Effects Of Dietary Energy Restriction Research Articles

Effect of Dietary Energy Restriction on the Expression of Genes Encoding the Enzymes Involved in Steroidogenesis in Normal Breast Epithelium and Abdominal Adipose Tissue of Women with Excessive Adiposity

Background: Weight gain and excessive adiposity are associated with an increased risk of breast cancer, especially in postmenopausal women with a high circulating level of testosterone. In this in silico study, the effect of the dietary energy restriction (DER) on the expression of a panel of genes coding for the main enzymes involved in androgens and estrogens production was investigated in breast epithelium and abdominal adipose tissue of healthy women with excessive adiposity and randomly allocated to DER for one menstrual cycle or asked to continue their usual diet. Methods: Wilcoxon-Signed-Rank test was used to assess the difference between paired samples before and after DER in the breast epithelium or adipose tissue, and Spearman’s rank correlation was used to investigate the correlation between genes. Results: In response to DER, the expression of HSD17B12, the gene encoding the enzyme that catalyzes the conversion of inactive estrone into bioactive estradiol, was significantly (P=0.0059) downregulated in breast epithelium and adipose tissue. Furthermore, the expression of HSD17B12 negatively correlated with that of the LRP1 gene (r= -0.91, P<0.0001), which codes for a multitasking protein recently proposed as a tumor suppressor in breast epithelium. Conclusion: Present results showed that, in addition to the recognized systemic effects (reduction of the fatty mass and decrease of circulating biomarkers of breast cancer risk), DER could act locally by down-regulating the expression of some genes pivotally involved in the production of biologically active estrogens, thus contributing to prevent the estrogen-dependent initiation of breast cancer.

The independent effects of dietary energy restriction and circuit exercise training on fat oxidation in patients with NAFLD

The independent effects of dietary energy restriction and circuit exercise training on fat oxidation in patients with NAFLD

Effect of dietary energy restriction and subsequent compensatory feeding on testicular transcriptome in developing rams

Nutritional intake and reproductive allocation are strongly associated and dietary energy restriction (ER) or surpluses can affect reproductive capacity. The objective of this study was to investigate the effect of energy levels on sheep testicular development. Three-month old Hu sheep were assigned to four groups, and fed diets containing different levels of energy (Control, maintenance energy; ER1, 85% maintenance energy; ER2, 70% maintenance energy; ER3, 55% maintenance energy). Two months later, half the sheep in each group were euthanized, whereas the remaining sheep were euthanized after a further 3 months feeding on a compensatory energy diet. The testicular weight and reproductive hormone levels of the Hu sheep were investigated. Differences in the testes of ER3 and control group sheep were investigated at the transcriptional level using high-throughput sequencing. The results showed that the testicular weights had decreased in the energy-restricted rams compared with the controls, and that the testosterone concentration in ER3 group rams was significantly lower than that in other compared groups (P < 0.05). After the period of compensatory feeding, however, ER3 sheep testicular weight and testosterone concentrations were similar to those of the control group sheep. In addition, the RNA sequencing results revealed that 81 genes were upregulated and 180 genes were downregulated in the ER3 group compared with the control group. Moreover, based on the enriched steroidogenesis, meiosis and kinases pathways, a number of candidate genes potentially involved in the regulation of testicular development or reproduction of Hu sheep, including CYP11A1, ALDH3B1, FDFT1, WNT2, PGR and INSR, were screened. Quantitative real-time polymerase chain reaction analysis results correlated well with the sequencing data. Taken together, this study provides a first insight into the development of the testis with dietary energy restriction in sheep and shows that these changes are associated with alterations in transcriptomic. The sheep testis mRNA database were extended in this study will provides novel candidate regulators for future genetic and molecular studies on sheep testicular development associated with energy restriction, which will contribute to improving the reproductive performance of sheep.

Energy restriction affect liver development in Hu sheep ram lambs through Hippo signaling pathway.

This study aimed to evaluate the effects of dietary energy restriction on postnatal liver development in Hu sheep ram lambs. A total of 16 ram lambs were randomly divided into two groups: 100% energy requirements diet and 55% energy requirements diet, which were fed for 75 d. Results showed that the final body and liver weights decreased with energy restriction (p <0.05). Energy restriction caused a significant decrease in the levels of circulating insulin-like growth factor 1 (IGF1) and an increase in growth hormone secretion (p <0.05), which can be explained by the decreased mRNA expression levels of the growth hormone receptor (GHR) and IGF1 (p <0.05). The proliferating cell nuclear antigen (PCNA), Ki-67 and apoptosis-related proteins (BAX and BCL2) were mainly located in the nucleus and the cytoplasm of hepatocytes, respectively. The transcription and protein levels of PCNA and BAX were significantly decreased and increased by energy restriction, respectively (p <0.05). The caspase9 and caspase3 mRNA and activity were increased in energy restriction group (p <0.05). Moreover, Hippo signaling pathway proteins [mammalian sterile 20-like protein kinase 1 (MST1), large tumor suppressor kinase 1 (LATS1), and yes-associated protein 1 (YAP1)] were mainly observed around the hepatic portal area, and the expression levels of their mRNA and proteins were significantly decreased in energy restriction group (p <0.05). In summary, energy restriction in ram lambs impairs liver development by increasing apoptosis, which may occur via the Hippo signaling pathway.

Luteal activity and effect of dietary energy restriction on follicular development in lactating cows.

The aim of this research has been to evaluate the presence of anomalies in the ovarian cycle activity during postpartum and to verify whether 72-hr dietary fasting during the dominance phase, the phase before ovulation, might modify the ovarian follicle population. The presence of anomalies in ovarian cycle activity has been evaluated in 30 Italian Friesian cows starting from 20days postpartum until 211days of lactation. Long oestrus and brief dioestrus or scarce luteal activity have been the main anomalies found through measuring progesterone concentrations in the whey. Until 100days of lactation, the BCS values of the problematic animals have been significantly lower than those in animals with normal ovarian activity. After 100days of lactation, the ovarian anomalies continued to appear despite the fact that all the animals have reached comparable BCS values. Starting from the results of this trial, the effect of 72-hr dietary fasting on dominant follicles has been studied in six cows. Ultrasonography revealed that the diameter of the follicles at 71days postpartum has been significantly lower than at 181days. A 72-hr dietary restriction at 101 and 211days postpartum did not affect the size of the dominant follicle. However, at 101days postpartum, half of the animals presented follicular cysts. The effect of fasting differed if the animal has been in early postpartum or 211days of lactation. Further researches are necessary to understand how different metabolic conditions can modify the follicular population but on the other hand the study shows the utility for farmers and field veterinarians of monitoring the resumption of the ovarian cycle postpartum through the whey progesterone concentrations.

Chronic Dietary Administration of 2-Deoxy-D-Glucose Does Not Compromise Neurobehavioral Functions at Tumor Preventive Doses in Mice

Beneficial effects of dietary energy restriction (DER), including extension of life-span, reduction in cancer risk, anti-cancer effects and decrease in age related neurodegenerative diseases have been well established. Given that DER is difficult to implement in humans due to practical constraints, development of energy restriction mimetics (ERMs) is considered as a suitable alternative. Our recent studies have established the anti-tumor effects of the dietary administration of the glycolytic inhibitor 2-deoxy-D-glucose, a potential ERM, an alternative to DER; without any adverse effects on general physiology. Since functioning of the brain is critically dependent on glucose, we investigated the effects of chronic dietary 2-DG administration on the behavioural outcome in mice. Our findings based on a battery of neuro-behavioural tests clearly suggest that the chronic dietary administration of 2-DG that appreciably impairs the process of tumorigenesis has no adverse effect on the cognitive, affective and sensory-motor functions. Together with the maintenance of normal physiology reported by us earlier, these observations strengthen the potential of dietary 2-DG as a safe cancer preventive strategy.

Reducing milking frequency during nutrient restriction has no effect on the hepatic transcriptome of lactating dairy cattle

The objective of this study was to investigate if a reduced milking frequency altered the effect of dietary energy restriction on the hepatic transcriptome of grazing dairy cows during early lactation. Multiparous Holstein-Friesian and Holstein-Friesian × Jersey cows (n = 120) were milked twice daily (2×) from calving until 34 ± 6 days in milk (mean ± SD). Cows were then allocated to one of four treatments in a 2 × 2 factorial arrangement. Treatments consisted of two milking frequencies [2× or once daily (1×)] and two feeding levels for 3 wk: adequately fed (AF) or underfed (UF, 60% of AF). Liver tissue was biopsied from 12 cows per treatment after 3 wk of treatment, and the hepatic transcriptome was profiled with an Agilent 4 × 44k bovine microarray. Over 2,900 genes were differentially expressed in response to the energy restriction; however, no effects resulted from changes to milking frequency. This may indicate that after 3 wk of 1× milking, any changes to the liver transcriptome that may have occurred earlier have returned to normal. After 3 wk of energy restriction, gene expression patterns indicate that glucose-sparing pathways were activated, and gluconeogenesis was increased in UF cows. Genes involved in hepatic stress were upregulated in response to the energy restriction indicative of the pressure energy restriction places on liver function. Other pathways upregulated included "cytoskeletal remodeling," indicating that a 3 wk energy restriction resulted in molecular changes to assist tissue remodeling. Overall, 1× milking does not modify the hepatic transcriptome changes that occur in response to an energy restriction.

Defining the Role of Histone Deacetylases in the Inhibition of Mammary Carcinogenesis by Dietary Energy Restriction (DER): Effects of Suberoylanilide Hydroxamic Acid (SAHA) and DER in a Rat Model

Dietary energy restriction (DER) inhibits experimentally induced mammary cancer, an effect accompanied by elevated levels of silent information regulator 2 (SIRT1), a class III histone deacetylase (HDAC). However, the effect of DER on targets of other classes of HDACs has not been reported, a highly relevant issue given evidence that HDAC induction favors the development of cancer and tumor growth. Experiments were carried out to determine whether suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor with broad activity, would affect the anti-cancer activity of DER. Female Sprague Dawley rats (n = 30/group) were injected with 1-methyl-1-nitrosourea (50 mg/kg) at 21 days of age and 7 days thereafter were randomized to groups fed: (i) control diet (AIN-93G), (ii) 0.1% SAHA (w/w), (iii) 40% DER, or (iv) 0.1% SAHA + 40% DER. An additional group was fed 0.1% SAHA + 40%DER for 5 weeks and released to control diet for 3 weeks. DER significantly reduced mammary cancer incidence, multiplicity, and cancer burden and prolonged cancer latency (P < 0.01). Cancer inhibition was maintained in SAHA + DER, despite evidence that histone (H2A(Lys9), H2B(Lys5), and H4(Lys5/8/12/16), but not H3(Lys9); P < 0.001) and non-histone protein deacetylation (p53(Lys373) and p53(Lys382); P < 0.001) induced by DER was reversed by SAHA. This indicates that the inhibition of DER of cancer is not dependent on HDAC induction. After releasing rats from DER + SAHA, cancer multiplicity remained lower than control (P < 0.05), consistent with apoptosis-mediated cell deletion. These findings support further investigation of the hypothesis that HDAC induction by DER blunts its anti-carcinogenic impact.

Excess body weight, diabetes and cancer: epidemiologic evidence implicating hormonal and metabolic mechanisms

Epidemiological observations increasingly implicate nutritional energy balance as a key risk factor for cancer development. Excess body weight is associated with increased risks of cancers of the endometrium, breast (postmenopausal women), kidney (renal cell tumours), colon, pancreas and oesophagus (adenocarcinomas), and is also a well-documented risk factor for high-grade prostate cancer. By contrast, regular physical activity reduces the risks of breast and colorectal cancers and potentially other tumour types, and overall, excess weight and lack of physical activity have been estimated to potentially account for one quarter to half of the occurrence of the most frequent tumour types in affluent, industrialized societies. Animal experiments have shown uniformly protective effects of dietary energy restriction against tumor development. Physiologic mechanisms that are thought to account for these effects of nutritional energy balance on cancer risks include changes in the metabolism of endogenous hormones, growth factors and inflammation factors, as well as in energy and nutrient status at the level of single cells. Together, these physiologic changes may stimulate cell growth and proliferation, inhibit apoptosis, and favour the occurrence of genetic mutations through increased oxidative stress. The key mechanisms that underlie these relationships of nutritional energy balance with cancer development may strongly depend, however, on tumour type. Prospective cohort studies have shown relationships of risks of various cancer types with blood levels of glucose and insulin, and insulin-like growth factor-l. Furthermore, increased levels of circulating androgens and estrogens and reduced levels of progesterone are strongly implicated especially in the development of cancers of the endometrium and breast, among women with excess weight. The implication of specific hormonal or metabolic factors in cancer development is further increased by a growing body of evidence from human intervention studies – e.g. using selective estrogen receptor modulators [SERMS] and aromatase inhibitors – and epidemiological studies on the effects of specific hormonal medications (e.g., oral contraceptives and postmenopausal hormone replacement therapy; specific anti-diabetic drugs). Finally, several studies have shown increased risks of cancer, e.g. of the colorectum and endometrium, among subjects with higher than average serum levels of inflammation factors such as C-reactive protein, TNF-alpha or various interleukins. According to present estimates, excess weight and lack of physical activity are likely the most important risk factors after smoking for cancer occurrence overall in western countries, and better knowledge of the true magnitude of the problem and of physiologic mechanisms involved can help define guidelines for primary prevention. At the same time, the knowledge gained about the hormonal and other physiologic mechanisms that link nutritional energy balance to cancer increasingly is also informing translational research into cancer preventive and curative treatments.

Brain‐derived neurotrophic factor as a regulator of systemic and brain energy metabolism and cardiovascular health

Overweight sedentary individuals are at increased risk for cardiovascular disease, diabetes, and some neurological disorders. Beneficial effects of dietary energy restriction (DER) and exercise on brain structural plasticity and behaviors have been demonstrated in animal models of aging and acute (stroke and trauma) and chronic (Alzheimer's and Parkinson's diseases) neurological disorders. The findings described later, and evolutionary considerations, suggest brain-derived neurotrophic factor (BDNF) plays a critical role in the integration and optimization of behavioral and metabolic responses to environments with limited energy resources and intense competition. In particular, BDNF signaling mediates adaptive responses of the central, autonomic, and peripheral nervous systems from exercise and DER. In the hypothalamus, BDNF inhibits food intake and increases energy expenditure. By promoting synaptic plasticity and neurogenesis in the hippocampus, BDNF mediates exercise- and DER-induced improvements in cognitive function and neuroprotection. DER improves cardiovascular stress adaptation by a mechanism involving enhancement of brainstem cholinergic activity. Collectively, findings reviewed in this paper provide a rationale for targeting BDNF signaling for novel therapeutic interventions in a range of metabolic and neurological disorders.

Eating less suppresses microRNA assassins in the brain

The aging brain is perhaps the most fragile of tissues, with a population of structurally elaborate and metabolically demanding neurons that are seldom replenished during adulthood. Neurodegenerative disorders such as Alzheimer's disease (AD) and acute insults such as an ischemic stroke lead to seemingly irreparable damage that impairs cognitive and/or motor function. Considerable evidence suggests that the vulnerability of neurons to aging and associated disorders can be modified by environmental factors including dietary energy intake and exercise [1, 2, 3]. Energy (caloric) restriction (CR) and exercise induce the expression of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), cytoprotective protein chaperones, and proteins that can prevent apoptosis. The mechanisms responsible for the neuro-protective effects of CR and exercise are mediated, in part, by changes in gene transcription [3]. However, new findings reported in this issue of Aging suggest a role for down-regulation of microRNAs (miRNAs) that target mRNAs encoding cell survival proteins in the beneficial effects of CR on the aging brain [4]. miRNAs are short non-coding RNAs that typically bind to scores of transcripts an inhibit translation of the targeted mRNA [5]. Khanna et al found that levels of three miRNAs (miR-34a, miR30e and miR181-a-1*) are significantly lower in brain tissue samples from old mice (24-28 months-old) that had been maintained on a CR diet (40% CR beginning at 4 months of age) compared to mice on the usual ad libitum diet. Interestingly, all three of these miRNAs were predicted to have at least one target site for Bcl2, an anti-apoptotic protein previously been shown to increase with CR. The authors confirmed the repressive action of these miRNAs on Bcl2. These changes in miRNA expression were validated in both cortical and hippocampal tissues, suggesting a more global repression of these miRNAs in the CNS due to CR. Although the authors chose to solely investigate Bcl2 as a target of these three miRNAs in mediating the beneficial effects of CR, eight additional shared mRNA targets were found using multiple target-prediction algorithms. One of these targets, cAMP response element binding protein 1 (CREB1), is an important activator of several immediate response genes that are critical to synaptic plasticity [6]. Repression of CREB1 by these three age-dependent miRNAs could play a role in the cognitive decline observed with aging. Additionally, huntingtin (Htt) is a predicted target of these miRNAs, and this possible interaction may play a role in the beneficial effects of dietary energy restriction in Htt mutant mice, a model of Huntington's disease [7]. Moreover, previous research demonstrated that miR-30a, which has an identical seed region to miR-30e acts to functionally repress BDNF expression in the cortex [8]. Up-regulation of BDNF by CR has been shown to mediate, in part, the increased neurogenesis by CR and is also thought to play an important role in learning and memory [9, 10]. Although not explored by Khanna and colleagues in the context of CR, the regulation of BDNF by the miR-34 family represents yet another potential avenue for miRNAs as mediators of effects of dietary energy intake on neuronal vulnerability in aging and disease. It will be important to determine whether changes in the expression of miRNAs 34a, 30e, and 181a do in fact mediate effects of energy intake on neuronal vulnerability. This might be accomplished by overexpressing or knocking down each of these miRNAs in neurons of interest in animal models of Alzheimer's, Parkinson's and Huntington's diseases. Whether Bcl-2 is a pivotal target of this miRNAs could be determined in experiments with Bcl-2 deficient mice. It seems likely that the findings of Khanna et al. [4] represent the ‘tip of the iceberg’ with regards to miRNAs in brain aging and disease as there are undoubtedly numerous other miRNAs and target mRNAs involved in regulating neuronal survival and plasticity. This arena of research is a promising area for the development of novel therapeutic interventions in neurodegenerative disorders. Indeed, multiple miRNAs appear to be dysregulated in neurological diseases [5]. Initial studies in non-human primates have further emphasized the potential for miRNA-based theraputics [11]. The work by Khanna and colleagues suggest a potential for miRNA-based therapies that utilize the same anti-apoptic mechanisms as CR. However, as a single miRNA can regulate hundreds of transcripts, systemic delivery of a miRNA mimetic or sponge may result in undesirable off-target and tissue specific effects.

Effect of dietary energy intake on erythrocytic antioxidant defence in growing lambs fed a wheat straw-based diet

Twenty-four Muzaffarnagari lambs (~8 months, 26.56 ± 2.04 kg), consisting 12 each of male and female, were used for ascertaining the effect of dietary energy restriction on the erythrocytic antioxidant defence including lipid peroxidation. The lambs, allotted randomly into three equal groups, were fed on wheat straw-based diets to provide 100, 80 and 70% of calculated metabolisable energy (ME) requirements. Bodyweight gain and feed intake were recorded. Blood samples were collected at the start and thereafter at 60-day intervals during 180 days of experimental duration and analysed for malonyl dialdehyde (MDA), reduced glutathione (GSH) and total thiol groups in addition to catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione S-transferase. Dietary treatments imparted significant (P &lt; 0.001) effects on feed intake and average daily gain. The haemoglobin and haematocrit contents in blood reduced significantly (P &lt; 0.001) on reduction in dietary energy levels. The dietary alterations elicited no change in the activities of SOD, catalase and glutathione S-transferase, but reduced activities of GSH-Px (P &lt; 0.001) and GSH (P = 0.133) were evident on feeding the diet with 70% ME. Concentrations of total thiols decreased (P &lt; 0.001) with reduced energy level. Both the energy-restricted groups exhibited a significant (P &lt; 0.001) increase in MDA, indicative of increased lipid peroxidation. It was concluded that long-term energy malnutrition on a wheat straw-based diet reduces the erythrocytic antioxidant defence in growing lambs.

Effect of ultraviolet B radiation on activator protein 1 constituent proteins and modulation by dietary energy restriction in SKH-1 mouse skin.

The study examined the timing of modulation of activator protein 1(AP-1):DNA binding and production of AP-1 constituent proteins by ultraviolet B (UVB) radiation and effect of dietary energy restriction [DER, 40% calorie reduction from fat and carbohydrate compared to control ad libitum (AL) diet] in SKH-1 mouse epidermis. AP-1:DNA binding by electromobility shift assay (EMSA) was increased in a biphasic manner after treatment with a tumor-promoting suberythemal dose (750 mJ/cm(2)) of UVB light (311-313 nm) with peaks at 3 and 18 h postirradiation. DER overall reduced AP-1:DNA binding in mock-treated and UVB-treated skin at 3 and 18 h after UVB treatment. The timing of modulation of production of AP-1 constituent proteins by Western blot analysis was examined at 0 h (mock treatment), 3, 9, 18, and 24 h. We found that c-jun (9 h), jun-B (9 and 18 h), phosphorylated c-jun (3 h), and fra-1 (18 h) protein levels were increased after UVB treatment compared to mock controls. In a follow-up diet experiment, animals were placed on DER or AL diet for 10-12 wk and treated with UVB as before. DER was found to completely block the UVB-induced increase in phosphorylated c-jun protein levels and decrease in fra-2 protein levels at 18 h. In addition, DER enhanced UVB-induced increase in jun B levels and lowered basal levels of c-fos seen 18 h after UVB. These data suggest that DER may be able to assist in the prevention of UVB-induced skin carcinogenesis by modulating AP-1:DNA binding and AP-1 constituent protein levels.

Effects of Dietary Energy Restriction on Gene Regulation in Mammary Epithelial Cells

The objective of this study was to determine whether dietary energy restriction (DER) affects the pattern of gene expression in three interrelated energy metabolism pathways: glycolysis, gluconeogenesis, and the citric acid cycle. Mammary carcinogenesis was initiated by the i.p. injection of female Sprague-Dawley rats with 50 mg of 1-methyl-1-nitrosourea per kilogram of body weight. Five days following 1-methyl-1-nitrosourea administration, animals were fed ad libitum or 80% or 60% of the ad libitum intake. Epithelial cells were harvested from histologically confirmed adenocarcinomas (adenocarcinoma epithelial cell; ACEC) and uninvolved mammary gland (mammary gland epithelial cells; MGEC) via laser capture microdissection, whereas isolated RNA was arrayed on Affymetrix R230 2.0 genome chips. Principal components analysis revealed complete separation of the patterns of gene expression between ACEC versus MGEC. Further examination of the data set revealed an up-regulated pattern of expression in the ACEC of genes involved in glycolysis, whereas gluconeogenesis was suppressed. In general, genes involved in the citric acid cycle were not differentially expressed; however, pyruvate dehydrogenase expression was down-regulated and lactate dehydrogenase expression was increased in ACEC versus MGEC. Collectively, the observed patterns of expression were consistent with the Warburg effect. DER exerted no effect on the Warburg pattern of gene expression or on other aspects of these energy metabolism pathways. These findings imply that efforts to target the Warburg effect for cancer prevention are mechanistically distinct from those modulated by DER and provide a rationale for the combination of approaches that target basic defects in energy metabolism and energy-sensing pathways for the prevention of breast cancer.

O-29 Effect of dietary energy restriction on gene expression in normal breast and subcutaneous adipose tissues of overweight women at increased breast cancer risk

Breast cancer is recognised to be a heterogeneous disease with a range of morphological appearances and behaviours. The recently recognised basal phenotype (BP) is associated with poor survival, but the clinical implications of this class of breast cancers remain to be adequately defined.We have examined a well-characterised series of 1872 invasive breast carcinomas with a long term follow-up to assess the clinical significance of BP.A pragmatic definition of the BP as immunophenotypic evidence of basal cytokeratins CK5/6 and/or CK14 expression was used. These tumours were associated with shorter overall survival and disease-free interval in our series as a whole and in both the lymph node (LN) negative and LN positive subgroups. When stratified by histological grade, BP was of highly significant prognostic value in grade 3 but not in grades 1 or 2 tumours. Similarly, it was associated with poor survival in the moderate group of the Nottingham prognostic Index but not in the other groups. In a subgroup comprising LN negative grade 3 tumours, BP was the most powerful prognostic marker followed only by tumour size, while the other variables were non-significant. Patients with BP were more likely to respond to chemotherapy than those with non-basal tumours.Our results provide robust evidence that BP is an important class of breast cancers with a particularly aggressive behaviour in patients with LN negative grade 3 disease. We recommend routine identification of BP in breast cancer and the development of effective adjuvant treatment strategies. These are important observations as these tumours typically lack hormone receptor and HER-2 overexpression limiting the range of relevant adjuvant therapies.

Effect of dietary energy restriction on vascular density during mammary carcinogenesis.

Inhibition of mammary carcinogenesis by dietary energy restriction is associated with a decrease in cell proliferation and the induction of apoptosis. Although changes in the metabolism of insulin-like growth factor I and glucocorticoids have been proposed to modulate these cellular processes, limitations in blood supply could induce similar effects. To investigate this possibility, female Sprague Dawley rats were given an injection of 1-methyl-1-nitrosourea and fed purified diets ad libitum or at 60% ad libitum intake, i.e., 40% dietary energy restriction. Premalignant mammary pathologies and mammary adenocarcinomas obtained from these rats were processed for vascular density analysis via CD-31 immunostaining. Vascular density, measured as vessels/unit area, of premalignant mammary pathologies and adenocarcinomas from dietary energy restriction rats was reduced 31 and 39%, respectively (P < 0.01). This effect, which was observed in a 50-microm wide band of tissue surrounding each pathology, was exerted on blood vessels > 25 microm2. Conversely, intratumoral vascular density was unaffected by dietary energy restriction. cDNA microarray and Western blot analyses of adenocarcinomas for evidence of dietary energy restriction-mediated effects on vascularization revealed that only the level of vascular endothelial growth factor receptor protein Flk-1 was significantly reduced (P < 0.001). It appears that dietary energy restriction imposes limitations in the supply of blood to developing pathologies, an effect that could directly inhibit the carcinogenic process. The vascular density data imply that dietary energy restriction inhibited the growth of endothelial cells but leave unresolved the question of whether dietary energy restriction had a specific effect on angiogenesis. The factors that account for the failure of dietary energy restriction to limit intratumoral vascularization are not obvious and merit additional investigation.

Adrenalectomy Does Not Block the Inhibition of Mammary Carcinogenesis by Dietary Energy Restriction in Rats

Dietary energy restriction (DER) has been shown to reproducibly inhibit chemically induced mammary carcinogenesis. The inhibitory activity of DER has been reported to be associated with an increase in circulating corticosterone as well as a decrease in insulin-like growth factor 1 (IGF-1). To determine whether the adrenal glands are required for cancer inhibitory activity, the effects of DER were investigated in adrenalectomized (ADX) rats. Female Sprague-Dawley rats, 29-31 per group, were injected with 0.05 g 1-methyl-1-nitrosourea/kg body wt at 21 d of age, sham operated (SHAM) or bilaterally ADX at 24 d of age, and after 3 d adapted to meal feeding during which rats ate ad libitum (AL) or were restricted to 60% of AL energy intake. ADX resulted in a marked reduction in serum corticosterone in both AL and DER rats. Whereas the carcinogenic response in the mammary gland was not statistically different in SHAM-AL and ADX-AL rats, ADX did not block the cancer inhibitory activity of DER. In fact, cancer inhibitory activity was greatest in ADX-DER rats. Circulating levels of glucose, insulin, IGF-1, and IGF binding protein 3 also were reduced in DER rats. Collectively, these findings indicate that adrenal glands are not required for manifestation of the cancer inhibitory activity of DER. If circulation-borne factors such as corticosterone or IGF-1 are involved in the inhibition of mammary carcinogenesis by DER, IGF-1 is likely to play a greater role than corticosterone.

Dietary energy restriction in breast cancer prevention.

Dietary energy restriction (DER) inhibits the development of spontaneous, chemically, genetically, and virally induced mammary cancer in rats and/or mice. DER inhibits the initiation and postinitiation stages of mammary carcinogenesis and the development of both ovarian-hormone-dependent and -independent mammary carcinomas. The predominant effect of DER appears to be suppression of the clonal expansion of transformed cells, and this effect is most likely mediated via the coordinated regulation of cell proliferation, apoptosis, and angiogenesis. The effects of DER on cell cycle regulation and apoptosis are consistent with the limitation of one or more cell survival factors. Evidence is presented that the chemical mediators of this effect, glucocorticoids, insulin, and/or insulin-like growth factors, are elicited in response to the limitation in glucose availability imposed by DER. Investigation of DER is highly relevant to the misregulation of body weight which has been identified as a human health problem of global proportion. Mechanistic studies hold the promise of leading to the identification of DER mimetic approaches that can be used in the prevention and control of breast cancer.

Dietary energy restriction inhibits estrogen-induced mammary, but not pituitary, tumorigenesis in the ACI rat

Because of the suggested role of energy consumption and the well-documented role of estrogens in the etiology of breast cancer, we have examined the effect of a 40% restriction of dietary energy consumption on the ability of administered 17beta-estradiol (E2) to induce mammary tumorigenesis in female ACI rats. Experiments herein test the hypothesis that at least part of the inhibitory effect of energy restriction on mammary tumorigenesis is exerted downstream of potential effects of dietary manipulation on the production of estrogens by the ovaries. Ovary-intact ACI rats were fed a control or a 40% energy-restricted diet and were either treated continuously with E2 from subcutaneous Silastic tubing implants or received no hormone treatment. Mammary cancers rapidly developed in E2-treated rats fed the control diet; within 216 days of initiation of E2 treatment 100% of the population at risk exhibited palpable mammary tumors. Dietary energy restriction markedly inhibited E2-induced mammary tumorigenesis, as evidenced by significant reductions in cancer incidence and tumor burden as well as a significant increase in the latency to the appearance of the first palpable cancer. The inhibitory actions of dietary energy restriction on E2-induced mammary tumorigenesis were associated with an inhibition of E2-stimulated mammary cell proliferation. However, this inhibition was insufficient to block induction of lobuloalveolar hyperplasia or appearance of focal regions of atypical epithelial hyperplasia. These data suggest that dietary energy restriction inhibits E2-induced mammary cancer by attenuating or retarding the progression of atypical hyperplasia to carcinoma. Expression of progesterone receptor (PR) was up-regulated within the focal regions of atypical hyperplasia and the carcinomas induced by E2, regardless of whether the rats were fed the control or energy-restricted diet. However, circulating progesterone was reduced by dietary energy restriction, suggesting a possible mechanism for inhibition of mammary tumorigenesis. Dietary energy restriction did not inhibit the ability of administered E2 to induce prolactin (PRL)-producing pituitary tumors and associated hyperprolactinemia, indicating that the inhibitory effects of dietary energy restriction on mammary tumorigenesis are tissue specific and independent of circulating E2 and PRL.

Inhibition of phorbol ester-induced AP-1-DNA binding, c-Jun protein and c-jun mRNA by dietary energy restriction is reversed by adrenalectomy in SENCAR mouse epidermis.

The aim of this study was to determine the effects of 40% dietary energy restriction (DER) relative to ad libitum feeding on AP-1-DNA binding and expression of c-Jun protein and c-jun mRNA in SENCAR mouse skin treated with acetone or 12-O-tetradecanoylphorbol 13-acetate (TPA). The role of the glucocorticoid hormone corticosterone (CCS) was investigated by adding CCS or vehicle control to the drinking water of adrenalectomized mice. AP-1-DNA binding, measured by electrophoretic mobility shift assay, showed that TPA treatment for 4 h increased AP-1-DNA binding by 2-fold over acetone controls (P < 0.05) and that DER reduced basal and TPA-induced AP-1-DNA binding in comparison with ad libitum fed groups in sham-operated mice (P < 0.05). TPA treatment increased c-Jun protein levels in control fed mice (4-fold) and in DER mice (2-fold) over basal levels 4 h post-treatment (P < 0.05). Analyzed over all groups, DER reduced c-Jun protein levels (P < 0.01) and this effect was reversed by adrenalectomy. TPA induction of c-jun mRNA was also reduced by DER compared with ad libitum fed mice (P < 0.05). Adrenalectomy and CCS supplementation demonstrated that the effects of DER on AP-1-DNA binding were mediated in part by CCS. Measurement of blood plasma CCS concentrations showed that: (i) DER increased CCS 5-fold over ad libitum fed mice in sham-operated animals (P < 0.05); (ii) adrenalectomy decreased CCS over sham-operated mice (P < 0.05); (iii) TPA treatment had no effect on CCS. Blood plasma IGF-I concentrations were unaffected by CCS modulation or TPA treatment but were decreased by DER compared with ad libitum fed mice (P < 0.05). Thus, dietary energy restriction may inhibit cancer mechanistically by reducing overall AP-1 transcription through a process that is mediated in part by glucocorticoid hormones.