αMUPA Mice Research Articles

Potential Nephroprotective Effect of uPA against Ischemia/Reperfusion-Induced Acute Kidney Injury in αMUPA Mice and HEK-293 Cells.

Background/Objectives: The incidence of acute kidney injury (AKI) has been steadily increasing. Despite its high prevalence, there is no pathogenetically rational therapy for AKI. This deficiency stems from the poor understanding of the pathogenesis of AKI. Renal ischemia/hypoxia is one of the leading causes of clinical AKI. This study investigates whether αMUPA mice, overexpressing the urokinase plasminogen activator (uPA) gene are protected against ischemic AKI, thus unraveling a potential renal damage treatment target. Methods: We utilized an in vivo model of I/R-induced AKI in αMUPA mice and in vitro experiments of uPA-treated HEK-293 cells. We evaluated renal injury markers, histological changes, mRNA expression of inflammatory, apoptotic, and autophagy markers, as compared with wild-type animals. Results: the αMUPA mice exhibited less renal injury post-AKI, as was evident by lower SCr, BUN, and renal NGAL and KIM-1 along attenuated adverse histological alterations. Notably, the αMUPA mice exhibited decreased levels pro-inflammatory, fibrotic, apoptotic, and autophagy markers like TGF-β, IL-6, STAT3, IKB, MAPK, Caspase-3, and LC3. By contrast, ACE-2, p-eNOS, and PGC1α were higher in the kidneys of the αMUPA mice. In vitro results of the uPA-treated HEK-293 cells mirrored the in vivo findings. Conclusions: These results indicate that uPA modulates key pathways involved in AKI, offering potential therapeutic targets for mitigating renal damage.

Gender-Specific Renoprotective Pathways in αMUPA Transgenic Mice Subjected to Acute Kidney Injury.

Acute kidney injury (AKI) is a serious health concern with high morbidity and high mortality worldwide. Recently, sexual dimorphism has become increasingly recognized as a factor influencing the severity of the disease. This study explores the gender-specific renoprotective pathways in αMUPA transgenic mice subjected to AKI. αMUPA transgenic male and female mice were subjected to ischemia-reperfusion (I/R)-AKI in the presence or absence of orchiectomy, oophorectomy, and L-NAME administration. Blood samples and kidneys were harvested 48 h following AKI for the biomarkers of kidney function, renal injury, inflammatory response and intracellular pathway sensing of or responding to AKI. Our findings show differing responses to AKI, where female αMUPA mice were remarkably protected against AKI as compared with males, as was evident by the lower SCr and BUN, normal renal histologically and attenuated expression of NGAL and KIM-1. Moreover, αMUPA females did not show a significant change in the renal inflammatory and fibrotic markers following AKI as compared with wild-type (WT) mice and αMUPA males. Interestingly, oophorectomized females eliminated the observed resistance to renal injury, highlighting the central protective role of estrogen. Correspondingly, orchiectomy in αMUPA males mitigated their sensitivity to renal damage, thereby emphasizing the devastating effects of testosterone. Additionally, treatment with L-NAME proved to have significant deleterious impacts on the renal protective mediators, thereby underscoring the involvement of eNOS. In conclusion, gender-specific differences in the response to AKI in αMUPA mice include multifaceted and keen interactions between the sex hormones and key biochemical mediators (such as estrogen, testosterone and eNOS). These novel findings shed light on the renoprotective pathways and mechanisms, which may pave the way for development of therapeutic interventions.

The Protective Pathways Activated in Kidneys of αMUPA Transgenic Mice Following Ischemia\Reperfusion-Induced Acute Kidney Injury.

Despite the high prevalence of acute kidney injury (AKI), the therapeutic approaches for AKI are disappointing. This deficiency stems from the poor understanding of the pathogenesis of AKI. Recent studies demonstrate that αMUPA, alpha murine urokinase-type plasminogen activator (uPA) transgenic mice, display a cardioprotective pathway following myocardial ischemia. We hypothesize that these mice also possess protective renal pathways. Male and female αMUPA mice and their wild type were subjected to 30 min of bilateral ischemic AKI. Blood samples and kidneys were harvested 48 h following AKI for biomarkers of kidney function, renal injury, inflammatory response, and intracellular pathways sensing or responding to AKI. αMUPA mice, especially females, exhibited attenuated renal damage in response to AKI, as was evident from lower SCr and BUN, normal renal histology, and attenuated expression of NGAL and KIM-1. Notably, αMUPA females did not show a significant change in renal inflammatory and fibrotic markers following AKI as compared with wild-type (WT) mice and αMUPA males. Moreover, αMUPA female mice exhibited the lowest levels of renal apoptotic and autophagy markers during normal conditions and following AKI. αMUPA mice, especially the females, showed remarkable expression of PGC1α and eNOS following AKI. Furthermore, MUPA mice showed a significant elevation in renal leptin expression before and following AKI. Pretreatment of αMUPA with leptin-neutralizing antibodies prior to AKI abolished their resistance to AKI. Collectively, the kidneys of αMUPA mice, especially those of females, are less susceptible to ischemic I/R injury compared to WT mice, and this is due to nephroprotective actions mediated by the upregulation of leptin, eNOS, ACE2, and PGC1α along with impaired inflammatory, fibrotic, and autophagy processes.

Leptin modulates gene expression in the heart, cardiomyocytes and the adipose tissue thus mitigating LPS-induced damage

Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1β in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

Leptin modulates gene expression in the heart and cardiomyocytes towards mitigating ischemia-induced damage

Leptin, an adipocyte-derived satiety hormone, has been previously linked to cardioprotection. We have shown before that leptin conferred resistance to ischemic damage in the heart in long-lived transgenic αMUPA mice overexpressing leptin compared to the wild type (WT) FVB/N control mice. To better understand the contribution of leptin to the ischemic heart, we measured here the expression of genes encoding leptin and ischemia-related proteins in αMUPA and WT mice in the heart vs adipose tissue after MI. In addition, we investigated gene expression in neonatal rat cardiomyocytes under hypoxia in the absence and presence of exogenously added leptin or a leptin antagonist. We used real time RT-PCR and ELISA or Western blot assays to measure, respectively, mRNA and protein levels. The results have shown that circulating leptin levels and mRNA levels of leptin and heme oxygenase-1 (HO-1) in the heart were elevated in both mouse genotypes after 24 h myocardial infarction (MI), reaching higher values in αMUPA mice. In contrast, leptin gene expression in the adipose tissue was significantly increased only in WT mice, but reaching lower levels compared to the heart. Expression of the proinflammatory genes encoding TNFα and IL-1β was also largely increased after MI in the heart in both mouse types, however reaching considerably lower levels in αMUPA mice indicating a mitigated inflammatory state. In cardiomyocytes, mRNA levels of all aforementioned genes as well as HIF-1α and SOD2 genes were elevated after hypoxia. Pretreatment with exogenous leptin largely reduced the mRNA levels of TNFα and IL-1β after hypoxia, while enhancing expression of all other genes and reducing ROS levels. Pretreating the cells with a leptin antagonist increased solely the levels of leptin mRNA, suggesting a negative regulation of the hormone on the expression of its own gene. Overall, the results have shown that leptin affects expression of genes in cardiomyocytes under hypoxia in a manner that could mitigate inflammation and oxidative stress, suggesting a similar influence by endogenous leptin in αMUPA mice. Furthermore, leptin is likely to function in the ischemic murine heart more effectively in an autocrine compared to paracrine manner. These results suggest that leptin can reduce ischemic damage by modulating gene expression in the heart.

Postnatal administration of leptin antagonist mitigates susceptibility to obesity under high‐fat diet in αMUPA male mice

Perturbations in postnatal leptin signaling have been associated with altered susceptibility to diet‐induced obesity (DIO), under high‐fat‐diet (HFD), albeit with contradicting evidence. Previous studies have shown that Alpha murine urokinase‐type plasminogen activator (αMUPA) mice have a higher and longer postnatal leptin surge compared with their wild types (WT), as well as lower body weight and food intake under regular diet (RD). Here we explore αMUPA’s propensity to DIO and the effect of attenuating postnatal leptin signaling, using leptin antagonist, on energy homeostasis under both RD and HFD. Four‐day‐old αMUPA pups were treated on alternate days until P‐18 with either vehicle or leptin antagonist (LA; 10 or 20 mg d−1 kg−1) and weaned into RD or HFD. Compared with RD‐fed αMUPA males, HFD‐fed αMUPA males showed higher energy intake, even when corrected for bodyweight difference, and became hyperinsulinemic and obese. Additionally, HFD‐fed αMUPA males gained bodyweight at a higher rate than their WTs due mainly to strain‐differences in energy expenditure. LA administration did not affect strain differences under RD, but attenuated αMUPA’s hyperinsulinemia and DIO under HFD, most likely by mediating energy expenditure. Together with our previous findings, these results suggest that αMUPA’s leptin surge underlies its higher susceptibility to obesity under HFD, highlighting the role of leptin‐related developmental processes in inducing obesity under post weaning obesogenic environment, at least in αMUPA males. This study, therefore, supports the use of αMUPA mice for elucidating developmental mechanisms of obesity and the efficacy of early‐life manipulations via leptin surge axis in attenuating DIO.Support or Funding InformationFrom the Union’s Seventh Framework Program FP7‐REGPOT‐2012‐2013‐1, Agreement no. 316157The effect of high‐fat diet (HFD) compared with regular diet (RD) on energy homeostasis of saline‐treated αMUPA and FVB/N wild‐type male pups. (A) Body weight; (B) body weight and composition at nine months of age; (C) energy balance. Abbreviations: FFM, fat‐free mass. N, 6–7 mice per treatment. Non‐matching lowercase letters indicate statistically significant differences among groups. *, statistically significant difference from zero, P<0.05. Results are presented as mean ± SEM.Figure 1The effect of leptin antagonist (LA) compared with saline on αMUPA under regular (RD) and high‐fat (HFD) diets, also compared HFD‐fed saline‐treated WT. Bodyweight (A) and composition at 9 mo of age (B); (C) Energy balance. Abbreviations: FFM, fat‐free mass; LA‐10, 10 mg·day‐1·kg‐1 of LA injections; LA‐20, 20 mg·day‐1·kg‐1 of LA injections. d, Day; Δ, change. Nonmatching letters indicate differences among groups. * and # difference from zero, P < 0.05 and P < 0.01.Figure 2

Postnatal administration of leptin antagonist mitigates susceptibility to obesity under high-fat diet in male αMUPA mice.

Perturbations in postnatal leptin signaling have been associated with altered susceptibility to diet-induced obesity (DIO) under high-fat-diet (HFD), albeit with contradicting evidence. Previous studies have shown that alpha murine urokinase-type plasminogen activator (αMUPA) mice have a higher and longer postnatal leptin surge compared with their wild types (WTs) as well as lower body weight and food intake under regular diet (RD). Here we explored αMUPA's propensity for DIO and the effect of attenuating postnatal leptin signaling with leptin antagonist (LA) on energy homeostasis under both RD and HFD. Four-day-old αMUPA pups were treated on alternate days until postnatal day 18 with either vehicle or LA (10 or 20 mg·day-1·kg-1) and weaned into RD or HFD. Compared with RD-fed αMUPA males, HFD-fed αMUPA males showed higher energy intake, even when corrected for body weight difference, and became hyperinsulinemic and obese. Additionally, HFD-fed αMUPA males gained body weight at a higher rate than their WTs mainly because of strain differences in energy expenditure. LA administration did not affect strain differences under RD but attenuated αMUPA's hyperinsulinemia and DIO under HFD, most likely by mediating energy expenditure. Together with our previous findings, these results suggest that αMUPA's leptin surge underlies its higher susceptibility to obesity under HFD, highlighting the role of leptin-related developmental processes in inducing obesity in a postweaning obesogenic environment, at least in αMUPA males. This study therefore supports the use of αMUPA mice for elucidating developmental mechanisms of obesity and the efficacy of early-life manipulations via leptin surge axis in attenuating DIO.

Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge.

We investigated whether long-lived weight-reduced αMUPA mice differ from their wild types in postnatal body composition and leptin level, and whether these differences are affected by maternal-borne factors. Newborn αMUPA and wild type mice had similar body weight and composition up to the third postnatal week, after which αMUPA mice maintained lower body weight due to lower fat-free mass. Both strains showed a surge in leptin levels at the second postnatal week, initiating earlier in αMUPA mice, rising higher and lasting longer than in the wild types, mainly in females. Leptin level in dams’ serum and breast milk, and in their pup’s stomach content were also higher in αMUPA than in the WT during the surge peak. Leptin surge preceded the strain divergence in body weight, and was associated with an age-dependent decrease in the leptin:fat mass ratio—suggesting that postnatal sex and strain differences in leptin ontogeny are strongly influenced by processes independent of fat mass, such as production and secretion, and possibly outside fat tissues. Dam removal elevated corticosterone level in female pups from both strains similarly, yet mitigated the leptin surge only in αMUPA–eliminating the strain differences in leptin levels. Overall, our results indicate that αMUPA’s postnatal leptin surge is more pronounced than in the wild type, more sensitive to maternal deprivation, less related to pup’s total adiposity, and is associated with a lower post-weaning fat-free mass. These strain-related postnatal differences may be related to αMUPA’s higher milk-borne leptin levels. Thus, our results support the use of αMUPA mice in future studies aimed to explore the relationship between maternal (i.e. milk-borne) factors, postnatal leptin levels, and post-weaning body composition and energy homeostasis.

Erratum: Wound healing and longevity: Lessons from long-lived αMUPA mice

Aging (Albany NY) 2015; 7(3): 167-176. PMCID: PMC4394728 PMID: 25960543 In this Article, the additional affiliation is added for Arie Budovsky, a co-author of this manuscript: http://www.impactaging.com/papers/v7/n3/pdf/100726.pdf. The additional affiliation 4 is provided here: Hagai Yanai1, Dimitri Toren1, Klemens Vierlinger2, Manuela Hofner2, Christa Nohammer2, Marco Chilosi3, Arie Budovsky1,4, and Vadim E. Fraifeld1 1The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel 2AIT - Austrian Institute of Technology, ATU14703506, Vienna, Austria 3Department of Pathology, University of Verona, Verona, Italy 4Judea Regional Research and Development Center, Carmel, Israel

Long-Lived αMUPA Mice Show Attenuation of Cardiac Aging and Leptin-Dependent Cardioprotection.

αMUPA transgenic mice spontaneously consume less food compared with their wild type (WT) ancestors due to endogenously increased levels of the satiety hormone leptin. αMUPA mice share many benefits with mice under caloric restriction (CR) including an extended life span. To understand mechanisms linked to cardiac aging, we explored the response of αMUPA hearts to ischemic conditions at the age of 6, 18, or 24 months. Mice were subjected to myocardial infarction (MI) in vivo and to ischemia/reperfusion ex vivo. Compared to WT mice, αMUPA showed functional and histological advantages under all experimental conditions. At 24 months, none of the WT mice survived the first ischemic day while αMUPA mice demonstrated 50% survival after 7 ischemic days. Leptin, an adipokine decreasing under CR, was consistently ~60% higher in αMUPA sera at baseline. Leptin levels gradually increased in both genotypes 24h post MI but were doubled in αMUPA. Pretreatment with leptin neutralizing antibodies or with inhibitors of leptin signaling (AG-490 and Wortmannin) abrogated the αMUPA benefits. The antibodies also reduced phosphorylation of the leptin signaling components STAT3 and AKT specifically in the αMUPA myocardium. αMUPA mice did not show elevation in adiponectin, an adipokine previously implicated in CR-induced cardioprotection. WT mice treated for short-term CR exhibited cardioprotection similar to that of αMUPA, however, along with increased adiponectin at baseline. Collectively, the results demonstrate a life-long increased ischemic tolerance in αMUPA mice, indicating the attenuation of cardiac aging. αMUPA cardioprotection is mediated through endogenous leptin, suggesting a protective pathway distinct from that elicited under CR.

Long-Lived αMUPA Mice Show Reduced Sexual Dimorphism in Lifespan, and in Energy and Circadian Homeostasis-Related Parameters.

Female αMUPA (alpha murine urokinase-like plasminogen activator) transgenic mice show increased lifespan, reduced body weight and food intake, and high-amplitude circadian rhythms with an endogenous period length (tau) of 24h, versus their wild types (WT) showing a 23.7-h tau. Our goal was to characterize αMUPA and WT male mice, and their in-strain sexual dimorphism, and to further understand the mechanisms underlying αMUPA's longevity. Male αMUPA mice showed increased lifespan, reduced body weight and food intake, and aligned endogenous rhythm with a tau of 24.0h versus a tau <24h in WT. However, no differences were found when intake was corrected for metabolic mass in male αMUPA mice. αMUPA's sexual dimorphism was damped or lacking in all studied traits, while WTs were sexually dimorphic, concluding that αMUPA's transgene overrides sex-dependent mechanisms involved in lifespan and in energy and circadian homeostasis. As enhanced resonance between tau and external circadian cycle correlates with increased lifespan and reduced body weight in other species, including humans, αMUPA's 24-h tau could contribute to their longevity. Focusing future research on the mechanistic interconnections between energy homeostasis, circadian homeostasis, sexual dimorphism, and aging, using αMUPA mice, may reveal mechanisms promoting reduced body weight and increased lifespan.

Wound healing and longevity: lessons from long-lived αMUPA mice.

Does the longevity phenotype offer an advantage in wound healing (WH)? In an attempt to answer this question, we explored skin wound healing in the long-lived transgenic αMUPA mice, a unique model of genetically extended life span. These mice spontaneously eat less, preserve their body mass, are more resistant to spontaneous and induced tumorigenesis and live longer, thus greatly mimicking the effects of caloric restriction (CR). We found that αMUPA mice showed a much slower age-related decline in the rate of WH than their wild-type counterparts (FVB/N). After full closure of the wound, gene expression in the skin of old αMUPA mice returned close to basal levels. In contrast, old FVB/N mice still exhibited significant upregulation of genes associated with growth-promoting pathways, apoptosis and cell-cell/cell-extra cellular matrix interaction, indicating an ongoing tissue remodeling or an inability to properly shut down the repair process. It appears that the CR-like longevity phenotype is associated with more balanced and efficient WH mechanisms in old age, which could ensure a long-term survival advantage.

Long‐lived and Obesity Resistant Mice Exhibit 24 h Locomotor Circadian Rhythms at Young and Old Age

Ageing is associated with disruption of circadian homeostasis, including reduced entraiment to light‐dark cycles, reduced amplitude and desynchronization of circadian physiological rhythms, and shortening or lengthening of the endogenous circadian period length (tau). αMUPA mice carry a transgene encoding the urokinase‐type plasminogen activator, an extracellular protease implicated in fibrinolysis, tissue remodeling, and brain plasticity. Female αMUPA mice spontaneously eat ~25% less, live ~20% longer, and show high‐amplitude circadian rhythms in food intake, body temperature, and in hepatic clock gene expression levels, compared with WT mice. Herein we examined the tau of locomotor activity of mature (8‐month old) and aged (18‐month old) female αMUPA and WT mice under total darkness. We show that tau changed in WT mice from a period &lt;24 h at 8 months to a period &gt;;24 h at 18 months. However, tau of αMUPA mice was ~24 h at both ages. Deviation of tau from 24 h has shown to be inversely related to life span of rodents and nonhuman primates. Hence, our results suggest that the sustainable endogenous period of ~24 h in αMUPA mice may contribute to their longevity. These results support the hypothesis that although a non‐24‐h tau has an adaptive significance in allowing a better response to natural daylight changes, such a lack of resonance between tau and external period length entails a life‐shortening metabolic cost.

A superactive leptin antagonist alters metabolism and locomotion in high-leptin mice

Transgenic alpha murine urokinase-type plasminogen activator (αMUPA) mice are resistant to obesity and their locomotor activity is altered. As these mice have high leptin levels, our objective was to test whether leptin is responsible for these characteristics. αMUPA, their genetic background control (FVB/N), and C57BL mice were injected s.c. every other day with 20 mg/kg pegylated superactive mouse leptin antagonist (PEG-SMLA) for 6 weeks. We tested the effect of PEG-SMLA on body weight, locomotion, and bone health. The antagonist led to a rapid increase in body weight and subsequent insulin resistance in all treated mice. Food intake of PEG-SMLA-injected animals increased during the initial period of the experiment but then declined to a similar level to that of the control animals. Interestingly, αMUPA mice were found to have reduced bone volume (BV) than FVB/N mice, although PEG-SMLA increased bone mass in both strains. In addition, PEG-SMLA led to disrupted locomotor activity and increased corticosterone levels in C57BL but decreased levels in αMUPA or FVB/N mice. These results suggest that leptin is responsible for the lean phenotype and reduced BV in αMUPA mice; leptin affects corticosterone levels in mice in a strain-specific manner; and leptin alters locomotor activity, a behavior determined by the central circadian clock.

Long-lived mice exhibit 24 h locomotor circadian rhythms at young and old age

αMUPA transgenic mice exhibit spontaneously reduced eating and increased life span compared with their wild type (WT) control FVB/N mice. αMUPA mice also show high-amplitude circadian rhythms in food intake, body temperature, and hepatic clock gene expression. Here we examined young and aged WT and αMUPA mice for the period of locomotor activity ( tau) under total darkness (DD). We show that tau changed in WT mice from a period < 24 h at 8 months to a period > 24 h at 18 months. However, the period of αMUPA mice was ~ 24 h at both 8 and 18 months. As deviation of tau from 24 h has been found to be inversely related to life span in a large number of rodents, our results suggest that the sustainable endogenous period of ~ 24 h in αMUPA mice may contribute to their prolonged life span.

Spontaneous caloric restriction associated with increased leptin levels in obesity-resistant αMUPA mice

αMUPA mice carry as a transgene the cDNA encoding urokinase-type plasminogen activator, a member of the plasminogen/plasmin system that functions in fibrinolysis and extracellular proteolysis. These mice spontaneously consume less food when fed ad libitum and live longer compared with wild-type (WT) control mice. αMUPA mice are obesity resistant and they share many similarities with calorically restricted animals. However, extensive metabolic characterization of this unique transgenic model has never been performed. Metabolism of αMUPA mice was analyzed by measuring hormone, lipid and glucose levels in the serum, as well as gene and protein expression levels in the liver, hypothalamus and brainstem. αMUPA mice were found to be leaner than WT mice mainly because of reduced fat depots. Serum analyses showed that αMUPA mice have high levels of the anorexigenic hormones insulin and leptin, and low levels of the orexigenic hormone ghrelin. Analyses of brain neuropeptides showed that the transcript of the anorexigenic neuropeptide Pomc is highly expressed in the brainstem, whereas the expression of the orexigenic neuropeptides Npy, Orexin and Mch is blunted in the hypothalamus of αMUPA mice. In addition, adenosine monophosphate (AMP)-activated protein kinase (AMPK) levels were higher in the liver and lower in the hypothalamus, thus promoting simultaneously central reduction in appetite and peripheral loss of fat. The levels of SIRT1 were low in the liver, but high in the hypothalamus, a feature that αMUPA mice share with calorically restricted animals. Taken together, αMUPA mice exhibit a unique metabolic phenotype of low-calorie intake and high leptin levels, and could serve as a model for both spontaneous calorie restriction and resistance to obesity.

The suprachiasmatic nuclei are involved in determining circadian rhythms during restricted feeding

The circadian clock in the suprachiasmatic nuclei (SCN) responds to light and regulates peripheral circadian rhythms. Feeding regimens also reset the clock, so that time-restricted feeding (RF) dictates rhythms in peripheral tissues, whereas calorie restriction (CR) affects the SCN clock. To better understand the influence of RF vs. CR on circadian rhythms, we took advantage of the transgenic αMUPA mice that exhibit spontaneously reduced eating, and can serve as a model for CR under ad libitum feeding, and a model for temporal CR under RF compared with wild type (WT) mice. Our results show that RF advanced and generally increased the amplitude of clock gene expression in the liver under LD in both mouse types. However, under disruptive light conditions, RF resulted in a different clock gene phase in WT mice compared with αMUPA mice, suggesting a role for the reduced calories in resetting the SCN that led to the change of phase in αMUPA mice. Comparison of the RF regimen in the two lighting conditions in WT mice revealed that mPer1, mClock, and mBmal1 increased, whereas mPer2 decreased in amplitude under ultradian light in WT mice, suggesting a role for the SCN in determining clock gene expression in the periphery during RF. In summary, herein we reinforce a role for calorie restriction in resetting the SCN clock, and unravel a role for the SCN in determining peripheral rhythms under RF.

Long-lived αMUPA transgenic mice exhibit pronounced circadian rhythms

Robust biological rhythms have been shown to affect life span. Biological clocks can be entrained by two feeding regimens, restricted feeding (RF) and caloric restriction (CR). RF restricts the time of food availability, whereas CR restricts the amount of calories with temporal food consumption. CR is known to retard aging and extend life span of animals via yet-unknown pathways. We hypothesize that resetting the biological clock could be one possible mechanism by which CR extends life span. Because it is experimentally difficult to uncouple calorie reduction from temporal food consumption, we took advantage of the murine urokinase-like plasminogen activator (alphaMUPA) transgenic mice overexpressing a serine protease implicated in brain development and plasticity; they exhibit spontaneously reduced eating and increased life span. Quantitative real-time PCR analysis revealed that alphaMUPA mice exhibit robust expression of the clock genes mPer1, mPer2, mClock, and mCry1 but not mBmal1 in the liver. We also found changes in the circadian amplitude and/or phase of clock-controlled output systems, such as feeding behavior, body temperature, and enteric cryptdin expression. A change in the light-dark regimen led to modified clock gene expression and abrogated circadian patterns of food intake in wild-type (WT) and alphaMUPA mice. Consequently, food consumption of WT mice increased, whereas that of alphaMUPA mice remained the same, indicating that reduced food intake occurs upstream and independently of the biological clock. Thus we surmise that CR could lead to pronounced and synchronized biological rhythms. Because the biological clock controls mitochondrial, hormonal, and physiological parameters, system synchronicity could lead to extended life span.

Long-lived αMUPA transgenic mice show reduced SOD2 expression, enhanced apoptosis and reduced susceptibility to the carcinogen dimethylhydrazine

Calorie restriction (CR) extends the life span of various species through mechanisms that are as yet unclear. Recently, we have reported that mitochondrion-mediated apoptosis was enhanced in αMUPA transgenic mice that spontaneously eat less and live longer compared with their wild-type (WT) control mice. To understand the molecular mechanisms underlying the increased apoptosis, we compared αMUPA and WT mice for parameters associated with SOD2 (MnSOD), a mitochondrial antioxidant enzyme that converts superoxide radicals into H 2O 2 and is also known to inhibit apoptosis. The SOD2-related parameters included the levels of SOD2 mRNA, immunoreactivity and enzymatic activity in the liver, lipid oxidation and aconitase activity in isolated liver mitochondria, and the sensitivity of the mice to paraquat, an agent that elicits oxidative stress. In addition, we compared the mice for the levels of SOD2 mRNA after treatment with bacterial lipopolysaccharides (LPS), and for the DNA binding activity of NFκB as a marker for the inflammatory state. We extended SOD2 determination to the colon, where we also examined the formation of pre-neoplastic aberrant crypt foci (ACF) following treatment with dimethylhydrazine (DMH), a colonic organotypic carcinogen. Overall, αMUPA mice showed reduced basal levels of SOD2 gene expression and activity concomitantly with reduced lipid oxidation, increased aconitase activity and enhanced paraquat sensitivity, while maintaining the capacity to produce high levels of SOD2 in response to the inflammatory stimulus. αMUPA mice also showed increased resistance to DMH-induced pre-neoplasia. Collectively, these data are consistent with a model, in which an optimal fine-tuning of SOD2 throughout a long-term regimen of reduced eating could contribute to longevity, at least in the αMUPA mice.

αMUPA mice: a transgenic model for longevity induced by caloric restriction

Caloric restriction (CR) is currently the only therapeutic intervention known to attenuate aging in mammals, but the underlying mechanisms of this phenomenon are still poorly understood. To get more insight into these mechanisms, we took advantage of the αMUPA transgenic mice that previously were reported to spontaneously eat less and live longer compared with their wild-type control mice. Currently, two transgenic lines that eat less are available, thus implicating the transgenic enzyme, i.e. the urokinase-type plasminogen activator (uPA), in causing the reduced appetite. This phenotypic change could have resulted from the ectopic transgenic expression that we detected in the adult αMUPA brain, or alternatively, from a transgenic interference in brain development. Here, we have summarized similarities and differences so far found between αMUPA and calorically restricted mice. Recently, we noted several changes in the αMUPA liver, at the mitochondrial and cellular level, which consistently pointed to an enhanced capacity to induce apoptosis. In addition, αMUPA mice showed a reduced level of serum IGF-1 and a reduced incidence of spontaneously occurring or carcinogen-induced tumors in several tissues. In contrast, αMUPA did not differ from wild type mice in the levels of low molecular weight antioxidants when compared in several tissues at a young or an old age. Overall, the αMUPA model suggests that fine-tuning of the threshold for apoptosis, possibly linked in part to modulation of serum IGF-1 and mitochondrial functions, could play a role in the attenuation of aging in calorically restricted mice.