Starvation-induced Alterations Research Articles

PSIII-36 The endocytic pathway plays a major role in the regulation of tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells

Abstract Postweaning pigs are subjected to nutrient deprivation during which intestinal epithelial cells undergo increased turnover. To preserve intestinal function, intestinal epithelial cells must activate adaptive mechanisms that allow them to cope with starvation-induced stress; most importantly, the preservation of intestinal barrier function. The objective of this study was to investigate the underlying mechanisms involved in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were subjected to total nutrient starvation in Krebs-Ringer bicarbonate (KRB) buffer for 0, 3, 6, 12 and 24 h. Abundance of tight junction proteins was determined by RT-PCR, western blotting and immunofluorescence. Compared with control group (0 h), the protein expression of claudin 1, claudin 3 and claudin 4 protein was downregulated up to 6 h of starvation and then increased thereafter (P < 0.01). However, there was no change in the protein level of occludin and ZO-1. To determine the contribution of the lysosome and the ubiquitin proteasome pathways to regulation of tight junction protein abundance, the lysosome (Bafilomycin A1) and the proteasome (MG132) inhibitors were used in nutrient starved cells. Results showed the degradation of claudin 1, 3 and 4 up to 6 h of starvation was through the lysosomal pathway. Surprisingly, re-synthesis of claudins 4 and claudin 3 after prolonged starvation (12 and 24 h) was prevented when cells were treated with bafilomycin A1 and MG132, respectively. The autophagy-lysosome pathway inhibitors (Wortmannin and MHY1485) and endosome-lysosome pathway inhibitors (Dynasore and Pitstop 2) were further used to determine the specific roles of these pathways. In summary, the degradation of claudin 3 and claudin 4 during short-term starvation (up to 6 h) was through the dynamin-dependent endocytic pathway. However, re-synthesis of these proteins after prolonged starvation relies on both the lysosome and proteasome pathways.

PSVIII-1 Nutrient starvation induces autophagy and alters intestinal barrier function in IPEC-J2 cells

Abstract Autophagy is a cellular process of controlled degradation of damaged organelles and cytoplasmic macromolecules during stress for maintaining homeostasis. Intestinal epithelial cells are barriers against microorganisms, toxins and food antigens. Whether autophagy plays a role in regulating intestinal barrier function is unclear. The objective of this study was to characterize the role of autophagy in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were nutrient starved in Krebs-Ringer bicarbonate (KRB) buffer for 0, 0.5, 1, 2, 3, 6, 9 and 12 h. Expression of genes implicated in autophagy regulation (AMPK, MDM2, p53 and DRAM) was determined by RT-PCR. The ratio of protein abundance of microtubule-associated protein light chain 3 (LC3-II/LC3-I) and p62, positive and negative markers of autophagy induction, respectively, was determined by western blotting. Compared with control group (0 h), the relative mRNA expression level of AMPK, MDM2, p53 and DRAM significantly decreased by an average of 52.5% (P < 0.01). Relative to 0 h, the mRNA expression of claudin 1 and claudin 4 significantly increased (108.0%) up to 6 h of starvation and then decreased (31.4%) thereafter (P < 0.01). On the contrary, abundance of claudin 1 and claudin 4 protein was downregulated (49.5%) up to 3 h of starvation and then increased (82.6%) thereafter (P < 0.01). Protein expression of claudin 3 was reduced (P < 0.01) with duration of starvation. There was no change in the protein level of occludin and ZO-1. The ratio of LC3-II/LC3-I significantly increased with duration starvation (P < 0.01), whereas p62 and phospho-AMPK levels decreased up to 6 h of starvation and then increased thereafter (P < 0.01). In summary, autophagy may be implicated in the regulation of tight junction integrity during nutrient starvation in IPEC-J2 cells.

Leptin Administration Restores the Fasting-Induced Increase of Hepatic Type 3 Deiodinase Expression in Mice

Decreased serum leptin has been proposed as a critical signal initiating the neuroendocrine response to fasting. Leptin administration partially reverses the fasting-induced suppression of the hypothalamus-pituitary-thyroid axis at the central level. It is, however, unknown to what extent leptin affects peripheral thyroid hormone metabolism. The aim of this study was to evaluate the effect of leptin administration on starvation-induced alterations of peripheral thyroid hormone metabolism in mice. Three types of experiments were performed: (i) mice were fasted for 24 hours while leptin was administered twice (at 0 and 8 hours, 1 μg/g body weight [BW]), (ii) mice were fasted for 24 hours and, subsequently, leptin was given once at 24 hours (killed at 28 and 32 hours), and (iii) mice were fasted for 48 hours. All groups had appropriate controls. Serum triiodothyronine and thyroxine, liver type 1 deiodinase (D1), type 3 deiodinase (D3), thyroid hormone receptor (TR)β1, TRα1 and α2 mRNA expression, and liver D1 and D3 activity were measured. Twenty-four hours of fasting decreased liver TRβ1 mRNA expression, while liver TRα1, TRα2, and D1 mRNA expression and activity did not change. In contrast, 24 hours of fasting increased liver D3 mRNA. Leptin administration after fasting restored liver D3 expression, while serum thyroid hormone levels and liver TRβ1 expression remained low. Leptin administration selectively restores starvation-induced increased hepatic D3 expression independently of serum thyroid hormone concentrations. The present study shows that fasting-induced changes in mRNA expression of genes involved in hepatic hormone metabolism are influenced not only by decreased serum thyroid hormone levels but also by serum leptin.

Role of Central Leptin Signaling in the Starvation-Induced Alteration of B-Cell Development

Nutritional deprivation or malnutrition suppresses immune function in humans and animals, thereby conferring higher susceptibility to infectious diseases. Indeed, nutritional deprivation induces atrophy of lymphoid tissues such as thymus and spleen and decreases the number of circulating lymphocytes. Leptin, a major adipocytokine, is exclusively produced in the adipose tissue in response to the nutritional status and acts on the hypothalamus, thereby regulating energy homeostasis. Although leptin plays a critical role in the starvation-induced T-cell-mediated immunosuppression, little is known about its role in B-cell homeostasis under starvation conditions. Here we show the alteration of B-cell development in the bone marrow of fasted mice, characterized by decrease in pro-B, pre-B, and immature B cells and increase in mature B cells. Interestingly, intracerebroventricular leptin injection was sufficient to prevent the alteration of B-cell development of fasted mice. The alteration of B lineage cells in the bone marrow of fasted mice was markedly prevented by oral administration of glucocorticoid receptor antagonist RU486 (11β-[p-(dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one). It was also effectively prevented by intracerebroventricular injection of neuropeptide Y Y(1) receptor antagonist BIBP3226 [(2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide], along with suppression of the otherwise increased serum corticosterone concentrations. This study provides the first in vivo evidence for the role of central leptin signaling in the starvation-induced alteration of B-cell development. The data of this study suggest that the CNS, which is inherent to integrate information from throughout the organism, is able to control immune function.

Unlike leptin, ciliary neurotrophic factor does not reverse the starvation-induced changes of serum corticosterone and hypothalamic neuropeptide levels but induces expression of hypothalamic inhibitors of leptin signaling.

Leptin mediates neuroendocrine responses to fasting and restores the starvation-induced changes of several hypothalamic neuropeptides. Ciliary neurotrophic factor (CNTF), a cytokine closely related to leptin, reduces food intake and reverses obesity, but its role in restoring the starvation-induced changes of hormones or hypothalamic neuropeptides remains largely unknown. To comparatively assess the roles of CNTF and leptin in reversing the starvation-induced changes of hypothalamic neuropeptides and endocrine function and in inducing expression of hypothalamic inhibitors of leptin and CNTF signaling (suppressor of cytokine signaling 3 [SOCS-3]) and mediators of energy expenditure (cyclo-oxygenase 2 [COX-2]), we studied the effect of CNTF and leptin administered by intraperitoneal injections (1 microg/g twice daily) in C57Bl/6J mice fasted for 48 h. Serum corticosterone levels increased with fasting, and leptin administration partially normalized them, whereas CNTF administration had no effect. Hypothalamic neuropeptide Y (NPY) and agouti-related protein (AgRP) mRNA expression increased and pro-opiomelanocortin (POMC) decreased in response to fasting. Leptin administration decreased NPY and AgRP and increased POMC mRNA levels toward baseline, but CNTF administration in fasted mice had no effect of comparable significance. Both leptin and CNTF administration in fasted mice resulted in an induction of SOCS-3 mRNA expression. CNTF also induced hypothalamic SOCS-2 mRNA expression. Finally, neither leptin nor CNTF administration in mice fasted for 48 h alters hypothalamic COX-2 expression. Our data suggest that only falling leptin levels mediate the starvation-induced alterations in corticosterone levels and expression of hypothalamic neuropeptides, but inhibitors of leptin signaling are induced by both leptin and CNTF. This may be of clinical importance because both agents are now being evaluated for the treatment of obesity in humans.

Neuropeptide abnormalities in anorexia nervosa

Starvation-induced alterations of neuropeptide activity probably contribute to neuroendocrine dysfunctions in anorexia nervosa. For example, CRH alterations contribute to hypercortisolemia and NPY alterations may contribute to amenorrhea. Alterations of these peptides as well as opioids, vasopressin, and oxytocin activity could contribute to other characteristic psychophysiological disturbances, such as reduced feeding, in acutely ill anorexies. Such neuropeptide disturbances could contribute to the vicious cycle that has been hypothesized to occur in anorexia nervosa. That is, the consequences of malnutrition perpetuate pathological behavior.

Kinetic behaviour of liver glucokinase in insulinopenic situations: effect of fructose 1-phosphate in fed and starved rats

In the liver postmicrosomal supernatant of starved rats, the high- K m glucose-phosphorylating enzymic activity, presumably attributable to glucokinase, is not solely decreased but also displays an apparently lower affinity and less pronounced temperature dependency than in fed rats. In the presence of exogenous d-glucose 6-phosphate and d-fructose 6-phosphate, the rate of d-glucose phosphorylation is enhanced by d-fructose 1-phosphate at low (10 mM) but not high (90 mM) hexose concentration and at high (30–37°C) but not low (10°C) temperature. The responsiveness of glucokinase to d-fructose 1-phosphate is apparently decreased in starved animals. Nevertheless, the latter ester does not suppress the starvation-induced alteration in both the apparent affinity and temperature dependency of liver glucokinase. It is proposed, therefore, that such an alteration may reflect changes in the intrinsic properties of the high- K m hepatic enzyme.

Starvation-Induced Alterations of the Ultrastructure of the Midgut of Daphnia magna Straus, 1820 (Cladocera)

Starvation-Induced Alterations of the Ultrastructure of the Midgut of Daphnia magna Straus, 1820 (Cladocera)

Starvation-induced alterations of ribosomal protein phosphorylation in Bombyx mori L.. Evidence for different phosphorylation kinetics in free and membrane-bound ribosomes

In the posterior silk gland of Bombyx mori, ribosomal protein S1, homologous to S6 in mammals, is partially phosphorylated in a normally fed animal. Before the first meal of the fifth larval instar, S1 is completely dephosphorylated. Likewise, starvation induces rapid dephosphorylation of the protein in both free and membrane-bound ribosomes. Upon refeeding after 48 h of starvation, S1 becomes phosphorylated again, first on membrane-bound ribosomes, then on free ribosomes, with a lag time of about 3 h. Following 48 h of refeeding, the most highly phosphorylated form of S1 predominates in both populations of ribosomes. These variations in phosphorylation are correlated with the level of protein synthesis in the posterior silk gland, 70% of the ribosomes occurring in polysomes upon feeding and only 30% upon starvation [Prudhomme, J.-C. & Couble, P. (1979) Biochimie (Paris) 61, 215-227]. After in vivo 32P labelling, the phosphopeptides of S1 from free and membrane-bound ribosomes were found to be identical and phosphoserine (only) was found in each S1. These results suggest the involvement of S1 phosphorylation in the regulation of protein synthesis at the translational level and the existence of at least two different pathways controlling this phosphorylation: one for the free ribosomes, the other for the membrane-bound ribosomes.

Response of heterogenous rat liver lysosome populations to starvation and refeeding

Starvation-induced alterations in liver lysosomes and their recovery pattern following refeeding were investigated. Fasting of adult rats for five days caused an increase in ‘free’ activities of acid hydrolyses in liver homogenates and loss in sedimentation of one of the heterogenous populations of lysosomes that could be isolated by differential centrifugation. Isopycnic sucrose gradient centrifugation revealed a decrease in the median and modal equilibration densities of all the forms of lysosomes in response to the dietary deprivation. Further, starvation also evoked a distinct bimodal distribution in a population that was rich in acid phosphatases, β-galactosidase and N-acetyl- βglucosaminidase. Realimentation of starved animals for 10 days was found to restore the enzyme levels and the sedimentation characteristics to normal profiles.

Starvation-induced alterations of circulating thyroid hormone concentrations in man

Serum concentrations of triiodothyronine (T3), thyroxine (T4), and TSH were examined in seven men and seven women of normal weight during a 60-hr fast. Similar studies were conducted in two women who received daily for 1 mo before and during a similar fast, 0.4 mg and 0.5 mg of l-thyroxine. The serum concentrations of T3 decreased in each of the untreated normal subjects (sign test of significance, p < 0.001). The mean control concentration of T3 in women was 152 ± 9 ng 100 ml ( X ± SEM); after 24 hr of fasting, 131 ± 31 ng 100 ml ; and at the termination of the fast, 90 ± 15 ng 100 ml . The latter value differed from the control value with a p value of < 0.01. Similar changes of T3 concentration occurred in men (mean basal T = 160 ± 11 ng 100 ml ; mean at termination of fast = 87 ± 16 ng 100 ml ). The range of decrease for T3 in all subjects varied from 24% to 55%. The mean T4 concentration at the beginning of the fast was 6.9 ± 0.9, and at the termination of the fast, 7.5 ± 0.6 ( p = NS). TSH concentrations remained unchanged (Control, 3.8 ± 0.45 μU/ml; at 60 hr, 4.0 ± 0.26 μU/ml, p = NS). Studies in two women who received, before and during a fast, T4, indicate that a decreased peripheral conversion of T4 to T3 is the most likely mechanism responsible for this change.