Transformation and action of extracellular NAD+ in perfused rat and mouse livers

Abstract

Aim: Transformation and possible metabolic effects of extracellular NAD + were investigated in the livers of mice (Mus musculus; Swiss strain) and rats (Rattus novergicus; Holtzman and Wistar strains). Methods: The livers were perfused in an open system using oxygen-saturated Krebs/Henseleit-bicarbonate buffer (pH 7.4) as the perfusion fluid. The transformation of NAD + was monitored using high-performance liquid chromatography. Results: In the mouse liver, the single-pass metabolism of 100 µmol/L NAD + was almost complete; ADP-ribose and nicotinamide were the main products in the outflowing perfusate. In the livers of both Holtzman and Wistar rats, the main transformation products were ADP-ribose, uric acid and nicotinamide; significant amounts of inosine and AMP were also identified. On a weight basis, the transformation of NAD + was more efficient in the mouse liver. In the rat liver, 100 µmol/L NAD + transiently inhibited gluconeogenesis and oxygen uptake. Inhibition was followed by a transient stimulation. Inhibition was more pronounced in the Wistar strain and stimulation was more pronounced in the Holtzman strain. In the mouse liver, no clear effects on gluconeogenesis and oxygen uptake were found even at 500 µmol/L NAD + . Conclusion:It can be concluded that the functions of extracellular NAD + are species-dependent and that observations in one species are strictly valid for that species. Interspecies extrapolations should thus be made very carefully. Actually, even variants of the same species can demonstrate considerably different responses.

Co‐regulation of primary mouse hepatocyte viability and function by oxygen and matrix

“…We posit this is the first reported value of per‐cell oxygen consumption from in vitro mouse hepatocyte cultures. These values are also of a similar magnitude to those reported for rat and human in vitro hepatocyte cultures, which range from 0.2 to 0.7 nmol/10 6 cells/s (Broetto‐Biazon et al, ; Domansky et al, ; Matsumara et al, ; Metzen et al, ). While mouse hepatocytes are found to be more highly metabolic than rat and human hepatocytes when tested in a single lab, there is a wide range of variability in measured oxygen consumption rates across labs, making it difficult to definitively assess how our observed consumption rates would compare to primary rat hepatocytes cultured similarly.…”

“…Under standard culture conditions (20% O 2 at either 1.3 or 2.6 mm medium depth), our observed per cell oxygen consumption rate of approximately 0.4 nmol/10 6 cells/s (assuming ∼1 mg protein/10 6 cells) is comparable to those rates measured in perfused livers and mitochondrial preparations from mice, which range from 0.3 to 0.8 nmol/10 6 cells/s (Broetto‐Biazon et al, ; Matoba et al, ; Porter and Brand, ). We posit this is the first reported value of per‐cell oxygen consumption from in vitro mouse hepatocyte cultures.…”

“…While estimates of cellular oxygen consumption derived from perfused liver measurements can be a useful guide for setting up in vitro culture conditions (Broetto‐Biazon et al, ; Matsumara and Thurman, ; Matsumara et al, ; Meren et al, ), metabolic rates are typically context‐dependent and in vitro measurements are desirable to obtain more precise control over culture variables that influence oxygen concentration in the hepatocellular microenvironment. The compendium of measured in vitro hepatocellular oxygen consumption rates under various culture configurations is growing as interest in 3D liver models increases (Domansky et al, ; Metzen et al, ).…”

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3‐dependent mechanism in male mice

“…NAD + , NMN, NR, and ATP were separated and measured using the Agilent Chemstation 1100 high‐performance liquid chromatography (HPLC) machine with a Jupiter C18 300A (250 × 4.6 mm, 5 micron) column. To the extracted metabolites from hippocampal tissue samples, a concentration gradient similar to (Broetto‐Biazon, Bracht, Bracht, Kelmer‐Bracht, & Bracht, ), see also (Long et al, ) was created with 50 mM sodium phosphate pH 6 and 50% of 50 mM sodium phosphate in HPLC grade methanol pH 7. The gradient consisted of following steps (in % methanol): 0 min, 0%; 6.5 min, 0.5%; 9 min, 3%; 11 min, 5%; 12 min, 12%; 14 min, 15%; 16 min, 20%; 21 min, 30%; 22 min, 50%.…”

Insulin degludec and glutamine dipeptide modify glucose homeostasis and liver metabolism in diabetic mice undergoing insulin-induced hypoglycemia

“…After 30-day treatment, 15 h fasted control and T1D mice received regular insulin to induce IIH and cortisol 15 min later. One hour after IIH, the non-recirculating liver perfusion was performed as previously described (Broetto-Biazon et al, 2009;Nunes Santiago et al, 2013). After an initial period of 10 min of perfusion to stabilize the preparation, samples of the effluent perfusion fluid were collected at five-minute intervals and analyzed for their content of glucose, lactate, pyruvate, ammonia and urea (Bergmeyer, 1974).…”