Michael H. Green

Signal Relay by Retinoic Acid Receptors α and β in the Retinoic Acid-induced Expression of Insulin-like Growth Factor-binding Protein-3 in Breast Cancer Cells

Neither retinoic acid receptor-β (RARβ) nor insulin-like growth factor-binding protein-3 (IGFBP-3) is expressed in breast cancer cell line MCF-7. The expression of both proteins can be induced in response to all-trans-retinoic acid (atRA). By using an RARα-selective antagonist (Ro 41-5253), we demonstrated that RARβ expression was induced by atRA through an RARα-dependent signaling pathway and that RARβ induction was correlated with IGFBP-3 induction. However, MCF-7 cells transfected with sense RARβ cDNA expressed IGFBP-3 even in the presence of the RARα-selective antagonist Ro 41-5253. On the other hand, antisense RARβ cDNA transfection of MCF-7 cells blocked atRA-induced IGFBP-3 expression, indicating that RARβ is directly involved in the mediation of IGFBP-3 induction by atRA. Induction of IGFBP-3 expression by atRA occurs at the transcriptional level, as measured by nuclear run-on assays. Finally, we showed that atRA-induced IGFBP-3 is functionally active in modulating the growth-promoting effect of IGF-I. These experiments indicate that RARα and RARβ, both individually and together, are important in mammary gland homeostasis and breast cancer development. By linking IGFBP-3 to RARβ, our experiments define the signal intersection between the retinoid and IGF systems in cell growth regulation and explain why loss of RARβ might be critical in breast cancer carcinogenesis/progression.

Chylomicron Margination, Lipolysis, and Vitamin A Uptake in the Lactating Rat Mammary Gland: Implications for Milk Retinoid Content

We have reported previously that the concentration of vitamin A (VA) in the milk of lactating rats varies with dietary VA intake, even when plasma retinol concentration is unaffected. In the current study, we investigated the role of lipolysis in the uptake of chylomicron (CM) VA into mammary tissue of lactating rats and estimated the proportion of CM-VA that is associated with the mammary gland during CM clearance. Chylomicrons containing [3H]VA, mainly as retinyl esters, were prepared in donor rats and administered intravenously to lactating recipient rats. Chylomicron VA rapidly disappeared from plasma and appeared in mammary tissue (maximum within 2–3 mins), followed by a decline. Concomitantly, uptake by liver increased continuously, reaching a plateau within 20–30 mins. Active lipolysis in mammary tissue was necessary for rapid VA uptake, as significantly less CM-VA was recovered in mammary tissue of postlactating rats than of lactating rats, after heparin treatment in lactating rats, or after injection of preformed CM remnants in lactating rats. [3H]Vitamin A uptake by mammary tissue increased linearly with CM-VA dose over a 150-fold dose range (R2 = 0.972, P = 0.0001), suggesting a high capacity for uptake and apparent first-order assimilation of CM-VA during CM remnant formation in situ. Model-based compartmental analysis using WinSAAM predicted that ~42% of CM-VA marginated, that is, were temporarily removed, from plasma to the mammary glands during lipolysis and that a total of 3.8% of CM-VA was transferred to mammary tissue. The model-predicted t1/2 for CM remnants was 3.04 mins. The metabolism of CM-VA in the lactating mammary gland, in proportion to VA absorption and CM-VA contents, may explain how milk VA concentration varies even when plasma retinol levels are unchanged. The mechanism of CM margination and mammary gland uptake described here for VA may be similar for other lipophilic substances.

Stable isotope dilution techniques for assessing vitamin A status and bioefficacy of provitamin A carotenoids in humans

Vitamin A deficiency is a major global public health problem. Among the variety of techniques that are available for assessing human vitamin A status, evaluating the provitamin A nutritional values of foodstuffs and estimating human vitamin A requirements, isotope dilution provides the most accurate estimates. Although the relative expense of isotope dilution restricts its applications, it has an important function as the standard of reference for other techniques. Mathematical modelling plays an indispensable role in the interpretation of isotope dilution data. This review summarises recent applications of stable isotope methodology to determine human vitamin A status, estimate human vitamin A requirements, and calculate the bioconversion and bioefficacy of food carotenoids.

Effects of dietary rhIGF-I in neonatal calves on the appearance of glucose, insulin, D-xylose, globulins and γ-glutamyl transferase in blood

Cow colostrum is rich in insulin-like growth factors IGF-I and II, thus the dietary effects of recombinant human (rh) rhIGF-I on the newborn were of interest. The objective of this study was to examine the effect of dietary IGF-I upon selected blood components and gut absorptive development. Calves were blocked by birth weight and fed two times per d for a total of four times with the initial restricted diet. The initial feeding was 1.5 l and the remaining three feedings were at 2 l with one of three experimental diets; 1) milk replacer plus isolated colostrum derived globulins (MR-), 2) same as 1 above plus 750 ng/ml rhIGF-I (MR+), 3) pooled cow colostrum (COL). Thereafter, all animals received only milk replacer at 5% of body weight (BW)/feeding two times per d with only treatment 2 having continued addition of 750 ng/ml rhIGF-I until experimental completion at 6 to 7 d after birth. At feeding three, animals were fed D-xylose (0.5 g/kg BW) and 5,000 U of bovine kidney membrane gamma-glutamyl transferase as indicators of gut absorptive capacity. Colostrum-fed animals received 5,000 U of natural occurring gamma-glutamyl transferase activity in the 1.5 l first feeding. Blood samples taken over time were collected and saved as frozen plasma. All diets were analyzed for nutrient composition and endogenous levels of test hormones. Colostrum fed calves had greater globulin concentration (P < 0.01) than MR- or MR+ fed calves. Recombinant hIGF-I feeding had no effect (MR- vs. MR+) upon total protein, albumin or globulin blood levels. Absorption of colostrum gamma-glutamyl transferase at first feeding resulted in a peak total blood U of 4.5% of that fed. Although enzyme absorption was greatly reduced by the third feeding (0.5% of total fed), MR+ fed calves exhibited no significant difference in enzyme absorption when compared with the controls (MR- vs. MR+). However, pharmacokinetic analysis of D-xylose absorption at the third feeding showed diet effect upon absorption of D-xylose. Dietary rhIGF-I may change development or activity of sugar transporters and also may alter absorption of macromolecules (closure) in neonatal calves.

Use of Model-Based Compartmental Analysis to Study Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic, widespread environmental contaminant that has dramatic adverse effects on the metabolism of vitamin A. We used model-based compartmental analysis to investigate sites and quantitative impacts of TCDD on vitamin A kinetics in rats given on oral loading dose of TCDD in oil (3.5 micrograms/kg) followed by weekly maintenance doses (0.7 microgram/kg) or oil only. [3H]Retinol in its plasma transport complex (experiment 1) of lymph containing chylomicrons labeled mainly with [3H]retinyl esters (experiment 2) were administered i.v., and tracer kinetics in plasma, liver, carcass, urine, and feces were measured for up to 42 days. TCDD treatment caused significant reductions in liver vitamin A levels and significant changes in tracer kinetics and tracer excretion. A four-compartment model was used to fit tracer data for experiment 1; for experiment 2, compartments were added to describe the metabolism of newly absorbed vitamin A. The compartmental models predict that TCDD caused a slight delay in plasma clearance (via an increased recycling to plasma), and in liver processing, of chylomicron-derived vitamin A. Models for both experiments predict that TCDD exposure did not affect the fractional uptake of plasma retinol from the rapidly turning-over extravascular pool, but it doubled the fractional transfer of recycled retinol from slowly turning-over pools of vitamin A to plasma. The residence time for vitamin A was reduced by 70% in TCDD-treated rats, transfer into urine and feces was tripled, and vitamin A utilization rates were significantly increased. Since our results do not indicate that retinol esterification is inhibited, we hypothesize that some of the significant effects of TCDD on vitamin A metabolism result from increased catabolism and mobilization of vitamin A from slowly turning-over pools (especially the liver).

c-Myc Is a Major Mediator of the Synergistic Growth Inhibitory Effects of Retinoic Acid and Interferon in Breast Cancer Cells

The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-α were investigated. All-trans-retinoic acid and interferon-α acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-α by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-α down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc·Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-α, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-α. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-α: all-trans-retinoic acid + interferon-α → ↑ double-stranded RNA-dependent protein kinase → ↓ c-Myc → cell growth inhibition.

Effects of Adiposity on Plasma Lipid Response to Reductions in Dietary Saturated Fatty Acids and Cholesterol

Dietary SFA and cholesterol are major targets for reducing plasma total and LDL cholesterol as a strategy to decrease cardiovascular disease risk. However, many studies show that excess adiposity attenuates the expected lipid and lipoprotein response to a plasma cholesterol-lowering diet. Diets low in SFA and cholesterol are less effective in improving the lipid profile in obese individuals and in patients with metabolic syndrome. In contrast, lean persons are more responsive to reductions in dietary SFA and cholesterol. Multiple mechanisms likely contribute to the altered plasma lipid responses to dietary changes in individuals with excess adiposity. The greater rate of hepatic cholesterol synthesis in obese individuals suppresses the expression of hepatic LDL receptors (LDLR), thereby reducing hepatic LDL uptake. Insulin resistance develops as a result of adipose-tissue induced inflammation, causing significant changes in enzymes necessary for normal lipid metabolism. In addition, the LDLR-mediated uptake in obesity is attenuated by alterations in neuroendocrine regulation of hormonal secretions (e.g. growth hormone, thyroid hormone, and cortisol) as well as the unique gut microbiota, the latter of which appears to affect lipid absorption. Reducing adipose tissue mass, especially from the abdominal region, is an effective strategy to improve the lipid response to dietary interventions by reducing inflammation, enhancing insulin sensitivity, and improving LDLR binding. Thus, normalizing adipose tissue mass is an important goal for maximizing the diet response to a plasma cholesterol-lowering diet.

The induction and activation of STAT1 by all-trans-retinoic acid are mediated by RARβ signaling pathways in breast cancer cells

Retinoic acid receptor-β (RARβ) and signal transducer and activator of transcription 1 (STAT1) are important mediators of the antiproliferative and apoptotic actions of retinoids and cytokines/growth factors, respectively. Expression of both RARβ and STAT1 is lost in most breast cancer cell lines but it can be induced by retinoids in estrogen receptor-positive cells. We investigated a possible functional connection between these two mediators and present evidence supporting RARβ as a tumor suppressor. First, by using different receptor-selective retinoids, we demonstrated that RARβ induction in MCF-7 cells by all-trans-retinoic acid (atRA) was associated with the activation of STAT1 gene transcription. The direct involvement of RARβ in atRA-induced STAT1 gene activation was further demonstrated by showing that transfection with an anti-sense RARβ construct blocked atRA-induced STAT1 expression in MCF-7 cells whereas introduction of a sense-RARβ construct resulted in STAT1 induction by atRA in MDA-MB 231 cells. In addition, we showed that STAT1 was phosphorylated/activated under atRA treatment of MCF-7 cells; this process required the involvement of RARβ and protein synthesis. STAT1 phosphorylation/activation was accompanied by increased tyrosine kinase activity that was not due to the activation of JAK1, JAK2 or Tyk 2, suggesting the possible involvement of an unidentified tyrosine kinase.

Use of Model‐Based Compartmental Analysis to Study Vitamin A Kinetics and Metabolism

We discuss the use of mathematical modeling, and specifically model-based compartmental analysis, to analyze vitamin A kinetic data obtained in rat and human studies over the past 25 years. Following an overview of whole-body vitamin A metabolism, a review of early kinetic studies, and an introduction to the approach and terminology of compartmental analysis, we summarize studies done in this laboratory to develop models of whole-body vitamin A metabolism in rats at varying levels of vitamin A status. Highlights of the results of these studies include the extensive recycling of vitamin A among plasma and tissues before irreversible utilization and the existence of significant extrahepatic pools of the vitamin. Our studies also document important differences in vitamin A kinetics as a function of vitamin A status and the importance of plasma retinol pool size in vitamin A utilization rate. Later we describe vitamin A kinetics and models developed for specific organs including the liver, eyes, kidneys, small intestine, lungs, testes, adrenals, and remaining carcass, and we discuss the effects of various exogenous factors (e.g., 4-HPR, dioxin, iron deficiency, dietary retinoic acid, and inflammation) on vitamin A dynamics. We also briefly review the retrospective application of model-based compartmental analysis to human vitamin A kinetic data. Overall, we conclude that the application of model-based compartmental analysis to vitamin A kinetic data provides unique insights into both quantitative and descriptive aspects of vitamin A metabolism and homeostasis in the intact animal.

Experimental and kinetic methods for studying vitamin A dynamics in vivo

Publisher Summary Many types of kinetic studies have been done to investigate the metabolism of retinoids. This chapter reviews the methods used to study vitamin A dynamics in the rat. The approach has been to administer radioactive vitamin A in a physiological form, so that tracer data can be extrapolated to the tracees of interest (retinol and retinyl esters). The chapter applies several kinetic methods to analyze the in vivo data, with the goal of developing a quantitative description of whole-body vitamin A dynamics at different levels of vitamin A status. Such techniques are applicable to studies of vitamin A metabolism in different physiological or nutritional states using rats or other species, as well as to experiments on the effects of alcohol and xenobiotics on vitamin A metabolism and to those on disposition of pharmacologically useful retinoids.