Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice.

Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene, first described in 2004 have now emerged as the most important genetic finding in both autosomal dominant and sporadic Parkinson's disease (PD). While a formidable research effort has ensued since the initial gene discovery, little is known of either the normal or the pathological role of LRRK2. We have created lines of mice that express human wild-type (hWT) or G2019S Lrrk2 via bacterial artificial chromosome (BAC) transgenesis. In vivo analysis of the dopaminergic system revealed abnormal dopamine neurotransmission in both hWT and G2019S transgenic mice evidenced by a decrease in extra-cellular dopamine levels, which was detected without pharmacological manipulation. Immunopathological analysis revealed changes in localization and increased phosphorylation of microtubule binding protein tau in G2019S mice. Quantitative biochemical analysis confirmed the presence of differential phospho-tau species in G2019S mice but surprisingly, upon dephosphorylation the tau isoform banding pattern in G2019S mice remained altered. This suggests that other post-translational modifications of tau occur in G2019S mice. We hypothesize that Lrrk2 may impact on tau processing which subsequently leads to increased phosphorylation. Our models will be useful for further understanding of the mechanistic actions of LRRK2 and future therapeutic screening.

A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease.

Pathogenic substitutions in leucine-rich repeat kinase 2 (LRRK2, Lrrk2) have been genetically linked to familial, late-onset Parkinsonism. End-stage disease is predominantly associated with nigral neuronal loss and Lewy body pathology, but patients may have gliosis, tau or ubiquitin inclusions (pleomorphic pathology). The anatomical distribution of Lrrk2 protein may provide insight into its function in health and neurodegeneration, thus we performed a comparative study with 'in-house' and commercially available Lrrk2 antibodies using brain tissue from wild type and human Lrrk2 transgenic bacterial artificial chromosome (BAC) mice, and from diffuse Lewy body disease (DLBD) patients. Lrrk2 protein was ubiquitously expressed and relatively abundant in most brain regions, including the substantia nigra, thalamus and striatum. Lrrk2 was not a major component of Lewy body or neuritic pathology associated with Parkinson's disease. However, selective loss of dopaminergic neurons in Lrrk2-associated Parkinsonism argues the protein may have regional-specific interactions. Lrrk2 immunohistochemical staining was present in the subventricular zone, a region containing stem cells that give rise to both neurons and glia. A role for Lrrk2 in neurogenesis might provide further insight into the aberrant role of mutant protein in age-associated neurodegeneration with pleomorphic pathology.

Anatomical localization of leucine-rich repeat kinase 2 in mouse brain.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been identified in autosomal dominant late-onset Parkinson's disease. Expression of LRRK2 has previously been reported in brain; however, no precise anatomical information is yet available. We have performed in situ hybridization and quantitative reverse transcription polymerase chain reaction to map LRRK2 mRNA expression in mouse brain. We find LRRK2 is highly expressed in the striatum, cortex and olfactory tubercle; however, little or no expression is found in the substantia nigra, where dopaminergic neurons preferentially degenerate in Parkinson's disease. These findings suggest that LRRK2 mRNA is expressed in dopamine-receptive areas rather than in the dopamine-synthesizing neurons. Consistent with a role LRRK2 in Parkinson's disease, dysfunction of leucine-rich repeat kinase 2 protein in dopamine-innervated areas may to lead to altered dopaminergic neurotransmission and degeneration of the nigro-striatal pathway.

Fiber type and citrate synthase activity in the human gastrocnemius and vastus lateralis with aging.

The purpose of this study was to determine whether enzymatic and histochemical characteristics of human skeletal muscle are altered with aging. Tissues from the vastus lateralis (VL) and gastrocnemius were analyzed for citrate synthase (CS) activity and fiber type in 55 sedentary men (age range 18-80 yr). In this population, CS activity in the gastrocnemius was negatively related to age (r = -0. 32, P < 0.05); there was no relationship in the VL. Treadmill-determined maximal oxygen consumption was positively related (r = 0.40, P < 0.05) to CS in the gastrocnemius but not in the VL. CS activity in the gastrocnemius was 24% lower in the oldest (>/=60 yr, n = 10) vs. the youngest (

Gender-dependent effects of exercise training on serum leptin levels in humans.

Leptin, the product of the ob gene, is elevated in obese humans and appears to be closely related to body fat content. The purpose of the present investigation was to determine the effect of aerobic exercise training on systemic leptin levels in humans. Eighteen sedentary middle-aged men (n = 9) and women (n = 9) who did not differ in aerobic capacity (29.4 +/- 1.2 vs. 27.5 +/- 1.2 ml x kg(-1) x min(-1)) or insulin sensitivity index (3.41 +/- 1.12 vs. 4.88 +/- 0.55) were studied. Fat mass was significantly lower in females vs. males (21.83 +/- 2.25 vs. 26.99 +/- 2.37 kg, P < 0.05). Despite this, fasting serum leptin was significantly higher in the females vs. males (18.27 +/- 2.55 vs. 9.88 +/- 1.26 ng/ml, P < 0.05). Serum leptin concentration decreased 17.5% in females (P < 0.05) after 12 wk of aerobic exercise training (4 day/wk, 30-45 min/day) but was not significantly reduced in males. Fat mass was not altered after training in either group. In contrast, both aerobic capacity (+13% males, +9.1% females) and insulin sensitivity (+35% males, +82% females) were significantly improved subsequent to training. These data suggest that 1) women have higher circulating leptin concentrations despite lower fat mass and 2) exercise training appears to have a greater effect on systemic leptin levels in females than in males.

Leptin is related to body fat content in male distance runners.

Leptin, the product of the ob gene, has been reported to be related to body fat in humans (Considine et al. N. Engl. J. Med. 334: 292, 1996). However, little is known about the physiology of this putative satiety signal in humans. The purpose of the present study was to determine whether leptin is related to body fat content in relatively lean endurance-trained adults. In addition, the effect of acute exercise on circulating leptin concentration was studied. Thirteen male runners, whose mean age, height, weight, %fat, and maximal oxygen consumption (VO2max) were 32.2 +/- 2.5 yr, 176.2 +/- 1.6 cm, 71.9 +/- 6.9 kg, 9.7 +/- 0.9%, and 62.9 +/- 2.2 ml.kg-1.min-1, respectively, were studied. Blood samples were obtained after an overnight fast and again immediately after the completion of a 20-mile run at 70% VO2max under controlled environmental conditions. Serum leptin was closely related to fat mass (r = 0.92) in the runners. Acute exercise had no detectable effect on serum leptin levels (PRE = 2.19 +/- 0.32 ng/ml, POST = 2.14 +/- 0.36 ng/ml). These data indicate that, even at a biological extreme of body fat, circulating leptin concentration is closely related to fat content. Furthermore, the data suggest that, in trained individuals with low leptin concentrations, acute exhaustive exercise has no immediate effect on circulating leptin concentration.

Gender differences in serum leptin levels in humans.

Leptin, the product of the ob gene, is an adipose tissue-derived hormone that appears to regulate both satiety and thermogenesis. In the present report, we have reexamined the relationship between circulating leptin concentration and body fat in humans using a more valid measure of adiposity (hydrodensitometry) and have extended these observations to examine the influence of regional body fat distribution and cardiorespiratory fitness. Fasting serum leptin concentration was 6.9 +/- 0.3 ng.ml-1 in males (N = 333) and 15.2 +/- 1.3 ng.ml-1 in females (N = 63). Interestingly, total fat mass did not differ between groups (males 20.5 +/- 0.5 kg; females 20.4 +/- 1.5 kg), suggesting that females have higher leptin levels per unit fat mass. In a multiple regression model, fat mass was the best predictor of serum leptin concentration in males, accounting for 51% of the variance in leptin concentration. In females, percentage body fat was the best predictor of leptin, accounting for 49% of the variance. In both groups, the relationship between leptin and adiposity remained significant after adjusting for age, maximal treadmill time, waist circumference, and fasting insulin concentration. These observations support previous conclusions that circulating leptin is primarily a function of adiposity and demonstrate for the first time that this relationship is independent of fat distribution or cardiorespiratory fitness. The data also suggest that there is a gender dichotomy in the relationship between leptin and body fat mass in humans.

Effect of reduced training and training cessation on insulin action and muscle GLUT-4.

This study examined the impact of a 50% reduction in training frequency or training cessation on insulin action and muscle GLUT-4 protein concentration. Middle-aged individuals were tested before and after 12 wk of exercise training (4 days/wk, 40-45 min/day). Subjects then either maintained training (n = 9), reduced training frequency by 50% (n = 11), or stopped exercising (n = 10) for the ensuing 2 wk. GLUT-4 protein concentration and insulin action (insulin sensitivity index, as determined by the minimal model) increased (P < or = 0.05) by an average of 1.6- and 1.9-fold, respectively, with the 12 wk of training. Insulin action and GLUT-4 did not increase further with the additional 2 wk of training in the maintained training group. Similarly, insulin sensitivity index and GLUT-4 concentration remained at trained levels when training frequency was reduced by 50% for 2 wk. GLUT-4 concentration and insulin action, however, were not different from sedentary values after 14 days of training cessation. These findings indicate that a 14-day 50% reduction in exercise frequency maintains the improvements in GLUT-4 protein concentration and insulin action gained with endurance training in moderately trained middle-aged adults; in contrast, these adaptations are largely lost with training cessation.

Cortisol, testosterone, and insulin action during intense swimming training in humans.

An increase in the amounts of circulating plasma cortisol or a decrease in testosterone can result in whole-body insulin resistance. The purpose of this study was to determine if the increase in cortisol and/or decrease in testosterone concentrations commonly evident with intense endurance training is associated with insulin resistance. Male (n = 9) and female (n = 10) swimmers were examined during the off-season, after 9 weeks (9 WKS) of training averaging 5,500 m* day(-1) and after an additional 9 weeks (18 WKS) of training averaging 8,300 m*day(-1). Resting plasma cortisol concentration was (P < or = 0.05) higher in the women compared to the men at 9 WKS; values were not significantly different between genders at 18 WKS. Plasma testosterone concentration decreased significantly (P < or = 0.05) in the men at 9 and 18 WKS, but did not change in the women. Whole-body insulin action, as determined by insulin and glucose responses during a 120 min, 75-g oral glucose tolerance test, did not change with training in either the men or women. These data indicated that plasma testosterone concentration can decrease in male swimmers during intense endurance training; this alteration does not affect wholebody insulin action. There would also appear to be a gender-specific response of plasma cortisol to endurance training, which does not influence insulin action.

Seven days of exercise increase GLUT-4 protein content in human skeletal muscle.

Insulin-responsive glucose transporter (GLUT-4) content increases by 1.8-fold in skeletal muscle with 14 wk of exercise training [Houmard et al. Am. J. Physiol. 264 (Endocrinol. Metab. 27): E896-E901, 1993]. The purpose of this study was to determine whether more short-term training (7 days) increases GLUT-4 protein content in human skeletal muscle. Seven sedentary men [25.0 +/- 1.1 (SE) yr, 44.1 +/- 2.2 ml.kg-1.min-1 maximal O2 uptake, 14.9 +/- 2.1% body fat] were examined before and after 7 days of cycle ergometer training (1 h/day, 76 +/- 2% maximal heart rate). Needle biopsy samples from the vastus lateralis were used to determine GLUT-4 protein content. Muscle GLUT-4 increased (P < 0.05) by an average of 2.8 +/- 0.5-fold with 7 days of training. GLUT-4 content in skeletal muscle thus increases substantially with short-term exercise training.

The insulin action-fiber type relationship in humans is muscle group specific.

The purpose of the present investigation was to determine the relationship between skeletal muscle characteristics, adiposity, and in vivo insulin action. Percutaneous muscle biopsies of the vastus lateralis (VL) and gastrocnemius (G) muscles were obtained from twenty-two sedentary male subjects. Insulin sensitivity (SI) and glucose effectiveness (SG) were determined from minimal model analysis, and indexes of regional and overall adiposity were obtained. SI was positively related to the citrate synthase activity from the VL (r = 0.50, P < 0.01) but unrelated to the citrate synthase activity from the G (r = 0.28). Similarly, SI was inversely related to the percentage of type IIb fibers in the VL (r = -0.47, P < 0.01) but unrelated to the percentage of type IIb fibers in the G (r = 0.06). SG was unrelated to fiber type, oxidative capacity, or adiposity. These data suggest that oxidative capacity and other characteristics related to VL skeletal muscle fiber type are determinants of in vivo insulin action but that this relationship cannot be extended to all muscle groups. Finally, neither skeletal muscle characteristics nor adiposity appears to be a determinant of SG in sedentary males.

Skeletal muscle GLUT4 protein concentration and aging in humans.

The insulin resistance of aging has been attributed to a postreceptor defect in skeletal muscle. The present study examined whether a reduction in the concentration of the insulin-stimulated glucose transporter (GLUT4) in skeletal muscle was associated with advancing age in men (n = 55) and women (n = 29). Insulin sensitivity (minimal model) was negatively associated (P < 0.001) with age (range, 18-80 years) in men (r = -0.44) and women (r = -0.58). GLUT4 protein concentration in the vastus lateralis was also negatively associated (P < 0.05) with age (men, r = -0.28; women, r = -0.51). There was no relation (P > 0.15) between GLUT4 content in the gastrocnemius and age. GLUT4 concentration in the vastus lateralis was positively associated (P < 0.01) with insulin sensitivity in both sexes (r = 0.42); this relationship persisted in the men after adjusting for overall adiposity, regional adiposity, and cardiorespiratory fitness. These findings suggest that a decrement in GLUT4 protein concentration in skeletal muscle may at least partially contribute to the insulin resistance of aging in humans.

Which anthropometric indices of regional adiposity are related to the insulin resistance of aging?

OBJECTIVE: To determine which anthropometric methods of assessing body fat distribution are the most predictive of the insulin resistance of aging. DESIGN: Commonly used indices of regional adiposity were correlated with whole-body insulin sensitivity in 55 men and 29 women (age range 18-80 years). MEASUREMENTS: Insulin sensitivity (SI, minimal model), waist and hip girths, waist-to-hip ratios, conicity index, skinfolds, body fat percentage, maximal oxygen consumption. RESULTS: There was a wide disparity in the strength of relationships between commonly used anthropometric indices of fat distribution and whole-body insulin sensitivity. In both sexes minimal waist girth, however, was the primary variable in multivariate regression analysis, accounting for approximately 37% of the variance in SI. CONCLUSION: These findings indicate that anthropometric measures of regional adiposity are not interchangeable and/or equivalent when attempting to discern relationships between body fat distribution, insulin sensitivity, and aging.

External cardiac pacing for out-of-hospital bradyasystolic arrest.

Cardiac pacing has been used successfully in patients with asystole or bradycardia compromising hemodynamics when it was applied soon after the onset of the event. An external cardiac pacemaker was used as part of initial resuscitative efforts for patients in primary, out-of-hospital, cardiac arrest who arrived in the emergency department in asystole, agonal rhythm, pulseless idioventricular rhythm, or bradycardia with hemodynamic compromise. A pulse was successfully generated in only one of twelve patients. That patient developed complete atrioventricular dissociation while in the emergency department. The nonresponding patients were in asystole or pulseless idioventricular rhythm when the pacemaker was applied. Pacing was initiated 1-13 minutes (mean 7 minutes) after arrival in the emergency department, but 27-90 minutes (mean 59 minutes) after arrest. The interval between arrest and application of the pacemaker was prolonged because of long periods for ambulance response, field resuscitation, and transport. It is concluded that the external cardiac pacemaker is a useful instrument for the treatment of bradyarrhythmias. While it may also be useful in the first few minutes after development of asystole, pulseless idioventricular rhythm, or agonal rhythm, it is of no benefit if applied long after the event.