Amino acids contribute to adaptive thermogenesis. New insights into the mechanisms of action of recent drugs for metabolic disorders are emerging.

Adaptive thermogenesis is the heat production by muscle contractions (shivering thermogenesis) or brown adipose tissue (BAT) and beige fat (non-shivering thermogenesis) in response to external stimuli, including cold exposure. BAT and beige fat communicate with peripheral organs and the brain through a variegate secretory and absorption processes - controlling adipokines, microRNAs, extracellular vesicles, and metabolites - and have received much attention as potential therapeutic targets for managing obesity-related disorders. The sympathetic nervous system and norepinephrine-releasing adipose tissue macrophages (ATM) activate uncoupling protein 1 (UCP1), expressed explicitly in brown and beige adipocytes, dissolving the electrochemical gradient and uncoupling tricarboxylic acid cycle and the electron transport chain from ATP production. Mounting evidence has attracted attention to the multiple effects of dietary and endogenously synthesised amino acids in BAT thermogenesis and metabolic phenotype in animals and humans. However, the mechanisms implicated in these processes have yet to be conclusively characterized. In the present review article, we aim to define the principal investigation areas in this context, including intestinal microbiota constitution, adipose autophagy modulation, and secretome and metabolic fluxes control, which lead to increased brown/beige thermogenesis. Finally, also based on our recent epicardial adipose tissue results, we summarise the evidence supporting the notion that the new dual and triple agonists of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon (GCG) receptor - with never before seen weight loss and insulin-sensitizing efficacy - promote thermogenic-like amino acid profiles in BAT with robust heat production and likely trigger sympathetic activation and adaptive thermogenesis by controlling amino acid metabolism and ATM expansion in BAT and beige fat.

Human epicardial adipose tissue expresses glucose-dependent insulinotropic polypeptide, glucagon, and glucagon-like peptide-1 receptors as potential targets of pleiotropic therapies.

AIMS: Human epicardial adipose tissue (EAT) plays a crucial role in the development and progression of coronary artery disease, atrial fibrillation, and heart failure. Microscopically, EAT is composed of adipocytes, nerve tissues, inflammatory, stromovascular, and immune cells. Epicardial adipose tissue is a white adipose tissue, albeit it also has brown fat-like or beige fat-like features. No muscle fascia divides EAT and myocardium; this allows a direct interaction and crosstalk between the epicardial fat and the myocardium. Thus, it might be a therapeutic target for pharmaceutical compounds acting on G-protein-coupled receptors, such as those for glucose-dependent insulinotropic polypeptide (GIP), glucagon (GCG), and glucagon-like peptide-1 (GLP-1), whose selective stimulation with innovative drugs has demonstrated beneficial cardiovascular effects. The precise mechanism of these novel drugs and their tissue and cellular target(s) need to be better understood. We evaluate whether human EAT expresses GIP, GCG, and GLP-1 receptors and whether their presence is related to EAT transcriptome. We also investigated protein expression and cell-type localization specifically for GIP receptor (GIPR) and glucagon receptor (GCGR). METHODS AND RESULTS: Epicardial adipose tissue samples were collected from 33 patients affected by cardiovascular diseases undergoing open heart surgery (90.9% males, age 67.2 ± 10.5 years mean ± SD). Microarray and immunohistochemistry analyses were performed. Microarray analysis showed that GIPR and GCGR messenger ribonucleic acids (mRNAs) are expressed in EAT, beyond confirming the previously found GLP-1 [3776 ± 1377 arbitrary unit (A.U.), 17.77 ± 14.91 A.U., and 3.41 ± 2.27 A.U., respectively]. The immunohistochemical analysis consistently indicates that GIPR and GCGR are expressed in EAT, mainly in macrophages, isolated, and in crown-like structures. In contrast, only some mature adipocytes of different sizes showed cytoplasmic immunostaining, similar to endothelial cells and pericytes in the capillaries and pre-capillary vascular structures. Notably, EAT GIPR is statistically associated with the low expression of genes involved in free fatty acid (FFA) oxidation and transport and those promoting FFA biosynthesis and adipogenesis (P < 0.01). Epicardial adipose tissue GCGR, in turn, is related to genes involved in FFA transport, mitochondrial fatty acid oxidation, and white-to-brown adipocyte differentiation, in addition to genes involved in the reduction of fatty acid biosynthesis and adipogenesis (P < 0.01). CONCLUSIONS: Having reported the expression of the GLP-1 receptor previously, here, we showed that GIPR and GCGR similarly present at mRNA and protein levels in human EAT, particularly in macrophages and partially adipocytes, suggesting these G-protein-coupled receptors as pharmacological targets on the ongoing innovative drugs, which seem cardiometabolically healthy well beyond their effects on glucose and body weight.

Human epicardial adipose tissue (EAT) is a unique and multifunctional fat compartment of the heart. Microscopically, EAT is composed of adipocytes, nerve tissues, inflammatory, stromovascular, and immune cells. Epicardial adipose tissue is a white adipose tissue, albeit it also has brown fat-like or beige fat-like features. No muscle fascia divides EAT and myocardium; this allows a direct interaction and crosstalk between the epicardial fat and the myocardium. Due to its distinctive transcriptome and functional proximity to the heart, EAT can play a key role in the development and progression of coronary artery disease, atrial fibrillation, and heart failure. Clinically, EAT, given its rapid metabolism and simple measurability, can be considered a novel therapeutic target, owing to its responsiveness to drugs with pleiotropic and clear beneficial cardiovascular effects such as the glucagon-like peptide-1 receptor (GLP-1R) agonists.Human EAT is found to express the genes encoding the receptors of glucose-dependent insulinotropic polypeptide receptor (GIPR), glucagon receptor (GCGR), and GLP-1. The immunohistochemistry indicates that GIP and GCG receptor proteins are present in EAT samples. Epicardial adipose tissue GIPR is inversely associated with genes involved in free fatty acid (FFA) oxidation and transport and with genes promoting FFA biosynthesis and adipogenesis. Epicardial adipose tissue GCGR is correlated with genes promoting FFA transport and activation for mitochondrial beta-oxidation and white-to-brown adipocyte differentiation and with genes reducing FFA biosynthesis and adipogenesis.As the myocardium relies mostly on FFAs as fuel and is in direct contiguity with EAT, these findings may have a great importance for the modulation of the myocardial activity and performance. Given the emerging use and cardiovascular effects of GLP-1R agonists, dual GIPR/GLP-1R agonists, and GLP-1R/GIPR/GCGR triagonists, we believe that pharmacologically targeting and potentially modulating organ-specific fat depots through G-protein­coupled receptors may produce beneficial cardiovascular and metabolic effects.

The three facets of the SARS-CoV-2 pandemic during the first two waves in the northern, central, and southern Italy.

BACKGROUND: There is a scarcity of information in literature regarding the clinical differences and comorbidities of patients affected by Coronavirus disease 2019 (COVID-19), which could clarify the different prevalence of the outcomes (composite and only death) between several Italian regions. OBJECTIVE: This study aimed to assess the heterogeneity of clinical features of patients with COVID-19 upon hospital admission and disease outcomes in the northern, central, and southern Italian regions. METHODS: An observational cohort multicenter retrospective study including 1210 patients who were admitted for COVID-19 in Infectious diseases, Pulmonology, Endocrinology, Geriatrics and Internal Medicine Units in Italian cities stratified between north (263 patients); center (320 patients); and south (627 patients), during the first and second pandemic waves of SARS-CoV-2 (from February 1, 2020 to January 31, 2021). The data, obtained from clinical charts and collected in a single database, comprehended demographic characteristics, comorbidities, hospital and home pharmacological therapies, oxygen therapy, laboratory values, discharge, death and Intensive care Unit (ICU) transfer. Death or ICU transfer were defined as composite outcomes. RESULTS: Male patients were more frequent in the northern Italian region than in the central and southern regions. Diabetes mellitus, arterial hypertension, chronic pulmonary and chronic kidney diseases were the comorbidities more frequent in the southern region; cancer, heart failure, stroke and atrial fibrillation were more frequent in the central region. The prevalence of the composite outcome was recorded more frequently in the southern region. Multivariable analysis showed a direct association between the combined event and age, ischemic cardiac disease, and chronic kidney disease, in addition to the geographical area. CONCLUSIONS: Statistically significant heterogeneity was observed in patients with COVID-19 characteristics at admission and outcomes from northern to southern Italy. The higher frequency of ICU transfer and death in the southern region may depend on the wider hospital admission of frail patients for the availability of more beds since the burden of COVID-19 on the healthcare system was less intense in southern region. In any case, predictive analysis of clinical outcomes should consider that the geographical differences that may reflect clinical differences in patient characteristics, are also related to access to health-care facilities and care modalities. Overall, the present results caution against generalizability of prognostic scores in COVID-19 patients derived from hospital cohorts in different settings.

The density of crown-like structures in epicardial adipose tissue could play a role in cardiovascular diseases.

PURPOSE: The visceral fat of patients affected by abdominal obesity is inflamed, and the main histopathologic feature is the high density of crown-like structures (CLS). Epicardial adipose tissue (EAT) is a visceral fat of paramount importance for its relationships with coronary vessels and myocardium. Its inflammation in patients with abdominal obesity could be of clinical relevance, but histopathological studies on CLS density in EAT are lacking. This study aimed to assess the histopathology of EAT biopsies obtained from patients undergoing open-heart surgery. METHODS: We collected EAT biopsies from 10 patients undergoing open-heart surgery for elective coronary artery bypass grafting (CABG) (n = 5) or valvular replacement (VR) (n = 5). Biopsies were treated for light microscopy and immunohistochemistry. We quantify the CLS density in each EAT sample. RESULTS: Despite all patients having abdominal obesity, in EAT samples, no CLS were detected in the VR group; in contrast, CLS were detected in the CABG group (about 17 CLS/104 adipocytes vs. 0.0 CLS/104 adipocytes, CABG vs. VR group, respectively). An impressive density of CLS (100 times that of other patients) was found in one patient (LS) in the CABG group that had a relevant anamnestic aspect: relatively rapid increase of weight gain, especially in abdominal adipose tissue, coincident with myocardial infarction. CONCLUSIONS: CLS density could be an important predictive tool for cardiovascular diseases. Furthermore, the LS case implies a role for timing in weight gain. LEVEL OF EVIDENCE: No level of evidence; this is a basic science study.

Molecular and metabolic effects of extra-virgin olive oil on the cardiovascular gene signature in rodents.

BACKGROUND AND AIMS: Overweight and obesity are major risk factors for degenerative diseases, including cardiometabolic disorders and cancer. Research on fat and fatty acids' type is attracting less attention than that on carbohydrates. High adherence to a Mediterranean diet is associated with a better prognosis. One characteristic of the Mediterranean diet is extra-virgin olive oil (EVOO) as the foremost source of dietary fat. EVOO is different from other vegetable oils because it contains peculiar "minor" components, mainly phenolic in nature. Even though olive oil is highly caloric, unrestricted use of olive oil in the PREDIMED trial did not result in weight gain. We sought to study the effects of EVOO in an appropriate mouse model of increased body weight. Furthermore, we explored the biochemical and metabolomic responses to EVOO consumption. METHODS AND RESULTS: C57BL/6N male mice were weight-matched and fed ad libitum with the following diets, for 16 weeks: 1) saturated fatty acid diet (SFA) or 2) extra-virgin olive oil diet (EVOO), a custom-prepared diet, isocaloric compared to SFA, in which 82% of fat was replaced by high (poly)phenol EVOO. We evaluated glucose homeostasis, serum biochemistry and plasma metabolomics, in addition to cardiac and hepatic gene profile, and mitochondrial respiration rate. CONCLUSION: Replacing saturated fatty acids (e.g. lard) with EVOO translates into moderate yet beneficial cardiometabolic and hepatic effects. Future research will further clarify the mechanisms of action of EVOO (poly)phenols and their role in a balanced diet.

An amino acid-defined diet impairs tumour growth in mice by promoting endoplasmic reticulum stress and mTOR inhibition.

OBJECTIVE: Profound metabolic alterations characterize cancer development and, beyond glucose addiction, amino acid (AA) dependency is now recognized as a hallmark of tumour growth. Therefore, targeting the metabolic addiction of tumours by reprogramming their substrate utilization is an attractive therapeutic strategy. We hypothesized that a dietary approach targeted to stimulate oxidative metabolism could reverse the metabolic inflexibility of tumours and represent a proper adjuvant therapy. METHODS: We measured tumour development in xenografted mice fed with a designer, casein-deprived diet enriched in free essential amino acids (EAAs; SFA-EAA diet), or two control isocaloric, isolipidic, and isonitrogenous diets, identical to the SFA-EAA diet except for casein presence (SFA diet), or casein replacement by the free AA mixture designed on the AA profile of casein (SFA-CAA diet). Moreover, we investigated the metabolic, biochemical, and molecular effects of two mixtures that reproduce the AA composition of the SFA-EAA diet (i.e., EAAm) and SFA-CAA diet (i.e., CAAm) in diverse cancer and non-cancer cells. RESULTS: The SFA-EAA diet reduced tumour growth in vivo, promoted endoplasmic reticulum (ER) stress, and inhibited mechanistic/mammalian target of rapamycin (mTOR) activity in the tumours. Accordingly, in culture, the EAAm, but not the CAAm, activated apoptotic cell death in cancer cells without affecting the survival and proliferation of non-cancer cells. The EAAm increased branched-chain amino acid (BCAA) oxidation and decreased glycolysis, ATP levels, redox potential, and intracellular content of selective non-essential amino acids (NEAA) in cancer cells. The EAAm-induced NEAA starvation activated the GCN2-ATF4 stress pathway, leading to ER stress, mTOR inactivation, and apoptosis in cancer cells, unlike non-cancer cells. CONCLUSION: Together, these results confirm the efficacy of specific EAA mixtures in promoting cancer cells' death and suggest that manipulation of dietary EAA content and profile could be a valuable support to the standard chemotherapy for specific cancers.

The European Association for the Study of Obesity (EASO) Endorses the Milan Charter on Urban Obesity.

The Milan Charter on Urban Obesity highlights the challenges of urban environments as a battleground for human health, as cities are often organized to subvert public health goals, and promote rather than prevent the development of obesity and consequent non-communicable diseases. The Charter articulates ten principles which detail actions and strategies through which general practitioners, diverse medical specialists, related healthcare professionals, administrators and healthcare practice managers, policy actors - within health systems and at a national level - along with experts across disciplines, and citizens, can work in cooperation to meet this challenge and improve public health. The Charter urges the adoption of decisions that deliver the following: (i) policies which enable our cities to become healthier and less obesogenic, more supportive of well-being and less health-disruptive in general, and (ii) policies that fully support primary prevention strategies, that address social stigma, and that ensure fair access to treatment for people living with obesity. The Milan Charter on Urban Obesity aims to raise awareness of our shared responsibility for the health of all citizens, and focuses on addressing the health of people living with obesity - not only as a challenge in its own right, but a gateway to other major non-communicable diseases, including cardiovascular diseases, type 2 diabetes, and some cancers.

A Special Amino-Acid Formula Tailored to Boosting Cell Respiration Prevents Mitochondrial Dysfunction and Oxidative Stress Caused by Doxorubicin in Mouse Cardiomyocytes.

Anthracycline anticancer drugs, such as doxorubicin (DOX), can induce cardiotoxicity supposed to be related to mitochondrial damage. We have recently demonstrated that a branched-chain amino acid (BCAA)-enriched mixture (BCAAem), supplemented with drinking water to middle-aged mice, was able to promote mitochondrial biogenesis in cardiac and skeletal muscle. To maximally favor and increase oxidative metabolism and mitochondrial function, here we tested a new original formula, composed of essential amino acids, tricarboxylic acid cycle precursors and co-factors (named 5), in HL-1 cardiomyocytes and mice treated with DOX. We measured mitochondrial biogenesis, oxidative stress, and BCAA catabolic pathway. Moreover, the molecular relevance of endothelial nitric oxide synthase (eNOS) and mechanistic/mammalian target of rapamycin complex 1 (mTORC1) was studied in both cardiac tissue and HL-1 cardiomyocytes. Finally, the role of Krüppel-like factor 15 (KLF15), a critical transcriptional regulator of BCAA oxidation and eNOS-mTORC1 signal, was investigated. Our results demonstrate that the 5 mixture prevents the DOX-dependent mitochondrial damage and oxidative stress better than the previous BCAAem, implying a KLF15/eNOS/mTORC1 signaling axis. These results could be relevant for the prevention of cardiotoxicity in the DOX-treated patients.

A specific amino acid formula prevents alcoholic liver disease in rodents.

Chronic alcohol consumption promotes mitochondrial dysfunction, oxidative stress, defective protein metabolism, and fat accumulation in hepatocytes (liver steatosis). Inadequate amino acid metabolism is worsened by protein malnutrition, frequently present in alcohol-consuming patients, with reduced circulating branched-chain amino acids (BCAAs). Here we asked whether dietary supplementation with a specific amino acid mixture, enriched in BCAAs (BCAAem) and able to promote mitochondrial function in muscle of middle-aged rodents, would prevent mitochondrial dysfunction and liver steatosis in Wistar rats fed on a Lieber-DeCarli ethanol (EtOH)-containing liquid diet. Supplementation of BCAAem, unlike a mixture based on the amino acid profile of casein, abrogated the EtOH-induced fat accumulation, mitochondrial impairment, and oxidative stress in liver. These effects of BCAAem were accompanied by normalization of leucine, arginine, and tryptophan levels, which were reduced in liver of EtOH-consuming rats. Moreover, although the EtOH exposure of HepG2 cells reduced mitochondrial DNA, mitochondrial transcription factors, and respiratory chain proteins, the BCAAem but not casein-derived amino acid supplementation halted this mitochondrial toxicity. Nicotinamide adenine dinucleotide levels and sirtuin 1 (Sirt1) expression, as well as endothelial nitric oxide (eNOS) and mammalian/mechanistic target of rapamycin (mTOR) signaling pathways, were downregulated in the EtOH-exposed HepG2 cells. BCAAem reverted these molecular defects and the mitochondrial dysfunction, suggesting that the mitochondrial integrity obtained with the amino acid supplementation could be mediated through a Sirt1-eNOS-mTOR pathway. Thus a dietary activation of the mitochondrial biogenesis and function by a specific amino acid supplement protects against the EtOH toxicity and preserves the liver integrity in mammals. NEW & NOTEWORTHY Dietary supplementation of a specific amino acid formula prevents both fat accumulation and mitochondrial dysfunction in hepatocytes of alcohol-consuming rats. These effects are accompanied also by increased expression of anti-reactive oxygen species genes. The amino acid-protective effects likely reflect activation of sirtuin 1-endothelial nitric oxide synthase-mammalian target of rapamycin pathway able to regulate the cellular energy balance of hepatocytes exposed to chronic, alcoholic damage.

Family lifestyle and childhood obesity in an urban city of Northern Italy.

PURPOSE: Over the last decades, the prevalence of overweight and obesity in elementary school children has steadily increased worldwide. This phenomenon is also linked to food habits. The main purpose of our study was to understand the role that environmental factors may play in this context; in particular, we investigated how and to what extent family food habits and children lifestyle are associated with the spread of children obesity. METHODS: One hundred and nine primary schools, with 6-11-year-old children (n = 14,500), were recruited for this cross-sectional study in Milan (Italy). Children anthropometric data were measured and reported by parents; citizenship, fruit and vegetable consumption data of both parents and children were collected. Time spent watching television and doing physical activity was also investigated in children. RESULTS: The study revealed that children's vegetable (not fruit) consumption was positively associated with physical activity, while negatively associated with time watching TV; in particular, fewer hours spent watching television were a stronger protective factor than more hours spent doing physical activity. Moreover, the parental feeding style was associated with children's attitudes toward consumption of fruit and vegetable. Family characteristics (family size and level of parents' education) and children gender were associated to the risk of being overweight/obese. CONCLUSIONS: Our findings support the relevance of environmental factors in childhood food consumption and BMI distribution among children in an urban city. This is the reason why we stress the need to design ad hoc interventions, which should be developed in accordance with the socio-economic peculiarities of a cosmopolitan city suburb.

Muscle uncoupling protein 3 expression is unchanged by chronic ephedrine/caffeine treatment: results of a double blind, randomised clinical trial in morbidly obese females.

UNLABELLED: Ephedrine/caffeine combination (EC) has been shown to induce a small-to-moderate weight loss in obese patients. Several mechanisms have been proposed, among which an increased thermogenic capacity of skeletal muscle consequent to the EC-induced up-regulation of uncoupling protein 3 (UCP3) gene expression. We did a parallel group double-blind, placebo-controlled, 4-week trial to investigate this hypothesis. Thirteen morbidly obese women (25-52 years of age, body-mass index 48.0±4.0 kg/m2, range 41.1-57.6) were randomly assigned to EC (200/20 mg, n = 6) or to placebo (n = 7) administered three times a day orally, before undergoing bariatric surgery. All individuals had an energy-deficit diet equal to about 70% of resting metabolic rate (RMR) diet (mean 5769±1105 kJ/day). The RMR analysed by intention to treat and the UCP3 (long and short isoform) mRNA levels in rectus abdominis were the primary outcomes. Body weight, plasma levels of adrenaline, noradrenaline, triglycerides, free fatty acids, glycerol, TSH, fT4, and fT3 were assessed, as well as fasting glucose, insulin and HOMA index, at baseline and at the end of treatments. Body weight loss was evident in both groups when compared to baseline values (overall -5.2±3.2%, p<0.0001) without significant differences between the treated groups. EC treatment increased the RMR (+9.2±6.8%, p = 0.020), differently from placebo which was linked to a reduction of RMR (-7.6±6.5%, p = 0.029). No significant differences were seen in other metabolic parameters. Notably, no changes of either UCP3 short or UCP3 long isoform mRNA levels were evident between EC and placebo group. Our study provides evidence that 4-week EC administration resulted in a pronounced thermogenic effect not related to muscle UCP3 gene expression and weight loss in morbidly obese females under controlled conditions. TRIAL REGISTRATION: ClinicalTrials.gov NCT02048215.

Cannabinoid receptor stimulation impairs mitochondrial biogenesis in mouse white adipose tissue, muscle, and liver: the role of eNOS, p38 MAPK, and AMPK pathways.

OBJECTIVE: Cannabinoid type 1 (CB1) receptor is involved in whole-body and cellular energy metabolism. We asked whether CB1 receptor stimulation was able to decrease mitochondrial biogenesis in different metabolically active tissues of obese high-fat diet (HFD)-fed mice. RESEARCH DESIGN AND METHODS: The effects of selective CB1 agonist arachidonyl-2-chloroethanolamide (ACEA) and endocannabinoids anandamide and 2-arachidonoylglycerol on endothelial nitric oxide synthase (eNOS) expression were examined, as were mitochondrial DNA amount and mitochondrial biogenesis parameters in cultured mouse and human white adipocytes. These parameters were also investigated in white adipose tissue (WAT), muscle, and liver of mice chronically treated with ACEA. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation was investigated in WAT and isolated mature adipocytes from eNOS(-/-) and wild-type mice. eNOS, p38 MAPK, adenosine monophosphate-activated protein kinase (AMPK), and mitochondrial biogenesis were investigated in WAT, muscle, and liver of HFD mice chronically treated with ACEA. RESULTS: ACEA decreased mitochondrial biogenesis and eNOS expression, activated p38 MAPK, and reduced AMPK phosphorylation in white adipocytes. The ACEA effects on mitochondria were antagonized by nitric oxide donors and by p38 MAPK silencing. White adipocytes from eNOS(-/-) mice displayed higher p38 MAPK phosphorylation than wild-type animals under basal conditions, and ACEA was ineffective in cells lacking eNOS. Moreover, mitochondrial biogenesis was downregulated, while p38 MAPK phosphorylation was increased and AMPK phosphorylation was decreased in WAT, muscle, and liver of ACEA-treated mice on a HFD. CONCLUSIONS: CB1 receptor stimulation decreases mitochondrial biogenesis in white adipocytes, through eNOS downregulation and p38 MAPK activation, and impairs mitochondrial function in metabolically active tissues of dietary obese mice.

Amino acids and mitochondrial biogenesis.

Mitochondria are sources of energy production through their role in producing adenosine triphosphate for cell metabolism. Defective mitochondrial biogenesis and function play relevant roles in the pathophysiology of relevant diseases, including obesity, diabetes mellitus, myopathies, and neurodegenerative diseases. Their function is the product of synthesis of macromolecules within the mitochondria and import of proteins and lipids synthesized outside the organelles. Both are required for mitochondrial proliferation and may also facilitate the growth of preexisting mitochondria. Recent evidence indicates that these events are regulated in a complex way by several agonists and environmental conditions, through activation of specific signaling pathways and transcription factors. Nitric oxide (NO) appears to be a novel modulator of mitochondrial biogenesis. High levels of NO acutely inhibit cell respiration by binding to cytochrome c oxidase. Conversely, chronic, low-grade increases of NO stimulate mitochondrial biogenesis in diverse cell types. Here, we suggest that some types of nutrients, including specific mixtures of amino acids, may improve mitochondrial biogenesis and energy production in energy-defective conditions by increasing endothelial NO synthase expression.

Cannabinoid type 1 receptor blockade promotes mitochondrial biogenesis through endothelial nitric oxide synthase expression in white adipocytes.

OBJECTIVE: Cannabinoid type 1 (CB1) receptor blockade decreases body weight and adiposity in obese subjects; however, the underlying mechanism is not yet fully understood. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) induces mitochondrial biogenesis and function in adipocytes. This study was undertaken to test whether CB1 receptor blockade increases the espression of eNOS and mitochondrial biogenesis in white adipocytes. RESEARCH DESIGN AND METHODS: We examined the effects on eNOS and mitochondrial biogenesis of selective pharmacological blockade of CB1 receptors by SR141716 (rimonabant) in mouse primary white adipocytes. We also examined eNOS expression and mitochondrial biogenesis in white adipose tissue (WAT) and isolated mature white adipocytes of CB1 receptor-deficient (CB1(-/-)) and chronically SR141716-treated mice on either a standard or high-fat diet. RESULTS: SR141716 treatment increased eNOS expression in cultured white adipocytes. Moreover, SR141716 increased mitochondrial DNA amount, mRNA levels of genes involved in mitochondrial biogenesis, and mitochondrial mass and function through eNOS induction, as demonstrated by reversal of SR141716 effects by small interfering RNA-mediated decrease in eNOS. While high-fat diet-fed wild-type mice showed reduced eNOS expression and mitochondrial biogenesis in WAT and isolated mature white adipocytes, genetic CB1 receptor deletion or chronic treatment with SR141716 restored these parameters to the levels observed in wild-type mice on the standard diet, an effect linked to the prevention of adiposity and body weight increase. CONCLUSIONS: CB1 receptor blockade increases mitochondrial biogenesis in white adipocytes by inducing the expression of eNOS. This is linked to the prevention of high-fat diet-induced fat accumulation, without concomitant changes in food intake.

TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents.

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

Nitric oxide and mitochondrial biogenesis.

The characteristic structural organization of mitochondria is the product of synthesis of macromolecules within the mitochondria together with the import of proteins and lipids synthesized outside the organelle. Synthetic and import processes are required for mitochondrial proliferation and might also facilitate the growth of pre-existing mitochondria. Recent evidence indicates that these events are regulated in a complex way by several agonists and environmental conditions, through activation of specific signaling pathways and transcription factors. A newly discovered role of this organelle in retrograde intracellular signaling back to the nucleus has also emerged. This is likely to have far-reaching implications in development, aging, disease and environmental adaptation. Generation of nitric oxide (NO) appears to be an important player in these processes, possibly acting as a unifying molecular switch to trigger the whole mitochondrial biogenesis process. High levels of NO acutely inhibit cell respiration by binding to cytochrome c oxidase. Conversely, chronic, smaller increases in NO levels stimulate mitochondrial biogenesis in diverse cell types. NO-induced mitochondrial biogenesis seems to be linked to proliferation and differentiation of normal and tumor cells, as well as in aging.

Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS.

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Emerging aspects of pharmacotherapy for obesity and metabolic syndrome.

Obesity is a multifactorial, chronic disorder that has reached epidemic proportions in most industrialized countries and is threatening to become a global epidemic. Obese patients are at higher risk from coronary artery disease, hypertension, hyperlipidemia, diabetes mellitus, cancers, cerebrovascular accidents, osteoarthritis, restrictive pulmonary disease, and sleep apnoea. In particular, visceral fat accumulation is usually accompanied by insulin resistance or type 2 diabetes mellitus, hypertension, hypertriglyceridemia, high uremic acid levels, low high density lipoprotein (HDL) cholesterol to define a variously named syndrome or metabolic syndrome. Metabolic syndrome is now considered a major cardiovascular risk factor in a large percentage of population in worldwide. Both obesity and metabolic syndrome are particularly challenging clinical conditions to treat because of their complex pathophysiological basis. Indeed, body weight represents the integration of many biological and environmental components and relationships among fat and glucose tolerance or blood pressure are not completely understood. Efforts to develop innovative anti-obesity drugs, with benefits for metabolic syndrome, have been recently intensified. In general two distinct strategies can be adopted: first, to reduce energy intake; second, to increase energy expenditure. Here we review some among the most promising avenues in these two fields of drug therapy of obesity and, consequently, of metabolic syndrome.