Assessing the use of piceid (resveratrol-glucoside)-accumulating transgenic alfalfa against colon cancer

Biotechnological approaches have been applied to introduce resveratrol-synthase (RS ) (aka. Stilbene synthase ) into novel crops such as apple, kiwi, tomato and wheat. As with natural sources of resveratrol, genetically-modified plants, which constitutively express RS, accumulate resveratrol primarily in the form of piceid (RG). The chemopreventive activity of a RG-accumulating transgenic alfalfa (variety Regen SY) against colon cancer was assessed in an azoxymethane (AOM)-induced aberrant crypt foci (ACF) rodent model of carcinogenesis. Our initial studies revealed that the transgenic alfalfa (TA) at 20% in the diet by weight had no affect on the number, size or multiplicity of ACF in CF-1 mice after a five-week feeding trial. However, the number of AOM-induced ACF was reduced in mice fed these diets supplemented with a β-glucosidase enzyme relative to mice fed TA alone. To test that RG is not bioavailable to the colon, the colonic concentrations of resveratrol-aglycone (Rag) and RG were measured following five-week administration of the diets. Rag, but not RG, was detected by liquid chromatography/mass spectrometry in the colons of mice fed transgenic alfalfa, but these concentrations tended to be lower in mice fed TA without α-galactosidase relative to mice fed these diets supplemented with enzyme. The use of a lactase inhibitor in transport studies conducted in everted, jejunal sacs from CF-1 mice suggested that RG was a substrate for lactase-phlorizin hydrolase and can be absorbed intact in the intestines, also. However, comparative studies with Rag suggest that the intestinal absorption of the Rag was much more efficient than that of RG. Rag could be detected in the plasma and the epithelia of the colon and small intestine of CF-1 mice intragastrically fed 25 mg (111 μM) Rag/kg body weight 1-7 hours post-gavage, but neither Rag nor RG could be detected in mice fed an equal dose of piceid at these time points. Collectively, these data suggest that the intestinal bioavailability of RG was much less than that of Rag. The findings of these studies raise doubt on the ability to achieve the purported health benefits of resveratrol through natural sources alone where resveratrol is present as RG

Transgenic Alfalfa That Accumulates Piceid (Trans-Resveratrol-3-O−β-D-glucopyranoside) Requires the Presence of β -Glucosidase to Inhibit the Formation of Aberrant Crypt Foci in the Colon of CF-1 Mice

Plants have been genetically enhanced to produce a number of products for agricultural, industrial and pharmaceutical purposes. This technology could potentially be applied to providing chemoprevention strategies to the general population. Resveratrol (3,5,4′-trihydroxystilbene) is a compound that has been shown to have protective activity against a number of cancers and could be an ideal candidate for such an application. Alfalfa that was genetically modified to express resveratrol-synthase was used as a model in applying biotechnological approaches to cancer prevention. The transgenic alfalfa, which accumulates resveratrol as a glucoside (piceid = trans-resveratrol-3-O−β-D-glucopyranoside) (152 ± 17.5 μ g piceid/g dry weight), was incorporated into a standard mouse diet at 20% of the diet by weight and fed for 5 wk to 6-wk-old, female CF-1 mice (N = 17–30) that were injected with a single dose of azoxymethane (5 mg/kg body weight). While the addition of resveratrol-aglycone (20 mg/kg diet) to the basal diet reduced the number of aberrant crypt foci/mouse, the transgenic alfalfa did not inhibit the number, size, or multiplicity of aberrant crypt foci in the colon of the CF-1 mice relative to control alfalfa which does not accumulate resveratrol-glucoside. However, diets containing transgenic alfalfa with an exogenous β -glucosidase (860 U/kg diet) did significantly inhibit the number of aberrant crypt foci in the distal 2 cm of the colon of the mice relative to mice fed diets containing the transgenic alfalfa without the enzyme (P \u3c 0.05; Fisher\u27s Combination of p-values). The β -glucosidase alone appeared to have no effect on the inhibition of aberrant crypt foci. These results suggest that piceid in transgenic piceid-accumulating alfalfa was not bioavailable

Imaging and manipulating pituitary function in the awake mouse

International audienceExtensive efforts have been made to explore how the activities of multiple brain cells combine to alter physiology through imaging and cell-specific manipulation in different animal models. However, the temporal regulation of peripheral organs by the neuroendocrine factors released by the brain is poorly understood. We have established a suite of adaptable methodologies to interrogate in vivo the relationship of hypothalamic regulation with the secretory output of the pituitary gland, which has complex functional networks of multiple cell types intermingled with the vasculature. These allow imaging and optogenetic manipulation of cell activities in the pituitary gland in awake mouse models, in which both neuronal regulatory activity and hormonal output are preserved. These methodologies are now readily applicable for longitudinal studies of short-lived events (e.g., calcium signals controlling hormone exocytosis) and slowly evolving processes such as tissue remodeling in health and disease over a period of days to weeks

A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with nativeghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cance

Growth hormone inhibits hepatic de novo lipogenesis in adult mice

Patients with nonalcoholic fatty liver disease (NAFLD) are reported to have low growth hormone (GH) production and/or hepatic GH resistance. GH replacement can resolve the fatty liver condition in diet-induced obese rodents and in GH-deficient patients. However, it remains to be determined whether this inhibitory action of GH is due to direct regulation of hepatic lipid metabolism. Therefore, an adult-onset, hepatocyte-specific, GH receptor (GHR) knockdown (aLivGHRkd) mouse was developed to model hepatic GH resistance in humans that may occur after sexual maturation. Just 7 days after aLivGHRkd, hepatic de novo lipogenesis (DNL) was increased in male and female chow-fed mice, compared with GHR-intact littermate controls. However, hepatosteatosis developed only in male and ovariectomized female aLivGHRkd mice. The increase in DNL observed in aLivGHRkd mice was not associated with hyperactivation of the pathway by which insulin is classically considered to regulate DNL. However, glucokinase mRNA and protein levels as well as fructose-2,6-bisphosphate levels were increased in aLivGHRkd mice, suggesting that enhanced glycolysis drives DNL in the GH-resistant liver. These results demonstrate that hepatic GH actions normally serve to inhibit DNL, where loss of this inhibitory signal may explain, in part, the inappropriate increase in hepatic DNL observed in NAFLD patients

The Pituitary Gland is a Novel Major Site of Action of Metformin in Non-Human Primates: a Potential Path to Expand and Integrate Its Metabolic Actions

Background/Aims: Biguanides are anti-hyperglycaemic agents used to treat diabetes by acting primarily on the liver, inhibiting hepatic gluconeogenesis. However, biguanides may target other key metabolic tissues to exert beneficial actions. As the “master endocrine gland”, the pituitary is a true homeostatic sensor that controls whole body homeostasis and metabolism by integrating central and peripheral signals. However, whether the pituitary is a primary site of biguanides action in normal adult humans/primates remains unknown. Therefore, we aimed to elucidate the direct effects of two biguanides (metformin/phenformin) on the expression and secretion of all anterior pituitary hormones in two non-human primate species (Papio anubis and Macaca fascicularis), and the molecular/signalling-mechanisms behind these actions. Methods: Primary pituitary cell cultures from baboons and macaques were used to determine the direct impact of metformin/phenformin (alone and combined with primary regulators) on the functioning of all pituitary cell-types (i.e. expression/secretion/signaling-pathways, etc). Results: Metformin/phenformin inhibited basal, but not GHRH/ghrelin-stimulated GH/ACTH/ FSH-secretion and GH/POMC-expression, without altering secretion or expression of other pituitary hormones (PRL/LH/TSH), FSH-expression or cell viability in both primate models. These biguanide actions are likely mediated through modulation of: 1) common (mTOR/PI3K/intracellular-Ca2+mobilization) and distinct (MAPK) signaling pathways; and 2) gene expression of key receptors regulating somatotrope/corticotrope/gonadotrope function (i.e. upregulation of SSTR2/SSTR5/INSR/IGF1R/LEPR). Conclusion: The pituitary gland is a primary target of biguanide actions wherein they modulate somatotrope/corticotrope/gonadotrope-function through multiple molecular/signaling pathways in non-human primate-models. This suggests that the well-known metabolic effects of biguanides might be, at least in part, influenced by their actions at the pituitary level

The GAIA theory: from Lovelock to Margulis. From a homeostatic to a cognitive autopoietic worldview

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Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest constitutive activity of any G protein coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin and, its activation increases transcriptional and electrical activity in hypothalamic neurons. It is known that GHSR1a is present at some specific GABAergic presynaptic terminals; however, its impact on neurotransmitter release remains elusive. The voltage gated calcium channels, CaV2.1 and CaV2.2, control neurotransmitter release at presynaptic terminals and their activities are modulated by many GPCRs. Here we show that constitutive as well as agonist-dependent GHSR1a activation trigger a strong impairment of both CaV2.1 and CaV2.2 currents in rat and mouse neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at plasma membrane, whereas, ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 current gating via a Gq-dependent pathway. Thus, we show that GHSR1a differentially inhibits CaV2 channels by Gi/o- or Gq-protein pathways depending on its activation mode. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 impairs GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation by the disinhibition of postsynaptic neurons

Metabolic Impact of Adult-Onset, Isolated, Growth Hormone Deficiency (AOiGHD) Due to Destruction of Pituitary Somatotropes

Growth hormone (GH) inhibits fat accumulation and promotes protein accretion, therefore the fall in GH observed with weight gain and normal aging may contribute to metabolic dysfunction. To directly test this hypothesis a novel mouse model of adult onset-isolated GH deficiency (AOiGHD) was generated by cross breeding rat GH promoter-driven Cre recombinase mice (Cre) with inducible diphtheria toxin receptor mice (iDTR) and treating adult Cre+/−,iDTR+/− offspring with DT to selectively destroy the somatotrope population of the anterior pituitary gland, leading to a reduction in circulating GH and IGF-I levels. DT-treated Cre−/−,iDTR+/− mice were used as GH-intact controls. AOiGHD improved whole body insulin sensitivity in both low-fat and high-fat fed mice. Consistent with improved insulin sensitivity, indirect calorimetry revealed AOiGHD mice preferentially utilized carbohydrates for energy metabolism, as compared to GH-intact controls. In high-fat, but not low-fat fed AOiGHD mice, fat mass increased, hepatic lipids decreased and glucose clearance and insulin output were impaired. These results suggest the age-related decline in GH helps to preserve systemic insulin sensitivity, and in the context of moderate caloric intake, prevents the deterioration in metabolic function. However, in the context of excess caloric intake, low GH leads to impaired insulin output, and thereby could contribute to the development of diabetes