A Role for Uric Acid in Fructose-induced Fat Accumulation
The observation that fructose-fed rats develop fatty liver and metabolic syndrome without requiring increased energy intake suggests that the metabolism of fructose may be different from that of other carbohydrates. Fructose is distinct from glucose only in its initial metabolism. The first enzyme to metabolize fructose is fructokinase (also known as ketohexokinase [KHK]). The metabolism of fructose to fructose-1-phosphate by KHK occurs primarily in the liver, is rapid and without any negative feedback, and results in a fall in intracellular phosphate and ATP levels.[14–16] This has been shown to occur in the liver in humans with relatively small doses of oral fructose (60 g fructose alone or 39 g fructose with 39 g glucose). The decrease in intracellular phosphate stimulates AMP deaminase (AMPD), which catalyzes the degradation of AMP to inosine monophosphate and eventually uric acid (Fig. 1). The increase in intracellular uric acid is followed by an acute rise in uric acid in the circulation likely due to its release from the liver. Fructose also stimulates uric acid synthesis from amino acid precursors, such as glycine.
Fructose-induced nucleotide turnover. Fructose is rapidly phosphorylated in the hepatocyte by KHK to fructose-1-phosphate (F-1-P), which uses ATP as a phosphate donor. Intracellular phosphate (PO4) levels decrease, stimulating the activity of AMP deaminase 2 (AMPD2). AMPD2 converts AMP to inosine monophosphate (IMP). IMP is metabolized to inosine by 5' nucleotidase (5'NT), which is further degraded to xanthine and hypoxanthine by xanthine oxidase (XO), ultimately generating uric acid.
Recent studies suggest that this "side event" in fructose metabolism may be critical for how fructose induces metabolic syndrome. First, there are actually two KHK isoforms, and they differ in their ability to activate this pathway. KHK-C phosphorylates fructose rapidly, consuming ATP with the generation of uric acid. In contrast, KHK-A phosphorylates fructose slowly and consumes minimal ATP. When both KHK-C and KHK-A are deleted, mice are fully protected from fructose-induced metabolic syndrome and fatty liver; however, when KHK-A is selectively deleted, there is increased fructose available for metabolism by KHK-C, and the metabolic syndrome and fatty liver are worsened compared with wild-type mice despite the same intake of total calories and fructose. These studies suggest that differences in nucleotide turnover might influence the metabolic response.
To examine the purine nucleotide pathway in the metabolic response, we silenced aldolase B in a hepatocyte line (HepG2 cells). Aldolase B is the second enzyme in fructose metabolism, and the genetic loss of aldolase B is the cause of hereditary fructose intolerance. When aldolase B is inhibited, fructose is phosphorylated by ATP but cannot be further metabolized, nevertheless fructose can be metabolized by other routes such as hexokinase. In this regard, subjects with hereditary fructose intolerance, are known to have hyperactive KHK and show enhanced ATP depletion and uric acid generation in response to fructose. As such, this is an interesting condition in which marked nucleotide turnover and ATP depletion occur but without the ability to be further metabolized by this primary enzymatic pathway to glucose, glycogen, or triglycerides. Nevertheless, the feeding of fructose to HepG2 cells lacking aldolase B resulted in a rapid accumulation of triglycerides, consistent with our findings that uric acid itself can induce triglyceride accumulation. These experiments explain why fatty liver and hyperuricemia are common complications of this disease and also why fatty liver and diabetes are complications in subjects with glycogen storage disease I, in which hepatic intracellular ATP depletion and hyperuricemia also occur.[22–25] Finally, it provides an explanation for why fructose is lipogenic (based on acetate labeling studies) despite little of the fructose molecule being incorporated into the triglyceride molecule itself.
We next addressed how the degradation of nucleotides might lead to fat accumulation. Specifically, our group and others have shown that AMPD counters the effects of AMP-activated protein kinase (AMPK).[26,27] Whereas activation of AMPK in hepatocytes induces oxidation of fatty acids and ATP generation, AMPD has opposite effects. Overexpression of AMPD in HepG2 cells blocks fatty acid oxidation and increases fat accumulation, whereas silencing AMPD blocks fructose-induced fat accumulation. The mechanism is mediated in part by the generation of uric acid, which inhibits AMPK.
In addition to inhibiting AMPK, uric acid may stimulate hepatic lipogenesis. The mechanism appears to be mediated by uric acid–dependent intracellular and mitochondrial oxidative stress. Although uric acid is a potent antioxidant in the extracellular environment, when uric acid enters cells via specific organic anion transporters, it induces an oxidative burst that has been shown in vascular smooth muscle cells, endothelial cells, adipocytes, islet cells, renal tubular cells, and hepatocytes.[29–31] Uric acid–induced oxidative stress appears to be mediated by the stimulation of NADPH oxidase, which translocates to mitochondria.[28,29,32] Uric acid can also generate triuretcarbonyl and aminocarbonyl radicals as well as alkylating species upon reaction with peroxynitrite and can also directly inactivate nitric oxide (NO) to 6-aminouracil.[33,34]
The induction of oxidative stress in the mitochondria causes a reduction in aconitase-2 activity in the Krebs cycle, resulting in citrate accumulation that is transported into the cytoplasm where it activates ATP citrate lyase, acetyl CoA carboxylase, and fatty acid synthase, leading to fat synthesis. Uric acid also causes a reduction in enoyl CoA hydratase-1, a rate-limiting enzyme in β-fatty acid oxidation. The consequence is fat accumulation in the hepatocyte.[19,35]
Recently, we identified another mechanism by which uric acid may increase the risk for hepatic fat accumulation and metabolic syndrome. Fructose (or sucrose) ingestion is known to increase hepatic KHK levels. The increased expression of KHK is driven in part by the production of uric acid from fructose A rise in intracellular uric acid activates the nuclear transcription factor, carbohydrate responsive element–binding protein. When KHK expression is increased in HepG2 cells by uric acid exposure, the triglyceride accumulation in response to fructose is enhanced.
This is relevant to subjects with nonalcoholic fatty liver disease (NAFLD). Subjects with NAFLD ingest more fructose-containing soft drinks than age, sex, and BMI-matched control subjects and have increased KHK expression in their liver. Subjects with NAFLD who have the highest fructose intake also show the greatest ATP depletion in response to a fructose load, and those subjects with the highest uric acid levels show a greater nadir in the ATP depletion. These data are consistent with an induction of KHK in the liver with subsequent increased sensitivity to the effects of fructose via a uric acid–dependent mechanism.
Rodents have lower serum uric acid than humans due to the presence of uricase in their liver, and hence show a lesser rise in serum uric acid in response to fructose. Nevertheless, lowering uric acid has also been found to block the development of hepatic steatosis in fructose-fed rats. Lowering uric acid also reduces hepatic steatosis in the desert gerbil (which spontaneously develops fatty liver on a normal diet), in alcoholic fatty liver (in which increased intrahepatic uric acid occurs), and in the Pound mouse (a mouse model of metabolic syndrome manifesting fatty liver, obesity, insulin resistance, and hypertension caused by a leptin receptor mutation). These studies supported the tight association of hyperuricemia with fatty liver; prospective studies have also reported that an elevated uric acid independently predicts the development of NAFLD. The ability of hyperuricemia to predict fatty liver is independent of obesity. Hyperuricemia is even associated with NAFLD in hemodialysis subjects who have a BMI below 20. A summary of how fructose and uric acid induce fatty liver is shown in Fig. 2.
Classic and alternative lipogenic pathways of fructose. In the classical pathway, triglycerides (TG) are a direct product of fructose metabolism by the action of multiple enzymes including aldolase B (Aldo B) and fatty acid synthase (FAS). An alternative mechanism was recently shown. Uric acid produced from the nucleotide turnover that occurs during the phosphorylation of fructose to fructose-1-phosphate (F-1-P) results in the generation of mitochondrial oxidative stress (mtROS), which causes a decrease in the activity of aconitase (ACO2) in the Krebs cycle. As a consequence, the ACO2 substrate, citrate, accumulates and is released to the cytosol where it acts as substrate for TG synthesis through the activation of ATP citrate lyase (ACL) and fatty acid synthase. AMPD2, AMP deaminase 2; IMP, inosine monophosphate; PO4, phosphate.
Diabetes. 2013;62(10):3307-3315. © 2013 American Diabetes Association, Inc.