Georgios K. Paschos

Obesity in mice with adipocyte-specific deletion of clock component Arntl

Adipocytes store excess energy in the form of triglycerides and signal the levels of stored energy to the brain. Here we show that adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component, results in obesity in mice with a shift in the diurnal rhythm of food intake, a result that is not seen when the gene is disrupted in hepatocytes or pancreatic islets. Changes in the expression of hypothalamic neuropeptides that regulate appetite are consistent with feedback from the adipocyte to the central nervous system to time feeding behavior. Ablation of the adipocyte clock is associated with a reduced number of polyunsaturated fatty acids in adipocyte triglycerides. This difference between mutant and wild-type mice is reflected in the circulating concentrations of polyunsaturated fatty acids and nonesterified polyunsaturated fatty acids in hypothalamic neurons that regulate food intake. Thus, this study reveals a role for the adipocyte clock in the temporal organization of energy regulation, highlights timing as a modulator of the adipocyte-hypothalamic axis and shows the impact of timing of food intake on body weight.

Circadian Clocks and Vascular Function

The circadian clock regulates many aspects of physiology, including cardiovascular function. Internal oscillators exist in endothelial, smooth muscle cells, and fibroblasts of the vasculature. Vascular tone and thrombus formation, 2 key elements of vascular function with regard to adverse cardiovascular events, exhibit diurnal rhythmicity. In this review, we describe changes in vascular function that result from genetic disruption of discrete elements of the circadian clock.

Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival

Postnatal knockout of a core clock gene in mice prompts reevaluation of the systemic role of the molecular clock in the biology of aging. For clock ticking, timing matters Ironically, antiaging product advertisements often promise to “slow down the clock.” But abolishing the circadian clock—for example, by knocking out Bmal1, a core clock gene—accelerates aging and shortens the life span in mice. As a result, Bmal1 knockout mice often serve as a model system in studies of the role of circadian rhythms in the aging process. Now Yang et al. show that the developmental timing of Bmal1 expression influences the circadian clock’s effects on aging and survival. To assess the role of circadian rhythms in the aging process, the authors made conditional Bmal1 knockout mice that are missing the BMAL1 protein only during adult life. Unlike knockout mice that perpetually lack Bmal1 expression, the new conditional Bmal1 knockout mice displayed loss of circadian rhythm in wheel-running activity, heart rate, and blood pressure, but exhibited normal life spans, fertility, body weight, blood glucose levels, and age-dependent arthropathy; in fact, atherosclerosis and hair growth actually improved, despite obliteration of clock function. Another surprising observation was little changes in overall gene expression in the livers of adult-life Bmal1 knockout mice, even though there’s a quelling of expression of oscillating genes. Both prenatal and postnatal knockout mice displayed similar ocular abnormalities and brain astrogliosis. Taken together, these findings reveal that many phenotypes thought to be caused by circadian rhythm disruption in conventional Bmal1 knockout mice apparently manifest as a result of clock-independent BMAL1 functions. Thus, the systemic role of the molecular clock in the biology of aging requires reinvestigation in order to increase the likelihood of translation for preclinical studies of the aging process. The absence of Bmal1, a core clock gene, results in a loss of circadian rhythms, an acceleration of aging, and a shortened life span in mice. To address the importance of circadian rhythms in the aging process, we generated conditional Bmal1 knockout mice that lacked the BMAL1 protein during adult life and found that wild-type circadian variations in wheel-running activity, heart rate, and blood pressure were abolished. Ocular abnormalities and brain astrogliosis were conserved irrespective of the timing of Bmal1 deletion. However, life span, fertility, body weight, blood glucose levels, and age-dependent arthropathy, which are altered in standard Bmal1 knockout mice, remained unaltered, whereas atherosclerosis and hair growth improved, in the conditional adult-life Bmal1 knockout mice, despite abolition of clock function. Hepatic RNA-Seq revealed that expression of oscillatory genes was dampened in the adult-life Bmal1 knockout mice, whereas overall gene expression was largely unchanged. Thus, many phenotypes in conventional Bmal1 knockout mice, hitherto attributed to disruption of circadian rhythms, reflect the loss of properties of BMAL1 that are independent of its role in the clock. These findings prompt reevaluation of the systemic consequences of disruption of the molecular clock.

The Role of Clock Genes in Pharmacology

The physiology of a wide variety of organisms is organized according to periodic environmental changes imposed by the earths rotation. This way, a large number of physiological processes present diurnal rhythms regulated by an internal timing system called the circadian clock. As part of the rhythmicity in physiology, drug efficacy and toxicity can vary with time. Studies over the past four decades present diurnal oscillations in drug absorption, distribution, metabolism, and excretion. On the other hand, diurnal variations in the availability and sensitivity of drug targets have been correlated with time-dependent changes in drug effectiveness. In this review, we provide evidence supporting the regulation of drug kinetics and dynamics by the circadian clock. We also use the examples of hypertension and cancer to show current achievements and challenges in chronopharmacology.

Novel Eicosapentaenoic Acid-derived F3-isoprostanes as Biomarkers of Lipid Peroxidation

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F2-iPs, PGF2α isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F3-iPs, 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI, derived from the ω-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in ω-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F3-iPs in mice and a shift in dietary ω-3/ω-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R2 median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of ω-3 versus ω-6 substrates. Clustered analysis of F2- and F3-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of ω-3/ω-6 PUFAs.

Knockout of Adamts7, a Novel Coronary Artery Disease Locus in Humans, Reduces Atherosclerosis in Mice

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-&agr; stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Peripheral Circadian Clock Rhythmicity Is Retained in the Absence of Adrenergic Signaling

Objective—The incidence of heart attack and stroke undergo diurnal variation. Molecular clocks have been described in the heart and the vasculature; however it is largely unknown how the suprachiasmatic nucleus (SCN) entrains these peripheral oscillators. Methods and Results—Norepinephrine and epinephrine, added to aortic smooth muscle cells (ASMCs) in vitro, altered Per1, E4bp4, and dbp expression and altered the observed oscillations in clock gene expression. However, oscillations of Per1, E4bp4, dbp, and Per2 were preserved ex vivo in the aorta, heart, and liver harvested from dopamine &bgr;-hydroxylase knockout mice (Dbh−/−) that cannot synthesize either norepinephrine or epinephrine. Furthermore, clock gene oscillations in heart, liver, and white adipose tissue phase shifted identically in Dbh−/− mice and in Dbh+/− controls in response to daytime restriction of feeding. Oscillation of clock genes was similarly preserved ex vivo in tissues from Dbh+/− and Dbh−/− chronically treated with both propranolol and terazosin, thus excluding compensation by dopamine in Dbh−/− mice. Conclusions—Although adrenergic signaling can influence circadian timing in vitro, peripheral circadian rhythmicity is retained despite its ablation in vivo.

Relationships between vitamin D status and cardio-metabolic risk factors in young European adults.

Background/Aims: To explore associations between vitamin D and cardiovascular disease risk factors in young European adults. Methods: This was a cross-sectional analysis of serum 25-hydroxyvitamin D [s25(OH)D], intact parathyroid hormone (iPTH) and biomarkers of cardiovascular disease risk in 195 healthy 20- to 40-year-olds (109 women) with a BMI between 27.5 and 32.5 from Iceland (64°N; n = 82), Ireland (51°N; n = 37) and Spain (42°N; n = 76) during mid-late winter. Results: The median s25(OH)D was 52.8 nmol/l (IQR 38.1–69.9) or 21.1 ng/ml (IQR 15.2–28.0) with a latitude-dependent gradient (p ≤ 0.0001): Iceland, 41.7 nmol/l (IQR 32.7–54.2) or 16.7 ng/ml (IQR 13.1–21.7); Ireland, 52.9 nmol/l (IQR 35.3–68.6) or 21.2 ng/ml (IQR 14.1–27.4), and Spain, 67.1 nmol/l (IQR 47.1–87.1) or 26.8 ng/ml (IQR 18.8–34.8). Eleven percent of Icelandic participants had s25(OH)D concentrations <25 nmol/l (10 ng/ml) and 66% of Icelandic, 43% of Irish, and 30% of Spanish volunteers had concentrations <50 nmol/l (20 ng/ml), respectively. Overall, 17% met 3 or more of the NCEP/ATP III criteria for cardio-metabolic syndrome (MetS). Participants in the lowest third of s25(OH)D [≤42.5 nmol/l (17 ng/ml)] were more likely to have MetS (OR 2.49, p = 0.045) and elevated TAG (OR 3.46, p = 0.019). Individuals with iPTH concentrations in the lowest third [2.34 pmol/l (22.2 pg/ml)] were more likely to have elevated fasting TAG (OR 4.17, p = 0.039), insulin (OR 3.15, p = 0.029) and HOMA-IR (OR 2.15, p = 0.031), and they were less likely to have elevated IL-6 (OR 0.24, p = 0.003). Conclusion: There were interactions between s25(OH)D, iPTH and cardio-metabolic risk factors which, given the increasing prevalence of overweight and obesity and a low vitamin D status among adults, require randomised controlled vitamin D intervention studies in overweight persons.

Young overweight and obese women with lower circulating osteocalcin concentrations exhibit higher insulin resistance and concentrations of C-reactive protein

The role of the skeleton in the regulation of energy metabolism in humans is not clear. This study investigates the hypothesis that biomarkers of bone turnover are associated with indices of glucose homeostasis and systemic inflammation in young adults. A cross-sectional study investigating the relationships between biomarkers of bone turnover (serum total and uncarboxylated osteocalcin, bone-specific alkaline phosphatase, C-telopeptide of type I collagen, urinary N-telopeptide of type I collagen) and glucose metabolism (fasting plasma glucose [FPG], insulin, insulin resistance [homeostatic model assessment of insulin resistance]), systemic inflammation (high-sensitivity C-reactive protein [hsCRP] and interleukin-6), adipokines (leptin and adiponectin), and body composition was conducted in 268 young, nondiabetic overweight and obese adults aged 20 to 40 years (116 men, 152 women; body mass index, 27.5-32.5 kg/m(2)). Data on diet, physical activity, serum 25-hydroxyvitamin D, and parathyroid hormone were also collected. In women, there was a stepwise increase in lean body mass (P < .05) and a decrease in serum hsCRP (P < .001) across tertiles of total osteocalcin. Multiple linear regression analysis showed significant inverse associations between total osteocalcin and FPG (β = -0.350; P = .016; 95% confidence interval [CI], -0.35 to -0.04), insulin (β = -0.455; P = .002; 95% CI, -1.9 to -0.46), and homeostatic model assessment of insulin resistance (β = -0.508; P = .001; 95% CI, -10.93 to -3.17) in women with total osteocalcin concentrations below the group median. Men in the lowest tertile of uncarboxylated osteocalcin had twice the concentration of hsCRP than did other men (P = .05). In this sample, women with less lean body mass had lower circulating total osteocalcin concentrations and exhibited higher FPG, insulin resistance, and hsCRP compared with their similarly sized counterparts, suggesting that associations between osteocalcin and systemic inflammation, glucose homeostasis, and insulin resistance may be influenced by differences in sex and body composition.

Knitting Up the Raveled Sleave of Care

Recent advances in our understanding of molecular clocks highlight their relevance to human physiology and disease. Recent advances in our understanding of molecular clocks highlight their relevance to human physiology and disease. This Review is based on the Franklin Epstein Lecture delivered at Beth Israel Deaconess Hospital on 25 April 2013. We discuss recent advances in our understanding of molecular clocks and highlight their relevance to human physiology and disease.