Beneficial effects of carotenoid-producing cells of Bacillus indicus HU16 in a rat model of diet-induced metabolic syndrome.

A well-established rat model of diet-induced metabolic syndrome was used to evaluate the effects of the oral administration of spores or cells of HU16, a carotenoid-producing strain of Bacillus indicus. Symptoms of metabolic syndrome were induced in 90-days old, male Sprague-Dawley rats maintained for eight weeks on a high-fat diet, as previously reported. Parallel groups of animals under the same diet regimen also received a daily dose of 1×1010 cells or spores of B. indicus HU16. Cells of strain HU16 were able to reduce symptoms of metabolic syndrome, plasma markers of inflammation and oxidative markers in plasma and liver to levels similar to those observed in rats under a standard diet. HU16 cells did not affect obesity markers or the accumulation of triglycerides in the liver of treated animals. Denaturing gradient gel electrophoresis analysis showed that the oral administration of HU16 cells did not significantly affect the gut microbiota of high fat-fed rats, suggesting that the observed beneficial effects are not due to a reshaping of the gut microbiota but rather to metabolites produced by HU16 cells.

The rs39335 polymorphism of the RELN gene is not associated with otosclerosis in a southern Italian population.

Otosclerosis, the single most common cause of hearing impairment in white adults, is characterised by bone dystrophy localized to the otic capsule and isolated endochondral bone sclerosis with alternating phases of bone resorption and formation. Conductive hearing loss develops when otosclerotic foci invade the stapedio-vestibular joint (oval window) and interfere with free motion of the stapes, but affected subjects frequently develop profound sensorineural hearing loss. The aetiology of otosclerosis is unknown. In the last years, several association studies have been performed and have suggested that single nucleotide polymorphisms in some genes may be implicated in development of otosclerosis. The strongest association has been demonstrated for the reelin gene, located on chromosome 7q22.1, which encodes an extracellular matrix protein. The involvement of reelin in the pathogenesis of otosclerosis is controversial; it was identified in European and North African populations, but was excluded in an Indian population. To analyze the role of reelin in otosclerosis, it has been studied in a case-control analysis for the polymorphism rs39335 in a southern Italy population. In this population, the pathogenic link between the rs39335 variant and otosclerosis was excluded.

Caloric restriction followed by high fat feeding predisposes to oxidative stress in skeletal muscle mitochondria.

The purpose of the present study was to assess the impact of previous period of caloric restriction on energy balance and skeletal muscle mitochondrial energetics in response to high-fat (HF) diet. To this end, 1 group of rats was subjected to 2 weeks of caloric restriction with nonpurified diet and then fed HF diet (430 kJ metabolizable energy/day) for 1 week, while the second group was fed ad libitum with nonpurified diet for 2 weeks and then fed HF diet (430 kJ metabolizable energy/day) for 1 week. Body composition, energy balance, and glucose homeostasis were measured. Mitochondrial mass, oxidative capacity and efficiency, parameters of oxidative stress, and antioxidant defense were evaluated in subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle. Body energy and lipid content, plasma insulin, and metabolic efficiency were significantly higher, while energy expenditure significantly decreased, in food-restricted rats fed HF diet compared to controls. Mitochondrial efficiency and oxidative damage in skeletal muscle were significantly increased, while antioxidant defence was significantly lower in food-restricted rats fed HF diet, compared with controls. Finally, food-restricted rats fed HF diet exhibited significant reduction in subsarcolemmal mitochondrial mass. In conclusion, caloric restriction elicits higher mitochondrial efficiency and predisposes skeletal muscle to high fat-induced oxidative damage, which in turn could lead to impaired glucose homeostasis in food-restricted rats fed HF diet.

Skeletal muscle subsarcolemmal mitochondrial dysfunction in high-fat fed rats exhibiting impaired glucose homeostasis.

OBJECTIVE: To investigate whether changes in body energy balance induced by long-term high-fat feeding in adult rats could be associated with modifications in energetic behaviour and oxidative stress of skeletal muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial populations. DESIGN: Adult rats were fed low-fat or high-fat diet for 7 weeks. MEASUREMENTS: Body energy balance and composition analysis together with plasma insulin and glucose level determination in the whole animal. Oxidative capacity, basal and induced proton leaks as well as aconitase and superoxide dismutase activities in SS and IMF mitochondria from skeletal muscle. RESULTS: High-fat fed rats exhibit increased body lipid content, as well as hyperinsulinemia, hyperglycaemia and higher plasma non-esterified fatty acids. In addition, SS mitochondria display lower respiratory capacity and a different behaviour of SS and IMF mitochondria is found in the prevention from oxidative damage. CONCLUSIONS: A deleterious consequence of decreased oxidative capacity in SS mitochondria from rats fed high-fat diet would be a reduced utilization of energy substrates, especially fatty acids, which may lead to intracellular triglyceride accumulation, lipotoxicity and insulin resistance development. Our results thus reveal a possible role for SS mitochondria in the impairment of glucose homeostasis induced by high-fat diet.

Heterogeneous bioenergetic behaviour of subsarcolemmal and intermyofibrillar mitochondria in fed and fasted rats.

This study was designed to examine energetic behaviour of skeletal muscle subsarcolemmal and intermyofibrillar mitochondrial populations. The data show that subsarcolemmal mitochondria exhibited a lower degree of coupling and efficiency than intermyofibrillar ones, and can therefore be considered less efficient at producing ATP. In addition, subsarcolemmal mitochondria showed an increased sensitivity to palmitate-induced uncoupling, in line with high adenine nucleotide translocator content and decreased oxidative damage. We then determined the effect of 24 h fasting on energetic characteristics of skeletal muscle mitochondrial populations. We found that fasting enhanced proton leak and decreased the degree of coupling and efficiency, both in the absence and in the presence of palmitate only in subsarcolemmal mitochondria. Moreover, this mitochondrial population showed lower oxidative damage, probably due to a counter-regulatory mechanism mediated by uncoupling protein 3. Subsarcolemmal and intermyofibrillar mitochondria appear to exhibit different energetic characteristics and can be differently affected by physiological stimuli.

Modulation of hepatic mitochondrial energy efficiency with age.

This study was designed to examine the effect of youth-adulthood transition on hepatic mitochondrial energy efficiency. The changes in basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, were evaluated in mitochondria isolated from the liver of young and adult rats. Alterations in mitochondrial cytochrome oxidase and aconitase specific activities, and in adenine nucleotide translocator content were also assessed. There was no difference in basal proton leak or thermodynamic coupling and efficiency of oxidative phosphorylation in liver mitochondria between the two rat groups. On the other hand, palmitate-induced proton leak increased significantly in adult rats. The function of this uncoupling could be avoidance of elevated formation of reactive oxygen species, which are known to accelerate ageing.

Skeletal muscle mitochondrial oxidative capacity and uncoupling protein 3 are differently influenced by semistarvation and refeeding.

We investigated, in skeletal muscle mitochondria isolated from semistarved and refed rats, the relation between the protein expression of uncoupling protein 3 (UCP3) and mitochondrial oxidative capacity, assessed as state 4 and state 3 respiration rates in presence of substrates that are either non-lipids (glutamate, succinate) or lipids (palmitoyl CoA, palmitoylcarnitine). During semistarvation, when whole-body thermogenesis is diminished, state 3 respiration was lower than in fed controls by about 30% independently of substrate types, while state 4 respiration was lower by 20% only during succinate oxidation, but UCP3 was unaltered. After 5 days of refeeding, when thermogenesis is still diminished, neither state 4, state 3 nor UCP3 were lower than in controls. Refeeding on a high-fat diet, which exacerbates the suppression of thermogenesis, resulted in a two-fold elevation in UCP3 but no change in state 4 or state 3 respiration. These results during semistarvation and refeeding, in line with those previously reported for fasting, are not in support of the hypothesis that UCP3 is a mediator of adaptive thermogenesis pertaining to weight regulation, and underscore the need for caution in interpreting parallel changes in UCP3 and mitochondrial oxidative capacity as the reflection of mitochondrial uncoupling by UCP3.

Skeletal muscle oxidative capacity in rats fed high-fat diet.

OBJECTIVE: To investigate whether young rats respond to high-fat feeding through changes in energy efficiency and fuel partitioning at the level of skeletal muscle, to avoid obesity development. In addition, to establish whether the two mitochondrial subpopulations, which exist in skeletal muscle, ie subsarcolemmal and intermyofibrillar, are differently affected by high-fat feeding. DESIGN: Weaning rats were fed a low-fat or a high-fat diet for 15 days. MEASUREMENTS: Energy balance and lipid partitioning in the whole animal. State 3 and state 4 oxygen consumption rates in whole skeletal muscle homogenate. State 3 and state 4 oxygen consumption rates, membrane potential and uncoupling effect of palmitate in subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle. RESULTS: Rats fed a high-fat diet showed an increased whole body lipid utilization. Skeletal muscle NAD-linked and lipid oxidative capacity significantly increased at the whole-tissue level, due to an increase in lipid oxidative capacity in subsarcolemmal and intermyofibrillar mitochondria and in NAD-linked activity only in intermyofibrillar ones. In addition, rats fed a high-fat diet showed an increase in the uncoupling effect of palmitate in both the mitochondrial populations. CONCLUSIONS: In young rats fed a high-fat diet, skeletal muscle contributes to enhanced whole body lipid oxidation through an increased mitochondrial capacity to use lipids as metabolic fuels, associated with a decrease in energy coupling.

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats.

We have investigated the effect of 24-h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24-h fasting, both at 24 and 29 degrees C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.

Acetyl-L-carnitine treatment stimulates oxygen consumption and biosynthetic function in perfused liver of young and old rats.

The effect of treatment with acetyl-L-carnitine on hepatic mitochondrial respiration and biosynthetic function in perfused liver from young (90 days) and old (22-24 months) rats was studied. Rats were given a 1.5% (w/v) solution of acetyl-L-carnitine in their drinking water for 1 month and oxygen consumption together with the rate of gluconeogenesis, urea synthesis, and ketogenesis with and without added substrates were measured in perfused liver. Mitochondrial oxygen consumption was also assessed in liver homogenate and isolated mitochondria to determine the maximal capacity for oxidative phosphorylation. Acetyl-L-carnitine treatment almost completely restored the age-dependent decline in oxygen consumption, gluconeogenesis, urea synthesis, and ketogenesis found in perfused liver of old rats to the levels found in young rats. In addition, acetyl-L-carnitine treatment increased oxygen consumption and biosynthetic function in perfused liver from young rats. After acetyl-L-carnitine treatment, we found detectable 3-oxoacyl-CoA-transferase activity associated with a consumption of ketone bodies in young and old rats. Finally, oxygen consumption measured in homogenate and isolated mitochondria did not change with age and acetyl-L-carnitine treatment. Our results show that in perfused liver, acetyl-L-carnitine treatment slows the age-associated decline in mitochondrial respiration and biosynthetic function. In addition, treatment of young rats with acetyl-L-carnitine has a stimulating effect on liver metabolism, probably through an increase in ATP production.

Fat balance and serum leptin concentrations in normal, hypothyroid, and hyperthyroid rats.

OBJECTIVE: To study the influence of thyroid hormones on the relationship between serum leptin and fat mass, as well as on energy and macronutrient balance. DESIGN: Rats with different thyroid states were obtained by 7 and 15 days of treatment with the antithyroid drug propylthiouracil or with triiodothyronine (T3). MEASUREMENTS: Energy balance, macronutrient balance and serum leptin concentrations. RESULTS: In hypothyroid rats we found a decrease in metabolizable energy (ME) intake and energy expenditure together with an increase in lipid gain/lipid intake ratio and a decrease in protein gain/protein intake ratio. Consequently, body lipid percentage significantly increased compared to euthyroid rats. Hyperthyroid rats first increased energy expenditure and later ME intake, so that increased metabolism was balanced by increased intake, and energy gain was similar to that found in euthyroid rats. CONCLUSION: These results indicate that T3 plays a major role in the maintenance of energy and lipid balance. Our results also indicate that an inverse relationship exists between T3 and leptin serum concentrations, and that this relationship is not only the result of changes in body fat stores induced by changed T3 concentrations.

Effect of cold exposure on energy balance and liver respiratory capacity in post-weaning rats fed a high-fat diet.

Variations in energy balance, body composition, and nutrient partitioning induced by high-fat feeding, cold exposure or by concomitant high-fat feeding and cold exposure were studied in young Wistar rats. Changes in hepatic metabolism as well as in serum free triiodothyronine and leptin levels were also evaluated. Rats were exposed to either 24 or 4 degrees C and fed either a low- or high-fat diet (10 % or 50 % energy respectively) for 2 weeks. Relative to low-fat feeding at 24 degrees C, both energy intake and expenditure were increased by high-fat feeding or by cold exposure, and these changes were accompanied by increased serum triiodothyronine levels. In response to concomitant high-fat feeding and cold exposure, serum triiodothyronine tended to be further elevated, but no further increases in energy intake or energy expenditure were observed. Independently of diet, the increased energy expenditure in cold-exposed rats was not completely balanced by adaptive hyperphagia, with consequential reductions in protein and fat gain, accompanied by marked decreases in serum leptin. Furthermore, unlike high-fat feeding at 24 degrees C, cold exposure enhanced hepatic mitochondrial oxidative capacity both in the low-fat- and high-fat-fed groups. It is concluded that in this strain of young Wistar rats, despite similarly marked stimulation of energy expenditure by high-fat feeding at 24 degrees C, by cold exposure and by concomitant high-fat feeding and cold exposure, an increased hepatic oxidative capacity occurred only in the presence of the cold stimulus.

Differential vulnerability of cortical and cerebellar neurons in primary culture to oxygen glucose deprivation followed by reoxygenation.

The effects of glucose and O2 deprivation (OGD) on the survival of cortical and cerebellar neurons were examined to characterize the biochemical mechanisms involved in OGD and OGD followed by reoxygenation. To this aim, neurons were kept for different time periods in a hypoxic chamber with a controlled atmosphere of 95% N(2) and 5% CO2 in a glucose-free medium. After OGD, reoxygenation was achieved by exposing the cells to normal O2 and glucose levels. Neither MTT, an index of mitochondrial oxidative phosphorylation, nor malondialdehyde (MDA) production, a parameter measuring lipid peroxidation, were affected by 1 hr of OGD in cortical neurons. When OGD was followed by 24 hr of reoxygenation, MTT levels were reduced by 40% and MDA was significantly increased, whereas cellular ATP content did not change. Cerebellar granule cells, on the other hand, did not show any reduction of mitochondrial activity after exposure to 1 hr OGD or to 1 hr OGD plus 24 hr of reoxygenation. When OGD was prolonged for 2 hr, a significant reduction of the mitochondrial activity and of cellular ATP content occurred, coupled to a significant MDA increase in cerebellar granule cells, whereas in cortical neurons a reduction of MTT levels after 2 hr OGD was not accompanied by a decrease of cellular ATP content nor by an increase of MDA production. Moreover, 24 hr of reoxygenation further reinforced lipid peroxidation, LDH release, propidium iodide positive neurons and the reduction of ATP content in cerebellar granule cells. The results of the present study collectively show that cortical and cerebellar neurons display different levels of vulnerability to reoxygenation followed by OGD. Furthermore, the impairment of mitochondrial activity and the consequent overproduction of free radicals in neurons were observed for the first time occurring not only during the reoxygenation phase, but already beginning during the OGD phase.

Effect of long-term high-fat feeding on energy balance and liver oxidative activity in rats.

The purpose of this work was to study the effect of early long-term high-fat feeding on energy balance and liver oxidative activity. To this end, rats aged about 30 d were fed a high-fat or a low-fat diet for 15, 30 or 60 d. Full energy balance and energy partitioning measurements were carried out. In addition, we measured hepatic mitochondrial and peroxisomal oxidative capacities. Serum levels of free triiodothyronine (T3) and leptin were also determined. Rats fed a high-fat diet showed an increase in metabolizable energy intake as well as in energy expenditure, while lipid gain over the whole period was lower than that expected due to a decrease in metabolic efficiency. An increase in serum free T3 levels was also found in rats fed a high-fat diet after 15 and 30 d. Statistically significant correlations between serum leptin levels and body fat mass were found after 15, 30 and 60 d of high-fat feeding. Finally, no variation in hepatic mitochondrial and peroxisomal fatty acid oxidation capacity was found in rats fed a high-fat diet for 15, 30 or 60 d. In conclusion, the results of the present study show that young Wistar rats fed a high-fat diet for up to 60 d are able to counteract, at least in part, obesity development.

Fat balance and hepatic mitochondrial function in response to fat feeding in mature rats.

OBJECTIVE: To study the effects of fat feeding on fat balance and hepatic mitochondrial function in postpubertal male rats. DESIGN: Rats were fed low fat, medium fat or high fat diet for 15 days. MEASUREMENTS: Energy balance, body composition, resting metabolic rate (RMR), mitochondrial state 3 and state 4 oxygen consumption rates, succinic dehydrogenase (EC 1.3.99.1) and mitochondrial alpha-glycerophosphate dehydrogenase (EC 1.1.1.8) activities. RESULTS: Rats fed medium fat or high fat diet, in comparison with rats fed low fat diet, showed a significantly greater metabolisable energy intake and energy expenditure. In addition, body energy and lipid gains were significantly higher in rats fed medium fat or high fat diet than in rats fed low fat diet. Mitochondrial respiration and enzymatic activities were not affected by fat feeding. CONCLUSION: These results indicate that in postpubertal rats fed high fat diets, the increase in energy expenditure counteracts only in part the excess fat deposition. This is probably due to the impairment in regulatory responses, and enhances thermogenesis.

Modulation of the K(+) channels encoded by the human ether-a-gogo-related gene-1 (hERG1) by nitric oxide.

The inhibition of nitric oxide synthase by N-nitro-L-arginine methyl ester (0.03-3 mM) dose-dependently reduced nitric oxide (NO(*)) levels and enhanced the outward currents carried by human ether-a-gogo-related gene-1 (hERG1) K(+) channels expressed in Xenopus laevis oocytes, whereas the increase in NO(*) levels achieved by exposure to L-arginine (0.03-10 mM) inhibited these currents. Furthermore, four NO(*) donors belonging to such different chemical classes as sodium nitroprusside (1-1000 microM), 3-morpholino-sydnonimine (100-1000 microM), (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1-300 microM), and S-nitroso N-acetylpenicillamine (1-300 microM) dose-dependently inhibited hERG1 outward K(+) currents. By contrast, the NO(*) donor NOC-18 (0.3 mM) did not affect other cloned K(+) channels such as rat neuroblastoma-glioma K(+) channel 2, rat delayed rectifier K(+) channel 1, bovine ether-a-gogo gene, rat ether-a-gogo-related gene-2, and rat ether-a-gogo-related gene-3. The inhibitory effect of NO(*) donors on hERG1 K(+) channels was prevented by the NO(*) scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and hemoglobin. The membrane permeable analog of cGMP, 8-bromo-cGMP (1 mM), failed to reproduce the inhibitory action of NO(*) donors on hERG1 outward currents; furthermore, the specific inhibitor of the NO(*)-dependent guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (50 microM), neither interfered with outward hERG1 K(+) currents nor prevented their inhibition by 0.3 mM NOC-18. Both L-arginine (10 mM) and NOC-18 (0.3 mM) counteracted the stimulatory effect on hERG1 outward currents induced by the radical oxygen species-generating system FeSO(4) (25 microM)/ascorbic acid (50 microM; Fe/Asc). Finally, L-arginine (10 mM) and NOC-18 (0.3 mM) inhibited both basal and Fe/Asc (0.1 mM/0.2 mM)-stimulated lipid peroxidation in X. laevis oocytes. Collectively, the present results suggest that NO(*), both endogenously produced and pharmacologically delivered, may exert in a cGMP-independent way an inhibitory effect on hERG1 outward K(+) currents via an interaction with radical oxygen species either generated under resting conditions or triggered by Fe/Asc.

Stimulation of oxygen consumption following addition of lipid substrates in liver and skeletal muscle from rats fed a high-fat diet.

We studied hepatic and skeletal muscle metabolic activity in rats fed a high-fat diet. Rats were fed a low-fat or high-fat diet for 15 days. At the end of the experimental period, full energy-balance determinations together with serum free triiodothyronine (FT3), leptin, and free fatty acid (FFA) measurements were performed. In addition, we assessed fatty acid-stimulated oxygen consumption in perfused liver and in skeletal muscle homogenate. Rats fed a high-fat diet showed a significant increase in energy intake but no variation in body energy gain, due to a significant increase in energy expenditure. Serum FT3 and FFA levels significantly increased in rats fed a high-fat diet versus rats fed a low-fat diet, while no variation was found in serum leptin levels. Perfused livers and skeletal muscle homogenates from rats fed a high-fat diet exhibited a significant increase in fatty acid-stimulated oxygen consumption. Our results suggest that the enhanced fatty acid oxidation rates in liver and skeletal muscle contribute to the maintenance of fat balance in response to increased fat intake, preventing excess fat deposition.

Energy intake and utilization vary during development in rats.

Energy intake, utilization, and partitioning were determined in male Wistar rats from 25 to 180 d of age. Serum free triiodothyronine, leptin, and free fatty acid concentrations were also measured. Energy balance measurements allowed us to identify a period from 25 to 90 d, characterized by a rapid body growth rate and another from 90 to 180 d, during which body growth rate slowed. From 25 to 180 d, we found decreases in daily energy intake and expenditure, which were faster before 90 d. The first period was characterized by storage of lipid and protein. In the second period, protein deposition approached zero and the excess of ingested energy was entirely stored as fat, so that age-associated obesity began to develop. The inability of rats to maintain a stable body weight after the cessation of growth of lean body mass is not due to decreased resting metabolism but rather to a partial leptin resistance.

Oxidative activity in mitochondria isolated from rat liver at different stages of development.

The purpose of this study was to evaluate the oxidative capacities in hepatic mitochondria isolated from prepubertal, young adult and adult rats (40, 90 and 180 days of age, respectively). In these rats, mitochondrial respiratory rates using FAD- and NAD-linked substrates as well as mitochondrial protein mass were measured. The results show that only the oxidative capacity of FAD-linked pathways significantly declined in mitochondria from 180-day-old rats compared with those from younger animals. When we consider FAD-linked respiration expressed per g liver, no significant difference was found among rats of different ages because of an increased mitochondrial protein mass found in 180-day-old rats. However, when FAD-linked and lipid-dependent respiratory rates were expressed per 100 g body weight, significant decreases occurred in 180-day-old rats. Therefore, the decrease in liver weight expressed per 100 g body weight rather than an impaired hepatic cellular activity may be the cause of body energy deficit in 180-day-old rats.