Effect of age and dietary restriction on protein synthesis by isolated kidney cells

The influence of age and food restriction on kidney protein synthesis was studied in Fischer F344 rats. The rate of total protein synthesis by suspensions of kidney cells declined 60% between 4 and 31 months of age. The rate of protein synthesis by kidney cells isolated from 19-month old rats fed a restricted diet (60% of diet consumed by rats fed ad libitum) was 45% higher than the rate of protein synthesis by kidney cells isolated from 19-month old rats fed ad libitum. The excretion of protein in the urine was measured to assess the effect of the age related decline in protein synthesis on kidney function. A dramatic increase in proteinuria was observed with increasing age, and rats fed the restricted diet excreted significantly less protein in the urine than rats fed ad libitum.     

Hormonal and developmental regulation of glycosylated alpha 2u-globulin synthesis

We have investigated the regulation of glycosylated α_2u-globulin synthesis by examining the appearance of these molecules in the medium of primary monolayer cultures of hepatocytes. Hepatocytes were isolated from male and female rats of various ages, as well as from castrated or ovariectomized animals. α_2u-Globulin was immunoprecipitated from the culture medium with rabbit antibody specific for α_2u-globulin, and the dissociated precipitates were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. We found that prepubescent male and female rats synthesized only the high molecular weight glycosylated forms of α_2u-globulin. Hepatocytes from 50-day-old intact and ovariectomized female rats, as well as from ovariectomized rats treated with 17β-estradiol, secreted only glycosylated α_2u-globulin. Hepatocytes from castrated male rats treated with dihydrotestosterone in vivo synthesized the 20,000-dalton nonglycosylated form of α_2u-globulin; the rate of glycosylated α_2u-globulin synthesis was reduced in these cells. The rate of synthesis of glycosylated α_2u-globulin by male rat hepatocytes declined concomitant with increases in the age of the rats, the level of serum testosterone, and the rate of synthesis of nonglycosylated α_2u-globulin. Under our conditions, dexamethasone administration to castrated male rats or ovariectomized female rats in vivo did not alter the species of α_2u-globulin that were synthesized subsequently by hepatocytes in vitro. Our results suggest that the synthesis of glycosylated α_2u-globulin is regulated differently than the synthesis of the 20,000-dalton nonglycosylated form of α_2u-globulin.     

Synthesis and processing of the dimorphic forms of rat alpha 2u-globulin

alpha 2u-Globulin the androgen-dependent male rat urinary protein, can be resolved into two distinct molecular forms by SDS-polyacrylamide slab gel electrophoresis. These two forms designated as alpha 2u-A (M, 18,800) and alpha 2u (Mr 18,100) are found both in urine and in the liver cells. Translation of rat liver mRNA in the rabbit reticulocyte lysate produced two preprotein forms of alpha 2u-globulin, designated as alpha 2uA' (Mr 20,300) and alpha 2uB' (Mr 19,600). Cell-free translation of rat liver mRNA in the presence of dog pancreas microsomal membrane or in Xenopus oocytes produced the two processed forms of alpha 2u-globulin (alpha 2uA and alpha 2uB). Quantitation of alpha 2uA and alpha 2uB within the in vitro translation products of the hepatic mRNA from albino rats of Yale, Sprague-Dawley and Fischer strains showed genetic differences in the proportion of translatable mRNA for alpha 2uA and alpha 2uB. The ratio of alpha 2uA: alpha 2uB in the translation products of liver mRNA from Yale rats was found to be 1:2.5 while in the case of both Sprague-Dawley and Fischer rats, the ratio was 1:4. A small portion of the alpha 2uA and alpha 2uB synthesized in the cultured hepatocytes, in the Xenopus oocytes or in the membrane-supplemented cell-free system appeared as two additional forms, designated as alpha 2uA" (Mr 21,200) and alpha 2uB" (Mr 20,600). Unlike alpha 2uA and alpha 2uB both alpha 2uA" and alpha 2uB" were found to bind to Con A-Sepharose, suggesting their glycoprotein nature.     

Monoclonal antibodies to alpha 2u-globulin and immunocytofluorometric analysis of alpha 2u-globulin-synthesizing hepatocytes during androgenic induction and aging

Stable hybridomas generated by fusion of spleen cells from hyperimmunized mice and mouse myeloma cells were cloned to prepare monoclonal antibodies to alpha 2u-globulin, an androgen-dependent urinary protein of hepatic origin. One of these monoclonal antibodies was used as a probe for immunocytofluorometric analysis of alpha 2u-globulin producing hepatocytes during androgenic induction and aging through fluorescence-activated cell sorting (FACS). FACS patterns of hepatocytes from mature male rats that produce high levels of alpha 2u-globulin showed tow distinct peaks, arbitrarily designated as peak I (weakly fluorescent) and peak II (brightly fluorescent). In the mature male rat, peak II represented about 40% of the total hepatocytes, and the fluorescence intensity of this subpopulation decreased in direct correspondence with the gradual decline of alpha 2u-globulin synthesis during aging. Similarly the androgenic induction of this protein in ovariectomized female rats was associated with an increase in the fluorescence intensity of the hepatocyte subpopulation under peak II rather than an increase in the relative number of these cells. From these results we conclude that the androgen-dependent synthesis of alpha 2u-globulin and its alteration during aging are confined to a specific subpopulation of hepatocytes within the liver.     

Transcortin and alpha 2u-globulin messenger RNA activities during turpentine-induced inflammation in the rat

Previously we have shown that the serum concentration of transcortin and alpha 2u-globulin markedly decreases during turpentine-induced inflammation. In the present study transcortin and alpha 2u-globulin mRNA from healthy rats and from animals with inflammation was translated in a rabbit reticulocyte lysate system. Female rats had higher levels of translatable transcortin mRNA than male animals and the level of mRNA for transcortin and alpha 2u-globulin decreased rapidly during inflammation. These results indicate that the sex difference in the serum level of transcortin and the changes in serum transcortin and alpha 2u-globulin during inflammation are mainly determined by differences in the mRNAs in the liver.     

Hormonal induction of alpha2u-globulin synthesis in isolated rat hepatocytes.

Hepatocytes freshly prepared with collagenase synthesize alpha 2u-globulin and other hepatic proteins in vitro at approximately the same rate throughout 30 h of incubation. The newly synthesized proteins are efficiently secreted into the medium throughout this period. That the secretion of proteins by hepatocytes is not due to cell leakage is shown by the fact that 30 micrometer colchicine prevents the appearance of labeled alpha2u-globulin and other proteins in the medium. Hepatocytes isolated from animals in different endocrine states synthesize alpha2u-globulin in vitro at rates consistent with the hormonal effects upon its in vivo biosynthesis. In vitro addition of androgens to hepatocytes isolated from castrated male rats evokes an elevation of general protein synthesis in these hepatocytes. Glucocorticoids, added in vitro, specifically induce and elevated rate of alpha2u-globulin synthesis.     

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.     

Renal reabsorptive capacity for alpha 2u-globulin in the adult male rat.

Adult male rats were maintained on 0, 5, 10, 15, and 20% casein diets to produce a series of animals having serum alpha 2u-globulin levels varying linearly from a normal of 31 micrograms/ml to a minimum of 13 micrograms/ml. In this way, it was possible to titrate endogenously the renal reabsorption and urinary excretion of this low molecular weight protein. The average maximal reabsorption rate (Tm) was established to be 9.7 micrograms/min and was reached at a renal filtered load (F alpha 2u) of 13.6 micrograms/min. These data were expressed in terms of a Tm:F alpha 2u ratio of 0.71. Below this value, the reabsorption declined from 70% to 50% of the F alpha 2u. Above 0.71, where F alpha 2u is less than the Tm, the reabsorption increased to 80-90%. It was observed that the fractional renal uptake of the alpha 2u-globulin varied linearly with the filtered load.     

Pretranslational regulation of alpha 2u-globulin in rat liver by growth hormone

Hypophysectomy is known to cause complete suppression of the hepatic synthesis alpha 2u-globulin. The effect of hypophysectomy on the synthesis of alpha 2u-globulin can be reversed by multiple hormone treatment. The role of pituitary growth hormone in the multihormonal regulation of alpha 2u-globulin in rat liver was examined in the hypophysectomized male rats with and without growth hormone supplementation. Daily treatment of hypophysectomized rats with 5 alpha-dihydrotestosterone, corticosterone, thyroxine, and growth hormone for 8 days caused about 80% recovery in the hepatic content of alpha 2u-globulin and its corresponding mRNA as determined by radioimmunoassay, in vitro translation, and liquid hybridization with a cloned cDNA probe. However, omission of growth hormone from the treatment regimen failed to raise hepatic alpha 2u-globulin and its mRNA to more than 5% of the normal control. The possible effect of growth hormone on the translation of the mRNA for alpha 2u-globulin was examined with cultured hepatocytes derived from growth hormone-deficient rats. Culture of these cells in the presence of growth hormone for 24 h did not turn on the synthesis of alpha 2u-globulin. These results indicate that growth hormone regulates the synthesis of alpha 2u-globulin by acting at a step antecedent to mRNA translation.     

Effect of age and dietary calcium on intestinal calbindin D-9k expression in the rat

The capacity of rats and humans to adapt to low dietary Ca by increasing intestinal Ca absorption declines with age. The intestinal calbindin-D-9k protein (calbindin) is thought to play a role in the transcellular transport of Ca across the mammalian intestine. The purpose of these studies was to determine the effect of age and diet on the expression of calbindin at the protein and mRNA levels. Young (2 month) and adult (12 month) male F344 rats were placed on either a high Ca diet (1.2%) or a low Ca diet (0.02%) for four weeks. In the duodenum, the level of intestinal calbindin protein induced by a low Ca diet was 8-fold higher in young rats compared to adult rats. In the ileum, expression of calbindin protein was only about 10% that of the duodenum. In addition, the adult ileum showed the same decreased adaptation to a low Ca diet that was seen in the adult duodenum. In both the duodenum and the ileum, the changes in calbindin protein expression were highly correlated with calbindin mRNA expression and the correlations in each segment were quantitatively similar. In the duodenum, the changes in calbindin protein levels were strongly correlated with both Ca transport and Ca uptake. This quantitative correlation suggests a role for calbindin protein in the age-related decline in Ca absorption. In the ileum, the decreased adaptation to a low Ca diet may also be important given the long transit time through the distal intestine. The changes in both intestinal segments may contribute to the negative Ca balance seen in adult rats fed a low Ca diet.     

The effects of age and dietary restriction on the tissue‐specific metabolome of Drosophila

Dietary restriction (DR) is a robust intervention that extends lifespan and slows the onset of age‐related diseases in diverse organisms. While significant progress has been made in attempts to uncover the genetic mechanisms of DR, there are few studies on the effects of DR on the metabolome. In recent years, metabolomic profiling has emerged as a powerful technology to understand the molecular causes and consequences of natural aging and disease‐associated phenotypes. Here, we use high‐resolution mass spectroscopy and novel computational approaches to examine changes in the metabolome from the head, thorax, abdomen, and whole body at multiple ages in Drosophila fed either a nutrient‐rich ad libitum (AL) or nutrient‐restricted (DR) diet. Multivariate analysis clearly separates the metabolome by diet in different tissues and different ages. DR significantly altered the metabolome and, in particular, slowed age‐related changes in the metabolome. Interestingly, we observed interacting metabolites whose correlation coefficients, but not mean levels, differed significantly between AL and DR. The number and magnitude of positively correlated metabolites was greater under a DR diet. Furthermore, there was a decrease in positive metabolite correlations as flies aged on an AL diet. Conversely, DR enhanced these correlations with age. Metabolic set enrichment analysis identified several known (e.g., amino acid and NAD metabolism) and novel metabolic pathways that may affect how DR effects aging. Our results suggest that network structure of metabolites is altered upon DR and may play an important role in preventing the decline of homeostasis with age.     

Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line

A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.     

Heterogeneity of alpha 2u-globulin gene products in different translational systems, plasma and urine

alpha 2u-Globulin, an androgen dependent rat urinary protein, displays considerable microheterogeneity. To explore whether this microheterogeneity of alpha 2u-globulin in male rat urine is related to the heterogeneity at the level of the genes encoding this protein, or whether it is due to post-translational processing we studied the alpha 2u-globulin mRNA translation products in rabbit reticulocyte and Xenopus oocytes. Comparison of the alpha 2u-globulin species produced in these two heterologous systems with those observed in plasma and urine indicates that the heterogeneity of this protein in urine is mainly due to heterogeneity at the level of the corresponding mRNAs.