Liver represents a suitable model for monitoring the effects of a diet, due to its key role in controlling the whole metabolism. Although no direct evidence has been reported so far that genetically modified (GM) food may affect health, previous studies on hepatocytes from young female mice fed on GM soybean demonstrated nuclear modifications involving transcription and splicing pathways. In this study, the effects of this diet were studied on liver of old female mice in order to elucidate possible interference with ageing. The morpho-functional characteristics of the liver of 24-month-old mice, fed from weaning on control or GM soybean, were investigated by combining a proteomic approach with ultrastructural, morphometrical and immunoelectron microscopical analyses. Several proteins belonging to hepatocyte metabolism, stress response, calcium signalling and mitochondria were differentially expressed in GM-fed mice, indicating a more marked expression of senescence markers in comparison to controls. Moreover, hepatocytes of GM-fed mice showed mitochondrial and nuclear modifications indicative of reduced metabolic rate. This study demonstrates that GM soybean intake can influence some liver features during ageing and, although the mechanisms remain unknown, underlines the importance to investigate the long-term consequences of GM-diets and the potential synergistic effects with ageing, xenobiotics and/or stress conditions.

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs ready to be exported to the cytoplasm. The molecular and structural apparatus for mRNA production is generally able to promptly respond to variations of metabolic demands. Hibernating mammals, which periodically enter a hypometabolic state, represent an interesting physiological model to investigate the adaptive morpho-functional modifications of the pre-mRNA transcriptional and processing machinery under extreme metabolic conditions. In this study, the subnuclear distribution of some transcriptional, splicing, and cleavage factors was investigated by ultrastructural immunocytochemistry in cell nuclei of the liver (a highly metabolizing organ involved in multiple regulatory functions) and the brown adipose tissue (responsible for nonshivering thermogenesis) from euthermic, hibernating, and arousing hazel dormice (Muscardinus avellanarius). Our observations demonstrate that, during hibernation, transcriptional activity significantly decreases and pre-mRNA processing factors undergo an intranuclear redistribution moving to domains usually devoid of such molecules; moreover, in hepatocytes, there is a preferential accumulation of pre-mRNAs at the splicing stage, whereas, in brown adipocytes, pre-mRNAs are mainly stored at the cleavage stage. Upon arousal, the pre-mRNAs at the cleavage stage are immediately utilized, while the maturation of pre-mRNAs at the splicing stage seems to be restored before transcription had taken place. Our data suggest a programmed intranuclear reorganization of the RNA maturation machinery aimed at efficiently and rapidly restoring the pre-mRNA processing, and, consequently, the specific cellular activities upon arousal. Once again natural hibernation appears as a highly programmed hypometabolic state rather than a simple fall of metabolic and physiological functions.

Several biochemical, physiological, and behavioral processes exhibit cyclic oscillations of about 24 h, which have been defined as circadian rhythms. In mammals, the primary circadian pacemaker resides in the suprachiasmatic nuclei; however, cell-autonomous circadian oscillators occur also in extraneural tissues, including the liver. CLOCK protein is a transcription factor essential for normal circadian rhythms and recent studies have demonstrated that it undergoes intranuclear redistribution in hepatocytes, along the daily cycle. It is known that aging leads to a progressive deterioration of the circadian rhythm at the behavioral, physiological, and cellular levels; in addition, aging affects the organization of nuclear structural components involved in transcription and splicing. In this view, we carried out ultrastructural immunocytochemical analyses on hepatocytes of adult and old rats, so as to investigate possible qualitative and quantitative modifications of CLOCK protein, in relation to the aging process. Our observations demonstrated that most CLOCK protein was always located in the cell nucleus, where it accumulated on perichromatin fibrils (the sites of premRNA transcription and early splicing); in addition, CLOCK showed daily oscillations in the different nuclear compartments, but these oscillations differed significantly between adult and old animals. This unusual distribution of CLOCK protein during aging could be related to the prolonged diurnal activity of old animals and/or to altered nuclear pathways.

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Fibrillar centers (FCs), dense fibrillar (DFC) and granular (GC) components in nucleoli, and perichromatin granules (PGs) in nucleoplasm were measured by morphometry. FC size and their nucleolar surface fraction significantly decreased in aging and vitamin E deficiency. The GC and DFC nucleolar fraction was unchanged in adult and old rats, but in vitamin E-deficient animals GC increased and DFC decreased significantly. PG density significantly increased in aging and decreased in vitamin E deficiency. The quantitative evaluation of immunolabeled transcription and splicing factors revealed that polymerase II and SC-35 significantly decreased in old and vitamin E-deficient versus adult animals. Fibrillarin and snRNPs did not change between adult and old rats, but were significantly lower in vitamin E-deficient rats. These data document altered RNA pathways in aging and vitamin E deficiency. Considering the antioxidant role of vitamin E, they lend further support to the importance of free radical production and control in the aging process.