The nutritional phenotype in the age of metabolomics.

The concept of the nutritional phenotype is proposed as a defined and integrated set of genetic, proteomic, metabolomic, functional, and behavioral factors that, when measured, form the basis for assessment of human nutritional status. The nutritional phenotype integrates the effects of diet on disease/wellness and is the quantitative indication of the paths by which genes and environment exert their effects on health. Advances in technology and in fundamental biological knowledge make it possible to define and measure the nutritional phenotype accurately in a cross section of individuals with various states of health and disease. This growing base of data and knowledge could serve as a resource for all scientific disciplines involved in human health. Nutritional sciences should be a prime mover in making key decisions that include: what environmental inputs (in addition to diet) are needed; what genes/proteins/metabolites should be measured; what end-point phenotypes should be included; and what informatics tools are available to ask nutritionally relevant questions. Nutrition should be the major discipline establishing how the elements of the nutritional phenotype vary as a function of diet. Nutritional sciences should also be instrumental in linking the elements that are responsive to diet with the functional outcomes in organisms that derive from them. As the first step in this initiative, a prioritized list of genomic, proteomic, and metabolomic as well as functional and behavioral measures that defines a practically useful subset of the nutritional phenotype for use in clinical and epidemiological investigations must be developed. From this list, analytic platforms must then be identified that are capable of delivering highly quantitative data on these endpoints. This conceptualization of a nutritional phenotype provides a concrete form and substance to the recognized future of nutritional sciences as a field addressing diet, integrated metabolism, and health.

Metabolomics in the opening decade of the 21st century: building the roads to individualized health.

It is rapidly becoming possible to measure hundreds or thousands of metabolites in small samples of biological fluids or tissues. This makes it possible to assess the metabolic component of nutritional phenotypes and will allow individualized dietary recommendations. ASNS has to take action to ensure that appropriate technologies are developed and that metabolic databases are constructed with the right inputs and organization. The relations between diet and metabolomic profiles and between those profiles and health and disease must be established. ASNS also should consider the social implications of these advances and plan for their appropriate utilization.

Glucose regulation of the acetyl-CoA carboxylase promoter PI in rat hepatocytes.

The rat acetyl-CoA carboxylase (ACC) alpha gene is transcribed from two promoters, denoted PI and PII, that direct regulated expression in a tissue-specific manner. Induction of ACC, the rate-controlling enzyme of fatty acid biosynthesis, occurs in the liver in response to feeding of a high carbohydrate, low fat diet, conditions that favor enhanced lipogenesis. This induction is mainly due to increases in PI promoter activity. We have used primary cultured hepatocytes from the rat to investigate glucose regulation of ACC expression. Glucose and insulin synergistically activated expression of ACC mRNAs transcribed from the PI promoter with little or no effect on PII mRNAs. Glucose treatment stimulated PI promoter activity in transfection assays and a glucose-regulated element was identified (-126/-102), homologous to those previously described in other responsive genes, including l-type pyruvate kinase, S(14) and fatty acid synthase. Mutation of this element eliminated the response to glucose. This region of the ACC PI promoter was able to bind a liver nuclear factor designated ChoRF that interacts with other conserved glucose-regulated elements. This ACC PI element is also capable of conferring a strong response to glucose when linked to a heterologous promoter. We conclude that induction of ACC gene expression under lipogenic conditions in hepatocytes is mediated in part by the activation of a glucose-regulated transcription factor, ChoRF, which stimulates transcription from the PI promoter. Similar mechanisms operate on related genes permitting the coordinate induction of the lipogenic pathway.

Actions and interactions of thyroid hormone and zinc status in growing rats.

Both thyroid hormone (triiodo-L-thyronine, T3) and zinc play important roles in growth and development. The T3 receptor is thought to require zinc to adopt its biologically active conformation. Some of the effects of zinc deficiency, therefore, may be due to loss of zinc from the T3 receptor and impairment of T3 action. This possibility was investigated in growing rats by examining the effects of hypothyroidism and hyperthyroidism in zinc-deficient, pair-fed and control rats. Measurement of serum zinc and T3 confirmed the efficacy of the treatments. Zinc deficiency and hypothyroidism resulted in lower food intake and growth failure, but no interaction was observed between the two treatments. Individual tissue weights were influenced by thyroid status as expected, regardless of zinc status. Both dietary and hormonal treatments influenced serum insulin-like growth factor (IGF)-I in an interactive manner. IGF-I was reduced to a greater extent in zinc-deficient than in pair-fed rats compared with controls. Both hypothyroidism and hyperthyroidism reduced serum IGF-I, and a greater reduction due to hyperthyroidism was apparent in zinc-deficient rats. IGF binding proteins were also influenced by diet and thyroid status. The hepatic expression of mRNA S14 was assessed as a direct index of the nuclear action of T3, but its response was not influenced by dietary treatment. Although confirming the role of both T3 and zinc in the regulation of growth and the somatotrophic axis, the growth failure of zinc deficiency does not appear to be due to impaired T3 function.

Nutrition: a reservoir for integrative science.

In the last twenty years, powerful new molecular techniques were introduced that made it possible to advance knowledge in human biology using a reductionist approach. Now, the need for scientists to deal with complexity should drive a movement toward an integrationist approach to science. We propose that nutritional science is one of the best reservoirs for this approach. The American Society for Nutritional Sciences can play an important role by developing and delivering a cogent message that convinces the scientific establishment that nutrition fills this valuable niche. The society must develop a comprehensive strategy to develop our image as the reservoir for life sciences integration. Our efforts can start with our national meeting and publications, with the research initiatives for which we advocate, with our graduate training programs and with the public relations image we project for ourselves. Defining the image and future directions of nutrition as the discipline that can integrate scientific knowledge from the cell and molecule to the whole body and beyond to populations can be the most important task that our society undertakes. If we do not effectively meet this challenge, a golden opportunity will pass to others and nutritional scientists will be left to follow them.

Chelation of zinc amplifies induction of growth hormone mRNA levels in cultured rat pituitary tumor cells.

Zinc is thought to be an integral part of nuclear receptor proteins, stabilizing them in a conformation required for binding to target genes. However, we have recently shown that restriction of zinc availability with a chelator (diethylenetriaminepenta-acetic acid, DTPA) enhances, rather than inhibits, the ability of thyroid hormone to induce growth hormone mRNA expression in GH3 rat pituitary tumor cells. In this report, we have extended these observations by showing that a prolonged (48 h) exposure to DTPA is required to see these effects. The induction by DTPA can be reversed by subsequent addition of zinc, but again, this reversal is slow. A second chelator, EDTA, can also induce growth hormone gene expression in the presence of thyroid hormone, though it is less potent than DTPA. Other agents which act via the nuclear receptor pathway, all-trans and 9-cis retinoic acid, also induce expression of growth hormone mRNA. Addition of DTPA amplifies these effects in a zinc-dependent manner. Thus chelation of zinc potentiates the action of ligands acting via nuclear receptors on growth hormone gene expression. The delayed nature of the response suggests an indirect effect.

A genetic mutation in PPAR gamma is associated with enhanced fat cell differentiation: implications for human obesity.

Human obesity may have genetic causes, but determining the specific genes involved has been difficult. The peroxisome proliferator-activated receptor gamma (PPAR gamma) gene encodes a protein that plays an important role in the differentiation of fat cells. A mutation has been discovered in this gene which leads to a receptor that cannot be inactivated. This mutation, while probably rare, is associated with extreme obesity.

Atorvastatin and simvastatin have distinct effects on hydroxy methylglutaryl-CoA reductase activity and mRNA abundance in the guinea pig.

The effects of atorvastatin and simvastatin on hydroxy methylglutaryl (HMG)-CoA reductase activity and mRNA abundance were studied in guinea pigs randomized to three groups: untreated animals and those treated with 20 mg/kg of atorvastatin or simvastatin. Guinea pigs were fasted for 0, 6, 12, or 18 h in an attempt to remove the drug from their systems. Reductase activity and mRNA levels were analyzed after each time point. Reductase inhibitor treatment resulted in 50-62% lower cholesterol concentrations compared to untreated guinea pigs (P < 0.0001), while plasma triacylglycerol (TAG) concentrations did not differ among groups. Plasma cholesterol and TAG were 50-70% lower after 18 h fasting in the three groups (P < 0.001). In the nonfasting state, simvastatin and atorvastatin treatment did not affect HMG-CoA reductase activity compared with untreated animals. However, after 6 h of fasting, simvastatin-treated guinea pigs had higher HMG-CoA reductase activity than untreated animals (P < 0.01), suggesting that the drug had been removed from the enzyme. In contrast, atorvastatin-treated guinea pigs maintained low enzyme activity even after 18 h of fasting. Further, HMG-CoA reductase mRNA abundance was increased by sevenfold after atorvastatin treatment and by twofold after simvastatin treatment (P < 0.01). These results suggest that simvastatin and atorvastatin have different half-lives, which may affect HMG-CoA reductase mRNA levels. The increase in reductase activity by simvastatin during fasting could be related to an effect of this statin in stabilizing the enzyme. In contrast, atorvastatin, possibly due to its longer half-life, prolonged inhibition of HMG-CoA reductase activity and resulted in a greater increase in mRNA synthesis.

Thyroid hormone regulates the acetyl-CoA carboxylase PI promoter.

The acetyl-CoA carboxylase-alpha gene has two promoters, PI and PII. A variety of mRNA products result from this gene, depending on promoter usage and splicing events. We have investigated thyroid hormone regulation of acetyl-CoA carboxylase-alpha gene expression, using the reverse-transcription polymerase chain reaction with PI- or PII-specific primers. RNA was extracted from a range of tissues taken from hypo-, eu-, or hyperthyroid rats. PII-generated products were found in all tissues examined at similar levels and were not affected by thyroid state. Products derived from PI were also widely found but with more variable levels of expression. PI mRNAs were reduced in hypo- and elevated in hyperthyroid livers. In brown adipose tissue, more PI products were found in hypothyroid animals. Thus, thyroid hormone regulates the activity of the acetyl-CoA carboxylase PI promoter to influence fatty acid synthesis in a tissue-specific manner.

Uncoupling proteins: beyond brown adipose tissue.

Uncoupling protein, originally described in the inner mitochondrial membrane of brown adipose tissue, permits the oxidation of fuels without the generation of adenosine triphosphate (ATP). Closely related proteins have now been found in many other tissues and shown to be regulated by thyroid hormones and dietary factors. These uncoupling proteins may play a significant role in energy expenditure, with implications for the development of human obesity.

Zinc chelation enhances thyroid hormone induction of growth hormone mRNA in GH3 cells.

The effects of restriction and addition of zinc on thyroid hormone responsiveness of the growth hormone gene were investigated in GH3, rat pituitary tumor cells. Addition of diethylenetriaminepenta-acetic acid (DTPA), a membrane-impermeable chelator, resulted in up to 10-fold increases in GH mRNA in the presence of 10 nM T3, with half-maximal induction at 50 microM DTPA. Only minor effects were seen in the absence of T3. Addition of zinc inhibited the stimulatory effect of DTPA in a dose-dependent manner. Equimolar concentrations of other divalent cations could not substitute for zinc, though inhibitions of the DTPA effect were observed at higher concentrations. In the absence of DTPA, exogenous zinc (100 microM) inhibited T3-induced GH mRNA by approximately 33%. Addition of DTPA or zinc did not affect T3 binding to its nuclear receptor. DTPA also enhanced the stimulatory effect of dexamethasone on GH mRNA. The results demonstrate that restricted zinc availability positively affects T3 induction of the GH gene in GH3 cells.

Promoter usage determines tissue specific responsiveness of the rat acetyl-CoA carboxylase gene.

The acetyl-CoA carboxylase gene contains two promoters, PI and PII which generate multiple mRNA forms. We have used the reverse transcription-polymerase chain reaction to investigate tissue specific promoter usage in rats either fed a standard chow diet, starved for 48 h, or starved and then refed a high carbohydrate, low fat diet. Expression of PII-generated mRNAs was seen in all tissues examined and was not dramatically changed by food removal or refeeding. PI-generated mRNAs were expressed at variable levels in a narrower range of tissues and were regulated by these dietary manipulations. Thus only the PI promoter is responsive to diet and the ability of a tissue to use this promoter determines whether it can alter fatty acid synthesis in response to nutritional challenges.

High carbohydrate diet and starvation regulate lipogenic mRNA in rats in a tissue-specific manner.

We have previously shown that the effects of a high carbohydrate, fat-free diet and 24-h starvation on fatty acid synthesis in rats are tissue specific. In the present study we examine the tissue-specific pretranslational effects of high carbohydrate feeding, starvation and refeeding a high carbohydrate diet after starvation on the lipogenic pathway by measuring the levels of mRNA encoding acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) using Northern analysis. Additionally, we measured mRNA S14, a sequence tightly associated with lipogenesis. In rats fed the high carbohydrate diet, hepatic levels of the three mRNA were 3-5 fold higher than in controls. The level of S14 mRNA was doubled in epididymal fat, but other effects of this diet in adipose tissues were not significant. Expression in kidney, heart, lung and brain was not altered. Starvation significantly reduced the level of these mRNA in all tissues examined except brain. In liver, refeeding the high carbohydrate diet induced the expression of ACC, FAS and S14 mRNA 20-30 fold compared with the values found in 48-h starved animals. Hyperinduction of ACC and FAS, but not S14 mRNA expression was also observed in adipose tissues. The tissue-specific nature of these effects is consistent with previous measurements of fatty acid synthesis and confirm that this regulation occurs at the pretranslational level.

Tissue-specific regulation of lipogenic mRNAs by thyroid hormone.

We have previously shown that triiodothyronine (T3) regulates rat fatty acid synthesis in a tissue specific manner. Here, we determined the effects of thyroid state on mRNAs encoding the lipogenic enzymes, acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS). S14 mRNA, a sequence tightly associated with lipogenesis, was also measured. Levels of the three mRNA were 9-13-fold higher in hyper- than hypothyroid liver. Limited expression in kidney and heart was also increased by thyroid hormone. In brown adipose tissue, highest levels were recorded in hypothyroid animals. Thyroid state did not affect expression in lung and brain. All these changes are consistent with those previously measured in fatty acid synthesis. In white adipose tissue, mRNA expression was increased by hyperthyroidism. This increase may not be reflected in fatty acid synthesis, since we recently showed lipogenesis to be reduced under these circumstances. All three mRNAs responded rapidly to T3 in liver, but more slowly in kidney and fat. Thus, T3 regulates lipogenesis by altering levels of ACC and FAS mRNAs. S14 mRNA changes in parallel.

Thermogenesis and thyroid function.

The past 10 years have seen tremendous progress in the definition of the nuclear mechanism of action of thyroid hormones. Although the way in which these nuclear mechanisms underlie the 3,5,3'-triiodo-L-thyronine (T3)-dependent stimulation of metabolic rate remains to be clarified, evidence favoring non-nuclear pathways is limited. Clearly, T3 stimulates both the production and consumption of energy within cells. It also exerts a number of parallel effects that result in increased oxygen consumption, e.g. on mitochondrial structure and composition; on the metabolism of lipids, carbohydrates, and proteins, and on cardiac function. Additionally, T3 may increase the proton permeability of the inner mitochondrial membrane, which implies that it may decrease the efficiency of energy production. These metabolic effects of T3 appear to be restricted to homeothermic-animals, representing a coordinated response to the challenge of maintaining body temperature.

Regulation of brown adipose tissue lipogenesis by thyroid hormone and the sympathetic nervous system.

Thyroid hormone regulates lipogenesis differently in rat liver and brown adipose tissue (BAT). In the hypothyroid state, lipogenesis is suppressed in liver but enhanced in BAT. Here we investigated the mechanisms underlying increased lipogenesis in hypothyroid BAT. Housing the animals at 28 degrees C decreased lipogenesis in hypothyroid BAT to euthyroid levels. Denervation resulted in a 90% reduction in lipogenesis in hypothyroid BAT such that levels were lower than in euthyroid tissue. Thyroid hormone treatment of hypothyroid rats stimulated fatty acid synthesis in denervated BAT, as in liver, but decreased it in intact BAT. Steady-state levels of mRNA encoding acetyl-CoA carboxylase, fatty-acid synthase, and spor 14 were measured in similar animals by Northern analysis. The expression of these mRNAs mirrored the lipogenic data, showing that both thyroid hormone and the sympathetic nervous system work at a pretranslational level in this tissue. These data suggest that the increased BAT lipogenesis found with hypothyroidism is mediated by the sympathetic nervous system to counter the reduction in metabolic rate in these animals.

Molecular biological approaches to studying trace minerals: why should clinicians care?

The approaches and tools of molecular biology have been enormously valuable to all branches of biological science over the last decade. Nutrition is no exception, where studies on the influence of nutrients on gene expression and of gene products on nutrient metabolism have resulted in a much more sophisticated and detailed understanding of nutritional biochemistry. An example of this as applied to trace minerals research can be seen in the area of thyroidology. Until recently, the sole link between thyroid hormones and trace minerals was iodide. Then the thyroid hormone receptor was cloned and analysis of the protein coding sequence showed it to be a member of a large family of gene activating receptor proteins. These all possess a region containing two clusters of cysteine residues, thought to chelate zinc, which is required for binding of the receptors to their target genes. Zinc appears to be necessary for the biological functioning of not only the thyroid hormone receptor but also many other nuclear proteins which regulate gene expression. The principal product of the thyroid gland is thyroxine from which the more active form of the hormone, triiodothyronine, is derived by peripheral monodeiodination. One of the two enzymes responsible, type I 5'-iodothyronine deiodinase, was recently cloned and shown to contain selenocysteine. Thus production of the active thyroid hormone is dependent on selenium status. These advances made with molecular biology have important implications for clinicians. The possibilities for understanding the clinical picture are immediately enhanced, improving both diagnosis and treatment. Molecular biology also provides the opportunity for developing more specific and sensitive tools for assessing nutritional status. Diseases with a genetic basis can be unequivocally diagnosed and perhaps even treated. A strength of nutrition is that it encompasses molecular biology and clinical practice and practitioners of each can benefit from an understanding of the complementary area.

Tissue-specific regulation of fatty acid synthesis by thyroid hormone.

It is generally agreed that thyroid hormone stimulates the hepatic synthesis of long chain fatty acids in the rat. However, there are conflicting data about its effects in white adipose tissue, while in brown adipose tissue, lipogenic rates are highest in hypothyroid animals. We have systematically examined the effect of thyroid state on lipogenesis in different rat tissues. Fatty acid synthesis was assessed in vivo, using the incorporation of tritiated water. Hepatic lipogenesis was induced 16-fold between hypothyroid (4.1 +/- 0.6 microns H incorporated/g.h) and hyperthyroid rats (66.5 +/- 13.2 microns H/g.h). Kidney and heart were much less lipogenically active, but also responded positively to thyroid hormone. Both hyper- and hypothyroidism diminished fatty acid synthesis in retroperitoneal fat and had similar, although not significant, effects in epididymal fat. However, epididymal adipocytes, taken from hyperthyroid rats and cultured in vitro, were 3 times more lipogenically active than cells from either hypo- or euthyroid animals. Lipogenesis in sc fat from hyperthyroid rats was enhanced when calculated per g tissue, but was not different when expressed per whole tissue. In brown adipose tissue, lipogenesis was inversely related to thyroid hormone status. Fatty acid synthesis in brain, lung, skin, and bone and muscle did not respond to changes in thyroid state. TLC confirmed that greater than 90% of the incorporated tritium was in fatty acids. Thus, in hypothyroid animals, lipogenesis primarily occurs in skin, bone, muscle, and other nonresponsive organs, whereas in hyperthyroid rats, the liver alone constitutes almost half of all fatty acid synthesis. The fatty acid synthetic pathway provides an excellent model for examining the tissue-specific regulation of gene expression by thyroid hormone.

The regulation of lipogenesis by thyroid hormone and its contribution to thermogenesis.

We have used the tritiated water method to quantitate the effects of thyroid hormone on lipogenesis in the rat and then determined the contribution of this process to thyroid hormone-induced thermogenesis. After thyroid hormone administration to hypothyroid animals, fatty acid synthesis rose after a lag time of 12-16 h and reached a plateau after 4-5 days. This is consistent with the kinetics of an increase in oxygen consumption measured by others in similar animals. A diurnal variation was maintained in all thyroid states, with the peak value in the middle of the dark period being 3-fold higher than the nadir. Fatty acid synthesis in the livers of hyperthyroid animals was 3- to 4-fold higher than that in euthyroid rats, which, in turn, was 3- to 5-fold higher than the rate observed in hypothyroid rats. Slightly smaller but similar fold increases were measured in epididymal fat. A stimulation of fatty acid synthesis by thyroid hormone was also measured in the rest of the carcass, with hyperthyroid rates being twice those in hypothyroid animals. The contribution of the liver was much greater in hyperthyroid rats (34% of total fatty acid synthesis) than in hypothyroid animals (5%). The energy costs of this synthesis were calculated and compared to published values for total oxygen consumption in different thyroid states. Thus, 6-10% of the total increment in oxygen consumption between hyperthyroid and hypothyroid animals could be attributed to lipogenesis, depending on which published figures were used. About 3% of this increment was due to the liver alone.

Differences in antibody recognition of the triiodothyronine nuclear receptor and c-erbA products.

The in vitro translated products of several c-erbA cDNAs have recently been shown to bind thyroid hormones with high affinity and have been termed thyroid hormone receptors. We have used a panel of five erbA-related antibodies to probe the relationship between c-erbA translated products and thyroid hormone receptors, as conventionally measured by 125I-T3 labeling of nuclear extracts. All five antibodies immunoprecipitated the chick c-erbA translated products, but only one of them recognized chick liver and brain T3 receptor, as judged by acceleration of sedimentation through sucrose gradients. None of the antibodies reacted with rat liver and brain or human liver T3 receptors, although one antibody did immunoprecipitate a human c-erbA translated product. We conclude that the T3 receptor, as conventionally measured from these sources, is related but not identical to recently cloned c-erbA sequences.