Metabolism in Hepatocytes and Adipocytes under inhibition of carbonic anhydrases studied with multinuclear NMR-spectroscopy

Abstract

Obesity is defined as an intense overweight condition and is an increasingly serious problem for the health of population, particularly in the modern Western countries. The excessive body weight is associated with various diseases, especially cardiovascular diseases, diabetes mellitus type 2, certain types of cancer, sleep apnea, and osteoarthritis.There are therefore already a number of therapeutic procedures aimed at the treatment or prophylaxis of obesity. A novel therapeutic procedure for the treatment and/or prophylaxis of obesity, which can complement the previously known form of therapy, intends the inhibition of de novo lipogenesis (DNL). DNL means the synthesis of endogenous fatty acids from carbohydrate in the mammalian organism. There are various conceivable possibilities to inhibite DNL in mammalian cells, all of which aim to reduce the turnover of the TCA cycle. The inhibition of carbonic anhydrases (CAs) isozymes involving in several steps of the de novo lipogenesis can inhibit the DNL. The CAs catalyze a very simple physiological reaction, the interconversion of carbon dioxide and bicarbonate. It is comprehensible that inhibition of mitochondrial CA (i.e. CA V) and/or cytosolic CA (i.e. CA II) subsequently affect pyruvate carboxylation. The carbohydrates are not only used to synthesis lipids, but also TCA cycle intermediates and TCA cycle related amino acids such as glutamate. Sulphonamide/ Sulphamate are carbonic anhydrase inhibitors (CAIs) and good candidates to inhibit DNL on the step of pyruvate carboxylation in mitochondria and/or in cytosol. Inhibition of CA will reduce the influx into the anaplerotic activity of pyruvate carboxylase (pc) and subsequently into the citric acid (TCA) cycle, which will modify the intermediates of the TCA cycle and its effluxes (in particular glutamate) and lipid synthesis. The inhibition of carboanhydrase affects also the synthesis of alanine and lactate. To study the effect of CAIs upon the synthesis of lipids and other metabolites in HEP-G2 and 3T3-L1 cells multinuclear NMR spectroscopy was used. To investigate the effect of CAIs on the cellular metabolism, the HEP-G2 and 3T3-L1 cells were incubated for 6, 12, or 24h in the presence or absence of CAIs, and metabolic alterations of labeled substrates were followed up using NMR spectroscopy. The data of the present study clearly show that topiramate (TPM) and acetazolamide (ACT) are nonspecific inhibitors of CA isozymes (IC50 ≥1000 à �à µM), but DNL in the presence of lower concentrations of ethoxyzolamide (ETZ) is more effectively inhibited. The results also show that compound 1 (a newly synthesised sulfonamide) is a very efficient candidate to inhibit CA II and CA V and possibly also other CA isozymes. The data of this study show also that compound 1 has no effect on DNL, if labelled acetate is used as tracer. Therefore, the effect of compound 1 on DNL operates at the level of CA V and/or CA II and not at the level of acetyl-CoA carboxylase (ACC).HEP-G2 cells were also incubated for 6h with compound 5 (another newly synthesised sulfonamide) and three follower compounds of compound 1 (i.e. compound 2, compound 3, compound 4) or drug vehicle (0.1% DMSO) to study the CA inhibitory effect on bicarbonate fixation in cultured HEP-G2 cells. The results of this study clearly show that compound 5 is the most efficient CAI whereas the followers of compound 1 are mediocer CAIs for DNL. DNL is inhibited to 100% in the presence of 50 à �à µM compound 5 or 100 à �à µM compound 1, thus the effects of 50 à �à µM compound 5 on bicarbonate fixation in cultured HEP-G2 cells is comparable with the inhibitory effect of 100 à �à µM compound 1. The HEP-G2 cells were incubated in the presence of CAIs or drug vehicle (0.1% DMSO) for control with [U-13C]glucose for 6 or 24h. The results of this study show that after 6h the effect of 50 à �à µM compound 5 and 100 à �à µM compound 1 on the glucose influx into the TCA cycle via pc and on pdh are similar. However after 24h in the presence of 50 à �à µM compound 5, the influx of labelled glucose into the TCA cycle via pc is decreased by only 20% and pdh activity is not affected, whereas after 24h the pc and pdh activities are reduced in the presence of 100 μM compound 1 to 100% and 80% of control respectively. The results of experiments with [2-13C]acetate in the presence of compound 5 show that compound 5 has possibly an indirect inhibitory effect on pdh. The results of this study also show that, similar to compound 1, the effect of compound 5 on DNL is at the level of CA and not at the level of acetyl-CoA carboxylase.