Caloric restriction alters the feeding response of key metabolic enzyme genes.

Differential 'fuel usage' has been proposed as a mechanism for life-span extension by caloric restriction (CR). Here, we report the effects of CR, initiated after weaning, on metabolic enzyme gene expression 0, 1.5, 5, and 12 h after feeding of 24-month-old mice. Plasma glucose and insulin were reduced by approximately 20 and 80%. Therefore, apparent insulin sensitivity, as judged by the glucose to insulin ratio, increased 3.3-fold in CR mice. Phosphoenolpyruvate carboxykinase mRNA and activity were transiently reduced 1.5 h after feeding, but were 20-100% higher in CR mice at other times. Glucose-6-phosphatase mRNA was induced in CR mice and repressed in control mice before, and for 5 h following feeding. Feeding transiently induced glucokinase mRNA fourfold in control mice, but only slightly in CR mice. Pyruvate kinase and pyruvate dehydrogenase activities were reduced approximately 50% in CR mice at most times. Feeding induced glutaminase mRNA, and carbamyl phosphate synthetase I and glutamine synthase activity (and mRNA). They were each approximately twofold or higher in CR mice. These results indicate that in mice, CR maintains higher rates of gluconeogenesis and protein catabolism, even in the hours after feeding. The data are consistent with the idea that CR continuously promotes the turnover and replacement of extrahepatic proteins.

Chaperone-mediated regulation of hepatic protein secretion by caloric restriction.

Calorie restriction (CR) delays age-related physiological changes, reduces cancer incidence, and increases maximum life span in mammals. Here we show that CR decreased the expression of many hepatic molecular chaperones and concomitantly increased the rate and efficiency of serum protein secretion. Hepatocytes from calorie-restricted mice secreted twice as much albumin, 63% more alpha1-antitrypsin, and 250% more of the 31.5-kDa protein 2 h after their synthesis. A number of trivial explanations for these results, such as differential rates of protein synthesis and cell leakage during the assay, were eliminated. These novel results suggest that CR may promote the secretion of serum proteins, thereby promoting serum protein turnover. This may reduce the circulating level of damaging, glycoxidated serum proteins.

Energy metabolism after 2 y of energy restriction: the biosphere 2 experiment.

BACKGROUND: An adaptive decrease in energy expenditure (EE) in response to 6 mo of severely restricted energy intake was shown in a classic semistarvation study-the Minnesota experiment. OBJECTIVE: Our objective was to examine whether such adaptation also occurs in response to less severe but sustained energy restriction. DESIGN: Body composition, 1-wk total EE (TEE), 24-h sedentary EE, and spontaneous physical activity were measured in 8 healthy subjects (4 men and 4 women) at the end of a 2-y confinement inside Biosphere 2. Unexpectedly, the food supply was markedly restricted during most of the confinement and all subjects experienced a marked, sustained weight loss (9.1 +/- 6.6 kg; P: < 0.001) from the low-energy (7000-11000 kJ/d), low-fat (9% of energy), but nutrient-dense, diet they consumed. RESULTS: The TEE inside Biosphere 2, assessed 3 wk before exit, averaged 10700 +/- 560 kJ/d (n = 8). Within 1 wk after exit, the adjusted 24-h EE and spontaneous physical activity were lower in the biospherians (n = 5) than in 152 control subjects (6% and 45%, respectively; both P: < 0.01). Six months after exit and return to an ad libitum diet, body weight had increased to preentry levels; however, adjusted 24-h EE and spontaneous physical activity were still significantly lower than in control subjects. CONCLUSIONS: In lean humans, an adaptive decrease in EE appears to occur not only in states of life-threatening undernutrition, but also in response to less severe energy restriction sustained over several years.

Calories and aging alter gene expression for gluconeogenic, glycolytic, and nitrogen-metabolizing enzymes.

We characterized the effects of calorie restriction (CR) on the expression of key glycolytic, gluconeogenic, and nitrogen-metabolizing enzymes in mice. Of the gluconeogenic enzymes investigated, liver glucose-6-phosphatase mRNA increased 1.7- and 2. 3-fold in young and old CR mice. Phosphoenolpyruvate carboxykinase mRNA and activity increased 2.5- and 1.7-fold in old CR mice. Of the key glycolytic enzymes, pyruvate kinase mRNA and activity decreased approximately 60% in CR mice. Hepatic phosphofructokinase-1 and pyruvate dehydrogenase mRNA decreased 10-20% in CR mice. Of the genes that detoxify ammonia generated from protein catabolism, hepatic glutaminase, carbamyl phosphate synthase I, and tyrosine aminotransferase mRNAs increased 2.4-, 1.8-, and 1.8-fold with CR, respectively. Muscle glutamine synthetase mRNA increased 1.3- and 2. 1-fold in young and old CR mice. Hepatic glutamine synthetase mRNA and activity each decreased 38% in CR mice. These CR-induced changes are consistent with other studies suggesting that CR may decrease enzymatic capacity for glycolysis and increase the enzymatic capacity for hepatic gluconeogenesis and the disposal of byproducts of muscle protein catabolism.

Physiologic changes in humans subjected to severe, selective calorie restriction for two years in biosphere 2: health, aging, and toxicological perspectives.

Biosphere 2 is a closed ecological space of 7-million cubic feet near Tucson, AZ, containing 7 biomes: rain forest, Savannah, ocean, marsh, desert, agricultural station, and habitat for humans and domestic animals. Sealed inside, 4 men and 4 women maintained themselves and the various systems for 2 years. All organic material, all water, and nearly all air was recycled, and virtually all food was grown inside. On the low calorie but nutrient-dense diet available, the men sustained 18% and the women 10% weight loss, mostly within the first 6 to 9 months. The nature of the diet duplicated rodent diets that had been shown to enhance health, lower disease incidence, and retard aging. Using blood specimens frozen at different points during and after the 2 years, determinations were made of a number of biochemical parameters judged to be pertinent based on past studies of rodents and monkeys on similar diets. These included blood lipids, glucose, insulin, glycosylated hemoglobin, renin, and others. The results clearly suggest that humans react to such a nutritional regime similarly to other vertebrates. In addition to these studies, and because this was a tightly closed, isolated environment, the levels of insecticides or pollutants or their derivatives were determined in the sera of 2 crew members. It was found that levels of the lipophilic toxicant DDE and the "total PCB" load increased with the loss of body fat during the first 12-18 months inside Biosphere 2, then decreased.

Caloric intake alters the efficiency of catalase mRNA translation in the liver of old female mice.

The free radical theory of aging predicts that calorie restriction, which extends life span, should reduce oxidant damage. In mammals, the oxidative processes centered in the liver are a major source of free radicals. Liver catalase has the dominant role in the intracellular detoxification of hydrogen peroxide. In male rodents, published studies indicate that aging decreases catalase gene transcription and that calorie restriction obviates this effect. In females, published studies are inconsistent, and no molecular mechanisms have been identified. Here we report that, in female mice, aging can lead to an increase in the translational efficiency of hepatic catalase mRNA, and that calorie restriction obviates this effect. Consideration of these results and published studies leads us to propose that the variability in catalase results in females may arise from the small number of studies or from unique aspects of female physiology, perhaps the estrous cycle and its cessation with age.

Changes in plasma lipids and lipoproteins in humans during a 2-year period of dietary restriction in Biosphere 2.

BACKGROUND: A cohort study was performed of 8 people sealed inside Biosphere 2 to evaluate the effects of dietary restriction in humans on lipid and lipoprotein levels and the relationship of these levels to energy, fat, and protein content of the diet, and body weight, weight change, and energy expenditure. METHODS: Eight healthy people aged 27 to 67 years, 4 women and 4 men, were sealed inside Biosphere 2 from September 26, 1991, to September 26, 1993, the longest sustained period in an "isolated confined environment" on record. They were studied throughout confinement and for more than 2 years after their exit and return to an ad libitum diet. Food available was severely restricted during most of the 2-year period inside Biosphere 2. High work output was maintained and food quality remained high, resulting in prolonged restriction of energy intake without malnutrition. RESULTS: Fasting plasma cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol levels; HDL subfraction distribution; dietary energy, fat, and protein content intake; and height, weight, weight change, and energy expenditure were measured. Total plasma cholesterol and triglyceride levels decreased 30% and 45%, respectively. The HDL and low-density lipoprotein levels also decreased and, in some participants, levels of HDL2 subfractions were increased. Multivariate analysis showed that the major cause of these changes was energy restriction. CONCLUSIONS: Energy restriction was the major factor leading to low lipid and lipoprotein levels. Energy restriction with adequate nutrition of young and middle-aged people may substantially reduce risk for atherosclerosis and consequent coronary artery and cerebrovascular disease.

Transgenic mice expressing a truncated Peromyscus leucopus TNF-alpha gene manifest an arthritis resembling ankylosing spondylitis.

Several studies have implicated tumor necrosis factor-alpha (TNF-alpha) in autoimmune diseases, such as rheumatoid arthritis (RA). To elucidate further the role of TNF-alpha in inflammatory arthritis, we generated transgenic mice harboring a truncated Peromyscus leucopus TNF-alpha (Pe-TNF) gene. An arthritic phenotype closely resembling human ankylosing spondylitis was observed only in transgenic lines expressing the Pe-TNF transgene at the mRNA level. We characterized the arthritic phenotype in detail by radiographic and histologic techniques. It consisted of severe axial skeletal kyphosis and ankylosis, accompanied by an inflammatory and fibrotic process at the end plates and enthesis. Peripheral joint lesions were absent in mice expressing the P. leucopus TNF-alpha gene, in contrast to the RA-like phenotype described in transgenic mice expressing a truncated human TNF-alpha gene. The Pe-TNF transgenic mouse model provides a unique opportunity to explore potential mechanisms whereby TNF-alpha may initiate an autoimmune arthritis resembling ankylosing spondylitis.

Dietary energy tissue-specifically regulates endoplasmic reticulum chaperone gene expression in the liver of mice.

A number of putative molecular chaperones seem to play essential roles in the correct folding, assembly and glycosylation of membrane and secreted proteins in the endoplasmic reticulum. We have shown that life span-extending dietary energy restriction significantly and specifically reduces GRP78 mRNA and protein by 50-75% in mice. Here, 5-mo-old female C3B10RF1 mice were given free access to food after being fed 50% less dietary energy since weaning. Hepatic GRP78 mRNA increased linearly, reaching the same level after 2 wk as was found in the liver of 20-mo-old mice with free access to food. This increase took place with no change in body weight. The mRNA levels of endoplasmic reticulum, cytosolic and mitochondrial chaperones were determined in young (7-mo-old) and old (21- or 28-mo-old) female C3B10RF1 mice. Each age group was either 50% energy restricted or was fed approximately 10% less energy than consumed by mice given free access to food. In young and old energy-restricted mice, hepatic expression of the endoplasmic reticulum chaperones ERp57 (37%), GRP170 (51%), ERp72 (43%), calreticulin (54%) and calnexin (23%) was significantly and specifically reduced. The GRP78, GRP94, GRP170, ERp57 and calnexin mRNA response to diet occurred reproducibly only in liver, and not in adipose, brain, heart, kidney, lung, muscle or small intestine. The mRNA for GRP75, a mitochondrial chaperone, HSC70, a cytoplasmic chaperone, protein disulfide isomerase, an endoplasmic reticulum chaperone, and C/EBPalpha, a transcription factor, was not regulated. Hepatic C/EBPbeta was 15% higher in old energy-restricted mice. Thus the expression of nearly all endoplasmic reticulum chaperones responded rapidly and specifically to dietary energy in mice.

The response to calorie restriction in mammals shows features also common to hibernation: a cross-adaptation hypothesis.

The marked elevation in hepatic carbamyl phosphate synthetase I (CPSI) in calorie-restricted mice, and the changes in erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and in hemoglobin oxygen affinity in calorie-restricted and hypoxic humans living in Biosphere 2 suggest similarities between physiologic events in calorie restriction and hibernation. Other data from the literature strengthen this comparison. Accordingly, we hypothesize that the response to the calorie restriction regime as studied by gerontologists, rather than being a laboratory artifact, is part of a spectrum of responses to food deprivation which have adaptive value in the wild, and whose triggering mechanism may primarily involve the neuroendocrine system.

The accumulation of non-replicative, non-functional, senescent T cells with age is avoided in calorically restricted mice by an enhancement of T cell apoptosis.

Peripheral blood lymphocytes of elderly humans show an increased percentage of T cells with characteristics of replicative senescence. Similarly, the overall decrease in T cell proliferation in aged mice reflects a progressively increasing proportion of non-functional cells rather than a uniform decline in function by all cells. The improved immune function of calorically restricted (CR) animals is, paradoxically, accompanied by a relative lymphopenia. To test whether the reduction in lymphocyte number in the CR mice might reflect more efficient elimination of T cells, we measured apoptosis in young, old and CR old mice. T cell apoptosis induced by irradiation, Staurosporine, anti-CD3, and heat shock was reduced by 62, 42, 32, and 30%, respectively, in old compared with young mice. Caloric restriction normalized apoptosis in T cells from aged mice. Enhanced elimination of non-functional T cells in CR mice may be, at least in part, responsible for their improved immune functional status relative to non-CR mice of the same age.

Calorie restriction inhibits the age-related dysregulation of the cytokines TNF-alpha and IL-6 in C3B10RF1 mice.

TNF-alpha and IL-6 are generally increased in the sera of aged humans and mice. The dysregulation of these cytokines may be critical in autoreactivity and immune dysfunction. In earlier studies we demonstrated that production of TNF-alpha and IL-6 following in vitro stimulation of peritoneal macrophages by LPS was reduced in old compared to young mice, and that dietary caloric restriction (CR) had no effect on the induction of TNF-alpha in this system. In the present study we examined the effects of age and calorie restriction on the constitutive production of both TNF-alpha and IL-6. Serum levels of both cytokines were significantly higher in old versus young mice. However, in old mice subjected to long term CR the serum levels were comparable to those of young mice. The potential involvement of normalization of TNF-alpha and IL-6 levels in the life extension effect of CR are discussed.

A tumor preventive effect of dietary restriction is antagonized by a high housing temperature through deprivation of torpor.

Energy restriction (ER) has proven to be the only effective means of retarding aging in mice. The mechanisms of multiplicity of effects of ER on aging remain, however, fragmentary. ER induces daily torpor, the induction of which is reduced by increasing the ambient temperature to 30 degrees C. The effects of preventing hypothermia in ER animals were studied in terms of the expected consequences of ER on survival, disease pattern and a number of physiological parameters in autoimmune prone MRL/lpr mice and lymphoma prone C57BL, 6 mice. The results demonstrate that torpor plays a crucial role in the prevention of lymphoma development but does not have an affect on other aspects of ER, such as prevention of autoimmune diseases.

"Biospheric medicine" as viewed from the two-year first closure of Biosphere 2.

Biosphere 2 is a 3.15-acre, 7-million ft. enclosed ecological space near Tucson, AZ. It contains five wilderness and two domestic biomes (rain forest, savanna, desert, ocean, marsh; agricultural station, living quarters), an original introduction of 3,800 species (approximately 20% extinctions have occurred), and a large basement "technosphere." Sealed inside Biosphere 2 in September 1991, four women and four men, including two of the authors, maintained themselves and the various systems for 2 yr, the longest-sustained "isolated confined environment" period on record. MMPI psychological profile scores for Biosphere 2 crewmembers correlated closely with those reported for astronauts and shuttle applicants. Major medical problems encountered during the 2 yr included adaptation to a low-calorie (1800-2200 kcal.d-1 per person) but otherwise nutritionally adequate diet, with substantial weight loss (18% for men, 10% for women), and a declining oxygen atmosphere (down to 14.2%). Life in a miniworld such as Biosphere 2 may differ substantially from life in a space station or temporary planetary base. These differences include multiple, shifting, sometimes opposing post-launch objectives; complete self-sustenance with recycling of virtually all materials within a highly complex biologic system; retooling of some areas of practical medicine; an attention to "culture" as a social dynamic and how that may influence crew and leadership selection in a societal rather than a quasi-military community. Assuming that long-term planetary colonies must be largely self-sustaining (due to costs of supply over great distances), they must of necessity approach the condition of biospheres. Subject to chaos dynamic (nonlinear dynamic) perturbations, the behavior of complex biospheres will be inherently non-predictable--as opposed to the linear dynamic situation of most space missions--and will require of the inhabitants, including the medical team, a wide range of coping abilities. Under the circumstances, and while strong similarities exist, important differences may serve to distinguish "biospheric medicine" from "space medicine."

Dietary energy restriction in mice negatively regulates hepatic glucose-regulated protein 78 (GRP78) expression at the posttranscriptional level.

Dietary energy restriction delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. We showed previously that 50% energy restriction in mice reduces hepatic expression of glucose-regulated protein mRNA by 50 to 80%. Changes in glucose-regulated protein 78 (GRP78) levels can either decrease or increase the rate of secretion of specific proteins. Therefore, energy restriction probably produces a global change in the spectrum of proteins secreted by the liver. These studies were initiated to investigate the molecular basis for the negative regulation of the gene. By use of transfection and nuclear run-on techniques, the strong induction of GRP78 gene transcription in cultured cells subjected to acute, extreme glucose deprivation has been well characterized. However, negative regulation of GRP78 gene expression in vivo by energy restriction is not as well understood. In our studies, a reduction in GRP78 protein levels determined using Western blotting closely paralleled a reduction in hepatic GRP78 mRNA measured by Northern and dot blotting. In each case the changes were statistically significant. This close correspondence indicates that energy restriction does not influence the translation rate or the stability of GRP78 protein. No statistically significant difference in the rate of transcription of the gene was detected in energy-restricted mice by use of transcription run-on assays. These results strongly suggest that energy restriction results in destabilization of GRP78 mRNA, thereby repressing hepatic expression of the gene.

Dietary calorie restriction in mice induces carbamyl phosphate synthetase I gene transcription tissue specifically.

Dietary calorie restriction (CR) delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. Here, we present the novel finding that chronic reduction of dietary calories by 50% without changing the intake of dietary protein induced the activity of mouse hepatic carbamyl phosphate synthetase I (CpsI) 5-fold. In liver, CpsI protein, mRNA, and gene transcription were each stimulated by approximately 3-fold. Thus, CR increased both the rate of gene transcription and the specific activity of the enzyme. Short-term feeding studies demonstrated that higher cpsI expression was due to CR and not consumption of more dietary protein. Intestinal CpsI activity was stimulated 2-fold, while its mRNA level did not change, suggesting enzyme activity or translation efficiency was stimulated. CpsI catalyzes the conversion of metabolic ammonia to carbamyl phosphate, the rate-limiting step in urea biosynthesis. cpsI induction suggests there is a shift in the metabolism of calorie-restricted animals toward protein catabolism. CpsI induction likely facilitates metabolic detoxification of ammonia, a strong neurotoxin. Enhanced protein turnover and metabolic detoxification may extend life span. Physiologic similarities between calorie-restricted and hibernating animals suggest the effects of CR may be part of a spectrum of adaptive responses that include hibernation.

Structure and regulation of the mouse GRP78 (BiP) promoter by glucose and calcium ionophore.

Dietary calorie restriction, also termed energy restriction, increases mean and maximum life span, reduces the incidence of tumors and increases the mean age of onset of diseases and tumors in every animal tested. Because life-span is genetically determined, we are studying the mechanisms by which energy restriction regulates the expression of genes. We found that energy restriction reduces hepatic glucose-regulated protein-78 (GRP78) and protein-94 mRNA levels by 2-3-fold in mice [Spindler et al., J. Nutr. 20 (1990) 1412-1417]. To investigate this down-regulation, we have cloned the mouse GRP78 promoter (pGRP78) and studied its regulation by glucose. The mouse pGRP78 and the previously cloned rat promoter mediate responsiveness to glucose deprivation, as well as to the calcium ionophore A23187. These studies are the first demonstration that cis-elements in the pGRP78 mediate responsiveness to glucose deprivation.

Caloric restriction and aging as viewed from Biosphere 2.

The low-calorie nutrient-dense diet consumed for 2 yr by the eight persons sealed inside the closed ecological space known as Biosphere 2, near Tucson, AZ, constituted a unique "experiment of nature," amounting to the first well-monitored application of a nutritional regimen proven in animals to substantially inhibit and delay time of onset of most age-related diseases, induce physiological changes characteristic of a functionally "younger" age, and extend both average and maximum lifespans. Over the 2 yr the eight persons demonstrated a substantial weight loss, remarkable fall in blood cholesterol, blood pressure, fasting blood sugar, and low white blood cell counts--exactly as seen in rodents on such a regimen. Studies in progress involving levels of cortisol, insulin, and glycosylated hemoglobin support the rodent similarity. Further studies will seek to determine whether additional among the large battery of physiologic changes induced in animals by caloric restriction are also induced in humans on a similar nutrient-dense, calorie-limited diet. Such evidence will pertain to the question whether the increased disease resistance and aging retardation shown by calorie-restricted rodents might also be expected to occur in humans.