Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro

Subcutaneous injections of hormone triiodothyronine in rats resulted in peak blood levels at 24 hr with return to baseline by 96 hr. The injections stimulated a liver regeneration response that resembled in timing and in magnitude of DNA synthesis (peak, 24 hr) that induced by 40% hepatic resection. The principal proliferation was of hepatocytes. Although there were some temporal differences from the gene expression of transforming growth factor-alpha, transforming growth factor-beta, and c-Ha-ras that are known to follow partial hepatectomy, the overall profile of these changes was similar to those after partial resection. The effect was liver specific and could be reproduced three times with no diminution in response in the same animal with injections at 10-day intervals. No response was detected in kidney or intestine. This effect in intact animals contrasted with the minimal ability of triiodothyronine to stimulate hepatocytes in culture. However, when the culture medium was enriched with epidermal growth factor, there was a dose-related response to triiodothyronine. The totality of these experiments provides a preliminary basis for the creation with pharmacological techniques of an in vivo hyperplastic hepatic condition permissive of transfection of new genes, as an alternative to partial hepatectomy. Although triiodothyronine was the test agent used, other hepatic growth factors singly or in combination could be candidates for this purpose.     

Tumor necrosis factor-alpha causes insulin receptor substrate-2-mediated insulin resistance and inhibits insulin-induced adipogenesis in fetal brown adipocytes

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.     

Differential transmembrane diffusion of triiodothyronine and thyroxine in liposomes: regulation by lipid composition

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3'triiodo l-thyronine and 3,5,3',5' tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2, 4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (125I) 3,5,3'triiodo l-thyronine and (125I) 3,5,3',5' tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3' triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3', 5' tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer.     

The regulation of pac gene expression by CRP and FNR

Effects of white and monochromatic (blue-434 nm, green-548 nm, and red-614 nm) lights on the nighttime retinal and pineal NAT activity were examined in chicks. The potency of the tested lights to suppress NAT activity was similar for the retina and pineal gland, with a following rank order: white > green > blue > or = red. The studied tissues of chick were far less sensitive to pulses of monochromatic light than the rat pineal gland. The potency of light to decrease pineal NAT activity of rat was: white > green > blue > red. In chicks, the suppression of the nocturnal NAT activity produced by a short 5-min pulse of monochromatic light was completely reversible in the pineal gland, and partially reversible in the retina. Our data suggest the existence of some differences between birds and mammals in terms of sensitivity and mechanisms involved in the light-induced suppression of melatonin biosynthesis.     

Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids

The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of insulin and corticosteroids to induce PPAR gamma expression in isolated human adipocytes in culture. We conclude that: (a) human PPAR gamma mRNA expression is most abundant in adipose tissue, but lower level expression of both splice variants is seen in skeletal muscle; to an extent that is unlikely to be due to adipose contamination. (b) RNA derived from adipose tissue of obese humans has increased expression of PPAR gamma 2 mRNA, as well as an increased ratio of PPAR gamma2/gamma1 splice variants that is proportional to the BMI; (c) a low calorie diet specifically down-regulates the expression of PPAR gamma2 mRNA in adipose tissue of obese humans; (d) insulin and corticosteroids synergistically induce PPAR gamma mRNA after in vitro exposure to isolated human adipocytes; and (e) the in vivo modulation of PPAR gamma2 mRNA levels is an additional level of regulation for the control of adipocyte development and function, and could provide a molecular mechanism for alterations in adipocyte number and function in obesity.     

Ovarian activin receptor subtype and follistatin gene expression in rats: reciprocal regulation by gonadotropins

The production of activin, follistatin (FS), and inhibin, proteins present in the ovary and involved in mammalian reproduction, is regulated by gonadotropins and estradiol. We report here gonadotropin regulation of ovarian activin receptor (ActR) subtype and FS mRNAs. Expression of ActRI, ActRIIA, ActRIIB, and FS mRNA was measured on the afternoon of proestrus (1800 h) and the morning of estrus (0800 h). ActRI and ActIIA subtype mRNA concentrations fell by approximately 50% (p < 0.05) following the proestrous gonadotropin surge (ActRIIB mRNA was undetectable), while FS mRNA was unchanged. To define the contribution of gonadotropins, hypophysectomized (HYPOX) female rats were given recombinant human (rh) FSH and hCG, which decreased both ActR mRNAs (by approximately 70% and aproximately 50% for ActRI and IIA, respectively) and increased FS mRNA by 2-fold. As gonadotropins could act via estradiol (E2), HYPOX rats were given E2; ActRI was decreased, but ActRIIA mRNA was increased. The actions of gonadotropins were preferential, as the combination of rhFSH and hCG with E2 reduced ActRIIA mRNA. FS mRNA was increased to a similar degree by E2 and/or gonadotropins. These data suggest that gonadotropins regulate ActR and FS gene expression via multiple mechanisms. Both a direct action on ActRIIA (inhibition) and an indirect action through E2 on ActRI (inhibition) and FS (stimulation) suggest potential physiologic mechanisms for the reciprocal regulation of ActR subtype and FS mRNAs.     

Nutrient regulation of gene expression: a methodological strategy

Nutrients should be viewed as the earliest of the hormone signals which allowed an organism to respond to changes in its nutrient environment. Defining nutrient function at the nuclear level will permit us to understand how our dietary environment is related to the development of nutritionally related pathophysiologies such as diabetes, heart disease, and cancer. Moreover, through the application of molecular techniques, we will begin to understand how specific nutrients govern the developmental pattern of specific organs such as the kidney. In this review, the fatty acid synthase gene is employed as a model to demonstrate how one sequentially approaches questions pertaining to the regulation of gene expression by a nutrient, and the article presents a nuclear explanation for how dietary polyunsaturated fats decrease blood triglycerides. More importantly, studies of this nature provide a basis for screening genetically susceptible populations and information that will allow the nutritionists of the 21st century to customize a diet for patients at risk.     

Regulation of multicatalytic enzyme activity by insulin and the insulin-degrading enzyme

The insulin-degrading enzyme (IDE) plays an important role in the cellular metabolism of insulin. Recent studies have also suggested a regulatory role for this protein in controlling the activity of cytoplasmic protein complexes, including the proteasome [multicatalytic proteinase (MCP)] and the glucocorticoid and androgen receptors. Binding of IDE to these complexes increases their activity, whereas the addition of substrates for IDE inhibits activity. This provides a potential mechanism of action for internalized insulin and other IDE substrates in the control of protein turnover. To examine further the interactions, partially purified IDE-MCP complex was treated with EDTA or EGTA, and activity was measured in the absence and presence of various divalent cations (Ca2+, Mn2+, Co2+, and Zn2+) and insulin. EDTA treatment reduced MCP activity and eliminated the effect of insulin on the complex. Divalent cations partially or completely restored MCP activity, but did not restore the effect of insulin. EGTA treatment had a lesser effect on MCP activity, but abolished insulin inhibition of activity. Divalent cations restored the insulin effect. Inhibitors of IDE also blocked the insulin effect on MCP activity, as did treatment with SDS. These findings suggest that conformational changes in the complex may play a role in the insulin control of MCP activity.     

Antilipolytic actions of vanadate and insulin in rat adipocytes mediated by distinctly different mechanisms

Vanadate, which mimics the biological effects of insulin, also inhibits lipolysis in rat adipocytes. Here we demonstrate that the antilipolytic effect of vanadate differs from that of insulin at least by the five following criteria: 1) vanadate inhibits lipolysis mediated by high (supraphysiological) concentrations of catecholamines; 2) vanadate antagonizes (Bu)2cAMP-mediated lipolysis; 3) vanadate antagonizes isobutylmethylxanthine-dependent lipolysis, 4) vanadate inhibits lipolysis mediated by okadaic acid; and 5) wortmannin, which blocks the antilipolytic effect of insulin, fails to block vanadate-mediated antilipolysis. Vanadate does activate phosphoinositol 3-kinase, and wortmannin blocks this activation. Our working hypothesis assumes that all of the insulin-like effects of vanadate, including antilipolysis, are initiated by the inhibition of protein phosphotyrosine phosphatases (PTPases). Among documented PTPase inhibitors we found that VOSO4 (oxidation state +4), several organic vanadyl compounds (+4), zinc (Zn2+), tungstate (W), and molybdate (Mo) also had antilipolytic activity. The order of potency was vanadyl acetylacetonate > or = VOSO4 > or = NaVO3 > or = vanadyl-dipicolinate > Zn2+ > W > Mo, and it correlated better with the inhibition of adipose membranal-PTPases in cell-free experiments. We have concluded that the antilipolytic effect of vanadate is 1) mechanistically distinct from that of insulin, 2) independent of phosphoinositol 3-kinase activation, and 3) independent of the lipolytic cascade. We also strongly suggest that the antilipolytic effect of vanadate emanates from inhibiting adipose membranal, rather than cytosolic PTPases, and present preliminary data showing distinct differences in catalysis between these two PTPase categories. Overall, the study indicates that antilipolysis can be manifested via alternative, insulin-independent, signal-transducing pathways.     

Differential regulation of gastrulation and neuroectodermal gene expression by Snail in the Drosophila embryo

The initiation of mesoderm differentiation in the Drosophila embryo requires the gene products of twist and snail. In either mutant, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutant is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such possibility, we analysed a series of snail mutant alleles. We found that different neuroectodermal genes respond differently in various snail mutant background. Due to the differential response of target genes, one of the mutant alleles, V2, that has reduced Snail function showed an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. Together with the data showing that the expression of some ventral genes disappear in snail mutants, we propose that Snail may repress or activate another set of target genes that are required specifically for gastrulation.     

Pituitary activin receptor subtypes and follistatin gene expression in female rats: differential regulation by activin and follistatin

The activins, hormones produced in the gonads and extragonadal tissues (including the pituitary), rapidly increase FSH beta messenger RNA (mRNA) and FSH secretion. In the rat, activin acts via a family of activin receptor (ActR) subunits that includes at least one type I (ActRI or ALK-2) and two homologous type II (IIA and IIB) subunits. We have previously reported that ActRIIA mRNA rises after ovariectomy (OVX). Potentially, the OVX-induced increases in ActR mRNAs could result from altered activin or the activin-binding protein follistatin. It was the purpose of the current studies to determine whether activin and/or follistatin regulated activin receptor subunit mRNAs. Adult female rat pituitaries were dissociated and plated for 48 h, transferred to wells containing follistatin or activin for 2 or 24 h, then RNA extracted for measurement of ActRI, IIA, and IIB and follistatin mRNAs. All three ActR mRNAs were easily detectable in pituitary RNA, with the relative abundance of ActRI > IIA > IIB (18:9:1). Between 2-24 h, levels of all three ActR mRNAs increased 2- to 3-fold in wells containing medium alone, whereas levels of follistatin mRNA were unchanged. Follistatin significantly reduced FSH secretion and follistatin mRNA, but not the ActR mRNAs. Activin increased ActRI (4-fold, at 2 h), ActRIIB (2-fold, at 24 h), and follistatin (2-fold, at 24 h) mRNAs and FSH release (2-fold, at 24 h), but did not alter ActRIIA mRNA levels. We conclude that 1) pituitary ActR mRNA expression is under inhibitory tone in vivo, as suggested by the effect of pituitary removal and cell dispersion and an earlier report after OVX. 2) Pituitary-derived activin stimulates follistatin (but not ActR) mRNA production, and additional increases in follistatin mRNA can be induced by exogenous activin. 3) Higher concentrations of activin differentially regulate pituitary ActR mRNA expression, suggesting that activin exerts a positive feedback effect on its own receptor.