Acid Metabolism In Hepatocytes Research Articles

Effects of in vivo phlorizin treatment and in vitro addition of carnitine, propionate, acetate, and 5-tetradecyloxy-2-furoic acid on palmitate metabolism in ovine hepatocytes

Modulatory effects of l-carnitine, acetate, propionate, and 5-tetradecyloxy-2-furoic acid (TOFA; an inhibitor of acetyl-CoA carboxylase) on oxidation and esterification of [1-14C]-palmitate were studied in hepatocytes isolated from phlorizin-treated and control wethers. Our hypotheses were that (1) palmitate oxidation would be greater in hepatocytes from sheep injected with phlorizin; (2) l-carnitine would increase palmitate oxidation more in hepatocytes from sheep injected with phlorizin; and (3) acetate and propionate would decrease oxidation in sheep hepatocytes partly through action of acetyl-CoA carboxylase. Palmitate metabolism did not differ between cells from control and those from phlorizin-treated wethers. Carnitine increased oxidation of palmitate to CO2 and acid-soluble products (ASP; mainly ketone bodies) and decreased esterification of palmitate in isolated hepatocytes from both groups of wethers, but the increase in oxidation to ASP was greater in cells from phlorizin-treated wethers. Propionate increased palmitate oxidation to CO2 in phlorizin-treated wethers. Propionate increased oxidation of palmitate to ASP in control wethers but decreased oxidation to ASP in phlorizin-treated wethers. Propionate increased esterification of palmitate to total esterified products and triglyceride, and the effect was larger in phlorizin-treated wethers. Acetate decreased palmitate esterification to total esterified products in control wethers, but the effect was blunted in phlorizin-treated wethers. Acetate did not affect palmitate oxidation. Addition of TOFA increased production of triglyceride from palmitate in the presence of propionate. The lack of interaction between TOFA and propionate indicates that propionate does not inhibit carnitine palmitoyltransferase I via cytosolic generation of methylmalonyl-CoA by acetyl-CoA carboxylase. In conclusion, although in vivo phlorizin treatment did not affect in vitro metabolism of palmitate by isolated ovine hepatocytes, phlorizin increased the stimulatory effect of carnitine on oxidation of palmitate to ASP and the inhibitory effect of propionate on oxidation of palmitate to ASP. Metabolism of acetate and propionate by acetyl-CoA carboxylase did not affect palmitate oxidation or esterification. Results provide additional insight into control of fatty acid metabolism in hepatocytes.

Evidence of a liver-alpha cell axis in humans: hepatic insulin resistance attenuates relationship between fasting plasma glucagon and glucagonotropic amino acids.

The secretion of glucagon is controlled by blood glucose and inappropriate secretion of glucagon contributes to hyperglycaemia in diabetes. Besides its role in glucose regulation, glucagon regulates amino acid metabolism in hepatocytes by increasing ureagenesis. Disruption of this mechanism causes hyperaminoacidaemia, which in turn increases glucagon secretion. We hypothesised that hepatic insulin resistance (secondary to hepatic steatosis) via defective glucagon signalling/glucagon resistance would lead to impaired ureagenesis and, hence, increased plasma concentrations of glucagonotropic amino acids and, subsequently, glucagon. To examine the association between glucagon and amino acids, and to explore whether this relationship was modified by hepatic insulin resistance, we studied a well-characterised cohort of 1408 individuals with normal and impaired glucose regulation. In this cohort, we have previously reported insulin resistance to be accompanied by increased plasma concentrations of glucagon. We now measure plasma levels of amino acids in the same cohort. HOMA-IR was calculated as a marker of hepatic insulin resistance. Fasting levels of glucagonotropic amino acids and glucagon were significantly and inversely associated in linear regression models (persisting after adjustment for age, sex and BMI). Increasing levels of hepatic, but not peripheral insulin resistance (p > 0.166) attenuated the association between glucagon and circulating levels of alanine, glutamine and tyrosine, and was significantly associated with hyperaminoacidaemia and hyperglucagonaemia. A doubling of the calculated glucagon-alanine index was significantly associated with a 30% increase in hepatic insulin resistance, a 7% increase in plasma alanine aminotransferase levels, and a 14% increase in plasma γ-glutamyltransferase levels. This cross-sectional study supports the existence of a liver-alpha cell axis in humans: glucagon regulates plasma levels of amino acids, which in turn feedback to regulate the secretion of glucagon. With hepatic insulin resistance, reflecting hepatic steatosis, the feedback cycle is disrupted, leading to hyperaminoacidaemia and hyperglucagonaemia. The glucagon-alanine index is suggested as a relevant marker for hepatic glucagon signalling.

Induction of peroxisomes by butyrate-producing probiotics.

We previously found that peroxisomal biogenesis factor 11a (Pex11a) deficiency is associated with a reduction in peroxisome abundance and impaired fatty acid metabolism in hepatocytes, and results in steatosis. In the present study, we investigated whether butyrate induces Pex11a expression and peroxisome proliferation, and studied its effect on lipid metabolism. C57BL/6 mice fed standard chow or a high-fat diet (HFD) were treated with tributyrin, 4-phelybutyrate acid (4-PBA), or the butyrate-producing probiotics (Clostridium butyricum MIYAIRI 588 [CBM]) plus inulin (dietary fiber), and the body weight, white adipose tissue, serum triglycerides, mRNA expression, and peroxisome abundance were evaluated. Tributyrin or 4-PBA treatment significantly decreased body weight and increased hepatic mRNA expression of peroxisome proliferator-activated receptor-α (PPARα) and Pex11a. In addition, 4-PBA treatment increased peroxisome abundance and the expression of genes involved in peroxisomal fatty acid β-oxidation (acyl-coenzyme A oxidase 1 and hydroxysteroid [17-beta] dehydrogenase 4). CBM and inulin administration reduced adipose tissue mass and serum triglycerides, induced Pex11a, acyl-coenzyme A oxidase 1, and hydroxysteroid (17-beta) dehydrogenase 4 genes, and increased peroxisome abundance in mice fed standard chow or an HFD. In conclusion, elevation of butyrate availability (directly through administration of butyrate or indirectly via administration of butyrate-producing probiotics plus fiber) induces PPARα and Pex11a and the genes involved in peroxisomal fatty acid β-oxidation, increases peroxisome abundance, and improves lipid metabolism. These results may provide a new therapeutic strategy against hyperlipidemia and obesity.

Pex11a deficiency is associated with a reduced abundance of functional peroxisomes and aggravated renal interstitial lesions.

Although proteinuria is known to be associated with the deterioration of chronic kidney disease, the molecular basis of this mechanism is not fully understood. We previously found that Pex11a deficiency was associated with a reduction of functional peroxisomes and impaired fatty acid metabolism in hepatocytes and resulted in steatosis. Proximal tubule cells are rich in peroxisomes. We assessed whether Pex11a deficiency might result in the derangement of peroxisome systems in proximal tubule cells and the aggravation of tubulointerstitial lesions in chronic kidney disease. Histological analyses showed that the number of functional peroxisomes in proximal tubule cells was reduced in Pex11a knockout (Pex11a(-/-)) mice. To clarify whether a decrease in the number of tubular peroxisomes might aggravate interstitial lesions, we assessed 2 models in which proximal tubule cells are overloaded with fatty acids (ie, deoxycorticosterone acetate and salt hypertension and the overload of fatty acid-bound albumin). Deoxycorticosterone acetate -salt-treated Pex11a(-/-) mice exhibited greater interstitial lesions than deoxycorticosterone acetate-salt-treated wild-type mice in terms of tubular lipid accumulation, blood pressure, urinary albumin, urinary N-acetyl-β-d-glucosaminidase, urinary 8-iso-prostane, and the histological evaluation of fibrosis and inflammation. An overload of fatty acid-bound albumin also resulted in more severe tubulointerstitial lesions in Pex11a(-/-) mice than in wild-type mice. Fenofibrate, a peroxisome proliferator-activated receptor-α agonist, restored the abundance of peroxisomes and reduced the tubulointerstitial lesions induced by deoxycorticosterone acetate-salt hypertension. In conclusion, our results indicate that proximal tubule peroxisomes play an important role in proteinuria-induced interstitial lesions. The activation of tubular peroxisomes might be an excellent therapeutic strategy against chronic kidney disease.

Downregulation of Fetuin-B and Zinc-α2-glycoprotein is Linked to Impaired Fatty Acid Metabolism in Liver Cells

Background: Our recent proteomic study has shown that plasma protein levels of fetuin-B (Ft-B) and zinc-α2-glycoprotein (ZAG) are significantly elevated in obesity-resistant (OR) rats exposed to a high fat diet. Time profiling of the plasma concentrations of Ft-B and ZAG in OR rats has shown stable regulation of these proteins throughout the entire period of rat breeding. Methods: To firmly establish roles for these proteins in lipogenesis, we efficiently knocked down (KD) the genes FETUB and AZGP1 encoding Ft-B and ZAG, respectively, using siRNA in Chang liver cells. Results: Reduced expression of FETUB and AZGP1 led to a significant increase in the expression of lipogenic genes, thereby resulting in higher lipid levels in both KD cells. Collectively with our previous findings, we confirmed that Ft-B was similarly regulated with Ft-A, in that their plasma protein levels were commonly reduced in diet-induced obese rats. Conclusion: Our results provide a possible relationship between reduced plasma protein levels of Ft-B and ZAG and higher risk of diet-induced obesity through impaired fatty acid metabolism in hepatocytes.

Lipin 2 is a Liver‐enriched Coactivator of Peroxisome Proliferator‐activated Receptor γ (PPARγ) Coactivator‐1α (PGC‐1α) and Regulates Fatty Acid Metabolism in Hepatocytes

Lipin 1 acts in the nucleus to regulate gene expression via interactions with DNA‐bound nuclear receptor transcription factors and the PGC‐1α coactivator. Interestingly, lipin 1 also functions as an enzyme, catalyzing the penultimate step in triglyceride (TG) synthesis. However, little is known about the second lipin family member (lipin 2). We found that the expression of lipin 2 mRNA and protein was quite high in murine and human liver, but low in adipose tissue and skeletal muscle, where lipin 1 was enriched. The hepatic expression of lipin 1 and 2 was induced by fasting, but unlike lipin 1, the increase in lipin 2 was preserved in mice lacking PGC‐1α. Moreover, adenoviral‐driven PGC‐1α overexpression increased the expression of lipin 1, but not lipin 2, in hepatocytes. Nevertheless, lipin 2 protein physically interacted with and coactivated PPARα and PGC‐1α. Lipin 2 overexpression also augmented palmitate oxidation rates via increased expression of mitochondrial fatty acid oxidation enzymes. Rates of TG synthesis were surprisingly unchanged by overexpression of either lipin 1 or 2 in hepatocytes. These data indicate that lipin 2 is a liver‐enriched and fasting‐induced PGC‐1α activator. Furthermore, although the effects of lipin 1 and 2 on hepatic fatty acid metabolism significantly overlap, the expression of these two lipin genes is under the control of distinct regulatory circuits.

The mammalian target of rapamycin regulates lipid metabolism in primary cultures of rat hepatocytes

The mammalian target of rapamycin (mTOR) is a conserved serine-threonine kinase that regulates cell growth and metabolism in response to nutrient signals. However, the specific involvement of mTOR in regulation of energy metabolism is poorly understood. To determine if signaling via mTOR might be directly involved in regulation of fatty acid metabolism in hepatocytes, we performed studies with rapamycin, a specific inhibitor of mTOR. Rapamycin-mediated inhibition of mTOR (18-48 hours) increased oxidation of exogenous fatty acids (46%-100%, respectively). In addition, esterification of exogenous fatty acids and de novo lipid synthesis were reduced (40%-60%, respectively). Consistent with inhibition of lipogenic pathways, rapamycin decreased expression of genes encoding acetyl–coenzyme A carboxylase I and mitochondrial glycerol phosphate acyltransferase. Non–insulin-dependent glucose transport and glycogen synthesis were decreased by 20% to 30%, whereas glucose utilization was unaffected by rapamycin. The data suggest that the hyperlipidemia observed with the drug in vivo is likely not the result of enhanced hepatic synthesis, but rather of delayed peripheral clearance. However, these results are consistent with the idea that mTOR may play a significant role, not only in “energy sensing,” but also in regulation of energy production through profound effects on hepatic fatty acid metabolism.

Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism in hepatocytes from ethanol-fed rats

The mechanisms by which ethanol causes accumulation of hepatic triacylglycerols are complex. It has been proposed that nitric oxide/cyclic GMP signaling pathway may be involved in regulation of fatty acid metabolism in the liver. Here, we investigated if this mechanism may have a role in adaptation to ethanol consumption. Hepatocytes were isolated from rats fed with an ethanol-containing liquid diet and pair-fed control rats, and incubated with a range of concentrations of 8-bromo-cyclic GMP. In both types of cells, this cyclic GMP analog inhibited in parallel fatty acid synthesis de novo and acetyl-CoA carboxylase activity. Addition of 8-bromo-cyclic GMP also decreased the rate of palmitate esterification to triacylglycerols and phospholipids, whereas palmitate oxidation was increased. However, in all these metabolic effects, hepatocytes from ethanol-fed rats were significantly less sensitive to the addition of 8-bromo-cyclic GMP. In order to know if this may be a more general mechanism of adaptation to ethanol, we also studied the effects on glucose metabolism. Similarly, hepatocytes from ethanol-fed rats showed a decreased sensitivity in the inhibition by 8-bromo-cyclic GMP of glycogen synthesis, fatty acid synthesis and the synthesis of glycerol backbone of hepatic triacylglycerols. These data suggest that ethanol consumption induces a desensitization of the regulatory effects mediated by cyclic GMP in fatty acid metabolism, contributing to triacylglycerol accumulation in the liver.

Fatty acid metabolism in hepatocytes isolated from rats adapted to high-fat diets containing long- or medium-chain triacylglycerols.

Fatty acid oxidation and synthesis were studied in isolated hepatocytes from adult rats adapted for 44 days on low-fat, high-carbohydrate (LF), diet or high-fat diets, composed of long-chain (LCT) or medium-chain (MCT) triacylglycerols. The rates of [1-14C]octanoate oxidation were almost similar in each group studied, whereas the oxidation of [1-14C]oleate was 50% lower in the LF group than in animals adapted to high-fat diets. The rates of oleate oxidation are inversely correlated with the rates of lipogenesis. However, it seems unlikely that [malonyl-CoA] itself represents the sole mechanism involved in the regulation of oleate oxidation during long-term LCT or MCT feeding, since: (1) despite a 3-fold higher concentration of malonyl-CoA in MCT-fed rats than in LCT-fed ones, the rates of oleate oxidation are similar; (2) when malonyl-CoA concentration is increased after stimulation of lipogenesis (by adding lactate + pyruvate) in MCT-fed rats, to a level comparable with that of the LF group, the rate of oleate oxidation remains 55% higher than that measured under similar conditions in the LF-fed rats; (3) in the LF group, the 90% decrease in malonyl-CoA concentration [by 5-(tetradecyloxy)-2-furoic acid] is not associated with a stimulation of oleate oxidation. By contrast, the sensitivity of carnitine palmitoyltransferase I (CPT I) to malonyl-CoA is markedly decreased in the LCT- and MCT-fed rats, by 90% and 70% respectively. The relevance of this decrease in the sensitivity of CPT I is discussed.

Amino acid metabolism in hepatocytes isolated from lactating rats

Parenchymal hepatocytes isolated from lactating rats had similar rates of amino acid incorporation into protein, but increased rates of urea formation compared to hepatocytes from non-lactating rats. The increased urea formation may be due to increased amino acid transport and degradation. The liver contributes to the increased utilization of amino acids during lactation.

The metabolism of fatty acids in hepatocytes isolated from triiodothyronine-treated rats

1. 1. The effect of triiodothyronine on the metabolism of palmitate, oleate and erucate in isolated rat hepatocytes was studied. 2. 2. In triiodothyronine-treated rats increased oxidation and decreased triacylglycerol formation from palmitate and oleate was observed. For erucate triiodothyronine caused increased oxidation, but had no significant effect on esterification. 3. 3. Glucagon had no effect on the fatty acid metabolism in hepatocytes from triiodothyronine-treated rats, whereas it stimulated the oxidation in hepatocytes from normal rats. Still, after treatment with triiodothyronine, the oxidation of fatty acids was significantly higher than in glucagon-stimulated normal hepatocytes. 4. 4. In isolated rat liver mitochodria triiodothyronine raised the activity of the outer carnitine palmitoyltransferase (EC 2.3.1.21). The activity of the total carnitine palmitoyltranferase was elevated only slightly in isolated mitochondria from triiodothyronine-treated rats. These effects were similar to those seen in fasted rats. 5. 5. Triiodothyronine had no significant influence on the concentration of long-chain acyl-CoA or α-glycerophosphate in isolated rat hepatocytes.