Necroptosis increases with age and is reduced by dietary restriction

Necroptosis is a newly identified programmed cell death pathway that is highly proinflammatory due to the release of cellular components that promote inflammation. To determine whether necroptosis might play a role in inflammaging, we studied the effect of age and dietary restriction (DR) on necroptosis in the epididymal white adipose tissue (eWAT), a major source of proinflammatory cytokines. Phosphorylated MLKL and RIPK3, markers of necroptosis, were increased 2.7‐ and 1.9‐fold, respectively, in eWAT of old mice compared to adult mice, and DR reduced P‐MLKL and P‐RIPK3 to levels similar to adult mice. An increase in the expression of RIPK1 (1.6‐fold) and MLKL (2.7‐fold), not RIPK3, was also observed in eWAT of old mice, which was reduced by DR in old mice. The increase in necroptosis was paralleled by an increase in 14 inflammatory cytokines, including the pro‐inflammatory cytokines IL‐6 (3.9‐fold), TNF‐α (4.7‐fold), and IL‐1β (5.1‐fold)], and 11 chemokines in old mice. DR attenuated the expression of IL‐6, TNF‐α, and IL‐1β as well as 85% of the other cytokines/chemokines induced with age. In contrast, inguinal WAT (iWAT), which is less inflammatory, did not show any significant increase with age in the levels of P‐MLKL and MLKL or inflammatory cytokines/chemokines. Because the changes in biomarkers of necroptosis in eWAT with age and DR paralleled the changes in the expression of pro‐inflammatory cytokines, our data support the possibility that necroptosis might play a role in increased chronic inflammation observed with age.     

Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver

Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0-500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.     

BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.     

Hepatic monocyte chemoattractant protein-1 is upregulated by dietary cholesterol and contributes to liver steatosis

To assess the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) in the development of fatty liver, we have used LDLr^−/− mice as an animal model of high-fat, high-cholesterol diet-induced liver steatosis. The rapid dietary induction of hepatic mRNA MCP-1 expression was paralleled by a concomitant increase in plasma MCP-1 that was strongly associated with the degree of liver steatosis. Hepatocytes showed an intense immunoreactivity for MCP-1 that was mainly located surrounding the hepatic lipid droplets. The intake of cholesterol also increased the concentration of MCP-1 in liver homogenates. This was accompanied by a differential expression of members of the PPAR family. Additionally, complete MCP-1 deficiency prevents the development of liver steatosis in LDLr^−/− mice and partial deficiency is accompanied by a certain protective effect. Our data also suggest that MCP-1 may be important in the regulation of hepatic insulin resistance and may represent a link between inflammation and metabolic diseases. We conclude that dietary cholesterol upregulation of hepatic MCP-1 may help to understand the role of circulating MCP-1 in conditions where liver derangements are clinically important and in the association of liver steatosis with the metabolic syndrome.     

PS2 protein expression is upregulated by sex steroids in the cerebral cortex of aging mice

Mutations in presenilin (PS) genes cause majority of early onset Alzheimer's disease (AD), an age related neurodegenerative disorder. PS proteins undergo proteolytic cleavage to produce biologically active fragments, which constitute the catalytic core of the gamma-secretase enzyme. This enzyme cleaves beta-amyloid precursor protein (betaAPP) to generate Abeta peptides, which are influenced by sex steroids. Recently we have reported the downregulation of PS1 expression by sex steroids in the brain of adult mice. Here we have examined the effect of gonadectomy and subsequent administration of gonadal hormones 17beta-estradiol and testosterone on the level of PS2 C-terminal fragment (CTF) in the cerebral cortex of adult and old AKR strain mice of both sexes. PS2 expression was downregulated following gonadectomy, but upregulated by supplementation of gonadal steroids in both age groups and sexes. Thus these results demonstrate up-regulation of PS2 protein expression by sex steroids, which in turn may influence PS2 associated brain functions.     

Mitochondrial turnover in liver is fast in vivo and is accelerated by dietary restriction: application of a simple dynamic model

'Mitochondrial dysfunction', which may result from an accumulation of damaged mitochondria in cells due to a slowed-down rate of mitochondrial turnover and inadequate removal of damaged mitochondria during aging, has been implicated as both cause and consequence of the aging process and a number of age-related pathologies. Despite growing interest in mitochondrial function during aging, published data on mitochondrial turnover are scarce, and differ from each other by up to one order of magnitude. Here we demonstrate that re-utilization of the radioactively labelled precursor in pulse-chase assays is the most likely cause of significant overestimation of mitochondrial turnover rates. We performed a classic radioactive label pulse-chase experiment using (14)C NaHCO(3), whose (14)C is incorporated into various amino acids, to measure mitochondrial turnover in mouse liver. In this system, the activity of the urea cycle greatly limited arginine dependent label re-utilization, but not that of other amino acids. We used information from tissues that do not have an active urea cycle (brain and muscle) to estimate the extent of label re-utilization with a dynamic mathematical model. We estimated the actual liver mitochondrial half life as only 1.83 days, and this decreased to 1.16 days following 3 months of dietary restriction, supporting the hypothesis that this intervention might promote mitochondrial turnover as a part of its beneficial effects.     

In vivo fractional P(i) absorption and NaPi-II mRNA expression in rainbow trout are upregulated by dietary P restriction

Mammalian type II sodium-phosphate cotransporter (NaPi-II) and inorganic phosphate uptake stimulator (PiUS) genes are upregulated by dietary phosphorus (P) restriction to increase intestinal and renal P transport, but little is known about NaPi-II and PiUS regulation in other vertebrates. We studied the 1). the tissue distribution and dietary regulation of NaPi-II, PiUS, and sodium-glucose cotransporter (SGLT1) mRNA and NaPi-II protein in juvenile rainbow trout (Oncorhynchus mykiss) and 2). effects of dietary P on intestinal Pi absorption in vivo. NaPi-II, PiUS, and SGLT1 mRNA were found in the proximal and distal intestine, pyloric ceca, and kidney. PiUS mRNA was also found in the heart, gill, blood, stomach, liver, skin, and muscle. Tissue distribution of NaPi-II protein correlated with that of NaPi-II mRNA except in gill ionocytes where NaPi-II antibodies recognized related epitopes. Chronic consumption of a low-P diet increased NaPi-II and PiUS but not SGLT1 mRNA abundance in the intestine and kidney. Unlike mammals, there was no detectable shift in tissue or cellular localization of NaPi-II protein in response to dietary P restriction. Regulation of NaPi and PiUS mRNA expression was observed only in fish grown under optimal aqueous oxygen concentrations. In vivo fractional absorption of Pi by the intestine decreased in fish fed high-P diets. Decreases in absorption were less pronounced in fish previously fed low-P diets, suggesting that diet history modulates acute regulation of P absorption. Regulation of dietary Pi absorption in vivo may involve a specific change in intestinal NaPi-II and PiUS gene expression.     

Long-term dietary restriction up-regulates activity and expression of renal arginase II in aging mice

Arginase II is a mitochondrial enzyme that catalyses the hydrolysis of L-arginine into urea and ornithine. It is present in other extra-hepatic tissues that lack urea cycle. Therefore, it is plausible that arginase II has a physiological role other than urea cycle which includes polyamine, proline, glutamate synthesis and regulation of nitric oxide production. The high expression of arginase II in kidney, among extrahepatic tissues, might have an important role associated with kidney functions. The present study is aimed to determine the age-associated alteration in the activity and expression of arginase II in the kidney of mice of different ages. The effect of dietary restriction to modulate the age-dependent changes of arginase II was also studied. Results showed that renal arginase II activity declines significantly with the progression of age (p<0.01 and p<0.001 in 6- and 18-month-old mice, respectively as compared to 2-month old mice) and is due to the reduction in its protein as well as the mRNA level (p<0.001 in both 6- and 18-month-old mice as compared to 2-month-old mice). Long-term dietary restriction for three months has significantly up-regulated arginase II activity and expression level in both 2- and 18-month-old mice (p<0.01 and p<0.001, respectively as compared to AL group). These findings clearly indicate that the reducing level of arginase II during aging might have an impact on the declining renal functions. This age-dependent down-regulation of arginase II in the kidney can be attenuated by dietary restriction which may help in the maintenance of such functions.     

Influence of dietary restriction on accumulation of heat-labile enzyme molecules in the liver and brain of mice

The influence of dietary restriction on alterations of two aminoacyl-tRNA synthetases was investigated in mouse tissues. Mice were fed a restricted diet (first 80% and then 60% of the ad libitum intake) from 23.5 months of age for 70 days. Before dietary restriction, about 35 and 25% of these enzymes in the brain and liver, respectively, were heat-labile. Dietary restriction resulted in a decrease in the percentage of heat-labile enzyme in both tissues: After 40 and 70 days of dietary restriction the percentage of heat-labile enzyme decreased to about 20 and 10%, respectively, in the brain and to undetectable or very low levels in the liver. These results suggest an interesting possibility that prolongation of the life span by dietary restriction is due to reduction in the level of altered enzyme molecules whose accumulation may be detrimental to cellular functions.     

Major urinary protein 5, a scent communication protein,is regulated by dietary restriction and subsequent re-feeding in mice

Major urinary proteins (Mups) are important for rodent scent communication and sexual behaviour. Recent evidence suggests that Mup1 may be regulated by fasting and re-feeding (RF). However, other Mup isoforms are poorly investigated, and data on the impact of long-term dietary restriction (DR) and ad libitum RF on Mup expression are missing. We investigated the effects of long-term 25 per cent DR and subsequent RF on Mup expression in male C57BL6 mice. DR significantly decreased Mup gene expression, hepatic and urinary protein levels compared with ad libitum (AL) fed control mice, with the greatest downregulation found for Mup5 expression. The decline in Mup expression was inverted by six months of RF. Because of inhibitory glucocorticoid response elements in the genomic sequence of the Mup5 gene, the observed inverse correlation of nuclear glucocorticoid receptor levels with Mup expression in response to DR and subsequent RF is a possible regulatory mechanism. Additionally, gene-expression-inhibiting histone deacetylation (H3K9) occurred in the region of the Mup5 gene in response to DR. We assume that Mup may act as a molecular switch linking nutritional status to sexual behaviour of mice, and thereby regulating male fertility and reproduction in response to food supply.     

Arginase as a useful factor for the diagnosis of colorectal cancer liver metastases

The present work is a continuation of studies on arginase as a marker in the diagnosis of colorectal cancer liver metastases (CRCLM). The purpose of the study was the evaluation of the arginase test in comparison with other colorectal cancer tests such as CEA, CA 19-9 and biochemical markers of liver function such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The studies were conducted on blood serum from 85 patients with CRCLM obtained one to two days before tumor resection. The control group comprised 140 healthy blood donors and 81 patients with various non-malignant gastrointestinal diseases. Raised arginase activity was observed in serum of 85% of CRCLM patients, whereas elevated levels of CEA and CA 19-9 were found in 63% and 42% of patients, respectively. The combination of CEA or CA 19-9 with the arginase assay improved their sensitivity, but the sensitivity of the combined parameters was not higher than that of the arginase test itself. AST and ALT activities were increased in about 30% of CRCLM patients. The specificity of the arginase test calculated for 221 control subjects was 76%. It can thus be concluded that the determination of serum arginase activity can be helpful in the diagnosis of patients with colorectal cancer liver metastases.     

The Cfr10I restriction enzyme is functional as a tetramer.

It is thought that most of the type II restriction endonucleases interact with DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation. The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.     

Induction of phosphoenolpyruvate carboxykinase by hydrocortisone in rat liver and brain as a function of age

The activity and induction pattern of phosphoenolpyruvate carboxykinase (PEPCK) in the liver and brain of young (6-), adult (30-) and old (90-weeks) male rats were studied. The activity of this enzyme increases in both tissues until adulthood and decreases gradually thereafter. Further, the activity of PEPCK is higher in the liver than the brain. Adrenalectomy decreases significantly the activity of this enzyme in the liver of rats of all ages. However, this treatment inhibits brain PEPCK in young and adult rats. Administration of hydrocortisone to adrenalectomized rats increases PEPCK in both tissues of young and adult rats. However, the magnitude of induction is higher in the young, as compared to the adult, rats. This hormone-mediated induction of the enzyme is actinomycin D-sensitive.     

Effects of dietary restriction on physical performance in mice

Dietary restriction is known to prolong life in laboratory animals. However, little is known about the effects of dietary restriction on physical performance. To evaluate physical performance, we measured four item indices: time to climb out of obstacles, time to escape restraint by gummed tape, time hanging from a bar, and ability to resist slipping every week. The diets of ICR mice were restricted from the age of 7 weeks through 24 weeks. Body weight of the diet-restricted mice decreased during the 7th to 9th weeks of age. After the 10th week, weight gain resumed. In response to assigned tasks, the diet-restricted mice performed better in all activities: they climbed out of obstacles faster, freed themselves sooner from restraint by gummed tape, hung from a bar longer, and better resisted slipping down a slope. These results suggest that diet-restricted mice have superior physical abilities, such as those required to overcome or avoid risks to life, than do ad-libitum-fed mice.     

Percent satellite DNA as a function of tissue and age of mice

A selective loss of satellite DNA was found to occur to different extents as a function of tissue and age of mice using several common DNA extraction and purification procedures. This result emphasizes a serious problem that may be encountered in comparative studies of DNA structure and composition if selective loss of specific DNA sequences occurs. We have developed a DNA extraction and purification procedure that is simple and reliable and gives a high percent DNA yield, which substantially reduces the selective loss of heterochromatin DNA sequences. The method features a centrifugation step of a proteolytic digest of chromatin in 2.4 M CsCl. Percent DNA yield of 82–98% are routinely obtained with no apparent loss of satellite DNA sequences from different tissues or ages of mice. Utilizing this method, percent satellite DNA was found to remain essentially constant at 11 ± 1% for spleen, kidney, and brain tissues obtained from mice of 10–780 days of age. However, for liver, percent satellite DNA remained at about 7–8% from 10 to 300 days of age and then increased to about 12–13% from 300 to 600 days of age. During this latter time interval (300–600 days), an increase of DNA per nucleus of about 3-fold occurred, due to the formation of tetra- and octaploid cell types. A steady loss in the total number of nuclei per gram of liver as a function of age was also found. These two opposing effects resulted in a nearly constant amount of DNA per gram and per organ for liver throughout the lifespan of the mouse.