Protein restriction cycles reduce IGF‐1 and phosphorylated Tau,and improve behavioral performance in an Alzheimer's disease mouse model

In laboratory animals, calorie restriction (CR) protects against aging, oxidative stress, and neurodegenerative pathologies. Reduced levels of growth hormone and IGF‐1, which mediate some of the protective effects of CR, can also extend longevity and/or protect against age‐related diseases in rodents and humans. However, severely restricted diets are difficult to maintain and are associated with chronically low weight and other major side effects. Here we show that 4 months of periodic protein restriction cycles (PRCs) with supplementation of nonessential amino acids in mice already displaying significant cognitive impairment and Alzheimer's disease (AD)‐like pathology reduced circulating IGF‐1 levels by 30–70% and caused an 8‐fold increase in IGFBP‐1. Whereas PRCs did not affect the levels of β amyloid (Aβ), they decreased tau phosphorylation in the hippocampus and alleviated the age‐dependent impairment in cognitive performance. These results indicate that periodic protein restriction cycles without CR can promote changes in circulating growth factors and tau phosphorylation associated with protection against age‐related neuropathologies.     

Long-term effects of calorie or protein restriction on serum IGF-1 and IGFBP-3 concentration in humans

Reduced function mutations in the insulin/IGF-I signaling pathway increase maximal lifespan and health span in many species. Calorie restriction (CR) decreases serum IGF-1 concentration by ~40%, protects against cancer and slows aging in rodents. However, the long-term effects of CR with adequate nutrition on circulating IGF-1 levels in humans are unknown. Here we report data from two long-term CR studies (1 and 6 years) showing that severe CR without malnutrition did not change IGF-1 and IGF-1 : IGFBP-3 ratio levels in humans. In contrast, total and free IGF-1 concentrations were significantly lower in moderately protein-restricted individuals. Reducing protein intake from an average of 1.67 g kg(-1) of body weight per day to 0.95 g kg(-1) of body weight per day for 3 weeks in six volunteers practicing CR resulted in a reduction in serum IGF-1 from 194 ng mL(-1) to 152 ng mL(-1). These findings demonstrate that, unlike in rodents, long-term severe CR does not reduce serum IGF-1 concentration and IGF-1 : IGFBP-3 ratio in humans. In addition, our data provide evidence that protein intake is a key determinant of circulating IGF-1 levels in humans, and suggest that reduced protein intake may become an important component of anticancer and anti-aging dietary interventions.     

lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.     

Ketone body 3‐hydroxybutyrate mimics calorie restriction via the Nrf2 activator,fumarate, in the retina

Calorie restriction (CR) being the most robust dietary intervention provides various health benefits. D‐3‐hydroxybutyrate (3HB), a major physiological ketone, has been proposed as an important endogenous molecule for CR. To investigate the role of 3HB in CR, we investigated potential shared mechanisms underlying increased retinal 3HB induced by CR and exogenously applied 3HB without CR to protect against ischemic retinal degeneration. The repeated elevation of retinal 3HB, with or without CR, suppressed retinal degeneration. Metabolomic analysis showed that the antioxidant pentose phosphate pathway and its limiting enzyme, glucose‐6‐phosphate dehydrogenase (G6PD), were concomitantly preserved. Importantly, the upregulation of nuclear factor erythroid 2 p45‐related factor 2 (Nrf2), a regulator of G6PD, and elevation of the tricarboxylic acid cycle's Nrf2 activator, fumarate, were also shared. Together, our findings suggest that CR provides retinal antioxidative defense by 3HB through the antioxidant Nrf2 pathway via modification of a tricarboxylic acid cycle intermediate during 3HB metabolism.     

Long‐term effects of calorie restriction on serum sex‐hormone concentrations in men

Calorie restriction (CR) slows aging and consistently reduces circulating sex hormones in laboratory animals. However, nothing is known regarding the long‐term effects of CR with adequate nutrition on serum sex‐hormone concentration in lean healthy humans. In this study, we measured body composition, and serum total testosterone, total 17‐β‐estradiol, sex hormone–binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEA‐S) concentrations in 24 men (mean age 51.5 ± 13 years), who had been practicing CR with adequate nutrition for an average of 7.4 ± 4.5 years, in 24 age‐ and body fat–matched endurance runners (EX), and 24 age‐matched sedentary controls eating Western diets (WD). We found that both the CR and EX volunteers had significantly lower body fat than the WD volunteers (total body fat, 8.7 ± 4.2%; 10.5 ± 4.4%; 23.2 ± 6.1%, respectively; P = 0.0001). Serum total testosterone and the free androgen index were significantly lower, and SHBG was higher in the CR group than in the EX and WD groups (P ≤ 0.001). Serum 17β‐estradiol and the estradiol:SHBG ratio were both significantly lower in the CR and EX groups than in the WD group (P ≤ 0.005). Serum DHEA‐S concentrations were not different between the three groups. These findings demonstrate that, as in long‐lived CR rodents, long‐term severe CR reduces serum total and free testosterone and increases SHBG concentrations in humans, independently of adiposity. More studies are needed to understand the role of this CR‐mediated reduction in sex hormones in modulating the pathogenesis of age‐associated chronic diseases such as cancer and the aging process itself.     

Effects of IGF‐1 isoforms on muscle growth and sarcopenia

The decline in skeletal muscle mass and strength occurring in aging, referred as sarcopenia, is the result of many factors including an imbalance between protein synthesis and degradation, changes in metabolic/hormonal status, and in circulating levels of inflammatory mediators. Thus, factors that increase muscle mass and promote anabolic pathways might be of therapeutic benefit to counteract sarcopenia. Among these, the insulin‐like growth factor‐1 (IGF‐1) has been implicated in many anabolic pathways in skeletal muscle. IGF‐1 exists in different isoforms that might exert different role in skeletal muscle. Here we study the effects of two full propeptides IGF‐1Ea and IGF‐1Eb in skeletal muscle, with the aim to define whether and through which mechanisms their overexpression impacts muscle aging. We report that only IGF‐1Ea expression promotes a pronounced hypertrophic phenotype in young mice, which is maintained in aged mice. Nevertheless, examination of aged transgenic mice revealed that the local expression of either IGF‐1Ea or IGF‐1Eb transgenes was protective against age‐related loss of muscle mass and force. At molecular level, both isoforms activate the autophagy/lysosome system, normally altered during aging, and increase PGC1‐α expression, modulating mitochondrial function, ROS detoxification, and the basal inflammatory state occurring at old age. Moreover, morphological integrity of neuromuscular junctions was maintained and preserved in both MLC/IGF‐1Ea and MLC/IGF‐1Eb mice during aging. These data suggest that IGF‐1 is a promising therapeutic agent in staving off advancing muscle weakness.     

Identification of genetic determinants of IGF‐1 levels and longevity among mouse inbred strains

The IGF‐1 signaling pathway plays an important role in regulating longevity. To identify the genetic loci and genes that regulate plasma IGF‐1 levels, we intercrossed MRL/MpJ and SM/J, inbred mouse strains that differ in IGF‐1 levels. Quantitative trait loci (QTL) analysis of IGF‐1 levels of these F2 mice detected four QTL on chromosomes (Chrs) 9 (48 Mb), 10 (86 Mb), 15 (18 Mb), and 17 (85 Mb). Haplotype association mapping of IGF‐1 levels in 28 domesticated inbred strains identified three suggestive loci in females on Chrs 2 (13 Mb), 10 (88 Mb), and 17 (28 Mb) and in four males on Chrs 1 (159 Mb), 3 (52 and 58 Mb), and 16 (74 Mb). Except for the QTL on Chr 9 and 16, all loci co‐localized with IGF‐1 QTL previously identified in other mouse crosses. The most significant locus was the QTL on Chr 10, which contains the Igf1 gene and which had a LOD score of 31.8. Haplotype analysis among 28 domesticated inbred strains revealed a major QTL on Chr 10 overlapping with the QTL identified in the F2 mice. This locus showed three major haplotypes; strains with haplotype 1 had significantly lower plasma IGF‐1 and extended longevity (P < 0.05) than strains with haplotype 2 or 3. Bioinformatic analysis, combined with sequencing and expression studies, showed that Igf1 is the most likely QTL gene, but that other genes may also play a role in this strong QTL.     

SUMO‐modified insulin‐like growth factor 1 receptor (IGF‐1R) increases cell cycle progression and cell proliferation

Increasing number of studies have shown nuclear localization of the insulin‐like growth factor 1 receptor (nIGF‐1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF‐1R have, however, still not been disclosed. Previously, we reported that IGF‐1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple‐SUMO‐site‐mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R‐). Cell clones (R‐WT and R‐TSM) expressing equal amounts of IGF‐1R were selected for experiments. Phosphorylation of IGF‐1R, Akt, and Erk upon IGF‐1 stimulation was equal in R‐WT and R‐TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R‐WT proliferated substantially faster than R‐TSM, which did not differ significantly from the empty vector control. Upon IGF‐1 stimulation G1‐S‐phase progression of R‐WT increased from 12 to 38%, compared to 13 to 20% of R‐TSM. The G1‐S progression of R‐WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO‐IGF‐1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO‐IGF‐1R dependent mechanisms seem important.     

Cross‐talk between IGF‐1 and estrogen receptors attenuates intracellular changes in ventral spinal cord 4.1 motoneuron cells because of interferon‐gamma exposure

Insulin‐like growth factor‐1 (IGF‐1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF‐1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon‐gamma (IFN‐γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl‐2 ratios, and expression of apoptosis‐related proteases (caspase‐3 and ‐12) in motoneurons rendered by IFN‐γ in a dose‐dependent manner. Post‐treatment with IGF‐1 attenuated these changes. In addition, IGF‐1 treatment of motoneurons exposed to IFN‐γ decreased expression of inflammatory markers (cyclooxygenase‐2 and nuclear factor‐kappa B:inhibitor of kappa B ratio). Furthermore, IGF‐1 attenuated the loss of expression of IGF‐1 receptors (IGF‐1Rα and IGF‐1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN‐γ. To determine whether the protective effects of IGF‐1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ‐silenced VSC4.1 motoneurons following IFN‐γ and IGF‐1 exposure. These results suggest that IGF‐1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ.

     

Synergistic effect of DHT and IGF‐1 hyperstimulation in human peripheral blood lymphocytes

The abuse of mixed or combined performance‐enhancing drugs is widespread among athletes and amateurs, adults and adolescents. Clinical studies demonstrated that misuse of these doping agents is associated with serious adverse effects to many organs in human. Previously, we demonstrated in human peripheral blood lymphocytes that high doses of anabolic androgenic steroids, such as dihydrotestosterone (DHT) and growth factors, such as insulin‐like growth factor‐1 (IGF‐1), have effects at gene and protein levels. Supraphysiological treatments of DHT and IGF‐1 affected the expression of genes involved in skeletal muscle disorders as well as in cell‐mediated immunological response. At protein level, DHT hyperdosage affects cell motility and apoptosis; IGF‐1 hyperstimulation triggers an active cytoskeletal reorganization and an overproduction of immune response‐ and inflammation‐related cytokines. In this study, we investigate the combined effects of DHT and IGF‐1 hyperdosage in peripheral blood lymphocytes using a differential proteomic approach. DHT and IGF‐1 combined treatment affects cell adhesion, migration, and survival through modulation of expression levels of cytokines and paxillin‐signaling‐related proteins, and activation of several pathways downstream focal adhesion kinase. Our results indicate a synergistic effect of DHT and IGF‐1 which has potential implications for health risk factors.     

Overexpression of CYB5R3 and NQO1, two NAD+‐producing enzymes,mimics aspects of caloric restriction

Calorie restriction (CR) is one of the most robust means to improve health and survival in model organisms. CR imposes a metabolic program that leads to increased stress resistance and delayed onset of chronic diseases, including cancer. In rodents, CR induces the upregulation of two NADH‐dehydrogenases, namely NAD(P)H:quinone oxidoreductase 1 (Nqo1) and cytochrome b₅ reductase 3 (Cyb5r3), which provide electrons for energy metabolism. It has been proposed that this upregulation may be responsible for some of the beneficial effects of CR, and defects in their activity are linked to aging and several age‐associated diseases. However, it is unclear whether changes in metabolic homeostasis solely through upregulation of these NADH‐dehydrogenases have a positive impact on health and survival. We generated a mouse that overexpresses both metabolic enzymes leading to phenotypes that resemble aspects of CR including a modest increase in lifespan, greater physical performance, a decrease in chronic inflammation, and, importantly, protection against carcinogenesis, one of the main hallmarks of CR. Furthermore, these animals showed an enhancement of metabolic flexibility and a significant upregulation of the NAD⁺/sirtuin pathway. The results highlight the importance of these NAD⁺ producers for the promotion of health and extended lifespan.     

Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2‐isoprostanes: The CALERIE 2 randomized clinical trial

Calorie restriction (CR) without malnutrition slows aging in animal models. Oxidative stress reduction was proposed to mediate CR effects. CR effect on urinary F2‐isoprostanes, validated oxidative stress markers, was assessed in CALERIE, a two‐year randomized controlled trial. Healthy volunteers (n = 218) were randomized to prescribed 25% CR (n = 143) or ad libitum control (AL, n = 75) stratifying the randomization schedule by site, sex, and BMI. F2‐isoprostanes were quantified using LC‐MS/MS in morning, fasted urine specimens at baseline, at 12 and 24 months. The primary measure of oxidative status was creatinine‐adjusted 2,3‐dinor‐iPF(2α)‐III concentration, additional measured included iPF(2α)‐III, iPF2a‐VI, and 8,12‐iso‐iPF2a‐VI. Intention‐to‐treat analyses assessed change in 2,3‐dinor‐iPF(2α)‐III using mixed models assessing treatment, time, and treatment‐by‐time interaction effects, adjusted for blocking variables and baseline F2‐isoprostane value. Exploratory analyses examined changes in iPF(2α)‐III, iPF(2α)‐VI, and 8,12‐iso‐iPF(2α)‐VI. A factor analysis used aggregate information on F2‐isoprostane values. In CR group, 2,3‐dinor‐iPF(2α)‐III concentrations were reduced from baseline by 17% and 13% at 12 and 24 months, respectively; these changes were significantly different from AL group (p < .01). CR reduced iPF(2α)‐III concentrations by 20% and 27% at 12 and 24 months, respectively (p < .05). The effects were weaker on the VI‐species. CR caused statistically significant reduction in isoprostane factor at both time points, and mean (se) changes were ?0.36 (0.06) and ?0.31 (0.06). No significant changes in isoprostane factor were at either time point in AL group (p < .01 between‐group difference). We conclude that two‐year CR intervention in healthy, nonobese men and women reduced whole body oxidative stress as assessed by urinary concentrations of F2‐isoprostanes.     

The evaluation of acute toxicity,antimicrobial activity of 1‐phenyl‐5‐p‐tolyl‐1H‐1, 2, 3‐triazole,and binding to human serum albumin

1‐Phenyl‐5‐p‐tolyl‐1H‐1, 2, 3‐triazole (PPTA) was a synthesized compound. The result of acute toxicities to mice of PPTA by intragastric administration indicated that PPTA did not produce any significant acute toxic effect on Kunming strain mice. It exhibited the various potent inhibitory activities against two kinds of bananas pathogenic bacteria, black sigatoka and freckle, when compared with that of control drugs and the inhibitory rates were up to 64.14% and 43.46%, respectively, with the same concentration of 7.06 mM. The interaction of PPTA with human serum albumin (HSA) was studied using fluorescence polarization, absorption spectra, 3D fluorescence, and synchronous spectra in combination with quantum chemistry and molecular modeling. Multiple modes of interaction between PPTA and HSA were suggested to stabilize the PPTA–HSA complex, based on thermodynamic data and molecular modeling. Binding of PPTA to HSA induced perturbation in the microenvironment around HSA as well as secondary structural changes in the protein.     

Hormone replacement therapy enhances IGF‐1 signaling in skeletal muscle by diminishing miR‐182 and miR‐223 expressions: a study on postmenopausal monozygotic twin pairs

MiRNAs are fine‐tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co‐twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen‐based hormone replacement therapy (HRT) to explore estrogen‐dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62‐years‐old monozygotic female twin pairs discordant for HRT (median 7 years). MCF‐7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR‐182, miR‐223 and miR‐142‐3p expressions in HRT using than in their nonusing co‐twins. Insulin/IGF‐1 signaling emerged one common pathway targeted by these miRNAs. IGF‐1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR‐182 and miR‐223 on IGF‐1R and FOXO3A mRNA as well as a dose‐dependent miR‐182 and miR‐223 down‐regulations concomitantly with up‐regulation of FOXO3A and IGF‐1R expression. Novel finding is the postmenopausal HRT‐reduced miRs‐182, miR‐223 and miR‐142‐3p expression in female skeletal muscle. The observed miRNA‐mediated enhancement of the target genes' IGF‐1R and FOXO3A expression as well as the activation of insulin/IGF‐1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.     

In a randomized trial in prostate cancer patients,dietary protein restriction modifies markers of leptin and insulin signaling in plasma extracellular vesicles

Obesity, metabolic syndrome, and hyperleptinemia are associated with aging and age‐associated diseases including prostate cancer. One experimental approach to inhibit tumor growth is to reduce dietary protein intake and hence levels of circulating amino acids. Dietary protein restriction (PR) increases insulin sensitivity and suppresses prostate cancer cell tumor growth in animal models, providing a rationale for clinical trials. We sought to demonstrate that biomarkers derived from plasma extracellular vesicles (EVs) reflect systemic leptin and insulin signaling and respond to dietary interventions. We studied plasma samples from men with prostate cancer awaiting prostatectomy who participated in a randomized trial of one month of PR or control diet. We found increased levels of leptin receptor in the PR group in total plasma EVs and in a subpopulation of plasma EVs expressing the neuronal marker L1CAM. Protein restriction also shifted the phosphorylation status of the insulin receptor signal transducer protein IRS1 in L1CAM+ EVs in a manner suggestive of improved insulin sensitivity. Dietary PR modifies indicators of leptin and insulin signaling in circulating EVs. These findings are consistent with improved insulin and leptin sensitivity in response to PR and open a new window for following physiologic responses to dietary interventions in humans.