Mechanisms underlying retardation of aging by dietary energy restriction.

Moderate restriction of dietary energy intake, referred to here as dietary restriction (DR), delays aging and extends lifespan in experimental animals compared with a diet of ad libitum feeding (AL) control animals. Basic knowledge of the mechanisms underlying the effects of DR could be applicable to extending the healthspan in humans. This review highlights the importance of forkhead box O (FoxO) transcription factors downstream of the growth hormone-insulin-like growth factor 1 signaling in the effects of DR. Our lifespan studies in mice with heterozygous Foxo1 or Foxo3 gene knockout indicated differential roles of FoxO1 and FoxO3 in the tumor-inhibiting and life-extending effects of DR. Subsequent studies suggested a critical role of FoxO3 in metabolic and mitochondrial bioenergetic adaptation to DR. Our studies also verified hypothalamic neuropeptide Y (Npy) as a vital neuropeptide showing pleiotropic and sexually dimorphic effects for extending the healthspan in the context of nutritional availability. Npy was necessary for DR to exert its effects in male and female mice; meanwhile, under AL conditions, the loss of Npy prevented obesity and insulin resistance only in female mice. Overnutrition disrupts FoxO- and Npy-associated metabolic and mitochondrial bioenergetic adaptive processes, causing the acceleration of aging and related diseases.

GALT carcinoma: three case reports with glycoprotein 2 immunohistochemistry and electron microscopic observations.

AIMS: Gut-associated lymphoid tissue (GALT) carcinoma is a rare colorectal tumour that arises in the epithelium covering GALT. GALT carcinoma is a differentiated tubular adenocarcinoma with dense lymphoid tissue with a characteristically well-demarcated margin. To date, 26 cases of GALT carcinoma, including the three cases discussed here, have been reported. Most (24 of 26) were discovered at early stages and none of the cases have documented any metastases. This suggests that GALT carcinoma may have a favourable prognosis. It is hypothesised that GALT carcinoma originates from M cells in specialised epithelia covering GALT. However, this hypothesis has yet to be confirmed. METHODS AND RESULTS: In this study, we examined three cases of GALT carcinoma by immunohistochemistry detection of glycoprotein 2, a specific marker for M cells, and electron microscopy. Our findings showed that the tumour cells of GALT carcinoma in all three cases were negative for M cells. We thus concluded that GALT carcinoma may be a tubular adenocarcinoma arising by chance in the GALT. This unique carcinoma is a diferentiated adenocarcinoma that grows slowly with the development of GALT. CONCLUSIONS: We propose that GALT carcinoma should be classified separately because of its histological setting and good prognosis.

Identification and functional analysis of inflammation-related miRNAs in skin wound repair.

Inflammation at a wound site is essential for preventing infection. However, misregulated inflammation leads to pathologies of the healing process, including chronic non-healing wounds and scarring. MicroRNAs (miRNAs) are key regulators of the inflammatory response and tissue repair, acting by translational processing of target mRNAs. In the final step of miRNA processing, Argonaute 2 (Ago2)-bound mature miRNA complexes bind to target mRNAs and inhibit their translation. A variety of wound healing-related miRNAs have been identified and their misregulation likely contributes to wound pathologies, including scarring and chronic healing. Recently, we have developed an Ago2-bound mature miRNA purification system that uses Ago2 antibody to analyze the expression of miRNAs from wound tissues by microarray and next generation sequencing. We have identified several wound inflammation-related miRNAs via Ago2-target immunoprecipitation assays and next generation sequencing of wound tissues from wild-type and PU.1 knockout mice, which exhibit no inflammatory response because of a lack of immune cell lineages. We demonstrated that miR-142, an identified inflammation-related miRNA, is essential role for neutrophilic chemotaxis via inhibition of small GTPase translation; its misregulation leads to susceptibility to infection against Staphylococcus aureus at skin wound sites. In this review, we summarize recent advances of miRNA studies in skin wound healing, introduce our miRNA purification system using an immunoprecipitation assay method, and discuss the function of miR-142 in skin wound healing.

Comparing the Effects of Melatonin with Caloric Restriction in the Hippocampus of Aging Mice: Involvement of Sirtuin1 and the FOXOs Pathway.

It has been suggested that age-related neurodegeneration might be associated with neuropeptide Y (NPY); sirtuin1 (SIRT1) and forkhead box transcription factors O subfamily (FOXOs) pathways. Melatonin, a hormone mainly secreted by the pineal gland, is another anti-aging agent associated with the SIRT1-FOXOs pathway. This study aimed to compare the effects of melatonin (Mel) and caloric restriction (CR) on the expression of Sirt1, FoxO1, FoxO3a and FOXOs target genes in the aging mouse hippocampus. Neuropeptide Y-knockout (NpyKO) and wild-type (WT) male mice aged 19 months were previously treated either with food ad libitum or CR for 16 months. WT old animals were divided into four groups: control, CR, Mel and CR+Mel treated groups. The Mel and CR+Mel were treated with melatonin 10 mg/kg, daily, subcutaneously for 7 consecutive days. Mel treatment upregulated the mRNA expression of Sirt1, FOXOs (FoxO1 and FoxO3a) target genes that regulated the cell cycle [e.g., cyclin-dependent kinase inhibitor 1B (p27)], Wingless and INT-1 (Wnt1) and inducible signaling pathway protein 1 (Wisp1) in the aged mouse hippocampus. CR treatment also showed the similar actions. However, the mRNA expression of Sirt1, FoxO1, FoxO3a, p27 or Wisp1 did not alter in the CR+Mel group when compared with CR or Mel group. Melatonin could not produce any additive effect on the CR treatment group, suggesting that both treatments mimicked the effect, possibly via the same pathway. NPY which mediates physiological adaptations to energy deficits is an essential link between CR and longevity in mice. In order to focus on the role of Npy in mediating the effects of melatonin, the gene expression between NpyKO and WT male mice were compared. Our data showed that, in the absence of Npy, melatonin could not mediate effects on those gene expressions, suggesting that Npy was required for melatonin to mediate the effect, possibly, on life extension.

Cancer Cell Interaction with Adipose Tissue: Correlation with the Finding of Spiculation at Mammography.

PURPOSE: To investigate the relationship between spiculated masses at mammography and marginal adipose tissue invasion at histologic examination. MATERIALS AND METHODS: The institutional review board approved this retrospective study, and the requirement to obtain informed consent was waived. A total of 478 patients with invasive breast cancer who underwent surgery between 1999 and 2009 were included in this study. Clinical-pathologic findings from patients with spiculated masses on mammograms were compared with those from patients without spiculated masses by using logistic regression models, Cox proportional hazards regression models, and the Kaplan-Meier method. RESULTS: There were 136 spiculated tumors and 342 nonspiculated tumors. All 136 spiculated tumors (100%) were positive for adipose tissue invasion, whereas only 264 of the 342 nonspiculated tumors (77%) were positive for adipose tissue invasion (P < .001). Multivariate analysis revealed that adipose tissue invasion (P < .001), histologic grade (P < .001), dense breast (P = .002), and body mass index (P = .02) were independent factors associated with spiculation. With regard to survival, although many patients with spiculated tumors had a hormone-sensitive (estrogen receptor positive: P = .004; progesterone receptor positive: P = .001) or low-grade (P < .001) tumor, in contrast to the patients without spiculated masses, the prognosis in the two groups was similar (disease-free survival: P = .09; overall survival: P = .23). CONCLUSION: Cancer cell interaction with adipose tissue is crucial for the finding of spiculation at mammography.

Reduced FOXO1 expression accelerates skin wound healing and attenuates scarring.

The forkhead box O (FOXO) family has been extensively investigated in aging and metabolism, but its role in tissue-repair processes remains largely unknown. Herein, we clarify the molecular aspect of the FOXO family in skin wound healing. We demonstrated that Foxo1 and Foxo3a were both up-regulated during murine skin wound healing. Partial knockout of Foxo1 in Foxo1(+/-) mice throughout the body led to accelerated skin wound healing with enhanced keratinocyte migration, reduced granulation tissue formation, and decreased collagen density, accompanied by an attenuated inflammatory response, but we observed no wound phenotype in Foxo3a(-/-) mice. Fibroblast growth factor 2, adiponectin, and notch1 genes were significantly increased at wound sites in Foxo1(+/-) mice, along with markedly altered extracellular signal-regulated kinase 1/2 and AKT phosphorylation. Similarly, transient knockdown of Foxo1 at the wound site by local delivery of antisense oligodeoxynucleotides enhanced skin wound healing. The link between FOXO1 and scarring extends to patients, in particular keloid scars, where we see FOXO1 expression markedly increased in fibroblasts and inflammatory cells within the otherwise normal dermis. This occurs in the immediate vicinity of the keloid by comparison to the center of the mature keloid, indicating that FOXO1 is associated with the overgrowth of this fibrotic response into adjacent normal skin. Overall, our data indicate that molecular targeting of FOXO1 may improve the quality of healing and reduce pathological scarring.

NPY antagonism reduces adiposity and attenuates age-related imbalance of adipose tissue metabolism.

An orexigenic hormone, neuropeptide Y (NPY), plays a role not only in the hypothalamic regulation of appetite, but also in the peripheral regulation of lipid metabolism. However, the intracellular mechanisms triggered by NPY to regulate lipid metabolism are poorly understood. Here we report that NPY deficiency reduces white adipose tissue (WAT) mass and ameliorates the age-related imbalance of adipose tissue metabolism in mice. Gene expression involved in adipogenesis/lipogenesis was found to decrease, whereas proteins involved in lipolysis increased in gonadal WAT (gWAT) of NPY-knockout mice. These changes were associated with an activated SIRT1- and PPARγ-mediated pathway. Moreover, the age-related decrease of de novo lipogenesis in gWAT and thermogenesis in inguinal WAT was inhibited by NPY deficiency. Further analysis using 3T3-L1 cells showed that NPY inhibited lipolysis through the Y1 receptor and enhanced lipogenesis following a reduction in cAMP response element-binding protein (CREB) and SIRT1 protein expression. Therefore, NPY appears to act as a key regulator of adipose tissue metabolism via the CREB-SIRT1 signaling pathway. Taken together, NPY deficiency reduces adiposity and ameliorates the age-related imbalance of adipose tissue metabolism, suggesting that antagonism of NPY may be a promising target for drug development to prevent age-related metabolic diseases.

Malignant transformation of breast ductal adenoma: a diagnostic pitfall.

We present what is believed to be the first report of heterogeneous carcinoma arising from breast ductal adenoma. A 57-year-old woman presented with a nodule in her right breast. Histological examination of a vacuum-assisted biopsy specimen revealed epithelial tubular proliferation and papillary apocrine epithelium. The myoepithelial cells lining the tubules were confirmed by immunohistochemistry. The nodule had increased in size 18 months later and tumorectomy was performed. The surgical specimen revealed proliferating apocrine epithelium with sheet-like and cribriform architecture within a mammary duct. Some myoepithelial cells showed irregular proliferation around the tubular epithelium. All three components, including apocrine, myoepithelial and glandular cells, showed prominent nuclear atypia and significant mitotic activity. The patient was diagnosed with malignant transformation of ductal adenoma. The malignant potential of ductal adenoma has not previously been discussed, but this heterogeneous carcinoma could represent a serious pitfall in the diagnosis of ductal adenomas.

Characterization of dsRNA-induced pancreatitis model reveals the regulatory role of IFN regulatory factor 2 (Irf2) in trypsinogen5 gene transcription.

Mice deficient for interferon regulatory factor (Irf)2 (Irf2(-/-) mice) exhibit immunological abnormalities and cannot survive lymphocytic choriomeningitis virus infection. The pancreas of these animals is highly inflamed, a phenotype replicated by treatment with poly(I:C), a synthetic double-stranded RNA. Trypsinogen5 mRNA was constitutively up-regulated about 1,000-fold in Irf2(-/-) mice compared with controls as assessed by quantitative RT-PCR. Further knockout of IFNα/ß receptor 1(Ifnar1) abolished poly(I:C)-induced pancreatitis but had no effect on the constitutive up-regulation of trypsinogen5 gene, indicating crucial type I IFN signaling to elicit the inflammation. Analysis of Ifnar1(-/-) mice confirmed type I IFN-dependent transcriptional activation of dsRNA-sensing pattern recognition receptor genes MDA5, RIG-I, and TLR3, which induced poly(I:C)-dependent cell death in acinar cells in the absence of IRF2. We speculate that Trypsin5, the trypsinogen5 gene product, leaking from dead acinar cells triggers a chain reaction leading to lethal pancreatitis in Irf2(-/-) mice because it is resistant to a major endogenous trypsin inhibitor, Spink3.

[Regulation of Aging Processes: A Perspective of Dietary Restriction Models].

The moderate restriction of dietary energy intake (dietary restriction: DR) extends the lifespan and health span of various laboratory animals, suggesting that it delays the aging process inherent in many animal species. Attenuated growth hormone and insulin-like growth factor-1 (IGF-1) signaling caused by mutations also increases the lifespan of mice, even those allowed to feed freely. In nematodes, the Daf16, mammalian Forkhead box O (FoxO) transcription factor, was shown to be required for lifespan extension in response to reduced IGF-1 signaling. Because DR also decreases the plasma concentration of IGF-1 in mammals, the IGF-1-FoxO axis may play a central role in the lifespan extension effect of DR and, thus, retardation of aging. Studies using knockout mice under DR conditions revealed the importance of FoxO1 and nuclear factor erythroid-derived 2-like 2 (Nrf2) in tumor suppression, and FoxO3 in lifespan extension. Human genomic studies also identified a strong association between a FOXO3 single nucleotide polymorphism and longevity. The aging mechanism is the most important risk factor for disease and frailty in aging humans. Therefore, further research on the application of DR to humans, the development of compounds and drugs that mimic the effects of DR, and mechanisms underlying FOXO3 polymorphisms for longevity is highly relevant to extending the human health span.

Influence of Adipokines on Metabolic Dysfunction and Aging.

Currently, 30% of the global population is overweight or obese, with projections from the World Obesity Federation suggesting that this figure will surpass 50% by 2035. Adipose tissue dysfunction, a primary characteristic of obesity, is closely associated with an increased risk of metabolic abnormalities, such as hypertension, hyperglycemia, and dyslipidemia, collectively termed metabolic syndrome. In particular, visceral fat accretion is considered as a hallmark of aging and is strongly linked to higher mortality rates in humans. Adipokines, bioactive peptides secreted by adipose tissue, play crucial roles in regulating appetite, satiety, adiposity, and metabolic balance, thereby rendering them key players in alleviating metabolic diseases and potentially extending health span. In this review, we elucidated the role of adipokines in the development of obesity and related metabolic disorders while also exploring the potential of certain adipokines as candidates for longevity interventions.

Release of TNF-α from macrophages is mediated by small GTPase Rab37.

Activated macrophages at wound sites release many cytokines which positively affect skin wound healing. However, the molecular mechanisms controlling cytokine secretion from macrophages have not been elucidated. In the present study, we performed an RT-PCR analysis and found that 19 small GTPase Rab isoforms were expressed at skin wound sites, with six of them (i.e. Rab3B, Rab27B, Rab30, Rab33A, Rab37, and Rab40C) being upregulated during the inflammation and proliferation/migration phase of skin repair. We also found that gene expression of Rab37 in murine primary and RAW264.7 macrophages was significantly induced after stimulation with LPS. Overexpression of wild type and constitutively active Rab37 in RAW264.7 cells significantly increased TNF-α secretion, whereas knockdown of Rab37 by siRNA significantly decreased it. We also identified 29 putative Rab37-interacting proteins, including the membrane fusion regulating Munc13-1, using liquid chromatography/linear ion trap mass spectrometry (LC-MS/MS). Immunocytochemical analysis further revealed that TNF-α-containing vesicles were colocalized with both Rab37 and Munc13-1 in activated macrophages. Knockdown of Munc13-1 by siRNA significantly decreased TNF-α secretion. Taken together, these findings demonstrate that Rab37 interacts with Munc13-1 to control TNF-α secretion from activated macrophages.

Protein reporter bioassay systems for the phenotypic screening of candidate drugs: a mouse platform for anti-aging drug screening.

Recent drug discovery efforts have utilized high throughput screening (HTS) of large chemical libraries to identify compounds that modify the activity of discrete molecular targets. The molecular target approach to drug screening is widely used in the pharmaceutical and biotechnology industries, because of the amount of knowledge now available regarding protein structure that has been obtained by computer simulation. The molecular target approach requires that the structure of target molecules, and an understanding of their physiological functions, is known. This approach to drug discovery may, however, limit the identification of novel drugs. As an alternative, the phenotypic- or pathway-screening approach to drug discovery is gaining popularity, particularly in the academic sector. This approach not only provides the opportunity to identify promising drug candidates, but also enables novel information regarding biological pathways to be unveiled. Reporter assays are a powerful tool for the phenotypic screening of compound libraries. Of the various reporter genes that can be used in such assays, those encoding secreted proteins enable the screening of hit molecules in both living cells and animals. Cell- and animal-based screens enable simultaneous evaluation of drug metabolism or toxicity with biological activity. Therefore, drug candidates identified in these screens may have increased biological efficacy and a lower risk of side effects in humans. In this article, we review the reporter bioassay systems available for phenotypic drug discovery.

Anticancer agent α-sulfoquinovosyl-acylpropanediol enhances the radiosensitivity of human malignant mesothelioma in nude mouse models.

Malignant pleural mesothelioma (MPM) is a highly malignant disease that develops after asbestos exposure. Although the number of MPM cases is predicted to increase, no effective standard therapies have been established. The novel radiosensitizer α-sulfoquinovosyl-acylpropanediol (SQAP) enhances the effects of γ-radiation in human lung and prostate cancer cell lines and in animal models. In this study, we explored the radiosensitizing effect of SQAP and its mechanisms in MPM. The human MPM cell lines MSTO-211H and MESO-4 were implanted subcutaneously into the backs and thoracic cavities of immunodeficient KSN/Slc mice, then 2 mg/kg SQAP was intravenously administered with or without irradiation with a total body dose of 8 Gy. In both the orthotopic and ectopic xenograft murine models, the combination of irradiation plus SQAP delayed the implanted human MSTO-211H tumor growth. The analysis of the changes in the relative tumor volume of the MSTO-211H indicated a statistically significant difference after 8 Gy total body combined with 2 mg/kg SQAP, compared to both the untreated control (P = 0.0127) and the radiation treatment alone (P = 0.0171). After the treatment in each case, immunostaining of the harvested tumors revealed decreased cell proliferation, increased apoptosis and normalization of tumor blood vessels in the SQAP- and irradiation-treated group. Furthermore, hypoxia-inducible factor (HIF) 1 mRNA and protein expression were decreased, indicating reoxygenation in this group. In conclusion, SQAP improved hypoxic conditions in tumor tissue and may elicit a radiosensitizing effect in malignant mesothelioma models.

Lack of grafted liver rejuvenation in adult-to-pediatric liver transplantation.

BACKGROUND: A grafted donor liver should grow and survive under the different conditions presented by a liver transplantation recipient. It has remained unclear, however, whether the age of a grafted liver can be modulated by recipient factors. AIMS: This study investigated whether a grafted aged donor liver can be rejuvenated in a pediatric recipient. METHODS: Of 119 living donor liver transplants, ten pairs were adult-to-pediatric combinations. Senescence marker protein-30 (SMP-30), which is a protein that is remarkably reduced upon aging, was used as a senescence marker. Immunohistochemical staining for SMP-30 was performed in biopsy specimen after living donor liver transplantation (LDLT). Re-expression of SMP-30 was investigated in a biopsied adult liver (n = 6) that had been transplanted in a pediatric recipient. RESULTS: A remarkable expression of SMP-30 was seen in a control pediatric normal liver in comparison with that in an aged adult donor biopsy. Re-expression or an increase in SMP-30 was not observed in the liver of any pediatric recipient who had received an adult liver. CONCLUSION: An adult grafted liver does not appear to rejuvenate in a pediatric recipient.

The Role of Neuropeptide Y in Adipocyte-Macrophage Crosstalk during High Fat Diet-Induced Adipose Inflammation and Liver Steatosis.

Obesity is associated with an increased risk of non-alcoholic fatty liver disease (NAFLD), which is initiated by adipocyte-macrophage crosstalk. Among the possible molecules regulating this crosstalk, we focused on neuropeptide Y (NPY), which is known to be involved in hypothalamic appetite and adipose tissue inflammation and metabolism. In this study, the NPY-/- mice showed a marked decrease in body weight and adiposity, and lower free fatty acid and adipose inflammation without food intake alteration during a high fat diet (HFD). Moreover, NPY deficiency increased the thermogenic genes expression in brown adipose tissue. Notably, NPY-mRNA expression was upregulated in macrophages from the HFD mice compared to that from the mice on a standard diet. The NPY-mRNA expression also positively correlated with the liver mass/body weight ratio. NPY deletion alleviated HFD-induced adipose inflammation and liver steatosis. Hence, our findings point toward a novel intracellular mechanism of NPY in the regulation of adipocyte-macrophage crosstalk and highlight NPY antagonism as a promising target for therapeutic approaches against obesity and NAFLD.

Primary intracranial aggressive fibromatosis arising in sella turcica: illustrative case.

BACKGROUND: Aggressive fibromatosis is a rare histologically benign but locally infiltrative myofibroblastic tumor. Primary intracranial aggressive fibromatosis (IAF) can exhibit a clinically malignant course. OBSERVATIONS: A 22-year-old otherwise healthy woman presented with left painful ophthalmoplegia. Magnetic resonance imaging (MRI) revealed a left sellar tumor with cavernous sinus invasion. Endoscopic transsphenoidal surgery was performed. The lesion could not be totally resected. An inflammatory myofibroblastic tumor was suspected, so steroid pulse therapy was introduced, but it was ineffective. The tumor recurred after a few months, and she complained of visual acuity loss, abducens nerve palsy, trigeminal neuralgia, and panhypopituitarism. The lesion was diagnosed as primary IAF by a pathological review. Gamma Knife radiosurgery was performed, and chemotherapies were introduced but ineffective. Her consciousness was disturbed, and MRI showed hypothalamic invasion of the tumor, occlusion and stenosis of carotid arteries, and cerebral stroke. Palliative care was introduced, and she died 32 months after the onset. The autopsy revealed tumor invasion to the cavernous sinus, optic nerve, hypothalamus, pituitary, and tonsillar herniation due to massive cerebral stroke. LESSONS: Radical resection can be impossible in patients with IAF. Radiotherapy and chemotherapy are not always effective for residual lesions. Adjuvant therapy for IAF remains to be explored.

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction.

Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4α and its regulator peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.

Modulation of oxidative phosphorylation machinery signifies a prime mode of anti-ageing mechanism of calorie restriction in male rat liver mitochondria.

Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such 'putatively hormetic' ageing-analogous effects of CR, besides the age-retarding ones.