Alberto Diaz-Ruiz

Growth hormone inhibits hepatic de novo lipogenesis in adult mice

Patients with nonalcoholic fatty liver disease (NAFLD) are reported to have low growth hormone (GH) production and/or hepatic GH resistance. GH replacement can resolve the fatty liver condition in diet-induced obese rodents and in GH-deficient patients. However, it remains to be determined whether this inhibitory action of GH is due to direct regulation of hepatic lipid metabolism. Therefore, an adult-onset, hepatocyte-specific, GH receptor (GHR) knockdown (aLivGHRkd) mouse was developed to model hepatic GH resistance in humans that may occur after sexual maturation. Just 7 days after aLivGHRkd, hepatic de novo lipogenesis (DNL) was increased in male and female chow-fed mice, compared with GHR-intact littermate controls. However, hepatosteatosis developed only in male and ovariectomized female aLivGHRkd mice. The increase in DNL observed in aLivGHRkd mice was not associated with hyperactivation of the pathway by which insulin is classically considered to regulate DNL. However, glucokinase mRNA and protein levels as well as fructose-2,6-bisphosphate levels were increased in aLivGHRkd mice, suggesting that enhanced glycolysis drives DNL in the GH-resistant liver. These results demonstrate that hepatic GH actions normally serve to inhibit DNL, where loss of this inhibitory signal may explain, in part, the inappropriate increase in hepatic DNL observed in NAFLD patients.

Cytochrome b 5 reductase and the control of lipid metabolism and healthspan

Cytochrome b5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan.

Genetic Ablation of miR-33 Increases Food Intake, Enhances Adipose Tissue Expansion, and Promotes Obesity and Insulin Resistance

SUMMARY While therapeutic modulation of miRNAs provides a promising approach for numerous diseases, the promiscuous nature of miRNAs raises concern over detrimental off-target effects. miR-33 has emerged as a likely target for treatment of cardiovascular diseases. However, the deleterious effects of long-term anti-miR-33 therapies and predisposition of miR-33−/− mice to obesity and metabolic dysfunction exemplify the possible pitfalls of miRNA-based therapies. Our work provides an in-depth characterization of miR-33−/− mice and explores the mechanisms by which loss of miR-33 promotes insulin resistance in key metabolic tissues. Contrary to previous reports, our data do not support a direct role for SREBP-1-mediated lipid synthesis in promoting these effects. Alternatively, in adipose tissue of miR-33−/− mice, we observe increased pre-adipocyte proliferation, enhanced lipid uptake, and impaired lipolysis. Moreover, we demonstrate that the driving force behind these abnormalities is increased food intake, which can be prevented by pair feeding with wild-type animals.

Hexokinases link DJ-1 to the PINK1/parkin pathway

BackgroundEarly onset Parkinson’s disease is caused by variants in PINK1, parkin, and DJ-1. PINK1 and parkin operate in pathways that preserve mitochondrial integrity, but the function of DJ-1 and how it relates to PINK1 and parkin is poorly understood.MethodsA series of unbiased high-content screens were used to analyze changes at the protein, RNA, and metabolite level in rodent brains lacking DJ-1. Results were validated using targeted approaches, and cellular assays were performed to probe the mechanisms involved.ResultsWe find that in both rat and mouse brains, DJ-1 knockout results in an age-dependent accumulation of hexokinase 1 in the cytosol, away from its usual location at the mitochondria, with subsequent activation of the polyol pathway of glucose metabolism in vivo. Both in the brain and in cultured cells, DJ-1 deficiency is associated with accumulation of the phosphatase PTEN that antagonizes the kinase AKT. In cells, addition of an inhibitor of AKT (MK2206) or addition of a peptide to dissociate association of hexokinases from mitochondria both inhibit the PINK1/parkin pathway, which works to maintain mitochondrial integrity.ConclusionHexokinases are an important link between three major genetic causes of early onset Parkinson’s disease. Because aging is associated with deregulated nutrient sensing, these results help explain why DJ-1 is associated with age-dependent disease.

Skeletal muscle ex vivo mitochondrial respiration parallels decline in vivo oxidative capacity, cardiorespiratory fitness, and muscle strength: The Baltimore Longitudinal Study of Aging

Mitochondrial function in human skeletal muscle declines with age. Most evidence for this decline comes from studies that assessed mitochondrial function indirectly, and the impact of such deterioration with respect to physical function has not been clearly delineated. We hypothesized that mitochondrial respiration in permeabilized human muscle fibers declines with age and correlates with phosphocreatine postexercise recovery rate (kPCr), muscle performance, and aerobic fitness. Mitochondrial respiration was assessed by high‐resolution respirometry in saponin‐permeabilized fibers from vastus lateralis muscle biopsies of 38 participants from the Baltimore Longitudinal Study of Aging (BLSA; 21 men, age 24–91 years) who also had available measures of peak oxygen consumption (VO2max) from treadmill tests, gait speed in different tasks, 31P magnetic resonance spectroscopy, isokinetic knee extension, and grip strength. Results indicated a significant reduction in mitochondrial respiration with age (p < .05) that was independent of other potential confounders. Mitochondrial respiratory capacity was also associated with VO2max, muscle strength, kPCr, and time to complete a 400‐m walk (p < .05). A negative trend toward significance (p = .074) was observed between mitochondrial respiration and BMI. Finally, transcriptional profiling revealed a reduced mRNA expression of mitochondrial gene networks with aging (p < .05). Overall, our findings reinforce the notion that mitochondrial function declines with age and may contribute to age‐associated loss of muscle performance and cardiorespiratory fitness.

Intermittent mTOR Inhibition Reverses Kidney Aging in Old Rats

Short-term administration of the rapamycin analogue RAD001 (everolimus), already approved for clinical use for different human disorders, has been found to counter-regulate age-related transcriptomic changes and kidney pathology in rats. The study offers a step forward in establishing a safer rapalog dosing regimen for the treatment of age-related diseases. The quest for therapies that slow aging and increase healthy life span is rapidly expanding, with a few compounds showing early promise in reaching this goal. The discovery that rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR), can extend the life span of several model organisms, including yeasts, worms, flies, and mice, has elicited major interest in aging research by providing a molecular target for a potential pharmacological antiaging intervention in humans. Rapamycin-based therapy has shown benefits for patients in a range of clinical applications by acting as an immunosuppressant to prevent rejection of kidney and liver transplants, and in the treatment of autoimmune disorders, certain types of cancers and lymphangioleiomyomatosis (a rare, progressive lung disease). However, the use of rapamycin as an antiaging molecule is unlikely to be approved for healthy individuals due to considerable side effects related to its ability to suppress the immune system (1). Moreover, rapamycin has been found to promote glucose intolerance, insulin resistance, and hyperlipidemia in mice and rats (2,3), and predispose transplant patients to new-onset diabetes mellitus (4). A number of other adverse events associated with rapamycin-based therapy has also been reported (5). mTOR is a protein serine/threonine kinase that is found in two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. It is now accepted that the beneficial effects of rapamycin are largely mediated by the inhibition of mTORC1, which is acutely sensitive to rapamycin, while many of the negative side effects of the drug stem from the inhibition of mTORC2 after long-term exposure to rapamycin in a cell- and tissue-specific manner. Several derivatives of rapamycin (known as rapalogs) with improved pharmacokinetics have been developed (6); some of these have been shown to have reduced metabolic side effects in mice, likely due to decreased disruption of mTORC2 due to their pharmacokinetics (7). Different dosing schedules have been tested that minimize side effects while providing a safer strategy that may enable the translation of rapamycin-based therapies to the clinic (7). The development of a pro-longevity drug is hampered by difficulties in defining physiological parameters that are true indicators of aging and by the amount of time required to evaluate the drugs’s ability to extend human life. One of the approaches used to overcome the limitation of lengthy clinical trials has been to test whether short-time intervention with putative pro-longevity drugs can slow down phenotypic changes associated with the aging process. A recent clinical trial demonstrated that treatment for 6 weeks with the rapamycin analogue RAD001 (everolimus) ameliorated immuno-senescence and improved the response of elderly humans to influenza vaccination (8). In this issue, Shavlakadze et al. (9) evaluated the outcome of an intermittent administration of RAD001, initiated at 22.5 months of age in rats (roughly equivalent to a 60-year-old person), at doses and treatment duration similar to those previously used in humans (8). The authors compared the transcriptional profiles in liver, skeletal muscle, and kidney between young and old rats on RAD001, and found a striking (37%) reversal in the expression of age-regulated genes in the kidney, many of which linked to inflammation and fibrosis, together with a reduction in the severity of nephropathy lesions in aged rats (9). Under these conditions, the expression of age-regulated genes in liver and gastrocnemius of RAD001-treated rats was minimally altered despite lower activating phosphorylation of two surrogate markers of mTORC1 activity. Additional studies are needed to establish the presence of alternative RAD001-dependent mechanisms in the control of the liver and gastrocnemius transcriptome. RAD001 (at a dose of 1 mg/kg once a week for 6 weeks) was well-tolerated by old rats, with no significant changes in fasting blood glucose and body weight trajectories compared to controls. The authors attribute the safe metabolic profile to a “fairly selective effect” of this low-dose and short-term treatment paradigm on gene expression in the kidney, a tissue in which mTORC2 signaling is strongly resistant to rapamycin (10). These findings reinforce the idea that selective mTORC1 inhibition might be a safe and effective strategy to counteract age-related kidney pathology. How does inhibition of TOR activity influence aging? The authors report the role of c-Myc as a possible target of mTORC1 and point at a mechanism whereby RAD001 may suppress the age-dependent increase in the expression of c-Myc downstream target genes. The c-Myc proto-oncogene functions as a transcriptional regulator modulated by the mTORC1-CREB2 pathway. A recent study reports on the fact that Myc haploinsufficiency (Myc+/–) confers extended life span and health span in mice resulting from changes in multiple cellular processes such as those implicated in nutrient and energy sensing pathways (eg, AKT, TOR, and S6K) (11). It is unclear whether administration of RAD001 will counteract age-related kidney pathology in this longevity mouse model. In this issue, Shavlakadze and coworkers further demonstrate in HEK293 cells that RAD001 may affect c-Myc protein turnover by promoting its degradation; however, the fact that c-Myc protein was below detectable levels in the kidney puts into question the relevance of this observation in vivo. Nonetheless, this study helps define a therapeutic window in which rapalogs confer beneficial antiaging effects and guard against age-related diseases through mTORC1 inhibition while minimizing mTORC2-related side effects. Are rapalogues suitable antiaging drugs for people? Further research is needed to determine whether intermittent mTOR inhibition will promote human longevity and protect against age-related diseases.

Benefits of Caloric Restriction in Longevity and Chemical-Induced Tumorigenesis Are Transmitted Independent of NQO1

Caloric restriction (CR) is the most potent nonpharmacological intervention known to both protect against carcinogenesis and delay aging in laboratory animals. There is a growing number of anticarcinogens and CR mimetics that activate NAD(P)H:quinone oxidoreductase 1 (NQO1). We have previously shown that NQO1, an antioxidant enzyme that acts as an energy sensor through modulation of intracellular redox and metabolic state, is upregulated by CR. Here, we used NQO1-knockout (KO) mice to investigate the role of NQO1 in both the aging process and tumor susceptibility, specifically in the context of CR. We found that NQO1 is not essential for the beneficial effects of CR on glucose homeostasis, physical performance, metabolic flexibility, life-span extension, and (unlike our previously observation with Nrf2) chemical-induced tumorigenesis.

Adult-onset hepatocyte GH resistance promotes NASH in male mice, without severe systemic metabolic dysfunction

Nonalcoholic fatty liver disease (NAFLD), which includes nonalcoholic steatohepatitis (NASH), is associated with reduced GH input/signaling, and GH therapy is effective in the reduction/resolution of NAFLD/NASH in selected patient populations. Our laboratory has focused on isolating the direct vs indirect effects of GH in preventing NAFLD/NASH. We reported that chow-fed, adult-onset, hepatocyte-specific, GH receptor knockdown (aHepGHRkd) mice rapidly (within 7 days) develop steatosis associated with increased hepatic de novo lipogenesis (DNL), independent of changes in systemic metabolic function. In this study, we report that 6 months after induction of aHepGHRkd early signs of NASH develop, which include hepatocyte ballooning, inflammation, signs of mild fibrosis, and elevated plasma alanine aminotransferase. These changes occur in the presence of enhanced systemic lipid utilization, without evidence of white adipose tissue lipolysis, indicating that the liver injury that develops after aHepGHRkd is due to hepatocyte-specific loss of GH signaling and not due to secondary defects in systemic metabolic function. Specifically, enhanced hepatic DNL is sustained with age in aHepGHRkd mice, associated with increased hepatic markers of lipid uptake/re-esterification. Because hepatic DNL is a hallmark of NAFLD/NASH, these studies suggest that enhancing hepatocyte GH signaling could represent an effective therapeutic target to reduce DNL and treat NASH.

Sirt1 protects from K‐Ras‐driven lung carcinogenesis

The NAD+‐dependent deacetylase SIRT1 can be oncogenic or tumor suppressive depending on the tissue. Little is known about the role of SIRT1 in non‐small cell lung carcinoma (NSCLC), one of the deadliest cancers, that is frequently associated with mutated K‐RAS. Therefore, we investigated the effect of SIRT1 on K‐RAS‐driven lung carcinogenesis. We report that SIRT1 protein levels are downregulated by oncogenic K‐RAS in a MEK and PI3K‐dependent manner in mouse embryo fibroblasts (MEFs), and in human lung adenocarcinoma cell lines. Furthermore, Sirt1 overexpression in mice delays the appearance of K‐RasG12V‐driven lung adenocarcinomas, reducing the number and size of carcinomas at the time of death and extending survival. Consistently, lower levels of SIRT1 are associated with worse prognosis in human NSCLCs. Mechanistically, analysis of mouse Sirt1‐Tg pneumocytes, isolated shortly after K‐RasG12V activation, reveals that Sirt1 overexpression alters pathways involved in tumor development: proliferation, apoptosis, or extracellular matrix organization. Our work demonstrates a tumor suppressive role of SIRT1 in the development of K‐RAS‐driven lung adenocarcinomas in mice and humans, suggesting that the SIRT1–K‐RAS axis could be a therapeutic target for NSCLCs.

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.