Rochelle Buffenstein

Genome sequencing reveals insights into physiology and longevity of the naked mole rat

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the ‘queen’, who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat’s exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

Protein stability and resistance to oxidative stress are determinants of longevity in the longest-living rodent, the naked mole-rat

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked mole-rats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging.

High oxidative damage levels in the longest‐living rodent, the naked mole‐rat

Oxidative stress is reputed to be a significant contributor to the aging process and a key factor affecting species longevity. The tremendous natural variation in maximum species lifespan may be due to interspecific differences in reactive oxygen species generation, antioxidant defenses and/or levels of accrued oxidative damage to cellular macromolecules (such as DNA, lipids and proteins). The present study tests if the exceptional longevity of the longest living (> 28.3 years) rodent species known, the naked mole‐rat (NMR, Heterocephalus glaber), is associated with attenuated levels of oxidative stress. We compare antioxidant defenses (reduced glutathione, GSH), redox status (GSH/GSSG), as well as lipid (malondialdehyde and isoprostanes), DNA (8‐OHdG), and protein (carbonyls) oxidation levels in urine and various tissues from both mole‐rats and similar‐sized mice. Significantly lower GSH and GSH/GSSG in mole‐rats indicate poorer antioxidant capacity and a surprisingly more pro‐oxidative cellular environment, manifested by 10‐fold higher levels of in vivo lipid peroxidation. Furthermore, mole‐rats exhibit greater levels of accrued oxidative damage to lipids (twofold), DNA (~two to eight times) and proteins (1.5 to 2‐fold) than physiologically age‐matched mice, and equal to that of same‐aged mice. Given that NMRs live an order of magnitude longer than predicted based on their body size, our findings strongly suggest that mechanisms other than attenuated oxidative stress explain the impressive longevity of this species.

Short-term effects of macronutrient preloads on appetite and energy intake in lean women

This study investigated the relative satiating hierarchy of the four energy-providing macronutrients (fat, carbohydrate (CHO), protein, and alcohol) in lean women. On four separate occasions, the composition of an iso-energetic lunch preload was manipulated in 12 lean (BMI < 25 kg/m2) women. The four treatments comprised a 1-MJ baseline meal and drink (40% fat, 48% CHO, 12% protein) to which was covertly added: 1) + 1MJ protein; 2) + 1MJ alcohol; 3) + 1MJ CHO; and 4) + 1MJ fat. Prior to and at 30-min intervals, subjects completed 100-mm visual analogue scales rating subjective hunger and satiety. Ninety min following the preload, an ad lib. lunch meal was given (40% fat, 48% CHO, and 12% protein) and energy intake (EI) measured. Energy intake at the lunch meal was 2195 (880, SD) kJ, 2772 (1191, SD) kJ, 2502 (681, SD), kJ and 2558 (1050, SD) kJ for the protein, alcohol, CHO, and fat preloads, respectively. There was no significant difference between the pleasantness of the preloads (p > 0.05). Macronutrient composition had a significant effect on short-term hunger (F = 3.19; p < 0.05), subjects being less hungry after the protein preload. Subjects also had a lower energy intake after the protein preload (F = 3.11; p < 0.05). We conclude that only protein has a differential short-term satiating effect when incorporated iso-energetically and at a similar energy density into the diet.

Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms

An inverse correlation between free radical production by isolated mitochondria and longevity in homeotherms has been reported, but previous comparative studies ignored possible confounding effects of body mass and phylogeny. We investigated this correlation by comparing rates of hydrogen peroxide (H2O2) production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum lifespans but similar body masses (small mammals, medium‐sized mammals, birds). During succinate oxidation, H2O2 production rates were generally lower in the longer‐lived species; the differences arose at complex I of the electron transport chain during reverse electron transport. Additional data were obtained from large species and the final dataset comprised mouse, rat, white‐footed mouse, naked mole‐rat, Damara mole‐rat, guinea pig, baboon, little brown bat, Brazilian free‐tailed bat, ox, pigeon and quail. In this dataset, maximum lifespan was negatively correlated with H2O2 production at complex I during reverse electron transport. Analysis of residual maximum lifespan and residual H2O2 production revealed that this correlation was even more significant after correction for effects of body mass. To remove effects of phylogeny, independent phylogenetic contrasts were obtained from the residuals. These revealed an inverse association between maximum lifespan and H2O2 production that was significant by sign test, but fell short of significance by regression analysis. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms.

Food intake and the menstrual cycle: A retrospective analysis, with implications for appetite research

The biological regulation of appetite is currently an important topic in nutrition, since hyperphagia has been implicated as the prime cause of obesity. Cyclical fluctuations in food intake occur in women across the menstrual cycle, with a periovulatory nadir and a peak in the luteal phase. These alterations in food intake, in response to ovarian steroid hormone changes may be more than 2.5 MJ/day, with the mean reported changes shown in 19 separate studies of 1.0 MJ/day. Hormonal induced fluctuations in food intake could, therefore, contribute to energy imbalance and consequent weight gain. Further, in nutrition studies involving women subjects where the menstrual cycle phase is not controlled, hormonally induced changes in food selection and intake may mask the often considerably smaller changes in response to experimental variables in appetite research.

Nrf2, a Guardian of Healthspan and Gatekeeper of Species Longevity

Although aging is a ubiquitous process that prevails in all organisms, the mechanisms governing both the rate of decline in functionality and the age of onset remain elusive. A profound constitutively upregulated cytoprotective response is commonly observed in naturally long-lived species and experimental models of extensions to lifespan (e.g., genetically-altered and/or experimentally manipulated organisms), as indicated by enhanced resistance to stress and upregulated downstream components of the cytoprotective nuclear factor erythroid 2-related factor 2 (Nrf2)-signaling pathway. The transcription factor Nrf2 is constitutively expressed in all tissues, although levels may vary among organs, with the key detoxification organs (kidney and liver) exhibiting highest levels. Nrf2 may be further induced by cellular stressors including endogenous reactive-oxygen species or exogenous electrophiles. The Nrf2-signaling pathway mediates multiple avenues of cytoprotection by activating the transcription of more than 200 genes that are crucial in the metabolism of drugs and toxins, protection against oxidative stress and inflammation, as well as playing an integral role in stability of proteins and in the removal of damaged proteins via proteasomal degradation or autophagy. Nrf2 interacts with other important cell regulators such as tumor suppressor protein 53 (p53) and nuclear factor-kappa beta (NF-κB) and through their combined interactions is the guardian of healthspan, protecting against many age-related diseases including cancer and neurodegeneration. We hypothesize that this signaling pathway plays a critical role in the determination of species longevity and that this pathway may indeed be the master regulator of the aging process.

Bone mineralization and turnover in preterm infants at 8-12 years of age: The effect of early diet

Our previous studies raised two hypotheses: first that suboptimal early nutrition and second that human milk have enhancing effects on long‐term bone mineralization. To test these hypotheses experimentally, we measured whole body and regional bone mineral content (BMC) and bone mineral density (BMD), using dual‐energy X‐ray absorptiometry and single‐photon absorptiometry, and bone turnover at 8–12 years in 244 preterm children (128 boys) who participated in a prospective randomized study of diet during the neonatal period. Dietary randomizations studied were: banked human milk (BBM, n = 87) versus preterm formula (PTF, n = 96) as the sole diet or as a supplement to mothers expressed breast milk (EBM); PTF (n = 25) versus term formula (TF, n = 36) as sole diet. Ninety‐five term children of the same age were also studied. First, preterm children were shorter and lighter than term children (height SD scores −0.49 (1.1) vs. +0.22 (0.9), weight SD scores −0.41 (1.2) vs. +0.38 (1.0) and had significantly lower whole‐body BMC than their peers; decrements were also evident at some regional sites. These differences disappeared after adjusting for bone area, body size, and pubertal status. Second, children previously randomized to BBM versus PTF or TF versus PTF showed no significant differences in anthropometry, BMC, BMD, or osteocalcin (OC). Third, there was no independent effect of the proportion of EBM on BMC, BMD, or OC and no interaction between randomized diet and the amount of EBM received. Fourth, plasma OC was significantly higher in preterm children than in term children (12.4 vs. 11.0 ng/ml, p < 0.005) and in preterm children who had received a low‐nutrient (BBM/TF) as opposed to a high‐nutrient diet (PTF) during the neonatal period (12.9 vs. 11.9 ng/ml, p = 0.03). In conclusion, preterm children are shorter, lighter, and have lower bone mass than their peers at age 8–12 years. The lower BMC is, however, appropriate for the bone and body size achieved. Despite large differences in early mineral intake, early diet does not affect bone mass in preterm children, and fresh human milk has no specific effect. However, poor nutrition during the neonatal period may result in higher bone formation rates during childhood.

Antioxidants do not explain the disparate longevity between mice and the longest-living rodent, the naked mole-rat

The maximum lifespan of naked mole-rats (NMRs; Heterocephalus glaber) is greater than that of any other rodent. These hystricognaths survive in captivity >28 years, eight-times longer than similar-sized mice. The present study tested if NMRs possess superior antioxidant defenses compared to mice and if age-related interspecies changes in antioxidants were evident. Activities of Cu/Zn superoxide dismutase (Cu/Zn, SOD), Mn SOD, catalase and cellular glutathione peroxidase (cGPx) were measured in livers of physiologically equivalent age-matched NMRs (30, 75 and 130 months) and CB6F1 mice (4, 12 and 18 months). In mice, Mn SOD activity increased with age, while the activity of catalase and cGPx declined. None of the antioxidants changed with age in mole-rats. cGPx activity of NMRs was 70-times lower (p < 0.0001) than in mice, and resembled that of cGPx knock-out animals. NMRs may partially compensate for the lower cGPx when compared to mice, by having moderately higher activities of the other antioxidants. It is nonetheless unlikely that antioxidant defenses are responsible for the eight-fold longevity difference between these two species. Maintenance of constant antioxidant defenses with age in NMRs concurs with previous physiological data, suggesting delayed aging in this species.

Is the naked mole-rat Hererocephalus glaber an endothermic yet poikilothermic mammal?

Abstract 1. 1. Thermoregulatory changes with ambient temperature (Ta) were monitored in Heterocephalus glaber between 12 and 37°C. 2. 2. At all Tas monitored, body temperature (Tb) was directly proportional to Ta (Tb = 0.568 + 10016 Ta). 3. 3. Below Ta 29°C, VO2 varied in a typically poikilothermic pattern, whereas above 29°C, VO2 followed a typically endothermic pattern. 4. 4. At Tas between 31 and 34°C, VO2 was minimal (1.00 ± 0.09 ml/g/h), skin temperature was 1.8 ± 0.6°C lower than Tb and evaporative water loss accounted for 173.7 ± 51.0% of the metabolic heat produced. 5. 5. The high rates of heat transfer are such that individual naked mole-rats cannot regulate Tb over the entire range of measured Tas and, as such, are poikilothermic mammals.