M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 3 ( 2 0 14 ) 84 1–8 5 0
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Effects of individual branched-chain amino acids deprivation on insulin sensitivity and glucose metabolism in mice Fei Xiao a , Junjie Yu a , Yajie Guo a , Jiali Deng a , Kai Li a , Ying Du a , Shanghai Chen a , Jianmin Zhu b,⁎, Hongguang Sheng b,⁎, Feifan Guo a,⁎ a
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, the Graduate School of the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, China 200031 b Shanghai Xuhui Central Hospital, 966 Huaihai Middle Road, Shanghai, China 200030
A R T I C LE I N FO Article history:
AB S T R A C T Objective. We recently discovered that leucine deprivation increases hepatic insulin
Received 17 January 2014
sensitivity via general control nondepressible (GCN) 2/mammalian target of rapamycin (mTOR)
Accepted 12 March 2014
and AMP-activated protein kinase (AMPK) pathways. The goal of the present study was to investigate whether the above effects were leucine specific or were also induced by deficiency
Keywords: Leucine deprivation
of other branched chain amino acids including valine and isoleucine. Methods. Following depletion of BCAAs, changes in metabolic parameters and the
Valine deprivation
expression of genes and proteins involved in regulation of insulin sensitivity and glucose
Isoleucine deprivation
metabolism were analyzed in mice and cell lines including human HepG2 cells, primary
Insulin signaling
mouse hepatocytes and a mouse myoblast cell line C2C12.
Glucose metabolism
Results. Valine or isoleucine deprivation for 7 days has similar effect on improving insulin sensitivity as leucine, in wild type and insulin-resistant mice models. These effects are possibly mediated by decreased mTOR/S6K1 and increased AMPK signaling pathways, in a GCN2-dependent manner. Similar observations were obtained in in vitro studies. In contrast to leucine withdrawal, valine or isoleucine deprivation for 7 days significantly decreased fed blood glucose levels, possibly due to reduced expression of a key gluconeogenesis gene, glucose-6-phosphatase. Finally, insulin sensitivity was rapidly improved in mice 1 day following maintenance on a diet deficient for any individual BCAAs. Conclusions. Our results show that while improvement on insulin sensitivity is a general feature of BCAAs depletion, individual BCAAs have specific effects on metabolic pathways, including those that regulate glucose level. These observations provide a conceptual framework for delineating the molecular mechanisms that underlie amino acid regulation of insulin sensitivity. © 2014 Elsevier Inc. All rights reserved.
Abbreviations: AKT, protein kinases B; AMPK, AMP-activated protein kinase; BCAAs, Branched-chain amino acids; GCN2, General Control Nonderepressible 2; IR, insulin receptor; mTOR: mammalian Target of Rapamycin; S6K1, p70 ribosomal protein S6 Kinase 1. ⁎ Corresponding author. Tel.: +86 21 54920945; fax: +86 21 54920291. E-mail addresses: [email protected] (F. Xiao), [email protected] (J. Yu), [email protected] (Y. Guo), [email protected] (J. Deng), [email protected] (K. Li), [email protected] (Y. Du), [email protected] (S. Chen), [email protected] (J. Zhu), [email protected] (H. Sheng), [email protected] (F. Guo). http://dx.doi.org/10.1016/j.metabol.2014.03.006 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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1.
M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL 6 3 ( 2 0 14 ) 84 1–8 50
Introduction
Diabetes is one of the major causes of morbidity and mortality worldwide. A common feature of type 2 diabetes is insulin resistance, which is characterized by reduced glucose uptake in muscle and adipose tissue, and increased glucose production in liver [1,2]. One of the important factors that contribute to insulin resistance is the unbalance of dietary macronutrients including fat, glucose and amino acids [3]. Aside from their role as the building blocks of proteins, amino acids are also critical mediators of intracellular signaling [4]. Branched-chain amino acids (BCAAs) that have non-linear aliphatic side-chains include leucine, valine and isoleucine, and are the most studied essential amino acids. Increased leucine concentration is reported to either improve or have no effect on glucose metabolism in mice [5,6]; however, there is increasing evidence of a correlation between increased levels of BCAAs and insulin resistance [7–9]. For example, metabolomic profiling of obese versus lean humans reveals a BCAAs-related metabolite signature that is suggestive of increased catabolism of BCAAs and correlated with insulin resistance [9]. Furthermore, Wang and colleagues found that quantification of BCAAs serum levels facilitates risk assessment for onset t of type 2 diabetes [10]. Though progress has been made in understanding the effect of BCAAs on insulin sensitivity and glucose metabolism, the effect of each individual BCAA remains largely unknown. In contrast to other studies examining relationships between increased levels of BCAAs and insulin sensitivity, our studies have focused on investigating the effects of elimination of dietary leucine and previously shown that leucine deprivation improves hepatic insulin sensitivity in vivo and in vitro [11]. It remains unclear, however, whether deficiency of other BCAAs, including valine and isoleucine, would have similar effects. A previous study showed that mice deleted for the BCATm gene encoding the enzyme catalyzing the first step in peripheral BCAAs metabolism, which would be expected to have low leucine, valine or isoleucine utilization, exhibited increased insulin sensitivity [12]. These results suggest that deficiency of valine or isoleucine may also modulate insulin sensitivity. The aim of our current study was to investigate these possibilities and elucidate underlying mechanisms. This study will help in understanding the unique feature of each individual BCAA. These observations are also important for understanding the molecular mechanisms underlying amino acid regulation of insulin sensitivity.
drate and lipid components. At the start of the feeding experiments, mice were acclimated to a control diet for 7 days and then randomly divided into control, (−) val or (−) ile diet group, with free access to each diet for 7 days. To determine the possible influences of reduced food intake in the BCAAsdeprived group, pair-fed (pf) groups were included. Mice in the pf group were provided with 18%, 30% or 40% less food, as determined in our preliminary experiments, compared to mice in the control group. A subset of mice underwent glucose tolerance test (GTT)/insulin tolerance test (ITT) prior to being killed by CO2 inhalation. As many things can change blood glucose quickly and significantly, we included corresponding control groups during every experiment. These experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (CAS).
2.2.
HepG2 cells were maintained in DMEM (Gibco, Grand Island, NY, USA) with 25 mmol/L glucose, 10% FBS, 50 μg/ml penicillin and streptomycin at 37 °C, 5% CO2–95% air. C2C12 myoblasts were obtained from the CAS Cell Bank of Type Culture Collection. Maintenance and induction of differentiation were performed as previously described [13]. Primary hepatocyte isolation was achieved by collagenase perfusion as described previously [14]. Control, (−) val or (−) ile medium were prepared by adding all the components of regular DMEM or lacking the corresponding amino acid.
2.3. Blood glucose, serum insulin, GTT, ITT and HOMA-IR index Blood glucose levels were measured using a Glucometer Elite monitor. Serum insulin levels were measured using the Mercodia Ultrasensitive Rat Insulin ELISA kit (Catalog Number: 80INSRTU-E01, ALPCO Diagnostic, Salem, NH, USA). GTT and ITT were performed by IP injection of 2 g/kg glucose after overnight fasting and 0.75 U/kg or 0.375 U/kg insulin after 4 h fasting, respectively. The HOMA-IR index was calculated according to the formula: [Fasting glucose levels (mmol/L)] × [Fasting serum insulin (μU/ml)]/22.5.
2.4.
2.
Methods
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Animals and treatments
Male C57BL/6 J mice were obtained from Shanghai Laboratory Animal (SLAC, Shanghai, China). GCN2 knockout (Gcn2−/−) and leptin receptor-mutated (db/db) mice were kindly provided by Dr. Douglas Cavener, Penn State University, USA and Dr. Xiang Gao, Nanjing University, China, respectively. Eight- to tenweek-old mice were maintained on a 12-h light/dark cycle at 25 °C. Control, (–) leu (leucine-deficient), (−) val (valine-deficient) and (−) ile (isoleucine- deficient) chow were obtained from Research Diets (New Brunswick, NJ, USA). All diets were isocaloric and compositionally the same in terms of carbohy-
Cell culture and treatments
In vivo insulin signaling assay
Mice maintained on different diets were fasted for 6 h prior to insulin injection as previously described [14]. Sections of liver and soleus muscle were excised from anesthetized mice and snap-frozen, as untreated controls. Three or five minutes after injection with 2 U/kg of insulin via the portal vein, pieces of tissue section were excised and snap-frozen for western blot analysis.
2.5.
Western blot analysis
Western blot analysis was performed as previously described [15]. Protein concentrations were assayed using BCA Kit (Pierce, Rockford, USA). Primary antibodies [anti-p-insulin receptor (Tyr1150/1151), anti-insulin receptor, anti-p-AKT (Ser473), antiAKT, anti-p-mTOR (Ser2448), anti-mTOR, anti-p-p70 S6K1
M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 3 ( 2 0 14 ) 84 1–8 5 0
(Thr389), anti-p70 S6K1, anti-p-S6 (Ser235/236), anti-S6, anti-pAMPK (Thr172), anti-t-AMPK (all the above from Cell Signaling Technology, Beverly, MA, USA)] were incubated overnight at 4 °C and specific proteins were visualized by ECL Plus (Amersham Biosciences, Buckinghamshire, UK). Band intensities were measured using Quantity One (Bio-Rad Laboratories) and normalized to total protein or actin.
2.6.
RNA isolation and relative quantitative RT-PCR
Total RNA was prepared from frozen tissues with TRIZOL (Life Technologies, Carlsbad, CA, USA) reagent. One microgram of RNA was reverse transcribed with random primer and M-MLV Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). Quantitative amplification by PCR was carried out using SYBR Green I Master Mix reagent by ABI 7500 system (Applied Biosystem). PCR products were subjected to a melting curve analysis. Cycle numbers of both GAPDH (as an internal control) and cDNAs of interest at a specific threshold within the exponential amplification range were used to calculate relative expression levels of the genes of interest. The sequences of primers used in this study are available upon request.
2.7.
examined the levels of phosphorylation of two key components in the insulin signaling pathways, insulin receptor (IR) and protein kinases B (AKT). As expected, insulin-stimulated phosphorylation of IR and AKT increased in the livers of valine-deprived or isoleucine- deprived mice compared with controls (Fig. 1C and D).
3.3. Valine or isoleucine deprivation increases insulin signaling in vitro To determine whether valine or isoleucine deprivation has a direct effect on insulin signaling in multiple cell types, we utilized the human hepatoma-derived cell line HepG2, primary mouse hepatocytes and C2C12, a mouse myoblast cell line. Consistent with our in vivo observations (Fig. 1C and D), we found that insulin-stimulated phosphorylation of IR and AKT was significantly elevated by valine or isoleucine deprivation in all three cell lines (Fig. 2A and B).
3.4. Valine or isoleucine deprivation decreases mammalian target of rapamycin (mTOR) and increases AMP-activated protein kinase (AMPK) signaling in vivo
Statistics
All data are expressed as mean ± SEM. Significant differences were assessed using a two-tailed student t-test. P < 0.05 was considered statistically significant.
3.
843
Results
3.1. Valine or isoleucine deprivation increases whole-body insulin sensitivity Mice were fed a control, valine- or isoleucine-deficient diet for 7 days, a period comparable to that we previously used for leucine deprivation tests. Glucose tolerance and clearance were examined by GTT and ITT, respectively. In GTT, mice fed a valine- or isoleucine-deficient diet exhibited significantly lower levels of blood glucose after overnight fasting. Fifteen minutes after injection of glucose, blood glucose levels were significantly lower in valine- or isoleucine-deprived mice compared with controls (Fig. 1A and B). Following administration of insulin, blood glucose levels decreased quickly in valineor isoleucine-deprived mice compared with mice maintained on a control diet (Fig. 1A and B). In contrast to the situation following valine deprivation, blood glucose levels were below the limit of detection in isoleucine-deprived mice when subjected to the same dose of insulin during the ITT. We therefore opted to use a 50% dose of insulin in the isoleucinedeprived mice and found much improved insulin sensitivity in these mice compared with control mice (Fig. 1B).
3.2. Valine or isoleucine deprivation increases insulin signaling in vivo Increased insulin sensitivity in mice upon valine or isoleucine deprivation suggests an increase in insulin signaling in peripheral tissues such as the liver. To test this possibility, we
The mTOR and AMPK signaling pathways play important roles in the development of insulin resistance, and both pathways are implicated in the regulation of insulin sensitivity during leucine deprivation. Consistent with changes in leucinedeprived mice, phosphorylation of mTOR and its downstream targets, including p70 ribosomal protein S6 Kinase 1 (S6K1) and ribosomal protein S6, was also significantly decreased in the livers of valine- or isoleucine-deprived mice compared with mice fed a control diet (Fig. 3A and B). In addition, AMPK phosphorylation was increased in the livers of mice fed a valine-deficient or isoleucine-deficient diet for 7 days compared with mice fed a control diet (Fig. 3A and B).
3.5. Valine deprivation increases insulin sensitivity by activation of GCN2 GCN2 is a serine protein kinase that senses amino acid deprivation and functions as an upstream regulator of mTOR to modulate hepatic insulin sensitivity upon leucine deprivation [11]. We thus speculated GCN2 might be involved in modulation of insulin sensitivity during valine deprivation. To examine this possibility, GCN2 phosphorylation was examined in the livers of mice maintained on a valinedeficient diet. Consistent with a previous study [16], GCN2 phosphorylation was increased in these mice (Fig. 4A). We then compared insulin sensitivity using ITT in Gcn2 +/+ and Gcn2 −/− mice maintained on a valine-deficient diet. Glucose clearance was decreased in Gcn2 −/− mice compared with their wild type counterparts upon valine deprivation (Fig. 4B). Consistent with these changes, insulin-stimulated phosphorylation of IR and AKT was decreased in the livers of Gcn2−/− mice (Fig. 4C). Since our previous study showed that the insulin response is normal in GCN2-null mice on a normal diet [17], these data indicate that GCN2 plays a specific role in sensing lack of dietary BCAAs.
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Fig. 1 – The effects of valine or isoleucine deprivation on insulin sensitivity in vivo. Mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 days, then subjected to glucose tolerance and insulin tolerance tests (GTT and ITT, respectively) in A and B. Insulin signaling in the liver was examined before (− Ins) and after (+ Ins) 2 U/kg insulin stimulation for 3 min in C and D. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of (−) val or (−) ile versus control diet (*p < 0.05). (A–B) GTT and ITT; (C–D) p-IR and p-AKT (left, western blot; right, quantitative measurements of p-IR and p-AKT proteins relative to their respective total protein levels).
3.6. Effects of individual BCAA deficiency on glucose metabolism Consistent with animals subjected to leucine deprivation, mice fed a valine- or isoleucine-deficient diet for 7 days also exhibited significantly lower levels of fasting blood glucose, fasting serum insulin and HOMA-IR index (Fig. 5A–C). In contrast to the unchanged fed blood glucose levels in leucinedeprived mice, fed blood glucose levels were also decreased in mice maintained on a valine- or isoleucine-deficient diet (Fig. 5D and E). To identify possible causes for the different blood glucose levels, we examined the expression of glucose-6phosphatase (g6pase), a key gluconeogenic gene in the liver. In contrast to leucine deprivation, which has no effect on g6pase expression, the levels of g6pase mRNA were decreased by both valine and isoleucine deprivation (Fig. 5F).
was performed. Blood glucose levels dropped more rapidly following administration of insulin in mice fed any individual BCAA-deficient diet compared with mice maintained on a control diet (Fig. 6A–C). As shown in our previous work, BCAAs deprivation also decreases food intake (18 % for leucine, 30 % for valine and 40 % for isoleucine,) compared with mice maintained on a control diet. As the insulin-sensitizing effect of isoleucine or valine deprivation may be related with the decreased food intake, pair-fed (pf) groups were included to distinguish the possible influence of the reduction in food intake in BCAAs-deprived groups. Mice in the pf group were provided with 18%, 30% or 40% less food compared to mice in the control group. As expected, no significant difference was observed between these groups when ITT was performed (Fig. 6D–F). Meanwhile, due to the differences in the amounts of food intake for each pf group, blood glucose levels were affected to different extent compared with control-diet fed mice.
3.7. Whole-body insulin sensitivity is increased in mice 1 day following maintenance on a diet deficient for any individual BCAAs
3.8. Valine or isoleucine deprivation increases insulin sensitivity under insulin-resistant condition
To investigate how quickly the effect of individual BCAAs deficiency on insulin sensitivity could occur, mice were fed a diet deficient for leucine, valine or isoleucine for 1 day and ITT
Our previous work has shown that leucine deprivation improves insulin sensitivity in insulin-resistant animal models, including high-fat diet fed mice and db/db mice [11].
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Fig. 2 – The effects of valine or isoleucine deprivation on insulin sensitivity in vitro. HepG2 cells and primary hepatocytes were incubated in control (+val) or valine-deficient (−val) medium for 48 h, or in control (+ile) or isoleucine-deficient (−ile) medium for 24 h after being treated with serum-free medium overnight. C2C12 cells were incubated in control, valine-deficient or isoleucin-deficient medium for 48 h. All the cells were then treated with (+ Ins) or without (− Ins) 100 nmol/L insulin stimulation for 20 min. Data are mean ± SEM of at least three independent experiments. Statistical significance is calculated using a two-tailed student t-test for the effects of (−) val or (−) ile versus control medium after insulin stimulation (*p < 0.05). (A and B) p-IR and p-AKT protein (top, western blot; bottom, quantitative measurements of p-IR and p-AKT proteins relative to their respective total protein levels).
We speculated that deprivation of valine and isoleucine may have the same effects. To test this possibility, db/db mice were fed a control, valine- or isoleucine-deficient diet for 7 or 5 days. Then insulin sensitivity was examined by ITT assay. As predicted, blood glucose levels decreased more quickly in valine- or isoleucine-deprived mice compared with mice maintained on a control diet following administration of insulin (Fig. 7A and B).
4.
Discussion
Increased serum levels of BCAAs are associated with insulin resistance in humans and mice [7–9], and infusion of isoleucine decreases glucose uptake in human forearm tissue [18], suggesting that decreased levels of valine or isoleucine may improve insulin sensitivity. Consistent with this possibility, we
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Fig. 3 – The effects of valine or isoleucine deprivation on mTOR/S6 and AMPK signaling in vivo. Mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 days. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–7 each group). Statistical significance is calculated using a two-tailed Student’s t-test for the effects of (−) val or (−) ile versus control diet (*p < 0.05). (A and B) p-mTOR, p-S6K1, p-S6 protein and p-AMPK (top, western blot; bottom, quantitative measurements of p-mTOR, p-S6K1 and p-S6 protein relative to their respective total protein levels).
found that valine or isoleucine deprivation increases wholebody insulin sensitivity and insulin signaling in liver and skeletal muscle (Supplementary Fig. 1) in vivo, and in several cell lines. These results are similar to those observed during leucine deprivation [11], suggesting enhanced insulin sensitivity is a general consequence of BCAAs deprivation. There are however some notable exceptions [19,20], suggesting that the effect of BCAAs could be very complicated. Intracellular amino acids levels can be sensed directly by aminoacyl tRNA formation, or indirectly by levels of their metabolites such as ATP and by intracellular proteins including mTOR, MAP4K3 and Rag [21,22]. Among them, mTOR and AMPK are the most extensively studied ones. mTOR is a Ser/Thr protein kinase, the activity of which is particularly regulated by leucine [23]. Furthermore, mTOR activity is closely related to the ability of the cell to respond to insulin; this is underscored by the observation that increased mTOR/S6K1 signaling contributes to the development of insulin resistance [7] and decreased mTOR/S6K1 signaling improves insulin sensitivity [24,25]. AMPK responds directly to fluctuations in the ratio of AMP:ATP [26]. Activation of AMPK stimulates cellular uptake of glucose and reduces glucose production [27–29]. For these reasons, AMPK activators, including metformin and rosiglitazone, have been widely used in the clinic to treat insulin resistance in diabetes patients [1]. Though both mTOR signaling and AMPK signaling are important
modulators of insulin sensitivity, whether they also perform this role in the context of valine or isoleucine deprivation has not been experimentally tested. In the current study, we observed that mTOR signaling decreases and AMPK phosphorylation increased in the liver during valine or isoleucine deprivation. Both signaling pathways are known to improve insulin sensitivity during leucine deprivation [11], and we thus speculate that valine or isoleucine deficiency may employ common mechanisms in regulating insulin sensitivity. Furthermore, our results strongly suggest that mTOR and AMPK activities are regulated by BCAA availability, which is consistent with results from other studies [30]. The upstream regulator responsible for decreased liver mTOR signaling during valine or isoleucine deprivation is unknown. GCN2 is a serine/threonine protein kinase. Lower intracellular amino acids levels lead to the accumulation of the corresponding amino acid uncharged tRNAs. GCN2 has a domain with homology with HisRS (histidyl-tRNA synthetase) that can bind to uncharged tRNAs, resulting in kinase activation through homodimerization and autophosphorylation [31]. Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2)α, concomitantly repressing general protein synthesis and increasing translation of proteins related to amino acid biosynthesis and transport [32–35]. In addition, our recent studies reveal that GCN2 is involved in regulating lipid metabolism and
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Fig. 4 – The role of GCN2 in valine deprivation—increased insulin sensitivity. (A) Mice were fed a control (ctrl) or valine-deficient [(−) val] diet for 7 days. (B and C) Both Gcn2+/+ and Gcn2−/− mice were fed a valine-deficient [(−) val] diet for 7 days, followed by an insulin tolerance test (ITT) in B and examination of insulin signaling before (− Ins) and after (+ Ins) 2 U/kg insulin stimulation for 3 min in C. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of (–) val versus control diet (p < 0.05) in A and Gcn2−/− versus Gcn2+/+ mice (*p < 0.05) in B and C. (A) p-GCN2 (top, western blot; bottom, quantitative measurements of p-GCN2 protein relative to its total protein levels). (B) ITT; (C) p-IR and p-AKT (left, western blot; right, quantitative measurements of p-IR and p-AKT protein relative to their respective total protein levels).
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Fig. 5 – Effects of individual BCAAs deprivation on glucose metabolism. Mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 days. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of BCAAs deprivation versus control diet (*p < 0.05). (A and B) Blood glucose and serum insulin levels after fasting overnight; (C) HOMA-IR index; (D and E) fed blood glucose and serum insulin levels; (F) g6pase mRNA.
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Fig. 6 – The effects of a 24 h dietary deficiency of individual BCAAs on insulin sensitivity. Mice were maintained 1 day on a control (ctrl), leucine-deficient [(−) leu], valine-deficient [(−) val], isoleucine-deficient [(−) ile] or pair-fed (pf) diet for each BCAA, followed by carrying out an insulin tolerance test (ITT). Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of BCAA deprivation or pf diet versus control diet (*p < 0.05). (A–C) ITT for ctrl and (−) BCAA group; (D–F) ITT for ctrl and pf group. insulin sensitivity during leucine deprivation [11,17]. Because GCN2 is supposed to be activated by valine or isoleucine deprivation and functions as an upstream regulator of mTOR in regulating hepatic insulin sensitivity during leucine deprivation [11], we speculated it may have a similar regulatory role upon withdrawal of valine or isoleucine. Consistent with this hypothesis, we found that GCN2 phosphorylation was increased in the livers of mice maintained
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on a valine-deficient diet compared with control mice. Furthermore, increased insulin sensitivity triggered by valine deprivation is impaired in GCN2−/− mice. Since GCN2 is activated by deficiency of any essential amino acids [16], we hypothesized that GCN2 is also involved in the regulation of insulin sensitivity during isoleucine deprivation. The role of GCN2 upon valine or isoleucine deprivation also highlights the critical role that GCN2 plays as a ‘master metabolite sensor’.
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Fig. 7 – Valine or isoleucine deprivation improves insulin sensitivity under insulin-resistant conditions. db/db mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 or 5 days, then subjected to insulin tolerance test (ITT). Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of (−) val or (−) ile versus control diet (*p < 0.05). (A and B) ITT.
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In contrast to the unaltered fed blood glucose levels in mice fed a leucine-deficient diet [11], fed blood glucose levels decreased during valine or isoleucine deprivation. One possible explanation for this differential effect could be the specific regulation of the key gluconeogenic enzyme G6Pase. This protein is not affected by leucine deprivation, but its level is reduced by valine or isoleucine deprivation. G6Pase catalyses the final step of the gluconeogenic pathway, which hydrolyzes glucose-6-phosphate, generating a phosphate group and free glucose [36]. Therefore, altered expression of G6Pase would lead to changes in blood glucose levels. In addition, valine and isoleucine are more readily used as substrates for gluconeogenesis when compared with leucine [37], which may also lead to the difference in fed blood glucose levels. Our previous work has shown that leucine deprivation also improves insulin sensitivity under insulin-resistant conditions, including high-fat diet fed mice and db/db mice [11]. Similar observations were obtained by valine or isoleucine deprivation. Besides, we found that insulin sensitivity is improved as early as 1 day following maintenance on a diet deficient for any of the individual BCAAs. As mice maintained on BCAA-deficient diet reduced their food intake, we included a pf group. However, we did not see any difference in ITT assay between pf group and control group at the same time. Furthermore, our previous study has shown that insulin sensitivity was increased in mice fed leucine-deficient diet, compared with control or pf mice [11]. Based on these results and our in vitro observations showing a direct effect of isoleucine or valine deficiency on insulin signaling, we speculate that the increased insulin sensitivity in isoleucine- or valine-deficient mice is caused primarily by a deficiency of BCAA, rather than a reduction in food intake. Because the effect of BCAA deficiency occurs very rapidly, these results provide important hints for the potential use of this diet in treating insulin resistance in diabetes patients. Determining the optimal concentration of BCAAs and the duration of therapeutic BCAAs-deficient diets will be important issues for future pre-clinical and clinical studies. Our current study provides important evidence demonstrating a general effect of BCAAs on improving insulin sensitivity, which would provide important hints for dietary control on insulin resistance. In addition, our unpublished results and those of others [38] reveal that individual amino acids have different effects on insulin sensitivity in primary adipocytes and HepG2 cells, suggesting that the regulation of insulin sensitivity and glucose metabolism by different amino acids is very complex. Furthermore, we can’t presently conclude that the effects on insulin sensitivity we observed are restricted to BCAAs; for this, a systematic study of BCAAs depletion versus lack of non-BCAA essential amino acids or nonessential amino acids will be required. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.metabol.2014.03.006.
Author contributions Fei Xiao designed the experiment, researched and analyzed data, contributed to discussion, wrote manuscript and reviewed/edited
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manuscript. Junjie Yu, Yajie Guo, Jiali Deng, Kai Li, Ying Du and Shanghai Chen researched data and contributed to discussion. Jianmin Zhu, Hongguang Sheng and Feifan Guo designed the experiment, contributed to discussion, wrote manuscript and reviewed/edited manuscript.
Funding This work was supported by grants from the Ministry of Science and Technology of China (973 Program 2010CB912502), China National Funds for Distinguished Young Scientists (81325005), National Natural Science Foundation (81130076, 31271269, 81100615, 30890043, 81390352 and 81300659), Basic Research Project of Shanghai Science and Technology Commission (13JC1409000), Key Program of Shanghai Scientific and Technological Innovation Action Plan (10JC1416900), the Knowledge Innovation Program of CAS(KSCX2-EW-R-09), Chinese Academy of Sciences-funded project (2011KIP307). Feifan Guo was also supported by the One Hundred Talents Program of the Chinese Academy of Sciences. Fei Xiao was supported by China Postdoctoral Science Foundation funded project (2012 M520950 and 2013 T60473) and a Chinese Academy of Sciences-funded project (2013KIP310).
Disclosure statement There is no conflict of interest that the authors should disclose.
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[10] Zhang J, Du YY, Lin YF, Chen YT, Yang L, Wang HJ, et al. The cell growth suppressor, mir-126, targets IRS-1. Biochem Biophys Res Commun 2008;377:136–40. [11] Xiao F, Huang Z, Li H, Yu J, Wang C, Chen S, et al. Leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways. Diabetes 2011;60:746–56. [12] She P, Reid TM, Bronson SK, Vary TC, Hajnal A, Lynch CJ, et al. Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle. Cell Metab 2007;6:181–94. [13] Sun C, Zhang F, Ge X, Yan T, Chen X, Shi X, et al. SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B. Cell Metab 2007;6:307–19. [14] Wang Q, Jiang L, Wang J, Li S, Yu Y, You J, et al. Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice. Hepatology 2009;49:1166–75. [15] Cheng Y, Meng Q, Wang C, Li H, Huang Z, Chen S, et al. Leucine deprivation decreases fat mass by stimulation of lipolysis in white adipose tissue and upregulation of uncoupling protein 1 (UCP1) in brown adipose tissue. Diabetes 2010;59:17–25. [16] Hinnebusch AG. The eIF-2 alpha kinases: regulators of protein synthesis in starvation and stress. Semin Cell Biol 1994;5:417–26. [17] Guo F, Cavener DR. The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid. Cell Metab 2007;5:103–14. [18] Schwenk WF, Haymond MW. Decreased uptake of glucose by human forearm during infusion of leucine, isoleucine, or threonine. Diabetes 1987;36:199–204. [19] Ikehara O, Kawasaki N, Maezono K, Komatsu M, Konishi A. Acute and chronic treatment of L-isoleucine ameliorates glucose metabolism in glucose-intolerant and diabetic mice. Biol Pharm Bull 2008;31:469–72. [20] Doi M, Yamaoka I, Nakayama M, Mochizuki S, Sugahara K, Yoshizawa F. Isoleucine, a blood glucose-lowering amino acid, increases glucose uptake in rat skeletal muscle in the absence of increases in AMP-activated protein kinase activity. J Nutr 2005;135:2103–8. [21] Kim E. Mechanisms of amino acid sensing in mTOR signaling pathway. Nutr Res Pract 2009;3:64–71. [22] Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006;124:471–84. [23] Lynch CJ. Role of leucine in the regulation of mTOR by amino acids: revelations from structure-activity studies. J Nutr 2001;131:861S–5S.
[24] Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 2004;431:200–5. [25] Krebs M, Brunmair B, Brehm A, Artwohl M, Szendroedi J, Nowotny P, et al. The mammalian target of rapamycin pathway regulates nutrient-sensitive glucose uptake in man. Diabetes 2007;56:1600–7. [26] Hardie DG. The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 2004;117:5479–87. [27] Ojuka EO. Role of calcium and AMP kinase in the regulation of mitochondrial biogenesis and GLUT4 levels in muscle. Proc Nutr Soc 2004;63:275–8. [28] Bergeron R, Russell III RR, Young LH, Ren JM, Marcucci M, Lee A, et al. Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol 1999;276:E938–44. [29] Rutter GA, Da Silva Xavier G, Leclerc I. Roles of 5'-AMPactivated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 2003;375:1–16. [30] Gleason CE, Lu D, Witters LA, Newgard CB, Birnbaum MJ. The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells. J Biol Chem 2007;282:10341–51. [31] Gallinetti J, Harputlugil E, Mitchell JR. Amino acid sensing in dietary-restriction-mediated longevity: roles of signal-transducing kinases GCN2 and TOR. Biochem J 2013;449:1–10. [32] Bruhat A, Jousse C, Fafournoux P. Amino acid limitation regulates gene expression. Proc Nutr Soc 1999;58:625–32. [33] Kilberg MS, Pan YX, Chen H, Leung-Pineda V. Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. Annu Rev Nutr 2005;25:59–85. [34] Averous J, Bruhat A, Mordier S, Fafournoux P. Recent advances in the understanding of amino acid regulation of gene expression. J Nutr 2003;133:2040S–5S. [35] Anthony TG, Reiter AK, Anthony JC, Kimball SR, Jefferson LS. Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats. Am J Physiol Endocrinol Metab 2001;281:E430–9. [36] Postic C, Dentin R, Girard J. Role of the liver in the control of carbohydrate and lipid homeostasis. Diabetes Metab 2004;30:398–408. [37] Brosnan JT. Interorgan amino acid transport and its regulation. J Nutr 2003;133:2068S–72S. [38] Traxinger RR, Marshall S. Role of amino acids in modulating glucose-induced desensitization of the glucose transport system. J Biol Chem 1989;264:20910–6.
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Effects of individual branched-chain amino acids deprivation on insulin sensitivity and glucose metabolism in mice Fei Xiao a , Junjie Yu a , Yajie Guo a , Jiali Deng a , Kai Li a , Ying Du a , Shanghai Chen a , Jianmin Zhu b,⁎, Hongguang Sheng b,⁎, Feifan Guo a,⁎ a
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, the Graduate School of the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, China 200031 b Shanghai Xuhui Central Hospital, 966 Huaihai Middle Road, Shanghai, China 200030
A R T I C LE I N FO Article history:
AB S T R A C T Objective. We recently discovered that leucine deprivation increases hepatic insulin
Received 17 January 2014
sensitivity via general control nondepressible (GCN) 2/mammalian target of rapamycin (mTOR)
Accepted 12 March 2014
and AMP-activated protein kinase (AMPK) pathways. The goal of the present study was to investigate whether the above effects were leucine specific or were also induced by deficiency
Keywords: Leucine deprivation
of other branched chain amino acids including valine and isoleucine. Methods. Following depletion of BCAAs, changes in metabolic parameters and the
Valine deprivation
expression of genes and proteins involved in regulation of insulin sensitivity and glucose
Isoleucine deprivation
metabolism were analyzed in mice and cell lines including human HepG2 cells, primary
Insulin signaling
mouse hepatocytes and a mouse myoblast cell line C2C12.
Glucose metabolism
Results. Valine or isoleucine deprivation for 7 days has similar effect on improving insulin sensitivity as leucine, in wild type and insulin-resistant mice models. These effects are possibly mediated by decreased mTOR/S6K1 and increased AMPK signaling pathways, in a GCN2-dependent manner. Similar observations were obtained in in vitro studies. In contrast to leucine withdrawal, valine or isoleucine deprivation for 7 days significantly decreased fed blood glucose levels, possibly due to reduced expression of a key gluconeogenesis gene, glucose-6-phosphatase. Finally, insulin sensitivity was rapidly improved in mice 1 day following maintenance on a diet deficient for any individual BCAAs. Conclusions. Our results show that while improvement on insulin sensitivity is a general feature of BCAAs depletion, individual BCAAs have specific effects on metabolic pathways, including those that regulate glucose level. These observations provide a conceptual framework for delineating the molecular mechanisms that underlie amino acid regulation of insulin sensitivity. © 2014 Elsevier Inc. All rights reserved.
Abbreviations: AKT, protein kinases B; AMPK, AMP-activated protein kinase; BCAAs, Branched-chain amino acids; GCN2, General Control Nonderepressible 2; IR, insulin receptor; mTOR: mammalian Target of Rapamycin; S6K1, p70 ribosomal protein S6 Kinase 1. ⁎ Corresponding author. Tel.: +86 21 54920945; fax: +86 21 54920291. E-mail addresses: [email protected] (F. Xiao), [email protected] (J. Yu), [email protected] (Y. Guo), [email protected] (J. Deng), [email protected] (K. Li), [email protected] (Y. Du), [email protected] (S. Chen), [email protected] (J. Zhu), [email protected] (H. Sheng), [email protected] (F. Guo). http://dx.doi.org/10.1016/j.metabol.2014.03.006 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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1.
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Introduction
Diabetes is one of the major causes of morbidity and mortality worldwide. A common feature of type 2 diabetes is insulin resistance, which is characterized by reduced glucose uptake in muscle and adipose tissue, and increased glucose production in liver [1,2]. One of the important factors that contribute to insulin resistance is the unbalance of dietary macronutrients including fat, glucose and amino acids [3]. Aside from their role as the building blocks of proteins, amino acids are also critical mediators of intracellular signaling [4]. Branched-chain amino acids (BCAAs) that have non-linear aliphatic side-chains include leucine, valine and isoleucine, and are the most studied essential amino acids. Increased leucine concentration is reported to either improve or have no effect on glucose metabolism in mice [5,6]; however, there is increasing evidence of a correlation between increased levels of BCAAs and insulin resistance [7–9]. For example, metabolomic profiling of obese versus lean humans reveals a BCAAs-related metabolite signature that is suggestive of increased catabolism of BCAAs and correlated with insulin resistance [9]. Furthermore, Wang and colleagues found that quantification of BCAAs serum levels facilitates risk assessment for onset t of type 2 diabetes [10]. Though progress has been made in understanding the effect of BCAAs on insulin sensitivity and glucose metabolism, the effect of each individual BCAA remains largely unknown. In contrast to other studies examining relationships between increased levels of BCAAs and insulin sensitivity, our studies have focused on investigating the effects of elimination of dietary leucine and previously shown that leucine deprivation improves hepatic insulin sensitivity in vivo and in vitro [11]. It remains unclear, however, whether deficiency of other BCAAs, including valine and isoleucine, would have similar effects. A previous study showed that mice deleted for the BCATm gene encoding the enzyme catalyzing the first step in peripheral BCAAs metabolism, which would be expected to have low leucine, valine or isoleucine utilization, exhibited increased insulin sensitivity [12]. These results suggest that deficiency of valine or isoleucine may also modulate insulin sensitivity. The aim of our current study was to investigate these possibilities and elucidate underlying mechanisms. This study will help in understanding the unique feature of each individual BCAA. These observations are also important for understanding the molecular mechanisms underlying amino acid regulation of insulin sensitivity.
drate and lipid components. At the start of the feeding experiments, mice were acclimated to a control diet for 7 days and then randomly divided into control, (−) val or (−) ile diet group, with free access to each diet for 7 days. To determine the possible influences of reduced food intake in the BCAAsdeprived group, pair-fed (pf) groups were included. Mice in the pf group were provided with 18%, 30% or 40% less food, as determined in our preliminary experiments, compared to mice in the control group. A subset of mice underwent glucose tolerance test (GTT)/insulin tolerance test (ITT) prior to being killed by CO2 inhalation. As many things can change blood glucose quickly and significantly, we included corresponding control groups during every experiment. These experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (CAS).
2.2.
HepG2 cells were maintained in DMEM (Gibco, Grand Island, NY, USA) with 25 mmol/L glucose, 10% FBS, 50 μg/ml penicillin and streptomycin at 37 °C, 5% CO2–95% air. C2C12 myoblasts were obtained from the CAS Cell Bank of Type Culture Collection. Maintenance and induction of differentiation were performed as previously described [13]. Primary hepatocyte isolation was achieved by collagenase perfusion as described previously [14]. Control, (−) val or (−) ile medium were prepared by adding all the components of regular DMEM or lacking the corresponding amino acid.
2.3. Blood glucose, serum insulin, GTT, ITT and HOMA-IR index Blood glucose levels were measured using a Glucometer Elite monitor. Serum insulin levels were measured using the Mercodia Ultrasensitive Rat Insulin ELISA kit (Catalog Number: 80INSRTU-E01, ALPCO Diagnostic, Salem, NH, USA). GTT and ITT were performed by IP injection of 2 g/kg glucose after overnight fasting and 0.75 U/kg or 0.375 U/kg insulin after 4 h fasting, respectively. The HOMA-IR index was calculated according to the formula: [Fasting glucose levels (mmol/L)] × [Fasting serum insulin (μU/ml)]/22.5.
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2.
Methods
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Animals and treatments
Male C57BL/6 J mice were obtained from Shanghai Laboratory Animal (SLAC, Shanghai, China). GCN2 knockout (Gcn2−/−) and leptin receptor-mutated (db/db) mice were kindly provided by Dr. Douglas Cavener, Penn State University, USA and Dr. Xiang Gao, Nanjing University, China, respectively. Eight- to tenweek-old mice were maintained on a 12-h light/dark cycle at 25 °C. Control, (–) leu (leucine-deficient), (−) val (valine-deficient) and (−) ile (isoleucine- deficient) chow were obtained from Research Diets (New Brunswick, NJ, USA). All diets were isocaloric and compositionally the same in terms of carbohy-
Cell culture and treatments
In vivo insulin signaling assay
Mice maintained on different diets were fasted for 6 h prior to insulin injection as previously described [14]. Sections of liver and soleus muscle were excised from anesthetized mice and snap-frozen, as untreated controls. Three or five minutes after injection with 2 U/kg of insulin via the portal vein, pieces of tissue section were excised and snap-frozen for western blot analysis.
2.5.
Western blot analysis
Western blot analysis was performed as previously described [15]. Protein concentrations were assayed using BCA Kit (Pierce, Rockford, USA). Primary antibodies [anti-p-insulin receptor (Tyr1150/1151), anti-insulin receptor, anti-p-AKT (Ser473), antiAKT, anti-p-mTOR (Ser2448), anti-mTOR, anti-p-p70 S6K1
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(Thr389), anti-p70 S6K1, anti-p-S6 (Ser235/236), anti-S6, anti-pAMPK (Thr172), anti-t-AMPK (all the above from Cell Signaling Technology, Beverly, MA, USA)] were incubated overnight at 4 °C and specific proteins were visualized by ECL Plus (Amersham Biosciences, Buckinghamshire, UK). Band intensities were measured using Quantity One (Bio-Rad Laboratories) and normalized to total protein or actin.
2.6.
RNA isolation and relative quantitative RT-PCR
Total RNA was prepared from frozen tissues with TRIZOL (Life Technologies, Carlsbad, CA, USA) reagent. One microgram of RNA was reverse transcribed with random primer and M-MLV Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). Quantitative amplification by PCR was carried out using SYBR Green I Master Mix reagent by ABI 7500 system (Applied Biosystem). PCR products were subjected to a melting curve analysis. Cycle numbers of both GAPDH (as an internal control) and cDNAs of interest at a specific threshold within the exponential amplification range were used to calculate relative expression levels of the genes of interest. The sequences of primers used in this study are available upon request.
2.7.
examined the levels of phosphorylation of two key components in the insulin signaling pathways, insulin receptor (IR) and protein kinases B (AKT). As expected, insulin-stimulated phosphorylation of IR and AKT increased in the livers of valine-deprived or isoleucine- deprived mice compared with controls (Fig. 1C and D).
3.3. Valine or isoleucine deprivation increases insulin signaling in vitro To determine whether valine or isoleucine deprivation has a direct effect on insulin signaling in multiple cell types, we utilized the human hepatoma-derived cell line HepG2, primary mouse hepatocytes and C2C12, a mouse myoblast cell line. Consistent with our in vivo observations (Fig. 1C and D), we found that insulin-stimulated phosphorylation of IR and AKT was significantly elevated by valine or isoleucine deprivation in all three cell lines (Fig. 2A and B).
3.4. Valine or isoleucine deprivation decreases mammalian target of rapamycin (mTOR) and increases AMP-activated protein kinase (AMPK) signaling in vivo
Statistics
All data are expressed as mean ± SEM. Significant differences were assessed using a two-tailed student t-test. P < 0.05 was considered statistically significant.
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Results
3.1. Valine or isoleucine deprivation increases whole-body insulin sensitivity Mice were fed a control, valine- or isoleucine-deficient diet for 7 days, a period comparable to that we previously used for leucine deprivation tests. Glucose tolerance and clearance were examined by GTT and ITT, respectively. In GTT, mice fed a valine- or isoleucine-deficient diet exhibited significantly lower levels of blood glucose after overnight fasting. Fifteen minutes after injection of glucose, blood glucose levels were significantly lower in valine- or isoleucine-deprived mice compared with controls (Fig. 1A and B). Following administration of insulin, blood glucose levels decreased quickly in valineor isoleucine-deprived mice compared with mice maintained on a control diet (Fig. 1A and B). In contrast to the situation following valine deprivation, blood glucose levels were below the limit of detection in isoleucine-deprived mice when subjected to the same dose of insulin during the ITT. We therefore opted to use a 50% dose of insulin in the isoleucinedeprived mice and found much improved insulin sensitivity in these mice compared with control mice (Fig. 1B).
3.2. Valine or isoleucine deprivation increases insulin signaling in vivo Increased insulin sensitivity in mice upon valine or isoleucine deprivation suggests an increase in insulin signaling in peripheral tissues such as the liver. To test this possibility, we
The mTOR and AMPK signaling pathways play important roles in the development of insulin resistance, and both pathways are implicated in the regulation of insulin sensitivity during leucine deprivation. Consistent with changes in leucinedeprived mice, phosphorylation of mTOR and its downstream targets, including p70 ribosomal protein S6 Kinase 1 (S6K1) and ribosomal protein S6, was also significantly decreased in the livers of valine- or isoleucine-deprived mice compared with mice fed a control diet (Fig. 3A and B). In addition, AMPK phosphorylation was increased in the livers of mice fed a valine-deficient or isoleucine-deficient diet for 7 days compared with mice fed a control diet (Fig. 3A and B).
3.5. Valine deprivation increases insulin sensitivity by activation of GCN2 GCN2 is a serine protein kinase that senses amino acid deprivation and functions as an upstream regulator of mTOR to modulate hepatic insulin sensitivity upon leucine deprivation [11]. We thus speculated GCN2 might be involved in modulation of insulin sensitivity during valine deprivation. To examine this possibility, GCN2 phosphorylation was examined in the livers of mice maintained on a valinedeficient diet. Consistent with a previous study [16], GCN2 phosphorylation was increased in these mice (Fig. 4A). We then compared insulin sensitivity using ITT in Gcn2 +/+ and Gcn2 −/− mice maintained on a valine-deficient diet. Glucose clearance was decreased in Gcn2 −/− mice compared with their wild type counterparts upon valine deprivation (Fig. 4B). Consistent with these changes, insulin-stimulated phosphorylation of IR and AKT was decreased in the livers of Gcn2−/− mice (Fig. 4C). Since our previous study showed that the insulin response is normal in GCN2-null mice on a normal diet [17], these data indicate that GCN2 plays a specific role in sensing lack of dietary BCAAs.
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Fig. 1 – The effects of valine or isoleucine deprivation on insulin sensitivity in vivo. Mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 days, then subjected to glucose tolerance and insulin tolerance tests (GTT and ITT, respectively) in A and B. Insulin signaling in the liver was examined before (− Ins) and after (+ Ins) 2 U/kg insulin stimulation for 3 min in C and D. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of (−) val or (−) ile versus control diet (*p < 0.05). (A–B) GTT and ITT; (C–D) p-IR and p-AKT (left, western blot; right, quantitative measurements of p-IR and p-AKT proteins relative to their respective total protein levels).
3.6. Effects of individual BCAA deficiency on glucose metabolism Consistent with animals subjected to leucine deprivation, mice fed a valine- or isoleucine-deficient diet for 7 days also exhibited significantly lower levels of fasting blood glucose, fasting serum insulin and HOMA-IR index (Fig. 5A–C). In contrast to the unchanged fed blood glucose levels in leucinedeprived mice, fed blood glucose levels were also decreased in mice maintained on a valine- or isoleucine-deficient diet (Fig. 5D and E). To identify possible causes for the different blood glucose levels, we examined the expression of glucose-6phosphatase (g6pase), a key gluconeogenic gene in the liver. In contrast to leucine deprivation, which has no effect on g6pase expression, the levels of g6pase mRNA were decreased by both valine and isoleucine deprivation (Fig. 5F).
was performed. Blood glucose levels dropped more rapidly following administration of insulin in mice fed any individual BCAA-deficient diet compared with mice maintained on a control diet (Fig. 6A–C). As shown in our previous work, BCAAs deprivation also decreases food intake (18 % for leucine, 30 % for valine and 40 % for isoleucine,) compared with mice maintained on a control diet. As the insulin-sensitizing effect of isoleucine or valine deprivation may be related with the decreased food intake, pair-fed (pf) groups were included to distinguish the possible influence of the reduction in food intake in BCAAs-deprived groups. Mice in the pf group were provided with 18%, 30% or 40% less food compared to mice in the control group. As expected, no significant difference was observed between these groups when ITT was performed (Fig. 6D–F). Meanwhile, due to the differences in the amounts of food intake for each pf group, blood glucose levels were affected to different extent compared with control-diet fed mice.
3.7. Whole-body insulin sensitivity is increased in mice 1 day following maintenance on a diet deficient for any individual BCAAs
3.8. Valine or isoleucine deprivation increases insulin sensitivity under insulin-resistant condition
To investigate how quickly the effect of individual BCAAs deficiency on insulin sensitivity could occur, mice were fed a diet deficient for leucine, valine or isoleucine for 1 day and ITT
Our previous work has shown that leucine deprivation improves insulin sensitivity in insulin-resistant animal models, including high-fat diet fed mice and db/db mice [11].
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Fig. 2 – The effects of valine or isoleucine deprivation on insulin sensitivity in vitro. HepG2 cells and primary hepatocytes were incubated in control (+val) or valine-deficient (−val) medium for 48 h, or in control (+ile) or isoleucine-deficient (−ile) medium for 24 h after being treated with serum-free medium overnight. C2C12 cells were incubated in control, valine-deficient or isoleucin-deficient medium for 48 h. All the cells were then treated with (+ Ins) or without (− Ins) 100 nmol/L insulin stimulation for 20 min. Data are mean ± SEM of at least three independent experiments. Statistical significance is calculated using a two-tailed student t-test for the effects of (−) val or (−) ile versus control medium after insulin stimulation (*p < 0.05). (A and B) p-IR and p-AKT protein (top, western blot; bottom, quantitative measurements of p-IR and p-AKT proteins relative to their respective total protein levels).
We speculated that deprivation of valine and isoleucine may have the same effects. To test this possibility, db/db mice were fed a control, valine- or isoleucine-deficient diet for 7 or 5 days. Then insulin sensitivity was examined by ITT assay. As predicted, blood glucose levels decreased more quickly in valine- or isoleucine-deprived mice compared with mice maintained on a control diet following administration of insulin (Fig. 7A and B).
4.
Discussion
Increased serum levels of BCAAs are associated with insulin resistance in humans and mice [7–9], and infusion of isoleucine decreases glucose uptake in human forearm tissue [18], suggesting that decreased levels of valine or isoleucine may improve insulin sensitivity. Consistent with this possibility, we
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Fig. 3 – The effects of valine or isoleucine deprivation on mTOR/S6 and AMPK signaling in vivo. Mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 days. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–7 each group). Statistical significance is calculated using a two-tailed Student’s t-test for the effects of (−) val or (−) ile versus control diet (*p < 0.05). (A and B) p-mTOR, p-S6K1, p-S6 protein and p-AMPK (top, western blot; bottom, quantitative measurements of p-mTOR, p-S6K1 and p-S6 protein relative to their respective total protein levels).
found that valine or isoleucine deprivation increases wholebody insulin sensitivity and insulin signaling in liver and skeletal muscle (Supplementary Fig. 1) in vivo, and in several cell lines. These results are similar to those observed during leucine deprivation [11], suggesting enhanced insulin sensitivity is a general consequence of BCAAs deprivation. There are however some notable exceptions [19,20], suggesting that the effect of BCAAs could be very complicated. Intracellular amino acids levels can be sensed directly by aminoacyl tRNA formation, or indirectly by levels of their metabolites such as ATP and by intracellular proteins including mTOR, MAP4K3 and Rag [21,22]. Among them, mTOR and AMPK are the most extensively studied ones. mTOR is a Ser/Thr protein kinase, the activity of which is particularly regulated by leucine [23]. Furthermore, mTOR activity is closely related to the ability of the cell to respond to insulin; this is underscored by the observation that increased mTOR/S6K1 signaling contributes to the development of insulin resistance [7] and decreased mTOR/S6K1 signaling improves insulin sensitivity [24,25]. AMPK responds directly to fluctuations in the ratio of AMP:ATP [26]. Activation of AMPK stimulates cellular uptake of glucose and reduces glucose production [27–29]. For these reasons, AMPK activators, including metformin and rosiglitazone, have been widely used in the clinic to treat insulin resistance in diabetes patients [1]. Though both mTOR signaling and AMPK signaling are important
modulators of insulin sensitivity, whether they also perform this role in the context of valine or isoleucine deprivation has not been experimentally tested. In the current study, we observed that mTOR signaling decreases and AMPK phosphorylation increased in the liver during valine or isoleucine deprivation. Both signaling pathways are known to improve insulin sensitivity during leucine deprivation [11], and we thus speculate that valine or isoleucine deficiency may employ common mechanisms in regulating insulin sensitivity. Furthermore, our results strongly suggest that mTOR and AMPK activities are regulated by BCAA availability, which is consistent with results from other studies [30]. The upstream regulator responsible for decreased liver mTOR signaling during valine or isoleucine deprivation is unknown. GCN2 is a serine/threonine protein kinase. Lower intracellular amino acids levels lead to the accumulation of the corresponding amino acid uncharged tRNAs. GCN2 has a domain with homology with HisRS (histidyl-tRNA synthetase) that can bind to uncharged tRNAs, resulting in kinase activation through homodimerization and autophosphorylation [31]. Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2)α, concomitantly repressing general protein synthesis and increasing translation of proteins related to amino acid biosynthesis and transport [32–35]. In addition, our recent studies reveal that GCN2 is involved in regulating lipid metabolism and
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Fig. 4 – The role of GCN2 in valine deprivation—increased insulin sensitivity. (A) Mice were fed a control (ctrl) or valine-deficient [(−) val] diet for 7 days. (B and C) Both Gcn2+/+ and Gcn2−/− mice were fed a valine-deficient [(−) val] diet for 7 days, followed by an insulin tolerance test (ITT) in B and examination of insulin signaling before (− Ins) and after (+ Ins) 2 U/kg insulin stimulation for 3 min in C. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of (–) val versus control diet (p < 0.05) in A and Gcn2−/− versus Gcn2+/+ mice (*p < 0.05) in B and C. (A) p-GCN2 (top, western blot; bottom, quantitative measurements of p-GCN2 protein relative to its total protein levels). (B) ITT; (C) p-IR and p-AKT (left, western blot; right, quantitative measurements of p-IR and p-AKT protein relative to their respective total protein levels).
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Fig. 5 – Effects of individual BCAAs deprivation on glucose metabolism. Mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 days. Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of BCAAs deprivation versus control diet (*p < 0.05). (A and B) Blood glucose and serum insulin levels after fasting overnight; (C) HOMA-IR index; (D and E) fed blood glucose and serum insulin levels; (F) g6pase mRNA.
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Fig. 6 – The effects of a 24 h dietary deficiency of individual BCAAs on insulin sensitivity. Mice were maintained 1 day on a control (ctrl), leucine-deficient [(−) leu], valine-deficient [(−) val], isoleucine-deficient [(−) ile] or pair-fed (pf) diet for each BCAA, followed by carrying out an insulin tolerance test (ITT). Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of BCAA deprivation or pf diet versus control diet (*p < 0.05). (A–C) ITT for ctrl and (−) BCAA group; (D–F) ITT for ctrl and pf group. insulin sensitivity during leucine deprivation [11,17]. Because GCN2 is supposed to be activated by valine or isoleucine deprivation and functions as an upstream regulator of mTOR in regulating hepatic insulin sensitivity during leucine deprivation [11], we speculated it may have a similar regulatory role upon withdrawal of valine or isoleucine. Consistent with this hypothesis, we found that GCN2 phosphorylation was increased in the livers of mice maintained
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on a valine-deficient diet compared with control mice. Furthermore, increased insulin sensitivity triggered by valine deprivation is impaired in GCN2−/− mice. Since GCN2 is activated by deficiency of any essential amino acids [16], we hypothesized that GCN2 is also involved in the regulation of insulin sensitivity during isoleucine deprivation. The role of GCN2 upon valine or isoleucine deprivation also highlights the critical role that GCN2 plays as a ‘master metabolite sensor’.
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Fig. 7 – Valine or isoleucine deprivation improves insulin sensitivity under insulin-resistant conditions. db/db mice were fed a control (ctrl), valine-deficient [(−) val], or isoleucine-deficient [(−) ile] diet for 7 or 5 days, then subjected to insulin tolerance test (ITT). Data are mean ± SEM of at least two independent experiments with mice of each group for each experiment (n = 5–6 each group). Statistical significance is calculated using a two-tailed student t-test for the effects of (−) val or (−) ile versus control diet (*p < 0.05). (A and B) ITT.
M ET ABOL I SM CL IN I CA L A N D EX PE RI ME N TA L 6 3 ( 2 0 14 ) 84 1–8 5 0
In contrast to the unaltered fed blood glucose levels in mice fed a leucine-deficient diet [11], fed blood glucose levels decreased during valine or isoleucine deprivation. One possible explanation for this differential effect could be the specific regulation of the key gluconeogenic enzyme G6Pase. This protein is not affected by leucine deprivation, but its level is reduced by valine or isoleucine deprivation. G6Pase catalyses the final step of the gluconeogenic pathway, which hydrolyzes glucose-6-phosphate, generating a phosphate group and free glucose [36]. Therefore, altered expression of G6Pase would lead to changes in blood glucose levels. In addition, valine and isoleucine are more readily used as substrates for gluconeogenesis when compared with leucine [37], which may also lead to the difference in fed blood glucose levels. Our previous work has shown that leucine deprivation also improves insulin sensitivity under insulin-resistant conditions, including high-fat diet fed mice and db/db mice [11]. Similar observations were obtained by valine or isoleucine deprivation. Besides, we found that insulin sensitivity is improved as early as 1 day following maintenance on a diet deficient for any of the individual BCAAs. As mice maintained on BCAA-deficient diet reduced their food intake, we included a pf group. However, we did not see any difference in ITT assay between pf group and control group at the same time. Furthermore, our previous study has shown that insulin sensitivity was increased in mice fed leucine-deficient diet, compared with control or pf mice [11]. Based on these results and our in vitro observations showing a direct effect of isoleucine or valine deficiency on insulin signaling, we speculate that the increased insulin sensitivity in isoleucine- or valine-deficient mice is caused primarily by a deficiency of BCAA, rather than a reduction in food intake. Because the effect of BCAA deficiency occurs very rapidly, these results provide important hints for the potential use of this diet in treating insulin resistance in diabetes patients. Determining the optimal concentration of BCAAs and the duration of therapeutic BCAAs-deficient diets will be important issues for future pre-clinical and clinical studies. Our current study provides important evidence demonstrating a general effect of BCAAs on improving insulin sensitivity, which would provide important hints for dietary control on insulin resistance. In addition, our unpublished results and those of others [38] reveal that individual amino acids have different effects on insulin sensitivity in primary adipocytes and HepG2 cells, suggesting that the regulation of insulin sensitivity and glucose metabolism by different amino acids is very complex. Furthermore, we can’t presently conclude that the effects on insulin sensitivity we observed are restricted to BCAAs; for this, a systematic study of BCAAs depletion versus lack of non-BCAA essential amino acids or nonessential amino acids will be required. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.metabol.2014.03.006.
Author contributions Fei Xiao designed the experiment, researched and analyzed data, contributed to discussion, wrote manuscript and reviewed/edited
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manuscript. Junjie Yu, Yajie Guo, Jiali Deng, Kai Li, Ying Du and Shanghai Chen researched data and contributed to discussion. Jianmin Zhu, Hongguang Sheng and Feifan Guo designed the experiment, contributed to discussion, wrote manuscript and reviewed/edited manuscript.
Funding This work was supported by grants from the Ministry of Science and Technology of China (973 Program 2010CB912502), China National Funds for Distinguished Young Scientists (81325005), National Natural Science Foundation (81130076, 31271269, 81100615, 30890043, 81390352 and 81300659), Basic Research Project of Shanghai Science and Technology Commission (13JC1409000), Key Program of Shanghai Scientific and Technological Innovation Action Plan (10JC1416900), the Knowledge Innovation Program of CAS(KSCX2-EW-R-09), Chinese Academy of Sciences-funded project (2011KIP307). Feifan Guo was also supported by the One Hundred Talents Program of the Chinese Academy of Sciences. Fei Xiao was supported by China Postdoctoral Science Foundation funded project (2012 M520950 and 2013 T60473) and a Chinese Academy of Sciences-funded project (2013KIP310).
Disclosure statement There is no conflict of interest that the authors should disclose.
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