Melanie Penke

FK866-induced NAMPT inhibition activates AMPK and downregulates mTOR signaling in hepatocarcinoma cells

BACKGROUND Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. RESULTS FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. CONCLUSION Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC.

Hepatic NAD salvage pathway is enhanced in mice on a high-fat diet.

Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for NAD salvage and the abundance of Nampt has been shown to be altered in non-alcoholic fatty liver disease. It is, however, unknown how hepatic Nampt is regulated in response to accumulation of lipids in the liver of mice fed a high-fat diet (HFD). HFD mice gained more weight, stored more hepatic lipids and had an impaired glucose tolerance compared with control mice. NAD levels as well as Nampt mRNA expression, protein abundance and activity were significantly increased in HFD mice. Enhanced NAD levels were associated with deacetylation of p53 and Nfκb indicating increased activation of Sirt1. Despite impaired glucose tolerance and increased hepatic lipid levels in HFD mice, NAD metabolism was significantly enhanced. Thus, improved NAD metabolism may be a compensatory mechanism to protect against negative impact of hepatic lipid accumulation.

Diabetes mellitus in children and adolescents - a global epidemic which has become neglected.

Incidences and prevalences of type 1 diabetes in children and adolescents are rising globally and continuously (1). This trend has almost reached epidemic proportions and occurs almost uncommented on outside the scientific and medical community of diabetes specialists. Diabetes will affect all parts of our societies and challenge societies financial resources and the health care systems worldwide (2). In this issue of the Journal of Pediatric Endocrinology and Metabolism ( JPEM ) we have therefore assembled a series of papers dealing with topics from several areas of diabetes research and clinical issues to draw attention to the increasingly important topic of diabetes in the young.

Childhood obesity at the crossroads

Overweight and obesity is frequently seen in children and adolescents across most developed, and developing, countries with a 20–35% prevalence. Although the prevalence of obesity at a young age seems to stabilise or even slightly decline in some countries, the number of adolescents with obesity is still increasing (1). This is a major concern, considering the well-described association of overweight and obesity with long-term health problems, such as cardiovascular disease, type 2 diabetes and cancer. In the majority of individuals, weight gain is the result of exposure to an ‘obesogenic’ environment, superimposed on a background of genetic susceptibility brought about through evolutionary adaptation (2). Approximately 40–70% of inter-individual differences in body weight and fat content is thought to be due to genetic variation (3). A large number of genes have been identified by genome wide association studies (GWAS) and candidate gene approaches that are associated with the regulation of body weight. According to the thrifty gene hypothesis, evolutionary selection pressure has selected genes which allow individuals to survive periods of food deprivation. Within our modern obesogenic environment, however, these same genetic susceptibility traits now appear to be detrimental by promoting obesity and its associated metabolic diseases (2). In this issue of our journal, three review papers, one on behavioural aspects of obesity (4), one on hypothalamic forms of obesity (5) and lastly and importantly one on the role of obesigenic environments address the important public health issue of childhood obesity. These reviews are followed by a series of papers that have been submitted recently to the journal and which cover a wide range of childhood obesity related topics mainly dealing with cardiovascular and metabolic sequelae of obesity. Scientific dilemma

Novel Insights in the Metabolic Syndrome in Childhood and Adolescence

Metabolic syndrome (MetS) is recognized as an escalating major health risk in adults as well as in children and adolescents. Its prevalence ranges from 6 to 39% depending on the applied definition criteria. To date, there is no consensus on a MetS definition for children and adolescents. However, most authors agree on essential components such as glucose intolerance, central obesity, hypertension, and dyslipidemia; each representing a risk for cardiovascular disease. Recently, associations between MetS and non-alcoholic fatty liver disease, hyperuricemia, and sleep disturbances have emerged. Biomarkers like adipocytokines are a subject of current research as they are implicated in the pathogenesis of the MetS. Epigenetics and gestational programming, especially the role of microRNA, comprise a novel, rapidly developing and promising research focus on the topic of MetS. MicroRNAs are increasingly valued for potential roles in the diagnosis, stratification, and therapeutics of MetS. Early detection of risk factors, screening for metabolic disturbances, and the identification of new therapies are major aims to reduce morbidity and mortality related to MetS. Dietary modification and physical activity are currently the only adopted treatment approaches. Pharmacological therapies and bariatric surgery are still contradictory and, therefore, are only recommended in selected high-risk cases.

Altered hepatic lipid metabolism in mice lacking both the melanocortin type 4 receptor and low density lipoprotein receptor.

Obesity is often associated with dyslipidemia and hepatosteatosis. A number of animal models of non-alcoholic fatty liver disease (NAFLD) are established but they significantly differ in the molecular and biochemical changes depending on the genetic modification and diet used. Mice deficient for melanocortin type 4 receptor (Mc4rmut) develop hyperphagia, obesity, and subsequently NAFLD already under regular chow and resemble more closely the energy supply-driven obesity found in humans. This animal model was used to assess the molecular and biochemical consequences of hyperphagia-induced obesity on hepatic lipid metabolism. We analyzed transcriptome changes in Mc4rmut mice by RNA sequencing and used high resolution 1H magic angle spinning NMR spectroscopy and MALDI-TOF mass spectrometry to assess changes in the lipid composition. On the transcriptomic level we found significant changes in components of the triacylglycerol metabolism, unsaturated fatty acids biosynthesis, peroxisome proliferator-activated receptor signaling pathways, and lipid transport and storage compared to the wild-type. These findings were supported by increases in triacylglycerol, monounsaturated fatty acid, and arachidonic acid levels. The transcriptome signatures significantly differ from those of other NAFLD mouse models supporting the concept of hepatic subphenotypes depending on the genetic background and diet. Comparative analyses of our data with previous studies allowed for the identification of common changes and genotype-specific components and pathways involved in obesity-associated NAFLD.

Non-alcoholic fatty liver disease in children and adolescents

Obesity is increasing in prevalence throughout the world and with this change there is a major increase in associated cardiac, metabolic and other diseases [1]. In fact, according to the definition that the World Health Organization proposed in 2000, overweight and obesity are seen as a social hindrance and it is known that both these conditions are associated with cardiovascular and metabolic diseases [2]. In particular, since 1980, the prevalence of childhood obesity has almost tripled in USA and in some European countries. In particular, it has been estimated that 200 million school-aged children were overweight or obese in 2010 worldwide, with a prevalence of > 20% in European states and > 30% in regions of North America [3]. The prevalence of obesity both in childhood and adulthood remains very high. Recently, several authors have suggested that in some Western countries such as the United States, Western Europe, Australia, and Japan, the levels of childhood obesity may have reached a plateau in the last decade and do not increase further while the number of obese adolescents is still increasing [4]. In addition, overweight and obesity during childhood and adolescence leads to adult obesity and the progression of the metabolic syndrome [5]. Definitions of the metabolic syndrome are wide-ranged but most often a cluster of different metabolic disorders leading to e.g. cardiovascular diseases, abdominal obesity, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD).

Vitamin D in health and disease: the global threat of vitamin D deficient rickets

Nowadays, infectious diseases are less prevalent in many countries around the world and chronic diseases including obesity, diabetes, behavioral problems and nutritional deficiencies are thought to have become the major and eminent health burdens in both the adult and the young population. Among these, vitamin D deficiency poses a major health problem in children and adolescents globally. In fact, even in countries with high sun and specifically high ultraviolet B (UVB) exposure such as Brasil and Australia vitamin D deficiency rickets has become very prevalent. In Australia this has been attributed to the use of sun protecting ointments and changes in life styles that lead to lesser sun exposure especially in children and young people [1]. In populations with dark skin living, for example, in Africa or the Middle East, the prevalence of vitamin D insufficiency with 25-hydroxyvitamin D (25(OH)D) levels ranging between 15 and 20 ng/ mL (37.5–50 nmol/L) ranges from 5% to 80%. Risk factors to develop nutritional rickets include traditional dress and avoidance of sunlight exposure, and also multiple dietary factors as a result of specific cultural beliefs in many of the affected countries as nutritional rickets may also be caused by inadequate dietary intake of calcium when, for example, milk and milk products are consumed less or are not available [2]. As a result of vitamin D and/or calcium deficiency severe bone disease, stunted growth and even the emergence of autoimmune disease and alterations of the immune system are being observed in many populations.

Thyroid disorders revisited.

Thyroid disorders are still a cause of health concerns, major illness, and comorbidities in neonates, children and adolescents around the world. Neonatal screening programs involve testing for congenital hypothyroidism usually using thyroid stimulating hormone (TSH) measurements in dried blood spots, and in developed countries, it is clinically and ethically mandatory to install neonatal screening programs for hypothyroidism. Also, as far as obese children are concerned, families and doctors alike tend to automatically ask about a child’s thyroid status. In addition, fatigue in a schoolchild is frequently met with concerns about whether or not there was an underlying thyroid dysfunction. However, most of the time, common causes such as psychosomatic illness or normal exhaustion are the reason for tiredness and thyroid function in these children are quite normal. Both hypothyroidism and hyperthyroidism may occur early in life, and thyroid cancer is of relevance in paediatric oncology and may be either sporadic or related to radiation be it after cancer treatments or environmental radiation exposure (Chernobyl accident and alike). However, overall clinically relevant disorders of the thyroid are still rather uncommon: congenital hypothyroidism occurs at a frequency of 1 in 3000, hypothyroidism in Hashimoto’s thyroiditis occurs in 1 in 2000, and lastly hyperthyroidism usually related to Graves’ disease is detected in 1 in 100,000 children and adolescents (1). Importantly, the relationship between different thyroid disorders is sometimes not completely understood as is shown by the cooccurrence of papillary thyroid cancer in patients with hyperthyroidism (2) and/or with autoimmune polyglandular syndrome (3). Lastly standardized reference values for serum concentrations of anti-thyroid peroxidase and anti-thyroglobulin antibodies and most importantly for TSH and thyroid hormones in relation to age, body mass index and to a much smaller extent to gender are to be employed when one investigates children’s thyroid status and health (4). The number of leucocytes in blood is related to thyroid hormone serum concentrations (5). In this issue of the Journal of Paediatric Endocrinology and Metabolism, scientific novelties and surprising clinical findings related to thyroid disorders are the focus of nine articles that will be introduced.

Hepatic NAD+ levels and NAMPT abundance are unaffected during prolonged high-fat diet consumption in C57BL/6JBomTac mice

Dietary supplementation of nicotinamide adenine dinucleotide (NAD+) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD+ is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD+ have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD+ salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD+ levels, but caused a decrease in NADP+ and NADPH levels. Decreased NADP+ content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD+ salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD+ pool in HFD-challenged C57BL/6JBomTac male mice.