Conservation of Atlantic salmon by supplementary stocking of juvenile fish

Wild salmon stocks in the northern Baltic rivers became endangered in the second half of the 20th century, mainly due to recruitment overfishing. As a result, supplementary stocking was widely practised, and supplementation of the Tornionjoki salmon stock took place over a 25 year period until 2002. The stock has been closely monitored by electrofishing, smolt trapping, mark-recapture studies, catch samples and catch surveys. Background information on hatchery-reared stocked juveniles was also collected for this study. Bayesian statistics was applied to the data as this method offers the possibility of bringing prior information into the analysis and an advanced ability for incorporating uncertainty, and also provides probabilities for a multitude of hypotheses. Substantial divergences between reared and wild Tornionjoki salmon were identified in both demographic and phenological characteristics. The divergences tended to be larger the longer the duration spent in hatchery and the more favourable the hatchery conditions were for fast growth. Differences in environment likely induced most of the divergences, but selection of brood fish might have resulted in genotypic divergence in maturation age of reared salmon. Survival of stocked 1-year old juveniles to smolt varied from about 10% to about 25%. Stocking on the lower reach of the river seemed to decrease survival, and the negative effect of stocking volume on survival raises the concern of possible similar effects on the extant wild population. Post-smolt survival of wild Tornionjoki smolts was on average two times higher than that of smolts stocked as parr and 2.5 times higher than that of stocked smolts. Smolts of different groups showed synchronous variation and similar long-term survival trends. Both groups of reared salmon were more vulnerable to offshore driftnet and coastal trapnet fishing than wild salmon. Average survival from smolt to spawners of wild salmon was 2.8 times higher than that of salmon stocked as parr and 3.3 times higher than that of salmon stocked as smolts. Wild salmon and salmon stocked as parr were found to have similar lifetime survival rates, while stocked smolts have a lifetime survival rate over 4 times higher than the two other groups. If eggs are collected from the wild brood fish, stocking parr would therefore not be a sensible option. Stocking smolts instead would create a net benefit in terms of the number of spawners, but this strategy has serious drawbacks and risks associated with the larger phenotypic and demographic divergences from wild salmon. Supplementation was shown not to be the key factor behind the recovery of the Tornionjoki and other northern Baltic salmon stocks. Instead, a combination of restrictions in the sea fishery and simultaneous occurrence of favourable natural conditions for survival were the main reasons for the revival in the 1990 s. This study questions the effectiveness of supplementation as a conservation management tool. The benefits of supplementation seem at best limited. Relatively high occurrences of reared fish in catches may generate false optimism concerning the effects of supplementation. Supplementation may lead to genetic risks due to problems in brood fish collection and artificial rearing with relaxed natural selection and domestication. Appropriate management of fisheries is the main alternative to supplementation, without which all other efforts for long-term maintenance of a healthy fish resource fail.

Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

Succinate and malate improve development of hamster eight-cell embryos in vitro: Confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% ± 3.9 vs. 54.5% ± 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% ± 7.2), but not pyruvate (85.8% ± 6.2) or glutamine (84.1% ± 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% ± 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 ± 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 ± 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 ± 1.1) or malate (24.7 ± 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (±4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% ± 2.0) or blastocysts (28.9% ± 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium. Mol. Reprod. Dev. 47:440-447, 1997. © 1997 Wiley-Liss, Inc.

Thyroid Hormone Control of Cardiac Substrate Metabolism

Thyroid hormone (TH) plays an important role in maintaining a homeostasis in all the cells of our body. It also has significant cardiovascular effects, and abnormalities of its concentration can cause cardiovascular disease and even morbidity. Especially development of heart failure has been connected to low levels of thyroid hormone. A decrease in TH levels or TH-receptor binding adversely effects cardiac function. Although, this occurs in part through alterations in excitation-contraction and transport proteins, recent data from our laboratory indicate that TH also mediates changes in myocardial energy metabolism. Thyroid dysfunction may limit the heart s ability to shift substrate pathways and provide adequate energy supply during stress responses. Our goals of these studies were to determine substrate oxidation pattern in systemic and cardiac specific hypothyroidism at rest and at higher rates of oxygen demand. Additionally we investigated the TH mediated mechanisms in myocardial substrate selection and established the metabolic phenotype caused by a thyroid receptor dysfunction. We measured cardiac metabolism in an isolated heart model using 13Carbon isotopomer analyses with MR spectroscopy to determine function, oxygen consumption, fluxes and fractional contribution of acetyl-CoA to the citric acid cycle (CAC). Molecular pathways for changes in cardiac function and substrate shifts occurring during stress through thyroid receptor abnormalities were determined by protein analyses. Our results show that TH modifies substrate selection through nuclear-mediated and rapid posttranscriptional mechanisms. It modifies substrate selection differentially at rest and at higher rates of oxygen demand. Chronic TH deficiency depresses total CAC flux and selectively fatty acid flux, whereas acute TH supplementation decreases lactate oxidation. Insertion of a dominant negative thyroid receptor (Δ337T) alters metabolic phenotype and contractive efficiency in heart. The capability of the Δ337T heart to increase carbohydrate oxidation in response to stress seems to be limited. These studies provided a clearer understanding of the TH role in heart disease and shed light to identification of the molecular mechanisms that will facilitate in finding targets for heart failure prevention and treatment.

The role of 25-hydroxyvitamin D deficiency in promoting insulin resistance and inflammation in patients with chronic kidney disease: A randomised controlled trial

BACKGROUND Approximately 50% of patients with stage 3 Chronic Kidney Disease are 25-hydroxyvitamin D insufficient, and this prevalence increases with falling glomerular filtration rate. Vitamin D is now recognised as having pleiotropic roles beyond bone and mineral homeostasis, with the vitamin D receptor and metabolising machinery identified in multiple tissues. Worryingly, recent observational data has highlighted an association between hypovitaminosis D and increased cardiovascular mortality, possibly mediated via vitamin D effects on insulin resistance and inflammation. The main hypothesis of this study is that oral Vitamin D supplementation will ameliorate insulin resistance in patients with Chronic Kidney Disease stage 3 when compared to placebo. Secondary hypotheses will test whether this is associated with decreased inflammation and bone/adipocyte-endocrine dysregulation. METHODS/DESIGN This study is a single-centre, double-blinded, randomised, placebo-controlled trial. Inclusion criteria include; estimated glomerular filtration rate 30-59 ml/min/1.73 m(2); aged >or=18 on entry to study; and serum 25-hydroxyvitamin D levels <75 nmol/L. Patients will be randomised 1:1 to receive either oral cholecalciferol 2000IU/day or placebo for 6 months. The primary outcome will be an improvement in insulin sensitivity, measured by hyperinsulinaemic euglycaemic clamp. Secondary outcome measures will include serum parathyroid hormone, cytokines (Interleukin-1beta, Interleukin-6, Tumour Necrosis Factor alpha), adiponectin (total and High Molecular Weight), osteocalcin (carboxylated and under-carboxylated), peripheral blood mononuclear cell Nuclear Factor Kappa-B p65 binding activity, brachial artery reactivity, aortic pulse wave velocity and waveform analysis, and indirect calorimetry. All outcome measures will be performed at baseline and end of study. DISCUSSION To date, no randomised controlled trial has been performed in pre-dialysis CKD patients to study the correlation between vitamin D status with supplementation, insulin resistance and markers of adverse cardiovascular risk. We remain hopeful that cholecalciferol may be a safe intervention, with health benefits beyond those related to bone-mineral homeostasis. TRIAL REGISTRATION Australian and New Zealand Clinical Trials Registry ACTRN12609000246280.

Probiotics modify tight junction proteins in an animal model of non-alcoholic fatty liver disease

Objectives We have investigated the effects of a multi–species probiotic preparation containing a combination of probiotic bacterial genera that included Bifidobacteria, Lactobacilli and a Streptococcus in a mouse model of high fat diet/obesity induced liver steatosis. Methods Three groups of C57B1/6J mice were fed either a standard chow or a high fat diet for 20 weeks, while a third group was fed a high fat diet for 10 weeks and then concomitantly administered probiotics for a further 10 weeks. Serum, liver and large bowel samples were collected for analysis. Results The expression of the tight junction proteins ZO-1 and ZO-2 was reduced (p < 0.05) in high fat diet fed mice compared to chow fed mice. Probiotic supplementation helped to maintain tight ZO-1 and ZO-2 expression compared with the high fat diet group (p < 0.05), but did not restore ZO-1 or ZO-2 expression compared with chow fed mice. Mice fed a high fat diet ± probiotics had significant steatosis development compared to chow fed mice (p < 0.05); steatosis was less severe in the probiotics group compared to the high fat diet group. Hepatic triglycerides concentration was higher in mice fed a high fat diet ± probiotics compared to the chow group (p < 0.05), and was lower in the probiotics group compared to the high fat diet group (p < 0.05). Compared to chow fed mice, serum glucose and cholesterol concentrations, and the activity of alanine transaminase were higher (p < 0.05), whereas serum triglyceride concentration was lower (p < 0.05) in mice fed a high fat diet ± probiotics. Conclusions Supplementation with a multi-species probiotic formulation helped to maintain tight junction proteins ZO-1 and ZO-2, and reduced hepatic triglyceride concentrations compared with a HFD alone.

Parathyroid hormone as an outcome indicator in old age

Serum parathyroid hormone (PTH) and vitamin D are the major regulators of extracellular calcium homeostasis. The inverse association between PTH and vitamin D and the common age-related elevation of the PTH concentration are well known phenomena. However, the confounding or modifying factors of this relationship and their impact on the response of PTH levels to vitamin D supplementation need further investigation. Clinical conditions such as primary hyperparathyroidism (PHPT), renal failure and vitamin D deficiency, characterized by an elevation of the PTH concentration, have been associated with impaired long-term health outcomes. Curative treatments for these conditions have also been shown to decreases PTH concentration and attenuate some of the adverse health effects. In PHPT it has also been commonly held that hypercalcaemia, the other hallmark of the disease, is the key mediator of the adverse health outcomes. In chronic kidney disease the systemic vascular disease has been proposed to have the most important impact on general health. Some evidence also indicates that vitamin D may have significant extraskeletal actions. However, the frank elevation of PTH concentration seen in advanced PHPT and in end-stage renal failure have also been suggested to be at least partly causally related to an increased risk of death as well as cognitive dysfunction. However, the exact mechanisms have remained unclear. Furthermore, the predictive value of elevated PTH in unselected older populations has been less well studied. The studies presented in this thesis investigated the impact of age and mobility on the responses of PTH levels to vitamin D deficiency and supplementation. Furthermore, the predictive value of PTH for long-term survival and cognitive decline was addressed in an unselected population of older people. The hypothesis was that age and chronic immobility are related to a persistently blunted elevation of PTH concentration, even in the presence of chronic vitamin D deficiency, and to attenuated responses of PTH to vitamin D supplementation. It was also further hypothesized that a slightly elevated or even high-normal PTH concentration is an independent indicator of an increased risk of death and cognitive decline in the general aged population. The data of this thesis are based on three samples: a meta-analysis of published vitamin D supplementation trials, a randomized placebo controlled six-month vitamin D supplementation trial, and a longitudinal prospective cohort study on a general aged population. Based on a PubMed search, a meta-analysis of 52 clinical trials with 6 290 adult participants was performed to evaluate the impact of age and immobility on the responses of PTH to 25-OHD levels and vitamin D supplementation. A total of 218 chronically immobile, very old inpatients were also enrolled into a vitamin D supplementation trial. Mortality data for these patients was also collected after a two-year follow-up. Finally, data from the Helsinki Aging Study, which followed three random age cohorts (75, 80 and 85 years) until death in almost all subjects, was used to evaluate the predictive value of PTH for long-term survival and cognitive decline. This series of studies demonstrated that in older people without overt renal failure or severe hypercalcaemia, serum 25-OHD and PTH were closely associated, but this relationship was also affected by age and immobility. Furthermore, a substantial proportion of old chronically bedridden patients did not respond to vitamin D deficiency by elevating PTH, and the effect of a high-dose (1200 IU/d) six-month cholecalciferol supplementation on the PTH concentration was minor. This study demonstrated longitudinally for the first time that the blunted PTH also persisted over time. Even a subtle elevation of PTH to high-normal levels predicted impaired long-term health outcomes. Slightly elevated PTH concentrations indicated an increased risk of clinically significant cognitive decline and death during the last years of life in a general aged population. This association was also independent of serum ionized calcium (Ca2+) and the estimated glomerular filtration rate (GFR). A slightly elevated PTH also indicated impaired two-year survival during the terminal years of frail elderly subjects independently of Ca2+, GFR, and of 25-OHD levels. The interplay between PTH and vitamin D in the regulation of calcium homeostasis is more complex than has been generally considered. In addition to muskuloskeletal health parathyroid hormone is also related to the maintenance of other important domains of health in old age. Higher PTH concentrations, even within conventional laboratory reference ranges, seem to be an independent indicator of an increased risk of all-cause and of cardiovascular mortality, independently of established cardiovascular risk factors, disturbances in mineral metabolism, and renal failure. Limited and inconsistent evidence supports the role of vitamin D deficiency-related lack of neuroprotective effects over the causal association between PTH and impaired cognitive functions. However, the causality of these associations remains unclear. The clinical implications of the observed relationships remain to be elucidated by future studies interfering with PTH concentrations, especially by long-term interventions to reduce PTH.

Effect of vitamins A and K on colon lysosomes

1. 1. Colon lysosome were separated by differential centrifugation and lysosomes with three different densities, probably arising from the three layers of colon, were found. 2. 2. Hypervitaminosis A resulted in a significant increase in prothrombin time which was restored to normal on vitamin K1 (20) supplementation. 3. 3. There was no appreciable change in the liver storage of vitamin A between hypervitaminotic rats receiving vitamin A and those rats receiving vitamin K1 (20) in addition to excess vitamin A. 4. 4. The colon lysosomes were unstable in hypervitaminosis A, showing an increased free activity of lysosomal enzymes like β-glucuronidase, acid phosphatase and arylsulphatase. This increase of free activity of lysoso3al enzymes in hypervitaminosis A could be prevented by oral supplementation of vitamin K1 (20). 5. 5. In "mild" vitamin A deficiency the release of arylsulphatase was significantly retarded, whereas the decreased free acid phosphatase activity was not significant. 6. 6. "Severe" vitamin A deficiency resulted in a significantly increased free activity of arylsulphatase and acid phosphatase, thus showing the instability of the lysosomal particles in this condition. 7. 7. Addition of vitamin K1 (20) to the incubation medium in vitro could prevent the vitamin A-induced release of arylsulphatase from liver lysosomes, whereas α-tocopherol was inactive. 8. 8. Retinol and retinoic acid were nearly twice as active as ethanol in the release of arylsulphatase from lysosomes in vitro, whereas 5,6-monoepoxyretinoic acid was inactive. 9. 9. The role of vitamins A and K on the lysosomal membrane structure is discussed.

Biosynthesis of sulfated mucopolysaccharides in relation to vitamins A and K

1.(1) Incorporation of Na235SO4 into acid mucopolysaccharides of intestine and colon tissue has been studied in normal, vitamin A-deficient and excess vitamin A-fed rats. 2. (2) Vitamin A deficiency resulted in a significant decrease in [35S]sulfate incorporation into mucopolysaccharides isolated from intestines of male rats. There was no significant change in the total mucopolysaccharides per gram of fresh tissue. 3. (3) When rats are fed excessive amounts of retinyl acetate, increased [35S]sulfate incorporation into mucopolysaccharides of rat intestine and colon is observed. 4. (4) Supplementation of vitamin K1 to rats fed excessive amounts of vitamin A restores the incorporation of [35S]sulfate into the acid mucopolysaccharides to the normal level. 5. (5) The implications of these findings with special reference to the role of vitamins A and K in the synthesis of sulfated mucopolysaccharides are discussed.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Molecular mechanisms of hypertension-induced heart failure : Experimental studies with special emphasis on local renin-angiotensin system, cardiac metabolism and levosimendan

Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.

Thyroidal control of hepatic release and metabolism of vitamin A

The inverse relationship that exists between thyroxine and the vitamin A level of plasma has been examined in chicken. Thyroxine treatment leads to a decrease in the level of vitamin A carrier proteins, retinol-binding protein and prealbumin-2 in plasma and liver. There is an accumulation of vitamin A in the liver, with a greater proportion of vitamin A alcohol being present compared to that of control birds. In thyroxine treatment there is enhanced plasma turnover of retinol-binding protein and prealbumin-2, while their rates of synthesis are marginally increased. Amino acid supplementation partially counteracts effects of thyroxine treatment. Amino acid supplementation of thyroxine-treated birds does not alter the plasma turnover rates of retinol-binding protein and prealbumin-2 but increases substentially their rates of synthesis. The release of vitamin A into circulation is interfered with in hyperthyroidism due to inadequate availability of retinol-binding protein being caused by enhanced plasma turnover rate not compensated for by synthesis.

Antioxidants, weight change and risk of type 2 diabetes

The incidence of type 2 diabetes has increased rapidly worldwide. Obesity is one of the most important modifiable risk factors of type 2 diabetes: weight gain increases and weight loss decreases the risk. However, the effects of weight fluctuation are unclear. Reactive oxygen species are presumably part of the complicated mechanism for the development of insulin resistance and beta-cell destruction in the pancreas. The association of antioxidants with the risk of incident type 2 diabetes has been studied in longitudinal prospective human studies, but so far there is no clear conclusion about protective effect of dietary or of supplementary antioxidants on diabetes risk. The present study examined 1) weight change and fluctuation as risk factors for incident type 2 diabetes; 2) the association of baseline serum alpha-tocopherol or beta-carotene concentration and dietary intake of antioxidants with the risk of type 2 diabetes; 3) the effect of supplementation with alpha-tocopherol or beta-carotene on the risk of incident type 2 diabetes; and on macrovascular complications and mortality among type 2 diabetics. This investigation was part of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a randomized, double-blind, placebo-controlled prevention trial, which has undertaken to examine the effect of alpha-tocopherol and beta-carotene supplementation on the development of lung cancer, other cancers, and cardiovascular diseases in male smokers aged 50-69 years at baseline. Participants were assigned to receive either 50 mg alpha-tocopherol, 20mg beta-carotene, both, or placebo daily in a 2 x 2 factorial design experiment during 1985-1993. Cases of incident diabetes were identified through a nationwide register of drug reimbursements of the Social Insurance Institution. At baseline 1700 men had a history of diabetes. Among those (n = 27 379) with no diabetes at baseline 305 new cases of type 2 diabetes were recognized during the intervention period and 705 during the whole follow-up to 12.5 years. Weight gain and weight fluctuation measured over a three year period were independent risk factors for subsequent incident type 2 diabetes. Relative risk (RR) was 1.77 (95% confidence interval [CI] 1.44-2.17) for weight gain of at least 4 kg compared to those with a weight change of less than 4 kg. The RR in the highest weight fluctuation quintile compared to the lowest was 1.64 (95% CI 1.24-2.17). Dietary tocopherols and tocotrienols as well as dietary carotenoids, flavonols, flavones and vitamin C were not associated with the risk of type 2 diabetes. Baseline serum alpha-tocopherol and beta-carotene concentrations were not associated with the risk of incident diabetes. Neither alpha-tocopherol nor beta-carotene supplementation affected the risk of diabetes. The relative risks for participants who received alpha-tocopherol compared with nonrecipients and for participants who received beta-carotene compared with nonrecipients were 0.92 (95% CI 0.79-1.07) and 0.99 (95% CI 0.85-1.15), respectively. Furthermore, alpha-tocopherol or beta-carotene supplementation did not affect the risk of macrovascular complications or mortality of diabetic subjects during the 19 years follow-up time. In conclusion, in this study of older middle-aged male smokers, weight gain and weight fluctuation were independent risk factors for type 2 diabetes. Intake of antioxidants or serum alpha-tocopherol or beta-carotene concentrations were not associated with the risk of type 2 diabetes. Supplementation with of alpha-tocopherol or beta-carotene did not prevent type 2 diabetes. Neither did they prevent macrovascular complications, or mortality among diabetic subjects.

Effect of specific FSH or LH deprivation on testicular function of the adult rat

While the need for FSH in initiating spermatogenesis in the immature rat is well accepted, its requirement for maintenance of spermatogenesis in adulthood is questioned. In the current study, using gonadotropin antisera to neutralize specifically either endogenous FSH or LH, we have investigated the effect of either FSH or LH deprivation for a 10-day period on (i) testicular macromolecular synthesis in vitro, (ii) the activities of testicular germ cell specific LDH-X and hyaluronidase enzymes, and finally (iii) on the concentration of sulphated glycoprotein (SGP-2), one of the Sertoli cell marker proteins. Both immature (35-day-old) and adult (100-day-old) rats have been used in this study. Since LH deprivation leads to a near total blockade of testosterone production, the ability of exogenous testosterone supplementation to override the effects of LH deficiency has also been evaluated. Deprivation of either of the gonadotropins significantly affected in vitro RNA and protein synthesis by both testicular minces as well as single cell preparations. Fractionation of dispersed testicular cells preincubated with labelled precursors of RNA and protein on Percoll density gradient revealed that FSH deprivation affected specifically the rate of RNA and protein synthesis of germ cell and not Leydig cell fraction. LH but not FSH deprivation inhibited [3H]thymidine incorporation into DNA. The inhibitory effect of LH could mostly be overriden by testosterone supplementation. LDH-X and hyaluronidase activities of testicular homogenates of adult rats showed significant reduction (50%; P less than .05) following either FSH or LH deprivation. Again testosterone supplementation was able to reverse the LH inhibitory effect.

Lower Gastrointestinal Microbiota in Health and Irritable Bowel Syndrome : Characterisation and Effect of Probiotic Intervention

The human gastrointestinal (GI) microbiota is a complex ecosystem that lives in symbiosis with its host. The growing awareness of the importance of the microbiota to the host as well as the development of culture-free laboratory techniques and computational methods has enormously expanded our knowledge of this microbial community. Irritable bowel syndrome (IBS) is a common functional bowel disorder affecting up to a fifth of the Western population. To date, IBS diagnosis has been based on GI symptoms and the exclusion of organic diseases. The GI microbiota has been found to be altered in this syndrome and probiotics can alleviate the symptoms, although clear links between the symptoms and the microbiota have not been demonstrated. The aim of the present work was to characterise IBS related alterations in the intestinal microbiota, their relation to IBS symptoms and their responsiveness to probiotic theraphy. In this thesis research, the healthy human microbiota was characterised by cloning and sequencing 16S rRNA genes from a faecal microbial community DNA pool that was first profiled and fractionated according to its guanine and cytosine content (%G+C). The most noticeable finding was that the high G+C Gram-positive bacteria (the phylum Actinobacteria) were more abundant compared to a corresponding library constructed from the unfractionated DNA pool sample. Previous molecular analyses of the gut microbiota have also shown comparatively low amounts of high G+C bacteria. Furthermore, the %G+C profiling approach was applied to a sample constructed of faecal DNA from diarrhea-predominant IBS (IBS-D) subjects. The phylogenetic microbial community comparison performed for healthy and IBS-D sequence libraries revealed that the IBS-D sample was rich in representatives of the phyla Firmicutes and Proteobacteria whereas Actinobacteria and Bacteroidetes were abundant in the healthy subjects. The family Lachnospiraceae within the Firmicutes was especially prevalent in the IBS-D sample. Moreover, associations of the GI microbiota with intestinal symptoms and the quality of life (QOL) were investigated, as well as the effect of probiotics on these factors. The microbial targets that were analysed with the quantitative real-time polymerase chain reaction (qPCR) in this study were phylotypes (species definition according to 16S rRNA gene sequence similarity) previously associated with either health or IBS. With a set of samples, the presence or abundance of a phylotype that had 94% 16S rRNA gene sequence similarity to Ruminococcus torques (R. torques 94%) was shown to be associated with the severity of IBS symptoms. The qPCR analyses for selected phylotypes were also applied to samples from a six-month probiotic intervention with a mixture of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99. The intervention had been previously reported to alleviate IBS symptoms, but no associations with the analysed microbiota representatives were shown. However, with the phylotype-specific assays applied here, the abundance of the R. torques 94% -phylotype was shown to be lowered in the probiotic-receiving group during the probiotic supplementation, whereas a Clostridium thermosuccinogenes 85% phylotype, previously associated with a healthy microbiota, was found to be increased compared to the placebo group. To conclude, with the combination of methods applied, higher abundance of Actinobacteria was detected in the healthy gut than found in previous studies, and significant phylum-level microbiota alterations could be shown in IBS-D. Thus, the results of this study provide a detailed overview of the human GI microbiota in healthy subjects and in subjects with IBS. Furthermore, the IBS symptoms were linked to a particular clostridial phylotype, and probiotic supplementation was demonstrated to alter the GI microbiota towards a healthier state with regard to this and an additional bacterial phylotype. For the first time, distinct phylotype-level alterations in the microbiota were linked to IBS symptoms and shown to respond to probiotic therapy.