Differential expression of thyroid hormone receptor isoforms is strikingly related to cardiac and skeletal muscle phenotype during postnatal development.

The genomic actions of thyroid hormones (THs) are mediated by receptors (TRs) that are encoded by two protooncogenes, c-erbA-alpha and c-erbA-beta. The precise functions of the TR isoforms are unclear and this study focuses on the potential roles of the TRalpha and TRbeta isoforms in mammalian striated muscles postnatally. The porcine TRalpha1, TRalpha2 and TRbeta1 cDNAs were first cloned, sequenced and characterised by Northern blotting. A quantitative analysis of TR isoform expression was then undertaken, using RNase protection analysis with novel riboprobes designed to detect relative expression levels of TRalpha1, TRalpha2, TRbeta1 and TRbeta2, in functionally distinct muscles from 7-week-old pigs kept under controlled conditions of nutrition and thermal environment. We found a striking muscle-specific pattern of TRalpha isoform distribution: in heart the mRNA level of TRalpha2 (non-TH binding) was markedly greater (P<0.01) than that of TRalpha1 (TH binding); in longissimus dorsi the opposite pattern of expression occurred (TRalpha1>TRalpha2, P<0.001); in soleus, diaphragm and rhomboideus there were no differences between the two isoforms. The overall abundance of TRbeta was very much lower than that of TRalpha, and TRbeta1 was expressed at a higher level than TRbeta2 in all muscles. Together with recent data from TR gene inactivation studies and the established role of TH in determining myosin heavy chain isoform expression and muscle phenotype, these results suggest a role for differential expression of TR isoforms in acquisition and maintenance of optimal cardiac and skeletal muscle function.

The porcine insulin-like growth factor-I gene: characterization and expression of alternate transcription sites.

Genomic DNA encoding the 5' region of the porcine IGF-I gene was cloned and sequenced and shown to be highly homologous to that of man, rats and sheep. Two leader exons (exons 1 and 2), which are alternately spliced to exon 3 (encoding part of the mature IGF-I molecule), were identified by RNase protection analysis. In both cases, transcription initiates upstream from exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes (1 and 2) which differ in the precursor peptides predicted from their individual leader sequences. The expression of class 1 and 2 transcripts was measured in liver and muscle RNA from two groups of 2-month-old pigs whose energy status had been manipulated within physiological limits to produce marked differences in plasma IGF-I levels and growth rates. For this purpose, RNase protection probes were developed that contained the individual leader exons 1 and 2 linked separately to the common exon 3, so that class-specific and total IGF-I gene expression could be determined in a single assay. At normal plasma IGF-I concentrations (200 ng/ml), class 1 and 2 transcripts comprised 81 and 19% respectively of total liver IGF-I mRNA, while at a lower plasma concentration (90 ng/ml) the corresponding values were 95 and 5% respectively. Although both classes of transcript declined with the decrease in plasma IGF-I, the relative drop in levels of class 2 transcripts (84%) was substantially greater than that of class 1 (54%). In longissimus dorsi, cardiac and soleus muscles IGF-I mRNA was predominantly of class 1 and did not change in response to decreased plasma IGF-I. This suggests that liver-derived endocrine IGF-I has an important function in the regulation of muscle growth and that class 2 IGF-I transcripts are more sensitive to conditions that promote optimal growth.

Short-term changes in the 3,5,3'-tri-iodothyronine nuclear receptor-binding capacity of porcine skeletal muscle following food intake.

The time-course of changes in the nuclear 3,5,3'-tri-iodothyronine (T3) receptor-binding capacity (Bmax) of longissimus dorsi muscle has been investigated in cold-acclimated young pigs after a single large meal. Measurement of Bmax values 4, 8, 12 and 24 h after feeding indicated a decline in receptor numbers after food intake with the lowest values occurring at 8 h. The receptor numbers then increased significantly, with the values at 12 h being more than 50% higher than those obtained at 8 h. The Bmax values reached their highest level 24 h after feeding. No significant changes in the dissociation constant were observed. Possible reasons for the changes in T3 receptor numbers are discussed and it is suggested that the increase in T3 receptor numbers 12-24 h after feeding may play a role in enhancing the thermogenic capacity of the tissues in response to food.

Regulation of porcine skeletal muscle nuclear 3,5,3'-tri-iodothyronine receptor binding capacity by thyroid hormones: modification by energy balance.

Thyroid hormones have been implicated in the regulation of nuclear 3,5,3'-tri-iodothyronine (T3) receptor binding capacity (Bmax) but, despite numerous in vivo and in vitro studies, there is considerable controversy regarding their exact role. Since changes in thyroid status alter energy balance and hence may influence T3 receptor numbers, the effects of chronic hypothyroidism and T4 treatment have been studied in young pigs under conditions of controlled energy intake. Four groups of animals comprising a hypothyroid, a euthyroid and a hyperthyroid group, all on the same level of food intake, and a hyperthyroid group on twice the amount of food were used. After 3 weeks on the treatment regimes, both the hypothyroid animals on the same level of food intake and the hyperthyroid animals on twice the amount of food had significantly increased Bmax values (97% and 137% higher respectively) compared with euthyroid controls. However, there was no difference between controls and the hyperthyroid animals on the same level of food intake. In a second study, the effects of short-term treatment of euthyroid animals with T3 was investigated. Results showed that in two groups of controls that received intravenous saline, those on a higher food intake had higher Bmax values (76% increase). Intravenous T3 administration to animals on a low food intake did not change the receptor numbers. In none of the studies was there any change in the dissociation constant of the receptors as a result of different treatments. It is suggested that, at least in postnatal life, thyroid hormones per se have no significant effect on nuclear T3 receptor numbers in skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of mechanisms mediating the increase in plasma concentrations of thyroid hormones after a meal in young growing pigs.

These studies investigated a number of possible mechanisms which could mediate the increase in plasma concentrations of thyroid hormones after a meal in young growing pigs. It has been established that in animals fed one meal a day, an immediate rise in plasma 3,5,3'-tri-iodothyronine (T3) and a slightly delayed increase in thyroxine (T4) levels are followed by a more sustained peak in both hormones several hours later. The increase in thyroid hormones involves both total and free T3 and T4, and there is no change in plasma albumin, the high-capacity thyroid hormone-binding protein in the pig. It has also been shown that the immediate rise in plasma T3 is not mediated either by an increase in plasma glucose concentration or by neural mechanisms associated with distension of the gastrointestinal tract. However, the finding that plasma T3 increases rapidly after feeding in thyroidectomized animals maintained on a replacement dose of T4 alone, indicates the source of T3 to be non-thyroidal. It is concluded that the rise in plasma thyroid hormones after a meal depends on the energy content of the food but not directly on the circulating glucose levels. The immediate increases in plasma T3 and T4 are probably due largely to a redistribution of the hormonal pools, and peripheral 5'-monodeiodination of T4 may also contribute significantly to the post-prandial rise in T3. The potential significance of these findings in relation to both the metabolic and growth-promoting effects of thyroid hormones is discussed.

Effects of thyroid status on insulin-like growth factor-I, growth hormone and insulin are modified by food intake.

Understanding the interactions between metabolic signals that regulate insulin-like growth factor-I (IGF-I) is crucial to a recognition of mechanisms that control mammalian growth. Thyroid hormones (THs) are essential for normal growth and development, and it has been suggested previously that they can modify circulating IGF-I concentrations. However, the fact that THs influence food intake, which can itself affect plasma IGF-I levels, has been ignored in previous studies. We have therefore investigated the effects of thyroid status on plasma IGF-I under conditions of controlled food intake in young growing pigs. Circulating IGF-I, growth hormone (GH) and insulin levels, were studied in hypo- and hyperthyroid animals on the same level of food intake as euthyroid controls. In addition, a separate group of hyperthyroid animals was given double the amount of food, in order to assess the influence of increased food intake, as would occur naturally in the hyperthyroid state. Hypothyroid animals and hyperthyroids with extra food had the greatest increase in body weight over the 3 weeks of treatment. These two groups had significantly higher circulating IGF-I and insulin concentrations than either the euthyroid or hyperthyroid animals on the same food intake. Integration of GH concentrations from samples taken every 20 min over a 9 h period showed that, by contrast with IGF-I and insulin levels, GH levels were significantly lower in hypothyroids and hyperthyroids on extra food compared with the euthyroids and the hyperthyroids on the same food intake. We conclude that the effects of thyroid status on IGF-I are mediated in part by the effects that THs have on energy balance, and that nutritional signals are capable of modifying the influence of thyroid status per se on circulating IGF-I concentrations.

Perinatal ontogeny of porcine growth hormone receptor gene expression is modulated by thyroid status.

The ontogeny of growth hormone receptors (GHR) represents a critical stage in growth and metabolism. We have investigated the perinatal ontogeny of hepatic and skeletal muscle GHR gene expression in piglets, and its modulation by GH and thyroid hormones. Test piglets were rendered hypothyroid in late gestation by feeding the sow a high-glucosinolate rapeseed meal. Plasma and tissue samples were obtained from test and control piglets at various ages between 80 days of fetal life (80F) and 2 days postnatally. Plasma hormone levels were determined by radioimmunoassay and GHR mRNA by RNase protection assays. In controls, plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels increased between 80F and birth and the early postnatal period was characterized by a marked surge in plasma T3. Test piglets were hypothyroid at 110F with total T4, total T3 and free T3 levels being reduced by 28, 53 and 33% respectively. By contrast, the postnatal increase in T3 was more marked in test than in control animals. Plasma GH levels decreased over the perinatal period and there was no effect of treatment. Hepatic GHR mRNA was at the lower limit of detection at 80F but by 110F was expressed in both groups of animals. However, fetal hypothyroidism at 110F resulted in a marked 70% decrease in hepatic GHR mRNA (p < 0.01). The higher postnatal rise in T3 in test piglets was accompanied by a recovery of hepatic GHR mRNA levels. By contrast with liver, skeletal muscle (longissimus dorsi) expressed high levels of GHR mRNA at 80F and hypothyroidism induced a 68% increase in GHR mRNA (p < 0.001). The present results suggest that thyroid hormones may modulate the perinatal ontogeny of GHR gene expression, in addition to other hormonal factors, and that this modulation is tissue-specific.

Ouabain sensitive Na+/K(+)-ATPase content is elevated in mdx mice: implications for the regulation of ions in dystrophic muscle.

Recent evidence indicates that in dystrophin-deficient muscle, intracellular sodium content (Na(i)) may be elevated and sodium regulation may be altered or impaired. If there is an elevation in Na(i), this could be due to decreased active pumping of sodium from the cell or increased passive influx of sodium. The present study has therefore determined the content of plasma membrane-bound Na+/K(+)-ATPase in the skeletal muscle of mdx mice; a genetically homologous model of Duchenne muscular dystrophy. Measurements were made on muscles from 5-6-month-old mdx mice and age-matched controls of the C57B1/10ScSn strain (n = 9 pairs), using the vanadate-facilitated ouabain-binding technique. The Na+/K(+)-ATPase concentration per unit weight increased by 2.3-fold in the longissimus dorsi and 1.4-fold in the gastrocnemius of mdx mice compared with controls. The increase in Na+/K(+)-ATPase content is of similar magnitude to the previously reported increase in ouabain-sensitive Na+/K(+)-ATPase activity in mdx muscle, suggesting that this elevated enzyme activity occurs largely through an increase in its concentration. This compensatory increase in the main regulator of internal sodium is likely to occur in an attempt to maintain homeostasis. Nevertheless, the elevated pump concentration is unable to compensate entirely for the increased Na(i). These results are consistent with a previously proposed hypothesis that sodium regulation is abnormal in dystrophin deficient muscles, and also that cell death in these muscles may be due to abnormal regulation of cell volume.

Behavioural energy regulation in lean and genetically obese (ob/ob) mice.

The role of behaviour in the control of energy regulation has been investigated in relation to environmental temperature, nutrition and genetics. Techniques of operant conditioning were used, with lean and genetically obese (ob/ob) mice being tested at three environmental temperatures (10, 20 and 30 degrees C) and on two feeding regimes (after a 24 hr fast and after feeding ad lib). They were allowed access to heat and food, although the design of the apparatus ensured that both were not available simultaneously. Both the lean and ob/ob showed an initial preference for heat when tested in a cold environment. At a low ambient temperature the ob/ob were dependent on the heater rather than food to increase rectal temperature, both when fasted and when fed. By contrast, the lean had a lower demand for heat than the obese and used the time to explore the environment and to feed. Food intake increased with an increase in ambient temperature in both genotypes. Possible reasons for this are discussed.

Postnatal development and differentiation of myofibres in functionally diverse porcine skeletal muscles.

Marked changes in muscle function occur after birth, with the response being dependent on developmental stage. Therefore, postnatal cellular ontogeny of functionally distinct skeletal muscles was investigated in the pig, a large mammal born at a relatively advanced stage of development. Assessment of myofibre contractile (type I slow/type II fast) and metabolic (oxidative/glycolytic) properties at Days 0, 2, 5 and 14 revealed type-specific differences in hypertrophy and differentiation. Type I fibre proportions increased significantly in soleus and diaphragm, especially between Days 0 and 5, and rhomboideus showed a similar trend, but in longissimus there was a slight decrease during Days 0-2. Cytochrome oxidase activity was relatively high and similar among myofibres in all muscles at birth, and fibres with low activity were not detected until Day 5. In contrast with previous reports, glycolytic fibres were present in all muscles at birth; postnatal changes in alpha-glycerophosphate dehydrogenase activity were both muscle- and myofibre-specific. Hence, although myosin ATPase activity and metabolic properties of porcine myofibres are well developed at birth, they continue to mature postnatally. This suggests that postnatal muscle development can be modulated by extrinsic factors, even in mammals born at a relatively advanced stage of development.

Effect of thyroid status in the perinatal period on oxidative capacities and mitochondrial respiration in porcine liver and skeletal muscle.

Regulatory thermogenesis is reduced in newborn piglets which have been made hypothyroid during late gestation by giving the sow a high glucosinolate rapeseed diet (test animals). Thereafter, the progressive increase in thermogenic capacity parallels the development of a marked postnatal hyperthyroid state. To explain these effects of thyroid hormones at the tissue and mitochondrial levels, we have examined both liver and skeletal muscle to determine possible underlying changes in (i) tissue oxidative capacities (cytochrome oxidase (CO) activity), between 80 d of gestation and 48 h after birth, and (ii) mitochondrial content and respiratory capacities at 24 h of life. In control piglets, CO activity increased sharply during late gestation and the first 2 d of life in liver and rhomboideus (RH) muscle (P < 0.01), whereas only a prenatal increase was observed in longissimus dorsi (LD) muscle. Test fetuses were hypothyroid and had lower CO activities than controls during late gestation in RH muscle (P < 0.06, at 110 d of gestation; P < 0.08, at birth) and in liver (P < 0.001, at birth). The postnatal increase in CO activity in RH muscle and liver was higher (P < 0.05) in test than in control piglets, and as a result the difference between the 2 groups was not significant by 24-48 h of life. There was no effect of treatment on LD muscle. At 24 h, hyperthyroid test piglets had lower amounts of mitochondrial proteins than controls (P < 0.05) in all three tissues, possibly reflecting reduced mitochondrial protein synthesis during fetal life and suggesting that high postnatal T3 levels did not bring about major increases in protein synthesis within 24 h. However, test piglets exhibited higher rates of mitochondrial respiration than controls in liver and RH muscle, as shown by increases in State III and FCCP-stimulated respirations (P < 0.05), and mitochondrial CO and creatine kinase activities (P < 0.05). In RH muscle, both subsarcolemmal and intermyofibrillar mitochondria showed the same trends. No changes were observed in LD muscle. Our results describe for the first time the effect of thyroid hormones on perinatal oxidative capacities and neonatal mitochondrial respiration in liver and skeletal muscle of the pig, through both the short-term regulation of mitochondrial respiration and the long-term control of mitochondrial biogenesis. The differential sensitivity of LD and RH muscles to thyroid hormones is discussed.

Evidence for the presence of brown adipose tissue in the pig.

Small quantities of tissue closely resembling brown adipose tissue have been found in pigs aged two to three months. The tissue, which was taken post mortem from near the great veins in the neck, in the subscapular area and close to the adrenal and thyroid glands, was embedded in connective tissue or white adipose tissue. Examination under the light microscope and electron microscope revealed the following features characteristic of brown adipocytes: multilocular fat: large, round nucleus; numerous mitochondria with cristae; the presence of large amounts of glycogen, and the close proximity of numerous unmyelinated nerve cells with synaptic vesicle filled varicosities. No mitochondrial inclusions were seen. It is suggested that this finding of brown adipose tissue could lead to a better understanding of the control of non-shivering thermogenesis.

Nutrition, the brain and cognitive decline: insights from epigenetics.

Nutrition affects the brain throughout life, with profound implications for cognitive decline and dementia. These effects are mediated by changes in expression of multiple genes, and responses to nutrition are in turn affected by individual genetic variability. An important layer of regulation is provided by the epigenome: nutrition is one of the many epigenetic regulators that modify gene expression without changes in DNA sequence. Epigenetic mechanisms are central to brain development, structure and function, and include DNA methylation, histone modifications and non-protein-coding RNAs. They enable cell-specific and age-related gene expression. Although epigenetic events can be highly stable, they can also be reversible, highlighting a critical role for nutrition in prevention and treatment of disease. Moreover, they suggest key mechanisms by which nutrition is involved in the pathogenesis of age-related cognitive decline: many nutrients, foods and diets have both immediate and long-term effects on the epigenome, including energy status, that is, energy intake, physical activity, energy metabolism and related changes in body composition, and micronutrients involved in DNA methylation, for example, folate, vitamins B6 and B12, choline, methionine. Optimal brain function results from highly complex interactions between numerous genetic and environmental factors, including food intake, physical activity, age and stress. Future studies linking nutrition with advances in neuroscience, genomics and epigenomics should provide novel approaches to the prevention of cognitive decline, and treatment of dementia and Alzheimer's disease.

Recent advances in nutrition, genes and brain health.

Molecular mechanisms underlying brain structure and function are affected by nutrition throughout the life cycle, with profound implications for health and disease. Responses to nutrition are in turn influenced by individual differences in multiple target genes. Recent advances in genomics and epigenomics are increasing understanding of mechanisms by which nutrition and genes interact. This review starts with a short account of current knowledge on nutrition-gene interactions, focusing on the significance of epigenetics to nutritional regulation of gene expression, and the roles of SNP and copy number variants (CNV) in determining individual responses to nutrition. A critical assessment is then provided of recent advances in nutrition-gene interactions, and especially energy status, in three related areas: (i) mental health and well-being, (ii) mental disorders and schizophrenia, (iii) neurological (neurodevelopmental and neurodegenerative) disorders and Alzheimer's disease. Optimal energy status, including physical activity, has a positive role in mental health. By contrast, sub-optimal energy status, including undernutrition and overnutrition, is implicated in many disorders of mental health and neurology. These actions are mediated by changes in energy metabolism and multiple signalling molecules, e.g. brain-derived neurotrophic factor (BDNF). They often involve epigenetic mechanisms, including DNA methylation and histone modifications. Recent advances show that many brain disorders result from a sophisticated network of interactions between numerous environmental and genetic factors. Personal, social and economic costs of sub-optimal brain health are immense. Future advances in understanding the complex interactions between nutrition, genes and the brain should help to reduce these costs and enhance quality of life.

New insights into nutrition and cognitive neuroscience.

Nutrition can affect the brain throughout the life cycle, with profound implications for mental health and degenerative disease. Many aspects of nutrition, from entire diets to specific nutrients, affect brain structure and function. The present short review focuses on recent insights into the role of nutrition in cognition and mental health and is divided into four main sections. First, the importance of nutritional balance and nutrient interactions to brain health are considered by reference to the Mediterranean diet, energy balance, fatty acids and trace elements. Many factors modulate the effects of nutrition on brain health and inconsistencies between studies can be explained in part by differences in early environment and genetic variability. Thus, these two factors are considered in the second and third parts of the present review. Finally, recent findings on mechanisms underlying the actions of nutrition on the brain are considered. These mechanisms involve changes in neurotrophic factors, neural pathways and brain plasticity. Advances in understanding the critical role of nutrition in brain health will help to fulfil the potential of nutrition to optimise brain function, prevent dysfunction and treat disease.

Activity-induced thermogenesis in lean and genetically obese (ob/ob) mice.

Motor activity was approximately 60% lower in genetically obese than in lean mice, during three consecutive hours at thermal neutrality. It is suggested that this must have contributed to the lower heat production measured in the obese mice and that activity-induced thermogenesis contributes significantly to differences in energy expenditure between the genotypes, at least in the short-term.

Activity and energy expenditure.

The influence of small changes in activity on energy expenditure and hence on energy requirements and energy balance is assessed. Evidence from direct and indirect calorimetry suggests that differences in spontaneous minor activity could readily alter 24-h energy expenditure by as much as 20%. This compares with values in the order of 10% for moderate overfeeding and somewhat less than this during mild cold exposure. Individual variability in 24-h energy expenditure can therefore be accounted for not only by differences in resting metabolism and the thermic responses to energy intake and temperature but also by differences in minor activity. Interactions between activity and environmental factors such as nutrition and temperature can modify the effect of activity on energy balance. Very little is known about mechanisms that could account for differences in spontaneous activity and these need to be the subject of future investigations.

Influence of mild cold on 24 h energy expenditure, resting metabolism and diet-induced thermogenesis.

UNLABELLED: 1. It has been suggested previously that people in developed countries do not expose themselves to cold severe enough to induce a metabolic response. The energy expenditure, at both heat production and total heat loss, of nine women was therefore measured continuously while each lived for 30 h in a whole-body calorimeter on two occasions, one at 28 degrees and the other at 22 degrees. All subjects followed a predetermined pattern of activity and food intake. The environmental conditions were judged by the subjects to be within those encountered in everyday life. In the standard clothing worn, 28 degrees was considered to be comfortably warm but not too hot, while 22 degrees was judged to be cool but not too cold. 2. Heat production for 24 h was significantly greater at the lower temperature, by (mean +/- SE) 7 . 0 +/- 1 . 1%. The range was between 2 and 12%. Total heat loss was also significantly greater, by 6%, and there was a large change in the partition of heat loss. At the lower temperature sensible heat loss increased by 29% while evaporative heat loss decreased by 39%. 3. Resting metabolism measured in the morning 12--13 h after the last meal was significantly greater at 22 degrees than at 28 degrees, whereas there was no difference when the resting measurement was made for 2 . 5 h following a meal. 4. IN CONCLUSION: (a) environmental temperature may play a more important role than was previously recognized in the energy balance of those living in this country, and (b) there is an indication of at least a partial replacement of cold-induced by diet-induced thermogenesis in man.