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* This work was supported, in whole or in part, by National Institutes of Health Grants DK090522 and AA017927 (to J. L. N.). 1 Present address: Dept. of Neurosurgery, University of Heidelberg, Heidelberg, Germany.
All-trans-retinoic acid (atRA), an autacoid derived from retinol (vitamin A), regulates energy balance and reduces adiposity. We show that energy status regulates atRA biosynthesis at the rate-limiting step, catalyzed by retinol dehydrogenases (RDH). Six h after re-feeding, Rdh1 expression decreased 80–90% in liver and brown adipose tissue and Rdh10 expression was decreased 45–63% in liver, pancreas, and kidney, all relative to mice fasted 16 h. atRA in the liver was decreased 44% 3 h after reduced Rdh expression. Oral gavage with glucose or injection with insulin decreased Rdh1 and Rdh10 mRNA 50% or greater in mouse liver. Removing serum from the medium of the human hepatoma cell line HepG2 increased Rdh10 and Rdh16 (human Rdh1 ortholog) mRNA expression 2–3-fold by 4 h, by increasing transcription and stabilizing mRNA. Insulin decreased Rdh10 and Rdh16 mRNA in HepG2 cells incubated in serum-free medium by inhibiting transcription and destabilizing mRNA. Insulin action required PI3K and Akt, which suppress FoxO1. Serum removal increased atRA biosynthesis 4-fold from retinol in HepG2 cells, whereas dominant-negative FoxO1 prevented the increase. Thus, energy status via insulin and FoxO1 regulate Rdh expression and atRA biosynthesis. These results reveal mechanisms for regulating atRA biosynthesis and the opposing effects of atRA and insulin on gluconeogenesis, and also suggest an interaction between atRA and insulin signaling related diseases, such as type II diabetes and cancer.
A wide scope of biological processes, including embryonic development, cell differentiation and proliferation, immune function, neurogenesis, and energy metabolism, depend on the vitamin A (retinol) metabolite all-trans-retinoic acid (atRA)
). atRA controls energy balance by inhibiting differentiation of pre-adipocytes into mature white adipose, regulating the function of white adipose cells, and by regulating whole body lipid and carbohydrate metabolism (
). Impairing atRA homeostasis causes abnormalities in intermediary metabolism. Mice with ablated cellular retinol-binding protein 1 (encoded by Rbp1), which regulates retinol homeostasis, experience glucose intolerance from enhanced gluconeogenesis, resulting from hyperglucagonemia, and also undergo increased adipocyte differentiation (
). During limited or normal vitamin A nutriture, retinol dehydrogenases (RDH) of the short-chain dehydrogenase/reductase gene family catalyze the first and rate-limiting reaction to produce all-trans-retinal. Retinal dehydrogenases, of the aldehyde dehydrogenase gene family, catalyze the second reaction to produce atRA. Multiple isoforms of retinoid-metabolizing enzymes occur in both families, often in the same cell types, but these enzymes differ in subcellular expression loci, and can show differential cell expression patterns. RDH1 and RDH10 are the most characterized RDH in the path of atRA biosynthesis: both are expressed early in embryogenesis and throughout life in multiple tissues, and both contribute to atRA biosynthesis from retinol in intact cells in the presence of retinal dehydrogenases (
). During feeding, liver catalyzes increased energy storage by generating glycogen and triacylglycerol. During fasting, liver generates glucose and ketone bodies from gluconeogenesis, glycogenolysis, and fatty acid oxidation. Glucose, insulin, and glucagon control the balance between energy storage during feeding and production of fuels during fasting.
A combination of portal vein glucose, insulin, and neuronal signal(s) causes liver to transition from catabolic to anabolic metabolism (
). Insulin binding to its cell surface receptors activates two canonical pathways in hepatocytes: the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K). The mitogen-activated protein kinase pathway includes extracellular-signal regulated kinase (ERK) signaling and regulates transcription involved in differentiation, growth, and survival (
). The PI3K pathway activates Akt (PKB) to mediate metabolic transitions, including promotion of glycogen and lipid synthesis, and suppression of gluconeogenesis. Effectors downstream of PI3K/Akt include: glycogen synthase kinase-3 (GSK-3), which regulates glycogen synthesis; mammalian target of rapamycin complex 1 (mTORC1), which regulates protein synthesis; BCL2-associated agonist of cell death (BAD), which regulates cell survival; and forkhead box other (FoxO), which regulates transcription of genes required for gluconeogenesis, including glucose-6-phosphatase (Glc-6-P) (
This report compares Rdh1 and Rdh10 expression in tissues of fasted versus re-fed mice, and focuses on atRA in liver. Liver served as the focus of this study because of its central contributions to both energy and retinoid homeostasis. We found that insulin, via inactivating FoxO1, represses Rdh1 and Rdh10 expression, with an associated decrease in atRA biosynthesis. These observations are consistent with the recent analysis that FoxO1 may link gluconeogenesis to retinoid homeostasis (
). Our results provide direct evidence showing that energy status regulates atRA biosynthesis, and provides new insight into the relationship between atRA biosynthesis and its regulation of energy balance.
A vitamin A-deficient diet results in glycogen deficiency because of impaired gluconeogenesis, caused by low Pck1 expression (
). Here we demonstrate reciprocal regulation by energy status of Rdh mRNA and atRA concentrations via insulin effects on FoxO1. Fasting-induced coordinate induction of both Rdh and Pck1 by FoxO1 stimulates gluconeogenesis (Fig. 8). In the fed state, insulin repression of FoxO1 suppresses gluconeogenesis via repressing both Rdh and Pck1. Although serum factor(s) other than insulin control Rdh expression and mRNA stability, we focused on insulin because of its critical role in regulating gluconeogenesis (
Tissue-specific changes in Rdh10 and Rdh1 expression during fasting and re-feeding are consistent with a complex contribution of atRA to regulating intermediary metabolism. Epididymal white adipose tissue and pancreata do not express Rdh1 (data not shown), but express Rdh10. The non-responsiveness of Rdh10 in epididymal white adipose tissue to changes in energy balance allows several interpretations, including the presence of another Rdh that responds to changes in energy status, or feeding versus fasting does not modify atRA epididymal white adipose tissue concentrations in adult mice. These possibilities are the subjects of ongoing studies. The decrease in Rdh10 expression in pancreas during re-feeding is consistent with the requirement for retinoids in pancreatic development (
), and also a subject of ongoing studies. Suppression of Rdh1 in liver by re-feeding, and Rdh10 in liver and kidney, relate insulin action with atRA signaling in two gluconeogenic tissues. Decreased Dhrs3 expression in liver of re-fed versus fasted mice is consistent with a mutually activating interaction of Rdh10 and Dhrs3 (
), and the reduction in atRA biosynthesis. Evidently, energy metabolism affects Rdh expression in multiple tissues to regulate retinoid signaling effects as feedback to regulation of energy disposition by atRA.
Oral dosing has a singular effect on glucose uptake and insulin action in liver (
). Glucose delivery by the portal vein combined with insulin, and an as yet unidentified neuronal signal, increases net hepatic glucose uptake compared with peripheral delivery, with a concomitant impact on insulin-regulated processes (
). A comparison of oral glucose dosing to infusion via the portal vein revealed similar enhancement in hepatic glucose uptake relative to peripheral presentation, which excludes a contribution from a gut-secreted factor, such as Glp-1 (
). Our data of oral but not peripheral glucose dosing reducing Rdh mRNA are consistent with these observations and support the physiological significance of the observation.
Reduced Rdh expression preceded the reduction in liver atRA, and was accompanied by a lower rate of atRA biosynthesis in HepG2 cells, consistent with a cause and effect relationship. atRA homeostasis autoregulates via induction of both catabolic cytochromes P-450 and the retinyl ester-forming lecithin:retinol acyltransferase (
). The former decreases atRA itself, whereas the latter decreases the amount of retinol available to support atRA biosynthesis. Changes in lecithin:retinol acyltransferase and cytochrome P-450 may have buffered atRA differences in the re-fed relative to the fasted liver. The 2-fold decrease in liver atRA resulting from insulin action seems remarkable in the context of this impetus to sustain atRA homeostasis.
FoxO (forkhead box “Other”) proteins constitute a subgroup of a family of evolutionarily conserved transcription factors that mediate insulin signaling in mammals, but also mediate metabolism and longevity in primitive organisms such as Caenorhabditis elegans and Drosophila (
). FoxO proteins contribute to metabolic regulation through effects in liver, muscle, adipose tissue, and pancreas. Of these, FoxO1 stimulates a committed step in hepatic gluconeogenesis, catalyzed by phosphoenolpyruvate carboxykinase (PCK1), and also the last step catalyzed by glucose-6-phosphatase (Glc-6-P) (
). A liver-specific FoxO1-null mouse crossed with an insulin receptor-null mouse has reduced glucose production and lacks the neonatal diabetes and hepatosteatosis that occur in insulin receptor-null mice (
). Inhibition of sirtuin1 in HepG2 cells with Ex-527 incubated in serum-free medium did not affect RDH10 expression (data not shown), indicating that acetylation is not involved in regulation of RDH10, at least under the conditions tested.
Recent insight into the functions of insulin receptor substrates IRS1 and IRS2 indicate that both coordinate responses to insulin via FoxO1 (
). During the re-fed state, insulin activates IRS1 to suppress FoxO1 and allow an increase in glucokinase and sterol regulatory element-binding transcription factor 1 (SREBF1, a.k.a. SREBP1) expression that promotes glycolysis and lipid biosynthesis, respectfully. In contrast, insulin inhibits IRS2. Because IRS2 stimulates gluconeogenesis by inducing PCK1 and G6PC in liver through FoxO1, the relatively low levels of insulin in the fasted state allow IRS2 to induce gluconeogenesis. Based on these insights, the higher insulin levels in the re-fed state would function through IRS1 to suppress RDH expression, whereas the lower insulin levels in the fasted state would allow IRS2 to induce RDH expression.
The actions of insulin and serum both prevented transcription and destabilized RDH mRNA. The effects differed in degree, and resulted in different levels of mRNA, but did not totally eliminate RDH mRNA. In both serum-free medium and insulin-containing serum-free medium, the amounts of RDH mRNA after 24 h remained 2–3-fold higher compared with serum-containing medium, because the initial concentrations were 2–3-fold higher. Destabilization of RDH mRNA by insulin depended on active PI3K, Akt, and inhibition of FoxO1. The reduction but not elimination of RDH mRNA by insulin and serum is compatible with the continued presence and biosynthesis of atRA, and a need for RDH to modulate basal gluconeogenesis and/or its other multiple functions, whether related or unrelated to energy balance, such as regulating proliferation (
FoxO1 binding sites have been identified in promoters of genes associated with retinoid metabolism, including dehydrogenase/reductase SDR family member 9 (Dhrs9), cellular retinol-binding protein type 1 (encoded by Rbp1), and Rdh8, but not in Rdh1 or Rdh10 (
). To address the impact of FoxO1-regulated expression of retinoid genes, atRA target genes Pck1 and Pdk4 were measured in cells with FoxO1 knocked down. Induction of both genes in response to atRA or to its precursor, retinol, were blunted. The dose (20 μm) of atRA used, however, exceeded those found in tissues by 400–4000-fold, and those used in vitro by 20-fold. Nevertheless, these results are also consistent with FoxO1 linking retinoid metabolism and hepatic gluconeogenesis. Multiple putative FoxO/A binding sites were found in the 5′-untranslated region of Rdh10 (Encyclopedia of DNA Elements Consortium, ENCODE; University of California Southern California genome browser). Future work will determine whether these or other sites are true response elements.
Regulation by insulin signaling prompts the question whether abnormal RDH expression associates with human disease. For example, impaired insulin secretion and insulin resistance during diabetes predicts elevated RDH expression and atRA synthesis (
). Reduced RDH expression would decrease atRA biosynthesis, which could affect tumor differentiation and/or aggressiveness. Various cancers show alterations in the atRA signaling pathway, such as loss of atRA receptors (
In summary, despite the importance of atRA in vertebrate physiology, little is known about regulation of the rate-limiting enzymes (Rdh) that catalyze its biosynthesis. This report reveals a reciprocal interaction in which energy status regulates atRA biosynthesis through insulin that would attenuate energy balance regulation by atRA. These data predict altered atRA concentrations in diseases characterized by dysfunctional insulin signaling, including diabetes and cancer.
We are grateful to Domenico Accili for a gift of the dominant-negative adenovirus construct (Δ256).
Vitamin A supplementation and retinoic acid treatment in the regulation of antibody responses in vivo.