Opposing Effects of Fructokinase C and a Isoforms on Fructose-Induced Metabolic Syndrome in Mice
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Opposing effects of fructokinase C and A isoforms on fructose-induced metabolic syndrome in mice Takuji Ishimotoa, Miguel A. Lanaspaa, MyPhuong T. Lea, Gabriela E. Garciaa, Christine P. Diggleb, Paul S. MacLeanc, Matthew R. Jackmanc, Aruna Asipub, Carlos A. Roncal-Jimeneza, Tomoki Kosugia, Christopher J. Rivarda, Shoichi Maruyamad, Bernardo Rodriguez-Iturbee, Laura G. Sánchez-Lozadaf, David T. Bonthronb, Yuri Y. Sauting, and Richard J. Johnsona,g,1,2 aDivision of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO 80045; bLeeds Institute of Molecular Medicine, University of Leeds, Leeds, LS9 7TF, United Kingdom; cDivision of Endocrinology, Colorado Nutrition Obesity Research Center, University of Colorado Denver, Aurora, CO, 80045; dDepartment of Nephrology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan; eInstituto Venezolano de Investigaciones Científicas- Zulia and Hospital Universitario y Universidad del Zulia, 4001-A, Maracaibo, Venezuela; fDepartment of Nephrology, Instituto Nacional de Cardiología Ignacio Chavez, Mexico City, 14080, Mexico; and gDivision of Nephrology and Hypertension, University of Florida, Gainesville, FL 32610 Edited by Kosaku Uyeda, University of Texas Southwestern Medical Center and Veterans Affairs Medical Center at Dallas, Dallas, TX, and accepted by the Editorial Board January 24, 2012 (received for review December 6, 2011) Fructose intake from added sugars correlates with the epidemic rise These latter findings suggest there may be unique features in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. involved in fructose metabolism that may predispose to the de- Fructose intake also causes features of metabolic syndrome in labo- velopment of metabolic syndrome. Although fructose can be ratory animals and humans. The first enzyme in fructose metabolism metabolized by hexokinase similarly to glucose (14), the relative is fructokinase, which exists as two isoforms, A and C. Here we show affinity for fructose is substantially less and under most con- that fructose-induced metabolic syndrome is prevented in mice lack- ditions fructose is metabolized by fructokinase (ketohexokinase, fi ing both isoforms but is exacerbated in mice lacking fructokinase A. KHK), an enzyme that is speci c for fructose. KHK is uniquely Fructokinase C is expressed primarily in liver, intestine, and kidney different from other hexokinases in its ability to induce transient and has high affinity for fructose, resulting in rapid metabolism and ATP depletion in the cell (15, 16). The mechanism is due to the marked ATP depletion. In contrast, fructokinase A is widely distrib- fact that KHK phosphorylates fructose to fructose-1-phosphate uted, has low affinity for fructose, and has less dramatic effects on rapidly, resulting in marked ATP depletion, coupled with the ATP levels. By reducing the amount of fructose for metabolism in the absence of a feedback inhibition (such as that which controls liver, fructokinase A protects against fructokinase C-mediated met- glucose catabolism). The ATP depletion is also associated with intracellular phosphate depletion and AMP generation, with abolic syndrome. These studies provide insights into the mecha- stimulation of AMP deaminase and the stepwise degradation of nisms by which fructose causes obesity and metabolic syndrome. AMP to purine products including uric acid (15, 16). Intracellular ATP depletion in response to fructose occurs with low concen- ketohexokinase | hepatic steatosis | insulin | leptin trations (1 mM) of fructose and has been shown in laboratory animals and humans (16–18). ructose, present in added sugars such as sucrose and high Fructokinase exists in two alternatively spliced isoforms con- Ffructose corn syrup, has been epidemiologically linked with sisting of fructokinase C (KHK-C) and fructokinase A (KHK-A) obesity and metabolic syndrome (1–3). Experimental studies differing in exon 3 (19, 20). KHK-C is expressed primarily in the have shown that fructose can induce leptin resistance and vir- liver, kidney, and intestines, whereas KHK-A is more ubiquitous tually all features of metabolic syndrome in rats, whereas glucose (21). Whereas both KHK-C and KHK-A can metabolize fructose, (or starch) intake does not (4). Clinical studies also support KHK-C is considered the primary enzyme involved in fructose fructose as a cause of metabolic syndrome, especially in over- metabolism due to its lower Km (22). Because of its higher Km, weight individuals. Thus, overweight subjects that consumed a KHK-A is not considered presently to be actively involved in 25% fructose-based diet for 10 wk developed insulin resistance, fructose metabolism but rather has been postulated to act on as postprandial hypertriglyceridemia, and visceral obesity, unlike yet unknown substrates (22). subjects given a glucose-based diet (5). Similarly, the additional Recently, we generated mice genetically lacking both KHK Δ Δ administration of 200 g of fructose per day to a normal diet could isoforms (the Khk / mouse; KHK-A/C KO), and mice lacking induce de novo metabolic syndrome in 25% of overweight men only KHK-A (the Khk3a/3a mouse; KHK-A KO) (21, 23). These in just 2 wk (6). Furthermore, a recent report showed that con- mice show a normal phenotype under normal diet (23). In the sumption of fructose-sweetened, but not glucose-sweetened, present study, we investigated how they would respond to a diet beverages, which provided 25% of energy requirement with usual supplemented with fructose. ad libitum diet could induce postprandial hypertriglyceridemia (not fasting triglycerides) and increased circulating LDL and apolipoprotein B (Apo-B) levels in young and overweight sub- Author contributions: T.I. and R.J.J. designed research; T.I., M.A.L., M.T.L., and G.E.G. jects (7). These data are consistent with a recent metaanalysis performed research; C.P.D., A.A., T.K., S.M., L.G.S.-L., and D.T.B. contributed new re- that found sugary soft drinks to be an independent risk factor for agents/analytic tools; P.S.M., M.R.J., C.A.R.-J., C.J.R., and B.R.-I. analyzed data; and T.I., obesity and diabetes (8). Y.Y.S., and R.J.J. wrote the paper. The mechanism by which fructose induces metabolic syndrome Conflict of interest statement: Based on the discoveries from this study, T.I., M.A.L., and R.J.J. are listed as inventors on a patent application from the University of Colorado has been shown to be independent of excessive energy intake. In the related to developing isoform-specific fructokinase inhibitors in the treatment of disor- study by Stanhope et al., the ability of fructose to induce features of ders associated with obesity and insulin resistance. No other authors have any conflicts metabolic syndrome compared with glucose was independent of of interest. changes in weight (5). Our group has also shown that fructose (or This article is a PNAS Direct Submission. K.U. is a guest editor invited by the Editorial sucrose) can exacerbate features of metabolic syndrome compared Board. with pair-fed glucose- or starch-fed controls, even under settings of 1Present address: Division of Renal Diseases and Hypertension, University of Colorado caloric restriction (9–11). Thus, whereas fructose intake may induce Denver, Box C281, 12700 E 19th Ave., Research 2 Room P15-7006, Aurora, CO 80045. leptin resistance with impaired satiety and weight gain (12, 13), 2To whom correspondence should be addressed. E-mail: [email protected]. fructose-induced metabolic syndrome may also occur, independent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of increases in energy intake or weight gain. 1073/pnas.1119908109/-/DCSupplemental. 4320–4325 | PNAS | March 13, 2012 | vol. 109 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1119908109 Downloaded by guest on September 26, 2021 Results elevated in fructose-fed KHK-A/C KO mice, overall energy bal- Energy Intake and Balance. At baseline (age 2 mo), there was no ance remained positive after correcting for the urinary fructose significant difference in body weight, liver, and epididymal fat excretion (Fig. 1 E and F), possibly related in part to a decrease in weight, blood pressure, and biochemical analyses between wild- total energy expenditure (Fig. 1D). Additionally, the fructose type (WT) mice, KHK-A/C KO mice, and KHK-A KO mice, balance in fructose-fed KHK-A/C KO mice was positive (fructose consistent with previous reports (Table S1). WT mice, KHK-A/C intake, 3.9 kcal/day; urinary fructose excretion, 1.2 kcal/day). KO mice, and KHK-A KO mice (8-wk-old male) were then fed KHK-A KO mice acted similarly to WT KO mice in their total normal chow diet ad libitum with 15 or 30% fructose in the ingestion of fructose, chow, and total cumulative energy intake. drinking water, or tap water, for 25 wk (n =8–9 per group). Although urinary fructose was slightly increased in KHK-A KO Energy balance (intake and expenditure) was assessed at 19 wk. and WT mice on 30% fructose compared with mice not receiving WT mice given 15 or 30% fructose received 32 and 45% of fructose, the levels obtained were much less than that observed their total energy intake as fructose, respectively (Fig. 1A). with KHK-A/C KO mice (Fig. 1E). Similar to WT mice, fructose Whereas WT mice given fructose in the water reduced their feeding in KHK-A KO mice increased total energy expenditure intake of chow, overall cumulative energy intake was increased and sustained a positive energy imbalance (Fig. 1 D and F). (Fig. 1A and Fig. S1). Indirect calorimetry performed at week 19 showed an increase in total energy expenditure and a positive Fructose Effects on Wild-Type Mice. Mice are relatively resistant to energy imbalance in WT mice receiving 30% fructose compared the effects of fructose to induce metabolic syndrome (24). Nev- with control WT mice (Fig.