Daniel Kraus

Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD+, an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine–spermine N1-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD+ levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD+-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

Leveraging leptin for type I diabetes

The discovery of insulin in 1922 is one of the miracles of modern medicine, and it turned a once deadly disease—insulin-deficient (autoimmune) type 1 diabetes mellitus (T1DM)—into a manageable one. It is now clear that insulin is a key metabolic regulator that is vital to glucose and lipid homeostasis and affects many aspects of growth and development. Insulin’s fundamental importance in biology is underscored by the conservation of insulin-like hormones and their receptors from flies and worms to humans. In PNAS, Wang et al. (1) extend previous findings (2–4) to suggest that another hormone, leptin, may substitute for or be used in combination with insulin to treat T1DM more effectively.

Quantitative assessment of adipocyte differentiation in cell culture

ABSTRACT Adipocyte cell culture is an important tool for mechanistic studies of energy metabolism. Many factors affect the differentiation of adipocytes in culture. Oil red O staining can be used to assess the degree of differentiation. However, the validity of this method for quantitative analysis has not yet been established. Here we show that a protocol with arbitrarily chosen parameters does not measure in the linear range and is not suitable for quantitative analysis (R2 = 0.077, p = 0.382), and develop and validate an optimized protocol for quantitative oil red O staining of cultured adipocytes. 3T3-L1 preadipocytes and adipocytes are fixed with 4% formaldehyde and stained with 0.2% oil red O solution in 40% 2-propanol for 30 minutes. Dye is eluted with 2-propanol, and absorption of the eluate is measured photometrically at 510 nm. This optimized protocol achieves excellent correlation between defined amounts of differentiated adipocytes on constant-size culture plates and photometric absorption (R2 = 0.972, p = 6.585E-14). The performance of the method is independent of the culture plates used. Thus, the optimized oil red O staining protocol can be universally employed to quantitatively assess adipocyte differentiation.

Body weight predicts Nicotinamide N-Methyltransferase activity in mouse fat

ABSTRACT Aim: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipose tissue. NNMT expression is higher in obese mice than in lean mice, and NNMT knockdown prevents diet-induced obesity. Little is known about the regulation of enzyme activity during the development of obesity. The aim of this study was to analyze NNMT activity in tissues of mice with incipient and established obesity. Methods: A fluorescence-based, sensitive, low-volume, high-throughput method was developed to assay NNMT activity. C57BL/6 mice were fed a high-fat diet for 4 weeks (incipient obesity) and for 12 weeks (established obesity). Tissues and serum were harvested and analyzed. Results: NNMT activity was highest in subcutaneous white fat (55.0 µU/mg), followed by epididymal white fat (35.6 µU/mg), brown adipose tissue (7.8 µU/mg), liver (7.6 µU/mg), and lung (7.3 µU/mg). Little activity was detected in heart, skeletal muscle, and kidney. No activity was found in serum samples. Body weight predicted NNMT activity in white fat, but not in brown fat or any other tissue, and only in incipient obesity. With established obesity, this association was lost. Conclusions: As obesity develops, body weight predicts NNMT activity in white adipose tissue, but not in any other tissue, consistent with a specific role of adipose-tissue NNMT in the regulation of body weight.

Uromodulin in the Bloodstream: Old Wine in a New Wineskin

“What is man, when you come to think upon him, but a minutely set, ingenious machine designed to turn, with infinite artfulness, the red wine of Shiraz into urine?,” wrote Karen Blixen (1885–1962) under her pen name, Isak Dinesen, in Seven Gothic Tails .[1][1] Urine, of course, is much more

Urinary neutrophil gelatinase-associated lipocalin and cystatin C compared to the estimated glomerular filtration rate to predict risk in patients with suspected acute myocardial infarction

INTRODUCTION Impaired renal function, reflected by estimated glomerular filtration rate (eGFR) or cystatin C, is a strong risk predictor in the presence of acute myocardial infarction (AMI). Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is an early marker of acute kidney injury. uNGAL might also be a good predictor of outcome in patients with cardiovascular disease. Aim of the present study was to evaluate the prognostic value of uNGAL compared to eGFR and cystatin C in patients with suspected AMI. METHODS 1818 patients were enrolled with suspected AMI. Follow-up information on the combined endpoint of death or non-fatal myocardial infarction was obtained 6months after enrolment and was available in 1804 patients. 63 events (3.5%) were registered. RESULTS While cystatin C and eGFR were strong risk predictors for the primary endpoint even adjusted for several variables, uNGAL was not independently associated with outcome: When applied continuously uNGAL was associated with outcome but did not remain a statistically significant predictor after several adjustments (i.e. eGFR). By adding cystatin C or uNGAL to GRACE risk score variables, only cystatin C could improve the predictive value while uNGAL showed no improvement. CONCLUSION We could show that cystatin C is an independent risk predictor in patients with suspected AMI and cystatin C can add improvement to the commonly used GRACE risk score. In contrast uNGAL is not independently associated with outcome and seems not to add further prognostic information to GRACE risk score.

A Janus-headed electrolyte and ‘RALES' disease’

In ancient Roman mythology, Janus is the god of beginnings and transitions. Janus has two faces, one looking back into the past and one looking ahead into the future (Figure 1). According to Ovid, having two faces also enabled him to never turn his back on his adored Cardea. In the figurative sense, something that is Janus‐headed is ambivalent and must be seen in context. In this regard, potassium is the Janus among electrolytes in heart failure. Potassium is abundant in cells and sparse in the extracellular space. This concentration gradient helps to build up the transmembrane potential that is indispensable for electrical signal conduction in the heart and in other organs. A decrease in extracellular potassium concentration augments, and an increase dampens excitability. Both conditions can severely impair normal physiological function. Serum potassium levels are largely determined by urinary potassium excretion. By acting on the expression of sodium and potassium transporters in the collecting tubules, the renin–angiotensin–aldosterone system (RAAS) causes sodium retention and potassium excretion. In patients with heart failure (HF), low levels of serum potassium are associated with increased mortality. Potassium depletion may result from neurohumoral RAAS activation in these patients, as well as from diuretic treatment, a mainstay in the management of heart failure. In the SOLVD trial cohort, the use of non‐potassium‐sparing diuretics was an independent risk factor for sudden cardiac death. The RALES, EPHESUS, and EMPHASIS‐HF trials have firmly established the role of potassium‐sparing mineralocorticoid receptor antagonists (MRA) in the treatment of heart failure. Aldosterone and eplerenone retain potassium and have direct effects on the myocardium. Because aldosterone levels are often increased despite RAAS blockade, simultaneous application of MRA has an additive effect over ACE inhibitors and angiotensin receptor blockers. On the other hand, the concomitant use of MRA and other RAAS blocking agents may put patients at risk of fatal arrhythmias due to hyperkalaemia—Janus’ second face. This is most commonly seen in those that exhibit reduced aldosterone levels in the first place, e.g. due to hyporeninaemic hypoaldosteronism. Elderly patients and those with diabetes mellitus or/and kidney disease are at highest risk. In the wake of the landmark MRA trials, the prescription rates for MRA have sharply increased. A concomitant increase in the rates of hyperkalaemia has been reported in one study (Figure 2), but not in another. However, the coincidence of hospital admissions due to very severe hyperkalaemia and the dual use of MRA and other RAAS blocking medications is now such a commonplace clinical experience that the term ‘RALES’ disease’ (Morbus RALES) has gained currency among emergency physicians. To assess the actual risk of patients on dual MRA and other RAAS blocking treatment, we queried Würzburg University Hospital’s Clinical Data Warehouse (Table 1). The Data Warehouse is a clinical registry that includes every inpatient and outpatient case managed by any department at this tertiary care centre. Out of ~5 million cases, we identified 118 691 cases (2.4%) between 2003 and January 2017 with at least one RAAS blocking agent (including an MRA). Dual ED ITOR IAL

Organersatzverfahren - Nierenersatzverfahren in der Intensivmedizin

Schwer kranke, intensivmedizinisch behandelte Patienten haben ein hohes Risiko, ein akutes Nierenversagen zu entwickeln. Jedes Nierenversagen verkurzt die weitere Lebenserwartung. Ein akutes Nierenversagen fuhrt aber nicht mehr unmittelbar zum Tod, weil die vielfaltigen Funktionen der Niere medikamentos oder maschinell ersetzt werden konnen. Hamodialyse und Hamofiltration sind die grundlegenden Verfahren der maschinellen Nierenersatztherapie; sie konnen kontinuierlich oder intermittierend durchgefuhrt werden. Die Entscheidung fur ein bestimmtes Verfahren muss vom Einzelfall abhangig gemacht werden. Die Peritonealdialyse ist eine Alternative zur Blutwasche, stellt aber besondere personelle und institutionelle Anforderungen. Rontgenkontrastmittel und Rhabdomyolyse sind haufige Ursachen eines toxischen akuten Nierenversagens auf der Intensivstation, konnen durch Nierenersatzverfahren aber nicht verhindert werden.