, 2.7 +/- 0.5 mL was ejected from the wrapped aortic segment. CONCLUSIONS: These data demonstrate that aortomyoplasty provides successful diastolic counterpulsation after muscle conditioning and heart failure.

Lactate-induced rage and panic in a select group of subjects who perpetrate acts of domestic violence.

BACKGROUND: Perpetrators of domestic violence frequently report symptoms of autonomic arousal and a sense of fear and/or loss of control at the time of the violence. Since many of these symptoms are also associated with panic attacks, we hypothesized that perpetrators of domestic violence and patients with panic attacks may share similar exaggerated fear-related behaviors. To test this hypothesis, we employed the panicogenic agent sodium lactate to examine the response of perpetrators to anxiety fear induced by a chemical agent. METHODS: Using a double-blind, placebo-controlled design, we infused 0.5 mol/L sodium lactate or placebo over 20 min on separate days to a select group of subjects who perpetrate acts of domestic violence and two nonviolent comparison groups. We compared their behavioral, neuroendocrine, and physiologic responses. RESULTS: Lactate administration elicited intense emotional responses in the perpetrators of domestic violence. Perpetrators evidenced more lactate-induced rage and panic and showed greater changes in speech, breathing, and motor activity than did nonviolent control subjects. There were no significant differences between the groups for any neuroendocrine or physiologic measure. CONCLUSIONS: These results are consistent with our hypothesis that some perpetrators of domestic violence have exaggerated fear-related behavioral responses.

Plasma total cholesterol concentrations do not predict cerebrospinal fluid neurotransmitter metabolites: implications for the biophysical role of highly unsaturated fatty acids.

Low concentrations of a metabolite of serotonin found in cerebrospinal fluid (CSF), 5-hydroxyindolacetic acid (5-HIAA), are strongly associated with suicidal and violent behaviors. Although lowering of plasma total cholesterol has been suggested to increase mortality from suicide and violence by decreasing concentrations of CSF 5-HIAA via changes in membrane biophysical properties, highly unsaturated fatty acids may play a more important role. Violent and nonviolent comparison groups, early- and late-onset alcoholics, and healthy comparison subjects were studied to control for alcohol use and predisposition to violence. Fasting plasma total cholesterol and CSF were assayed under stringently controlled conditions. When all groups were combined (n = 234), plasma cholesterol concentrations had a weak positive correlation with CSF 5-HIAA (r = 0.18, P < 0.01). However, age correlated with both plasma total cholesterol and CSF 5-HIAA concentrations. When age was included in multiple regression models, the correlation between cholesterol and CSF 5-HIAA concentrations was not significant. Cholesterol correlated weakly with CSF 5-HIAA concentrations only in late-onset alcoholics after age was controlled for, but the relation was not significant after correction for multiple testing. CSF homovanillic acid did not correlate with plasma total cholesterol in any group. Plasma total cholesterol had no apparent relation to CSF neurotransmitter metabolites in any group of subjects. Highly unsaturated essential fatty acids, which are also critical determinants of membrane biophysical properties and may be linked to brain serotonin concentrations, should also be considered in studies examining the effect of lowering fat intake on the incidence of suicide and violence.

Buspirone treatment of alcoholism: age of onset, and cerebrospinal fluid 5-hydroxyindolacetic acid and homovanillic acid concentrations, but not medication treatment, predict return to drinking.

Disturbances in central nervous system serotonin (5-HT) have been implicated in the pathophysiology of alcoholism. To test the hypothesis that increasing 5-HT function could promote treatment compliance, we randomized patients who had completed a 5-week inpatient treatment program for alcoholism to receive either buspirone or placebo for 1 year. Ten of the 49 patients remained in the study for the entire year. The days to relapse did not differ significantly between patients receiving buspirone or placebo. Regardless of the medication, late-onset alcoholics had a longer time to relapse than early-onset alcoholics. Cerebrospinal fluid showed that patients with high concentrations of both the 5-HT metabolite, 5-hydroxyindoleacetic acid, and the dopamine metabolite, homovanillic acid, were more likely to relapse, compared with patients with low concentrations of cerebrospinal fluid 5-hydroxyindoleacetic acid and homovanillic acid.

Selective stimulation of latissimus dorsi muscle for cardiac assist.

The contractile power of the latissimus dorsi muscle (LDM) is used in skeletal muscle cardiac assist (SMCA) to augment the blood pumping ability of a failing heart. The LDM has three anatomically distinct, independently innervate segments-the transverse, oblique, and lateral. There are potential advantages to selectively stimulating these LDM regions. We hypothesized that (1) the three nerve branches could be stimulated selectively to activate individual muscle regions with little or no functional overlap, (2) the three muscle regions would generate similar force, and (3) nerves stimulated in combinations would generate forces corresponding to the sum of forces generated by the individual regions. In acute studies of canine LDM (n=5), regional electromyogram (EMG) and isometric force were recorded while branches of the thoracodorsal nerve were stimulated (via nerve-cuff electrodes) individually and in combinations. Analysis of regional EMG and force confirmed selective activation. Stimulation of lateral, oblique, and transverse branches of thoracodorsal nerve activated 53+/-5%, 20+/-9%, and 36+/-9% of the muscle, respectively; with corresponding developed forces of 48+/-6%, 21+/-8%, and 31+/-8% of total muscle force (R=0.98, p<0.05). Selective activation of LDM is possible with little or no functional overlap; however, the muscle regions were nonuniform. Selective stimulation may ultimately facilitate the use of performance enhancing stimulus protocols for SMCA.

Triazolam-induced changes in alcoholic thought processes.

This study was designed to examine and contrast cognitive effects (explicit memory and access to semantic knowledge) of the benzodiazepine Halcion (triazolam) in ten normal volunteers and ten cognitively un-impaired detoxified alcoholics. The two groups were indistinguishable from one another under placebo conditions on all measures of cognitive functioning. Under Halcion test conditions (0.375 mg p.o.), both groups were about equally impaired in their recall of to-be-remembered information. However, alcoholics, were more likely to recall information that they were not asked to remember (intrusion errors) on all measures of explicit remembering. Alcoholics also generated relatively uncommon (low frequency) responses from semantic memory, rather than common, categorically related associations in response to stimuli such as types of vegetables, flowers, and fruit following the administration of Halcion, but were not different from normal volunteers in the types of responses generated under placebo conditions. These findings suggest that a drug challenge that simulates many of the effects of acute alcohol administration induces alcoholics to think and remember differently (qualitatively) from normal volunteers.

Essential fatty acids predict metabolites of serotonin and dopamine in cerebrospinal fluid among healthy control subjects, and early- and late-onset alcoholics.

BACKGROUND: Impulsive violence, suicide, and depression are strongly associated with low concentrations of cerebrospinal fluid 5-hydroxyindoleacetic acid (CSF 5-HIAA). Increased suicide and trauma reported in some cholesterol-lowering trials may be related to altered concentrations of polyunsaturated fatty acids rather than cholesterol, a possible surrogate marker. METHODS: CSF 5-HIAA and homovanillic acid (HVA), total cholesterol, and plasma fatty acid concentrations were examined in 176 subjects, including 49 healthy volunteers, and 88 early- and 39 late-onset alcoholics. RESULTS: Among each group, polyunsaturated fatty acids predicted both CSF 5-HIAA and CSF HVA concentrations, but total cholesterol was unrelated to either neurotransmitter metabolite. The relationships between plasma 22: 6n3 and CSF 5-HIAA were significantly different when healthy volunteers (r = .35) were compared to early-onset alcoholics (r = -.38) (p < .0002). CONCLUSIONS: Dietary studies are indicated to determine if essential fatty acid supplementation can influence central nervous system serotonin and dopamine metabolism and modify impulsive behaviors related to these neurotransmitters.

A replication study of violent and nonviolent subjects: cerebrospinal fluid metabolites of serotonin and dopamine are predicted by plasma essential fatty acids.

BACKGROUND: Among an independent group of subjects selected for their history of violent, impulsive behaviors and nonviolent control subjects, we attempted to replicate the finding that plasma docosahexaenoic acid concentrations were negatively correlated with cerebrospinal fluid 5-hydroxyindoleacetic acid (CSF 5-HIAA) concentrations. METHODS: CSF 5-HIAA and homovanillic acid (HVA), fasting total cholesterol, and plasma fatty acid concentrations were examined in violent and nonviolent subjects matched for their severity of alcohol dependence. RESULTS: Violent subjects had significantly higher lifetime violence and hostility ratings and lower concentrations of CSF 5-HIAA than nonviolent subjects. Plasma docosahexaenoic acid was negatively correlated with CSF 5-HIAA only among violent subjects. CONCLUSIONS: This observational study suggests that dietary essential fatty acids may change neurotransmitter concentrations. Prospective dietary intervention trials will be required to determine if increasing dietary intake of docosahexaenoic acid will increase or decrease either CSF 5-HIAA concentrations or impulsive and violent behaviors.

A versatile microprocessor-based multichannel stimulator for skeletal muscle cardiac assist.

A versatile, microprocessor-based stimulator for skeletal muscle cardiac assist (SMCA) has been designed, constructed, and used in several studies. The stimulator uses multiple bipolar electrodes to deliver arbitrarily specified electrical stimulus sequences to three nerve branches of the latissimus dorsi muscle. The electrodes are electrically isolated to effect regional stimulation of the muscle. The width, amplitude, and interpulse interval of each pulse in the stimulus sequence are independently variable, and the three channels are independently programmable, allowing a wide variety of stimulus patterns. Battery powered units have been used in studies for up to one year. In this paper, the stimulator and sample applications in SMCA are described.

Effect of alcoholism on the incidence of lactate-induced panic attacks.

BACKGROUND: Chronic alcohol use is associated with higher than expected rates of panic disorder. METHODS: To study the relationship between alcoholism and panic disorder, we administered the panicogenic agent, sodium lactate, to 26 alcoholics with either panic disorder or frequent panic attacks (ALCPAN), 20 nonalcoholics with panic disorder (PAN), 14 alcoholics without a history of panic attacks, and 14 healthy volunteers. RESULTS: PAN were significantly more likely to have a lactate-induced panic attack (65%) than ALCPAN (23%). ALCPAN who had the onset of panic attacks prior to alcoholism also had a reduced frequency of lactate-induced panic attacks (26.7%) compared to PAN. CONCLUSIONS: There is a reduced incidence of lactate-induced panic attacks in ALCPAN. This reduction does not appear to be explained by the relative onset of panic attacks to alcoholism. The role of excessive alcohol consumption in the decreased frequency of lactate-induced panic attacks seen in ALCPAN needs further study.

Effects of triazolam and ethanol on proactive interference: evidence for an impairment in retrieval inhibition.

The effects of two memory-impairing drugs, ethanol and triazolam, on proactive interference (PI) in memory were studied. Following ingestion of either one of these drugs or a placebo, subjects studied an A-B list (e.g., BEE-WASP) of paired associates, followed by an A-C list (e.g., BEE-HONEY) on the interference trial, and a D-E list (e.g., KING-QUEEN) followed by an A-C list on the control trial. A PI effect was found in the data, such that subjects produced fewer correct second list targets on the interference trial than on the control trial. Neither ethanol nor triazolam was found to influence the size of the PI effect. However, both drugs were found to increase B intrusions on the test of the A-C list, to impair subjects' ability to produce more than one studied response for each cue word, and to impair the subjective experience of retrieved memory information. These data suggest that ethanol and triazolam impair an inhibitory process that normally operates as one component of intentional retrieval, playing an important role in the suppression of unwanted information during a memory task.

Variable-frequency train stimulation of canine latissimus dorsi muscle during shortening contractions.

In cardiomyoplasty, the latissimus dorsi muscle (LDM) is wrapped around the heart ventricles and electrically activated with a constant-frequency train (CFT). This study tested the hypotheses that increased mechanical performance from the LDM could be achieved by activating the muscle with variable-frequency trains (VFTs) of shorter duration or containing fewer stimulus pulses than the CFT now used. The mechanical performance of the canine LDM (n = 7) during shortening contractions was measured while the muscle was stimulated with 5- and 6-pulse CFTs (of duration 132 and 165 ms, respectively) and 5- and 6-pulse VFTs (of duration 104 and 143 ms, respectively) that were designed to take advantage of the catchlike property of skeletal muscle. Measurements were made from fresh and fatigued muscles. For the fresh muscles, the VFTs elicited significantly greater peak power than did the 6-pulse CFT. When the muscles were fatigued, VFT stimulation significantly improved both the peak and mean power produced compared with stimulation by CFTs. These results show that stimulation of the LDM with shorter duration VFTs is potentially useful for application in cardiomyoplasty.

Cardiomyoplasty. Latissimus dorsi muscle function and blood flow during isolation.

Cardiomyoplasty is a new surgical treatment for heart failure in which skeletal muscle assists the heart. However, for the first 2 weeks postoperatively, the latissimus dorsi muscle (LDM) remains unstimulated, and during the next 2 weeks, the LDM is stimulated with only one pulse every other heart beat. Thus, for the initial 4 postoperative weeks, minimal systolic assistance is provided. The present study determined if the LDM is capable of providing early assistance. Cardiomyoplasty surgery involves severing the perforating intercostal arteries to the LDM, detaching the LDM from its distal insertion, and wrapping it around the heart. At each of these steps, we measured LDM force development, shortening, and blood flow in six dogs. At control, LDM shortening, work, and power decreased during a 2 min fatigue test: fatigue indices (final/ initial value) for shortening, work, and power were 47.6 +/- 6.9%, 47.5 +/- 7.1%, and 46.9 +/- 6.6%, respectively. Blood flow increased in the proximal (P), mid (M), and distal (D) LDM during the fatigue test. After partial vascular isolation, initial shortening, work, and power decreased by 29.4%, 32.5%, and 31.7% from their respective control values. During the fatigue test, fatigue indices for shortening, work, and power were 24.7 +/- 3.3%, 19.5 +/- 4.6%, and 22.2 +/- 4.7%, respectively, all significantly (p < 0.05) less than control values. Resting blood flows were unaltered. During exercise, flow to the P increased, whereas flow did not increase in M (p < 0.05). Loss of LDM function was most apparent after mobilizing and reattaching the muscle. Initial shortening, work, and power significantly decreased (p < 0.05) by 74.1%, 76.8%, and 74.4%, from their respective control values. During a fatigue test, final values for shortening, work, and power were all near zero. Resting blood flow decreased in the M and D (p < 0.05) and, during exercise, blood flow increased only in P. Thus, LDM function was severely depressed during the isolation procedure. This functional loss is associated with inadequate blood flow responses. Therefore, preconditioning and/or revascularization is needed if the LDM is to provide cardiac assistance shortly after cardiomyoplasty surgery.

Behavioral and neuroendocrine responses to m-chlorophenylpiperazine in subtypes of alcoholics and in healthy comparison subjects.

OBJECTIVE: The purpose of this study was to explore central serotonergic functions in subgroups of alcoholics and in healthy comparison subjects. METHOD: The mixed serotonin (5-HT) agonist/antagonist m-chlorophenylpiperazine (m-CPP) was administered to male alcoholic patients who were classified according to the criteria of von Knorring et al. as type I alcoholics (late onset) (N = 16) or type II alcoholics (early onset with antisocial traits) (N = 24) and to 22 healthy comparison subjects. Psychological, physiological, and neuroendocrine measures were obtained before and after the m-CPP infusion. RESULTS: m-CPP elicited subtype-related differential effects among the alcoholics; the type I alcoholics reported more anger and anxiety, and the type II alcoholics reported increased euphoria and a greater likelihood of drinking. The healthy comparison subjects exhibited a greater increase in plasma ACTH response to the m-CPP infusion than the alcoholics regardless of subtype. CONCLUSIONS: Differences in certain 5-HT receptor functions may explain some of the clinical characteristics that differentiate the type II and type I subgroups of alcoholic patients. Furthermore, alcoholics may have reduced sensitivity of 5-HT2C receptors in comparison with healthy subjects.