Independent and combined effects of acute physiological ...

Clinical Science (2013) 124, 675–684 (Printed in Great Britain) doi: 10.1042/CS20120481

Independent and combined effects of acute physiological hyperglycaemia and hyperinsulinaemia on metabolic gene expression in human skeletal muscle Kostas TSINTZAS*, Luke NORTON†, Kamal CHOKKALINGAM*, Nusrat NIZAMANI*, Scott COOPER*, Francis STEPHENS*, Rudolf BILLETER* and Andrew BENNETT*‡ *School of Biomedical Sciences, Nottingham University Medical School, Queens Medical Centre, Nottingham NG7 2UH, U.K. †Division of Diabetes, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, U.S.A. ‡FRAME Alternatives Laboratory, Nottingham University Medical School, Queens Medical Centre, Nottingham NG7 2UH, U.K.

Abstract Physiological hyperglycaemia and hyperinsulinaemia are strong modulators of gene expression, which underpins some of their well-known effects on insulin action and energy metabolism. The aim of the present study was to examine whether acute in vivo exposure of healthy humans to hyperinsulinaemia and hyperglycaemia have independent or additive effects on expression of key metabolic genes in skeletal muscle. On three randomized occasions, seven young subjects underwent a 4 h (i) hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (HIHG), (ii) hyperglycaemic (10 mmol/l) euinsulinaemic (5 m-units · m − 2 · min − 1 ) clamp (LIHG) and (iii) hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) euglycaemic (4.5 mmol/l) clamp (HING). Muscle biopsies were obtained before and after each clamp for the determination of expression of genes involved in energy metabolism, and phosphorylation of key insulin signalling proteins. Hyperinsulinaemia and hyperglycaemia exerted independent effects with similar direction of modulation on PI3KR1 (phosphatidylinositol 3-kinase, regulatory 1), LXRα (liver X receptor α), PDK4 (pyruvate dehydrogenase kinase 4) and FOXO1 (forkhead box O1A) and produced an additive effect on PI3KR1, the gene that encodes the p85α subunit of PI3K in human skeletal muscle. Acute hyperglycaemia itself altered the expression of genes involved in fatty acid transport and oxidation [fatty acid transporter (CD36), LCAD (long-chain acyl-CoA dehydrogenase) and FOXO1], and lipogenesis [LXRα, ChREBP (carbohydrate-responseelement-binding protein), ABCA1 (ATP-binding cassette transporter A1) and G6PD (glucose-6-phosphate dehydrogenase). Surperimposing hyperinsulinaemia on hyperglycaemia modulated a number of genes involved in insulin signalling, glucose metabolism and intracellular lipid accumulation and exerted an additive effect on PI3KR1. These may be early molecular events that precede the development of glucolipotoxicity and insulin resistance normally associated with more prolonged periods of hyperglycaemia and hyperinsulinaemia. Key words: additive effect, hyperglycaemia, hyperinsulinaemia, phosphatidylinositol 3-kinase, regulatory 1 (PI3KR1), skeletal muscle

INTRODUCTION Physiological hyperglycaemia and hyperinsulinaemia stimulate glucose uptake, storage and oxidation [1,2]. In addition to their well-known metabolic effects, insulin and glucose are strong

modulators of gene expression. Short-term insulin infusion in healthy humans was shown to regulate about 800 skeletal muscle genes involved in a number of processes including transcriptional regulation, intracellular signalling and energy metabolism [3,4].

Abbreviations: ABCA1, ATP-binding cassette transporter A1; CHO, carbohydrate; ChREBP, carbohydrate-response element-binding protein; FASN, fatty acid synthase; FATP1, fatty acid transport protein 1; FOXO1, forkhead box O1A; G6P, glucose 6-phosphate; G6PD, G6P dehydrogenase; GIR, glucose infusion rate; GLUT4, glucose transporter 4; HKII, hexokinase II; HMBS, hydroxymethylbilane synthase; IRS1, insulin receptor substrate-1; LCAD, long-chain acyl-CoA dehydrogenase; LDH, lactate dehydrogenase B; LXRα, liver X receptor α; NEFA, non-esterified fatty acid; PDK4, pyruvate dehydrogenase kinase 4; PDP1, pyruvate dehydrogenase phosphatase, isoenzyme 1; PI3KR1, phosphatidylinositol 3-kinase, regulatory 1; PP2A, protein phosphatase 2A; PPP, pentose phosphate pathway; RER, respiratory exchange ratio; SREBP1c, sterol-regulatory-element-binding protein 1c; TAG, triacylglycerol. Correspondence: Dr Kostas Tsintzas (email [email protected]).

www.clinsci.org

675

K. Tsintzas and others

While those studies investigated global changes in gene expression following insulin infusion, our laboratory has shown that in healthy humans the expression of insulin-inducible genes in human skeletal muscle [such as PDK4 (pyruvate dehydrogenase kinase 4), HKII (hexokinase II), SREBP1c (sterol-regulatoryelement-binding protein 1c) and facilitated GLUT4 (glucose transporter 4)] is altered in insulin resistant and dietary carbohydrate deprivation states [5–8]. However, these states are often accompanied by changes in blood glucose concentrations and/or metabolism and therefore one cannot fully distinguish the independent effects of changes in insulin signalling and availability of glucose as a substrate on gene expression under those conditions. Hence, many previously ascribed insulin sensitive genes are also regulated by glucose. Indeed, maintaining hyperglycaemia (∼ 10 mmol/l) during a 3 h clamp in healthy humans modified the expression of about 300 skeletal muscle genes involved in various processes including glucose metabolism (such as HKII and PDK4) [9]. Very little is known about the combined effects of physiological hyperinsulinaemia and hyperglycaemia on metabolic gene expression. Some of these effects might be additive or synergistic depending on: (i) the extent of convergence of the signalling pathways by which glucose and insulin control gene expression, and (ii) the extent to which these effects can be attributed to changes in intracellular glucose flux. The aim of the present study was to examine for the first time whether acute in vivo exposure of healthy humans to hyperinsulinaemia and hyperglycaemia have independent or additive effects on expression of genes encoding key enzymes and transcription factors involved in energy metabolism in human skeletal muscle. The adverse effects of hyperglycaemia on both insulin action and intramuscular lipid accumulation (glucolipotoxicity) are not acute and occur after at least 5 h of exposure to hyperglycaemia [10,11]. Therefore the present study sought to examine changes in gene expression during the first 4 h of hyperglycaemia with and without concomitant hyperinsulinaemia in an attempt to identify the early molecular events that may precede the development of glucolipotoxicity and insulin resistance.

MATERIALS AND METHODS Subjects Seven healthy non-obese males [age 22.7 + − 1.9 years; body mass, 2 + 78.8 + 3.2 kg, BMI (body mass index), 24.2 − − 0.8 kg/m ] participated in the study after providing written informed consent. All procedures were performed according to the Declaration of Helsinki and approved by the local Medical School Ethics Committee. In the absence of data on the additive effects of hyperinsulinaemia and hyperglyceamia on gene expression in humans, data from studies that reported their independent effects on key metabolic genes (such as PDK4, HKII, SREBP1c) in human skeletal muscle [5,8,9] was used to calculate the number of subjects required to provide 80 % probability at a 5 % significance level to detect at least a 50 % change in their mRNA content over the duration of the study.

676

C The Authors Journal compilation C 2013 Biochemical Society

Experimental design On three randomized occasions, 2 weeks apart, all subjects underwent: (i) a hyperinsulinaemic–hyperglycaemic clamp for 4 h (HIHG trial). The magnitude of hyperinsulinaemia– hyperglycaemia was designed to match the plasma insulin (∼ 80 m-units/l) and glucose (∼ 10 mmol/l) responses normally seen after ingestion of carbohydrate-rich meals; (ii) a euinsulinaemic (∼ 8–10 m-units/l)–hyperglycaemic (10 mmol/l) clamp for 4 h (LIHG trial); (iii) a hyperinsulinaemic–euglycaemic (4.5 mmol/l) clamp for 4 h (HING trial).

Experimental protocol On each experimental day, subjects arrived at the laboratory after an overnight fast (10–12 h) having abstained from smoking and caffeine on the day of the study, and from alcohol and heavy exercise for the previous 3 days. The subjects were asked to lie on a bed and sterile cannulae were inserted under local anaesthetic (1 % lidocaine) into an antecubital vein on one arm for glucose and insulin infusion, and into a dorsal vein on the non-dominant hand for sampling, with the hand placed in a hot-air box maintained at 50–55 ◦ C to arterialize the blood. A slow infusion of 0.9 % sterile saline was used to keep the sampling line patent. After resting for 15 min, a baseline blood sample was obtained followed by a muscle sample from the vastus lateralis of one leg using the percutaneous needle biopsy technique as previously described [7]. All muscle samples were kept in liquid nitrogen for subsequent analysis. After this, on two occasions, infusion of human soluble insulin (Actrapid, Novo) commenced at a rate of 50 munits · m − 2 · min − 1 and continued throughout each 4 h clamp, with 20 % (w/v) dextrose infused at a variable rate to maintain blood glucose concentrations at either 4.5 mmol/l (HING trial) or 10 mmol/l (HIHG trial), respectively. On both occasions, infusion of somatostatin at 500 μg/h (to inhibit endogenous insulin secretion), and replacement infusion of glucagon [0.7 ng · (kg − 1 of body mass) · min − 1 ] started 30 min before dextrose infusion. On a third occasion, 20 % (w/v) dextrose was infused at a variable rate to maintain blood glucose concentration at 10 mmol/l (LIHG trial). Infusion of somatostatin at 500 μg/h and basal replacement infusions of glucagon [0.7 ng · (kg − 1 of body mass) · min − 1 ] and insulin (5 m-units · m − 2 · min − 1 ; aiming to maintain plasma insulin levels at approximately 8–10 m-units/l) started 30 min before dextrose infusion. On all occasions, monitoring of blood glucose was carried out every 5 min to ensure that it remained at desired levels. Subjects remained in a semi-recumbent position for the entire period of infusion. Then 8 ml blood samples were also collected every 20 min. A ventilated canopy system linked to a metabolic cart (GEM; Nutren Technologies) was used to measure O2 consumption and CO2 production for 15 min immediately before and every hour during each clamp. Measurements were made while the subjects were lying supine, undisturbed and awake. RERs (respiratory exchange ratio) and rates of carbohydrate and fat oxidation [expressed as mg · (kg of body mass) − 1 · min − 1 ] were calculated from the CO2 and O2 measurements as described previously [12]. A second muscle sample was obtained at the end of the clamp (4 h) from the same leg but a different site at least 3 cm apart.

Hyperglycaemia and gene expression in human skeletal muscle

Following this, the insulin, somatostatin and glucagon infusions were discontinued. The 20 % (w/v) dextrose infusion was continued to prevent symptoms of hypoglycaemia. A meal was provided after which subjects were allowed to leave the laboratory.

bilane synthase)] was used to normalize the data to minimize variations in the expression of individual housekeeping genes. Real-time PCR was also performed using an ABI Prism 7000 Sequence Detection System (ABI Applied Biosystems) to confirm changes in patterns of expression of individual genes (PDK4, HKII, SREBP1c and α-actin; results not shown).

Blood analysis Whole-blood glucose concentrations were measured immediately after collection using a Yellow Springs Instrument Analyser (2300 STAT PLUS). Plasma and serum were separated by centrifugation (15 min at 3000 g). Plasma was analysed for NEFA (nonesterified fatty acid) concentrations using a commercially available kit (NEFA-C test; Wako Chemicals). Serum was analysed for insulin and glucagon concentrations by RIA (Diagnostics). The aliquot of whole blood used to derive the serum for the glucagon assay was treated with 100 μl of aprotinin containing 1000 kallikrein-inactivating units and the serum was subsequently stored in glass tubes at − 80 ◦ C until analysis.

Skeletal muscle analysis Long-chain acyl-CoA and G6P (glucose 6-phosphate) content were determined enzymatically ([13] and [14] respectively). Both measurements were performed in muscle samples from five subjects due to limited biopsy material. Skeletal muscle long-chain acyl-CoA content was measured to assess the extent of intramyocellular lipid accumulation in response to hyperglycaemia, which has been shown to impinge on insulin-stimulated glucose uptake [15]. Skeletal muscle G6P content was measured to assess whether its accumulation in response to hyperglycaemia is associated with the induction of genes involved in skeletal muscle lipogenesis under those conditions [16,17]. Total RNA was extracted from 15–20 mg of frozen muscle tissue according to the method described by Chomczynski and Sacchi [18] using TRIzol® reagent (Invitrogen). Quantification of RNA and its RT (reverse transcription) was carried out as described previously [7]. Taqman low-density custom array using Micro Fluidic cards (ABI Applied Biosystems) was used for the relative quantification of expression of 48 metabolic genes. Each card allowed for eight samples to be run in parallel against 48 Taqman gene expression assay targets that were pre-loaded into each of the wells on the card (Supplementary Table S1 at http://www.clinsci.org/cs/124/cs1240675add.htm). A control sample was loaded in each card to assess reproducibility of data between cards and normalize for variations in the expression of the target genes between cards. Briefly, 50 μl of Taqman Universal PCR master mix (2×) (ABI Applied Biosystems) was added to 200 ng of RNA equivalent of cDNA into an Eppendorf RNAsefree tube. RNAse-free water was added to make the total volume of the reaction mixture up to 100 μl. The reaction mixture was mixed, centrifuged and loaded into one of the fill reservoir of the Micro Fluidic card. The cards were centrifuged (MULTIFUGE 3 S-R; Heraeus) and ran on a 7900HT Fast Real-Time PCR System (ABI Applied Biosystems). Relative quantification of the genes of interest was performed using the comparative C T method. The average expression of three housekeeping genes [α-actin, 18S ribosomal RNA (18S) and HMBS (hydroxymethyl-

Protein extraction and Western blotting Total protein extracts were prepared from 20–30 mg of frozen biopsy tissue by homogenization in a Hepes phosphatase buffer as described previously [8]. The extracts were used for the determination of phospho-IRS1 (insulin receptor substrate-1) Ser302 and phospho-Akt Ser473 (Cell Signaling Technology) and the endogenous control α-actin (Sigma–Aldrich). Protein concentrations of tissue extracts were measured using the BCA (bicinchoninic acid) method (Pierce). Proteins were separated, blocked, Western blotted and quantified as described previously [7,8]. The phosphorylation of IRS1 at Ser302 and Akt at Ser473 was assessed as indices of activation of insulin signalling in response to treatments. Increased phosphorylation of IRS1 at Ser2 has been implicated in the development of lipid-induced insulin resistance [19], whereas impairment in Akt phosphorylation at Ser473 is associated with decreased skeletal muscle insulin-stimulated glucose uptake in Type 2 diabetes [20].

Statistical analysis All biochemical and molecular data were analysed using a repeated measures two-way [treatment (HING against HIHG against LIHG)×time (pre-clamp against post-clamp)] ANOVA. A one-way ANOVA was used to compare glucose disposal, RER and substrate oxidation rates between the three trials. When a significant difference was obtained with either two-way or oneway ANOVA, data were analysed further with paired Student’s t tests using the Bonferroni correction. Statistical significance was accepted at a 5 % level. Results are presented as means + − S.E.M.

RESULTS Whole-body responses The GIR (glucose infusion rate) during the last 2 h of the clamp was higher (P < 0.001) in HIHG than HING [16.7 + − 1.3 against −1 −1 8.9 + − 0.4 mg · (kg of body mass) · min ]. In both trials, GIR was higher (P < 0.001) than LIHG [2.6 + − 0.3 mg · (kg of body mass) − 1 · min − 1 ] over the same period of time. There was no decrease in the GIR required to maintain hyperinsulinaemia and/or hyperglycaemia during the latter stages of the three clamps (Figure 1). There was an increase (P < 0.001) in the RER in HIHG and HING (pre-clamp, 0.87 + − 0.02 and 0.85 + − 0.02 respectively; + clamp, 1.01 + 0.02 and 0.97 0.01 respectively), but not in the − − LIHG trial (pre-clamp, 0.87 + − 0.02; clamp, 0.90 + − 0.02). The average rate of CHO (carbohydrate) oxidation was lower in LIGH −1 −1 [2.80 + − 0.12 mg · (kg of body mass) · min ] than in both the −1 · min − 1 ; P < 0.001] HIHG [5.42 + 015 mg · (kg of body mass) − −1 −1 and HING [4.39 + − 0.11 mg · (kg of body mass) · min ; P < 0.05] trials. The CHO oxidation rate was higher (P < 0.05) in HIHG than in HING. On the other hand, the average rate of fat

www.clinsci.org

677


Figure 1

GIRs [mg · (kg of body mass) − 1 · min − 1 ] during 4 h of (i) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) euglycaemic (4.5 mmol/l) clamp (HING trial), (ii) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (HIHG trial) and (iii) a euinsulinaemic (5 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (LIHG trial) + S.E.M., n = 7. §P < 0.001 compared with HING over the last 2 h; ¥ P < 0.001 compared Values are expressed as means − with LIHG over the last 2 h.

Table 1

Blood metabolite concentrations before (pre-clamp) and during 240 min of (i) a hyperinsulinaemic (50 munits · m − 2 · min − 1 ) euglycaemic (4.5 mmol/l) clamp (HING), (ii) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (HIHG) and (iii) a euinsulinaemic (5 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (LIHG) Results are means (S.E.M.), n = 7. *P < 0.05 comapred with HIHG; #P < 0.0001 compared with LIHG and HIHG; †P < 0.001 from pre-clamp; ‡P < 0.01 compared with pre-clamp; § P < 0.0001 compared with HING and HIHG. Clamp

Parameter

Treatment

Pre-clamp

Blood glucose (mmol/l)

HING

4.2 (0.1)

4.3 (01)#

4.4 (0.1)#

HIHG

4.3 (0.1)

8.3 (0.8)

10. 2 (0.3)

Serum insulin (m-units/l)

Plasma NEFAs (mmol/l)

60 min

180 min 4.5 (0.1)# 9.9 (0.3)

240 min 4.2 (0.1)# 10.2 (0.2)

LIHG

4.4 (0.1)

11.2 (0.4)*

10.6 (0.4)

10.4 (0.2)

10.1 (0.2)

HING

6.5 (1.1)

74.6 (1.7)

77.0 (2.5)

73.1 (2.0)

78.4 (2.2)

HIHG

6.3 (0.6)

75.1 (3.4)

74.4 (2.9)

81.6 (3.4)

81.8 (3.9)

LIHG

7.2 (1.0)

3.4 (0.3)§

7.6 (1.5)§

8.5 (1.6)§

9.7 (1.5)§

HING

0.45 (0.06)

0.03 (0.004)†

0.01 (0.004)†

0.01 (0.003)†

0.01 (0.003)†

HIHG

0.35 (0.05)

0.02 (0.005)†

0.01 (0.002)†

0.01 (0.001)†

0.01 (0.003)†

LIHG

0.35 (0.04)

0.53 (0.04)§

0.32 (0.03)§

0.14 (0.03)§‡

0.08 (0.01)§†

oxidation was higher (P < 0.05) in LIHG [0.57 + − 0.05 mg · (kg of body mass) − 1 · min − 1 ] than in both the HING [0.17 + − 0.03 mg · (kg of body mass) − 1 · min − 1 ] and HIHG (where it was completely suppressed).

HING (P < 0.001) (Table 1). Circulating NEFAs were also suppressed during the last 2 h of LIHG (P < 0.01), although to a lesser extent compared with the other trials.

Blood metabolites

Skeletal muscle long-chain acyl-CoA and G6P content

Blood glucose concentrations were kept at similar levels in LIHG and HIHG (10.4 + − 0.1 and 10.1 + − 0.1 mmol/l respectively), whereas they were maintained at fasting levels (4.4 + − 0.1 mmol/l) throughout the HING clamp (Table 1). Serum insulin concentrations were maintained at 78 + − 3 and 76 + − 2 m-units/l during the clamps in HIHG and HING, respectively, and were higher (P < 0.0001) than the fasting levels maintained in LIHG (7.3 + − 1.1 m-units/l) (Table 1). Serum glucagon concentrations declined by about 33 % at the end of all trials (P < 0.001) from an average baseline value of 71.0 + − 8.5 pg/ml with no difference observed between treatments. Plasma NEFA concentrations were suppressed below baseline values at all times in both HIHG and

678

120 min


Muscle long-chain acyl-CoA concentrations were not different between the HING, HIHG and LIHG trials (preclamp, 17.5 + − 3.7, 19.4 + − 2.8 and 13.3 + − 3.3 μmol/kg of dry mass respectively; post-clamp, 14.4 + − 5.5, 15.2 + − 3.8 and 20.6 + − 3.5 μmol/kg of dry mass respectively). There was a trend (P = 0.08) for an increase in the G6P concentrations in HIHG and LIHG (pre-clamp, 3.5 + − 0.5 and 3.0 + − 0.8 mmol/kg of dry mass respectively; post-clamp, 5.9 + 0.7 and 4.7 + − − 0.7 mmol/kg of dry mass respectively), whereas there was no difference in HING (pre-clamp, 3.3 + − 0.3 mmol/kg of dry mass; post-clamp, 2.9 + 0.5 mmol/kg of dry mass). −


Figure 2

Skeletal muscle gene expression before (pre-clamp) and after (post-clamp) (A) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) euglycemic (4.5 mmol/l) clamp (HING trial); (B) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (HIHG trial); (C) an euinsulinaemic (5 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (LIHG trial) Values represent ratios of the mRNA content of target genes to the average content of three housekeeping genes (α-actin, 18S and HMBS) and are expressed as means + − S.E.M., n = 7. Two-way ANOVA revealed significant time effects (changes from pre-clamp to post-clamp values) for 17 genes across all three treatments: *P < 0.05 and **P < 0.01 compared with pre-clamp. Two-way ANOVA revealed significant interaction (between treatment and time) effects (P < 0.05) for LDH, IRS2, ABCA1, FATP1 and CD36. †P < 0.05 compared with HIHG; ‡P < 0.05 compared with HING; # P < 0.05 compared with LIHG. Both treatment and interaction effects were observed with two-way ANOVA for PI3KR1 (P < 0.01 and P < 0.001, respectively) and SREBP1c (P < 0.05 and P < 0.01, respectively): §P < 0.05 compared with HING and LIHG; ¥ P < 0.01 compared with LIHG.

Skeletal muscle gene expression Two-way ANOVA revealed significant time effects (changes from pre-clamp to post-clamp values) for 17 genes involved in glucose uptake, phosphorylation and metabolism [GLUT4, HKII, LDH (lactate dehydrogenase B), PDK4 and PDP1 (pyruvate dehydrogenase phosphatase, isoenzyme 1)], insulin signalling [IRS1, IRS2, PI3KR1 (phosphatidylinositol 3-kinase, regulatory 1) and FOXO1 (forkhead box O1A)], intracellular lipid formation and reverse cholesterol efflux [LXRα (liver X receptor α), SREBP1c, ChREBP (carbohydrate-response-elementbinding protein), ABCA1 (ATP binding cassette transporter A1)]

and G6PD (G6P dehydrogenase) and fatty acid transport and oxidation [CD36, FATP1 (fatty acid transport protein 1) and LCAD (long-chain acyl-CoA dehydrogenase)]. Specifically, post-hoc analysis between the pre-clamp and post-clamp values within each trial revealed an up-regulation of GLUT4, HKII, PI3KR1, FOXO1, LXRα and SREBP1c mRNA in the HING trial, whereas PDK4, IRS2 and FATP1 were suppressed (Figure 2) There was a trend for LDH (pre-clamp, 1.26 + − 0.24; post-clamp, 0.97 + − 0.15; P = 0.099) to decrease in HING. The HIHG clamp caused an increase in the mRNA content of GLUT4, HKII, PI3KR1, FOXO1, LXRα, SREBP1c and G6PD,

www.clinsci.org

679


whereas a decrease was observed for PDK4, PDP1, IRS1, IRS2 and FATP1 (Figure 2B). There was a trend for induction of LCAD (P = 0.08) in HIHG. The LIHG clamp resulted in up-regulation of PI3KR1, LXRα, ChREBP, ABCA1, CD36 and LCAD, whereas PDP1 was suppressed (Figure 2C). There was a strong trend for suppression of PDK4 (P = 0.06) and induction of FOXO1 and G6PD (both P = 0.08) in the LIHG trial. Two-way ANOVA also revealed significant interaction between treatment (HING against HIHG against LIHG) and time (pre-clamp against post-clamp) effects (P < 0.05) for IRS2, FATP1, LDH, ABCA1 and CD36. Hyperinsulinaemia resulted in suppression of IRS2 and FATP1 (Figures 2A and 2B), and this response was different (P < 0.05) when compared with the LIHG trial, which showed no change in the expression of those genes. In contrast, the hyperinsulinaemia-induced decrease in LDH gene expression in HING (Figure 2A) was different (P < 0.05) to the response observed in the HIHG trial (Figure 2B), suggesting that in the presence of hyperglycaemia the suppressive effect of hyperinsulinaemia on LDH was abolished in the HIHG trial. The expression of ABCA1 was up-regulated (P < 0.05) in LIHG (Figure 2C), whereas in the presence of hyperinsulinaemia the stimulatory effect of hyperglycaemia was abolished in the HIHG trial (P < 0.05 from LIHG). The effect of hyperglycaemia with and without concomitant hyperinsulinaemia (HIHG and LIHG trials, respectively; Figures 2B and 2C) on CD36 was greater (P < 0.05) than the effect of hyperinsulinaemia alone in the HING trial (Figure 2A). Both treatment and interaction (between treatment and time) effects were observed for PI3KR1 (P < 0.01 and P < 0.001, respectively) and SREBP1c (P < 0.05 and P < 0.01, respectively). SREBP1c was equally induced (P < 0.01) in the hyperinsulinaemic trials (HING and HIHG) (Figures 2A and 2B) and this effect was higher (P < 0.01) in both trials when compared with a small non-significant decrease in LIHG (Figure 2C). Hyperinsulinaemia and hyperglycaemia exerted an additive effect on PI3KR1, with both stimuli increasing its expression (P < 0.01 and P < 0.05, respectively) albeit the former was a more potent stimulus than the latter (P < 0.05). When combined, their effect was higher (P < 0.05) than either of the responses alone and similar to the sum of the individual effects (Figure 2B).

Skeletal muscle insulin signalling proteins Two-way ANOVA revealed a significant time (but not treatment or interaction) effect for the phosphorylation of IRS1 at Ser302 . Post-hoc analysis between the pre-clamp and post-clamp values showed a 25 % increase in the phosphorylation of IRS1 at Ser302 at the end of HING (P < 0.05), with no significant changes in HIHG and LIHG (Figure 3A). There was a 2.5–3.0-fold increase (P < 0.05) in the phosphorylation of Akt at Ser473 in response to HIHG and HING treatments, but no increase was seen in LIHG (Figure 4A). The phosphorylation of Akt in HIHG and HING was higher than in LIHG (P < 0.01 and P < 0.05, respectively; two-way ANOVA interaction effect). Representative blots for phospho-IRS1 and phospho-Akt are shown in Figures 3(B) and 4(B), respectively.

680


DISCUSSION The results of the present study have shown for the first time that superimposing hyperinsulinaemia on hyperglycaemia exerts an additive effect on PI3KR1, a key gene involved in insulinstimulated glucose transport in human skeletal muscle. Furthermore, hyperinsulinaemia, but not hyperglycaemia, altered the expression of genes involved in glucose metabolism and insulin signalling (SREBP1c, GLUT4, HKII, LDH and IRS2). Hyperglycaemia itself altered the expression of genes involved in fatty acid transport and oxidation (CD36, LCAD and FOXO1), lipid formation and reverse cholesterol efflux (LXRα, ChREBP, ABCA1 and G6PD). PI3K, a signal transduction protein in the insulin signalling pathway, is an important mediator of insulin-stimulated glucose transport in skeletal muscle and consists of a regulatory (p85) and a catalytic subunit (p110). Two isoforms of p85 (p85α and p85β) have been identified, which are encoded by distinct genes (PI3KR1 and PI3KR2 respectively). Free p85α plays a negative role in skeletal muscle PI3K/Akt signalling axis by competing with the p85/p110 dimer for binding to IRS1 and activation of PI3K [21,22]. Human skeletal muscle p85α gene (PI3KR1) expression increases in response to short-term hyperinsulinaemia in both healthy lean subjects and obese insulin resistant subjects, but this effect is blunted in Type 2 diabetic patients [23,24]. The present study confirmed the stimulatory effect of hyperinsulinaemia on the PI3KR1 gene which encodes the p85α subunit in human skeletal muscle, and showed for the first time that hyperglycaemia exerts a similar effect, which is enhanced in an additive manner when the two stimuli are combined. Since superimposing hyperglycaemia on hyperinsulinaemia resulted in substantial increases in the rates of glucose infusion and oxidation when compared with each stimulus itself, it is possible that their additive effect may have been brought about by two different mechanisms, namely, the activation of the PI3K/Akt signalling axis and the concurrent increase in glucose availability and intracellular metabolism. Although the IRS1-associated PI3K activity was not determined in the present study, the fact that there was no difference in the phosphorylation of serine Akt at Ser473 and IRS1 at Ser302 between the HIHG and HING trials, suggests that differences between trials in IRS1-associated PI3K activity would have been unlikely in this setting. In support of this, the adverse effects of hyperglycaemia on insulin signalling are not acute and occur after at least 5 h of exposure to hyperglycaemia [10,11]. Indeed, previous studies on rodents have shown that shortterm hyperglycaemia (3–5 h) does not affect the activation of insulin signalling proteins in skeletal muscle [16], whereas prolonged infusion of glucose (1–5 days) inhibited early steps in insulin signalling [25]. This might be related to a concomitant increase in intramuscular lipid formation under the latter conditions. Indeed, in rodent skeletal muscle [10] and human muscle culture [11], prolonged hyperglycaemia is associated with longchain acyl-CoA, DAG (diacylglycerol) and TAG (triacylglycerol) accumulation, which may impinge on insulin-stimulated glucose transport [15]. In the present study, the duration (i.e. 4 h) of hyperglycaemia in the combined trial (HIHG) was not long enough to cause impairment in the rate of glucose infusion required to


Figure 3

Phosphorylation of skeletal muscle IRS1 at Ser302 before (pre-clamp) and after (post-clamp) 4 h of (i) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) euglycemic (4.5 mmol/l) clamp (HING trial), (ii) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (HIHG trial) and (iii) a euinsulinaemic (5 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (LIHG trial) + S.E.M., n = 7. *P < 0.05 compared with pre-clamp within treatment (two-way ANOVA (A) Values are expressed as means − time effect only). (B) A representative immunoblot image for phospho-IRS1.

Figure 4

Phosphorylation of skeletal muscle Akt at Ser473 before (pre-clamp) and after (post-clamp) 4 h of (i) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) euglycemic (4.5 mmol/l) clamp (HING trial), (ii) a hyperinsulinaemic (50 m-units · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (HIHG trial) and (iii) a euinsulinaemic (5 munits · m − 2 · min − 1 ) hyperglycaemic (10 mmol/l) clamp (LIHG trial) (A) Values are expressed as means + − S.E.M., n = 7. *P < 0.05 compared with pre-clamp within treatment (two-way ANOVA time effect); ¥ P < 0.01 compared with LIHG; # P < 0.05 compared with LIHG (two-way ANOVA interaction effect). (B) A representative immunoblot image for phospho-Akt.

www.clinsci.org

681


maintain hyperglycaemia during the latter stages of the clamp. This is consistent with the fact that there was neither significant accumulation of intramuscular long-chain acyl-CoAs in response to that treatment, nor impairment in the activation of Akt or a significant phosphorylation of IRS1 on Ser302 . The latter has been implicated in the development of lipid-induced insulin resistance by interfering with the interaction between insulin receptor and IRS1 in most [19,26] but not all [27] studies. Activation of SREBP1c, LXRα and/or ChREBP may provide a potential mechanism by which prolonged hyperglycaemia promotes lipogenesis in skeletal muscle [28]. In the present study, acute hyperglycaemia did not alter the gene expression of SREBP1c, which is in line with a study using primary rat muscle culture exposed to hyperglycaemia (25 mmol/l) for 2 h and showed no effect on skeletal muscle SREBP1c mRNA content [29]. However, an increase in the precursor and mature forms of the SREBP1c protein was observed in the latter study suggesting that hyperglycaemia may regulate SREBP1c at a posttranslational level. In the present study, acute hyperinsulinaemia up-regulated the expression of SREBP1c and its transcriptional target HKII, which confirmed previous findings [5,30]. The observation that hyperglycaemia itself increased glucose disposal (albeit to a lesser extent than hyperinsulinaemia alone) but failed to activate Akt, suggests that an increase in glucose flux may not be sufficient to up-regulate the expression of SREBP1c and HKII and that, in line with previous observations, activation of the PI3K/Akt pathway is essential for induction of those genes by insulin [31]. Glucose was also shown to stimulate the transcriptional activity of LXR and induce expression of genes involved in lipid metabolism and lipogenesis [such as SREBP1c, PK (pyruvate kinase), ABCA1, ACC (acetyl-CoA carboxylase) and FASN (fatty acid synthase)] [32]. These effects are mediated, at least in part, through ChREBP, which is as a target for LXR [33] and appears to be necessary for the induction of glucose-regulated genes in mouse liver [34]. Interestingly, a study by Meugnier et al. [9] using a global gene array approach showed no effect of hyperglycaemia (3 h at 10 mmol/l) on ChREBP in human skeletal muscle. In contrast, the present study showed, for the first time in an in vivo setting, an increase in the expression of human skeletal muscle ChREBP, LXRα and one of its target genes (ABCA1, a reverse cholesterol transporter) in response to acute hyperglycaemia. Although both hyperglycaemia and hyperinsulinaemia increased the expression of LXRα, no additive effect was observed when the two stimuli were combined. In contrast, in the presence of hyperinsulinaemia the stimulatory effect of hyperglycaemia on ABCA1 was abolished. Glucose can activate ChREBP through dephosphorylation by activation of PP2A (protein phosphatase 2A) [35], which is mediated by accumulation of xylulose 5-phosphate [a metabolite in the PPP (pentose phosphate pathway)]. Accumulation of G6P is an alternative mechanism for activation of ChREBP by glucose independent of the PP2A-mediated pathway [17]. Interestingly, there was a trend for an increase in muscle G6P content in the hyperglycaemic trials in the present study. Previous studies have shown that chronic hyperglycaemia (up to 4 days) reduces insulin-stimulated glucose uptake and glycogen synthesis in human skeletal muscle cells [36], although acutely (up to

682


4 h) it may actually increase muscle G6P and glycogen levels [16]. Therefore it is conceivable that an increase in the formation of intramuscular G6P (through an increase in hyperglycaemiastimulated glucose disposal) along with activation of the PPP under those conditions may provide a hormone-independent dual mechanism for the induction of genes involved in intramuscular lipid accumulation (such as LXRα and ChREBP). One may argue that the early changes in expression of genes involved in lipogenesis observed in the present study may be transient and not reflected by changes in protein expression and therefore have little significance for events developing in response to more sustained levels of hyperglycaemia. The main reason total protein measurements were not included in the present study is the short duration of the intervention (i.e. 4 h), which in line with our main aim was employed to identify the early transcriptional events that precede the induction of glucolipotoxicity and insulin resistance under hyperglycaemic and hyperinsulinaemic conditions. We have shown previously [5,8] that most of the metabolic transcripts studied (such as GLUT4, HKII and PDK4) in human skeletal muscle are not translated into protein within that time frame. Therefore future studies using longer periods of hyperglycaemia will be required to investigate the translational changes in response to treatment. Recent unpublished evidence from our laboratory showed that incubating human primary myotubes in high compared with normal glucose media for up to 24 h increases the abundance of genes associated with lipogenesis and TAG storage such as CHREBP, FASN and ADFP (adipose differentiation-related protein/perilipin). The use of human primary skeletal muscle cells in future studies will provide the opportunity to investigate both transcriptional and translational changes over the time course of prolonged periods of hyperglycaemia (up to 4 days) that are difficult to achieve in human in vivo studies. Surprisingly, hyperglycaemia itself in the present study also altered the expression of genes involved in fatty acid transport and oxidation (CD36, LCAD and FOXO1) despite the fact that circulating NEFAs (and hence extracellular fat availability) were suppressed during the LIHG trial. This reduction in plasma NEFA levels may have been the result of either a direct effect of hyperglycaemia on suppression of adipose tissue lipolysis, although the literature on this topic is conflicting [37,38], or an indirect anti-lypolytic effect by a subtle non-significant increase (of ∼ 2 m-units/l) in insulin concentration during the LIHG trial [39]. The fatty acid translocase CD36 facilitates NEFA uptake by skeletal muscle and its overexpression induces expression of FOXO1 in a PPARδ (peroxisome-proliferator-activated receptor, isotype δ)-dependent mechanism, which promotes fat oxidation in skeletal muscle [40]. FOXO1 is regulated by both insulin (through phosphorylation-mediated inactivation) and glucose (through deacetylation-dependent activation). Liverspecific deletion of FOXO1 in hyperglycaemic mice exacerbated lipid abnormalities typically associated with hyperglycaemia [41], whereas increased deacetylation (and hence activation) of FOXO1 in rat hepatocytes promotes its nuclear retention and thus transcriptional activity [42]. Furthermore, deacetylation of FOXO1 in skeletal muscle mediates the transcriptional modulation of mitochondrial and fat utilization genes in response to


fasting and exercise [43]. In the present study, both hyperglycaemia and hyperinsulinaemia increased the expression of FOXO1 in skeletal muscle, but no additive effects were observed when the two stimuli were combined. The implication of these findings is that induction FOXO1 (along with that of CD36 and LCAD) under hyperglycaemic conditions may be an early molecular manifestation of a protective mechanism against excessive intracellular lipid formation by increasing intramuscular fat oxidation, although further studies are required to test this hypothesis. In conclusion, the results of the present study have shown that acute hyperglycaemia causes an early induction of key genes involved in intracellular lipid transport and formation in human skeletal muscle. Combining hyperinsulinaemia with hyperglycaemia exerted an additive effect on PI3KR1, a key gene in the insulin-signalling cascade. Collectively, the up-regulation of these genes may represent early molecular events that precede the development of glucolipotoxicity and insulin resistance normally associated with more prolonged periods of hyperglycaemia and hyperinsulinaemia.

FUNDING

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number 42/D1563 and postgraduate studentship number BBS/S/P/2003/10402 (to K.T.)].

REFERENCES 1

2

3

4

CLINICAL PERSPECTIVES

r

r

r

Sustained hyperinsulinaemia and hyperglycaemia promote ectopic fat accumulation in skeletal muscle and induce insulin resistance. In an attempt to identify the early transcriptional changes that precede these events, the present study examined changes in metabolic gene expression in human skeletal muscle during short-term hyperglycaemia with and without concomitant hyperinsulinaemia. The results from the present study show that (i) hyperglycaemia causes an early induction of key genes (including LXRα and ChREBP) involved in intramuscular lipid formation, and (ii) superimposing hyperinsulinaemia on hyperglycaemia exerts an additive effect on PI3KR1, which plays a negative role in insulin signalling. These may be early molecular events (and therefore potential targets for interventions) that precede the development of glucolipotoxicity and insulin resistance, two hallmarks of the metabolic syndrome that occur in response to chronic exposure to hyperinsulinaemia and hyperglycaemia.

5

6

7

8

9

AUTHOR CONTRIBUTION

Kostas Tsintzas, Luke Norton, Kamal Chokkalingam, Nusrat Nizamani, Scott Cooper and Francis Stephens researched the data. Rudi Billeter contributed to the statistical analysis. Andrew Bennett contributed to the discussion. Kostas Tsintzas wrote the paper. Francis Stephens, Rudi Billeter and Andrew Bennett reviewed/edited the paper.

ACKNOWLEDGEMENTS

We thank Professor Mark Boyett (University of Manchester) for allowing us access to the 7900 HT PCR system, and Dr James Tellez for his assistance.

10

11

12 13

Mandarino, L. J., Consoli, A., Jain, A. and Kelley, D. E. (1993) Differential regulation of intracellular glucose metabolism by glucose and insulin in human muscle. Am. J. Physiol. 265, E898–E905 Mandarino, L. J., Consoli, A., Jain, A. and Kelley, D. E. (1996) Interaction of carbohydrate and fat fuels in human skeletal muscle: impact of obesity and NIDDM. Am. J. Physiol. 270, E463–E470 Rome, S., Clement, K., Rabasa-Lhoret, R., Loizon, E., Poitou, C., Barsh, G. S., Riou, J. P., Laville, M. and Vidal, H. (2003) Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp. J. Biol. Chem. 278, 18063–18068 Coletta, D. K., Balas, B., Chavez, A. O., Baig, M., Abdul-Ghani, M., Kashyap, S. R., Folli, F., Tripathy, D., Mandarino, L. J., Cornell, J. E. et al. (2008) Effect of acute physiological hyperinsulinemia on gene expression in human skeletal muscle in vivo. Am. J. Physiol. Endocrinol. Metab. 294, E910–E917 Chokkalingam, K., Jewell, K., Norton, L., Littlewood, J., van Loon, L. J., Mansell, P., Macdonald, I. A. and Tsintzas, K. (2007) High-fat/low-carbohydrate diet reduces insulin-stimulated carbohydrate oxidation but stimulates nonoxidative glucose disposal in humans: an important role for skeletal muscle pyruvate dehydrogenase kinase 4. J. Clin. Endocrinol. Metab. 92, 284–292 Norton, L., Parr, T., Bardsley, R. G., Ye, H. and Tsintzas, K. (2007) Characterization of GLUT4 and calpain expression in healthy human skeletal muscle during fasting and refeeding. Acta Physiol. 189, 233–240 Tsintzas, K., Jewell, K., Kamran, M., Laithwaite, D., Boonsong, T., Littlewood, J., Macdonald, I. and Bennett, A. (2006) Differential regulation of metabolic genes in skeletal muscle during starvation and refeeding in humans. J. Physiol. 575, 291–303 Tsintzas, K., Chokkalingam, K., Jewell, K., Norton, L., Macdonald, I. A. and Constantin-Teodosiu, D. (2007) Elevated free fatty acids attenuate the insulin-induced suppression of PDK4 gene expression in human skeletal muscle: potential role of intramuscular long-chain acyl-coenzyme A. J. Clin. Endocrinol. Metab. 92, 3967–3972 Meugnier, E., Faraj, M., Rome, S., Beauregard, G., Michaut, A., Pelloux, V., Chiasson, J. L., Laville, M., Clement, K., Vidal, H. and Rabasa-Lhoret, R. (2007) Acute hyperglycemia induces a global down-regulation of gene expression in adipose tissue and skeletal muscle of healthy subjects. Diabetes 56, 992–999 Kraegen, E. W., Saha, A. K., Preston, E., Wilks, D., Hoy, A. J., Cooney, G. J. and Ruderman, N. B. (2006) Increased malonyl-CoA and diacylglycerol content and reduced AMPK activity accompany insulin resistance induced by glucose infusion in muscle and liver of rats. Am. J. Physiol. Endocrinol. Metab. 290, E471–E479 Aas, V., Kase, E. T., Solberg, R., Jensen, J. and Rustan, A. C. (2004) Chronic hyperglycaemia promotes lipogenesis and triacylglycerol accumulation in human skeletal muscle cells. Diabetologia 47, 1452–1461 Frayn, K. N. (1983) Calculation of substrate oxidation rates in vivo from gaseous exchange. J. Appl. Physiol. 55, 628–634 Cederblad, G., Carlin, J. I., Constantin-Teodosiu, D., Harper, P. and Hultman, E. (1990) Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle. Anal. Biochem. 185, 274–278

www.clinsci.org

683


14

15

16

17

18

19

20

21

22

23

24

25

26

27

Harris, R. C., Hultman, E. and Nordesjo, L. O. (1974) Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand. J. Clin. Lab. Invest. 33, 109–120 Chibalin, A. V., Leng, Y., Vieira, E., Krook, A., Bjornholm, M., Long, Y. C., Kotova, O., Zhong, Z., Sakane, F., Steiler, T. et al. (2008) Down-regulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance. Cell 132, 375–386 Hoy, A. J., Bruce, C. R., Cederberg, A., Turner, N., James, D. E., Cooney, G. J. and Kraegen, E. W. (2007) Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation. Am. J. Physiol. Endocrinol. Metab. 293, E1358–E1364 Li, M. V., Chen, W., Harmancey, R. N., Nuotio-Antar, A. M., Imamura, M., Saha, P., Taegtmeyer, H. and Chan, L. (2010) Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP). Biochem. Biophys. Res. Commun. 395, 395–400 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159 Werner, E. D., Lee, J., Hansen, L., Yuan, M. and Shoelson, S. E. (2004) Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302. J. Biol. Chem. 279, 35298–35305 Cozzone, D., Frojdo, S., Disse, E., Debard, C., Laville, M., Pirola, L. and Vidal, H. (2008) Isoform-specific defects of insulin stimulation of Akt/protein kinase B (PKB) in skeletal muscle cells from type 2 diabetic patients. Diabetologia 51, 512–521 Barbour, L. A., Rahman, S. M., Gurevich, I., Leitner, J. W., Fischer, S. J., Roper, M. D., Knotts, T. A., Vo, Y., McCurdy, C. E., Yakar, S. et al. (2005) Increased P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess. J. Biol. Chem. 280, 37489–37494 Brachmann, S. M., Ueki, K., Engelman, J. A., Kahn, R. C. and Cantley, L. C. (2005) Phosphoinositide 3-kinase catalytic subunit deletion and regulatory subunit deletion have opposite effects on insulin sensitivity in mice. Mol. Cell Biol. 25, 1596–1607 Laville, M., Auboeuf, D., Khalfallah, Y., Vega, N., Riou, J. P. and Vidal, H. (1996) Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle. J. Clin. Invest. 98, 43–49 Ducluzeau, P. H., Perretti, N., Laville, M., Andreelli, F., Vega, N., Riou, J. P. and Vidal, H. (2001) Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes. Diabetes 50, 1134–1142 Houdali, B., Nguyen, V., Ammon, H. P., Haap, M., Schechinger, W., Machicao, F., Rett, K., Haring, H. U. and Schleicher, E. D. (2002) Prolonged glucose infusion into conscious rats inhibits early steps in insulin signalling and induces translocation of GLUT4 and protein kinase C in skeletal muscle. Diabetologia 45, 356–368 Morino, K., Neschen, S., Bilz, S., Sono, S., Tsirigotis, D., Reznick, R. M., Moore, I., Nagai, Y., Samuel, V., Sebastian, D. et al. (2008) Muscle-specific IRS-1 Ser→Ala transgenic mice are protected from fat-induced insulin resistance in skeletal muscle. Diabetes 57, 2644–2651 Giraud, J., Leshan, R., Lee, Y. H. and White, M. F. (2004) Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling. J. Biol Chem. 279, 3447–3454

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

Kase, E. T., Thoresen, G. H., Westerlund, S., Hojlund, K., Rustan, A. C. and Gaster, M. (2007) Liver X receptor antagonist reduces lipid formation and increases glucose metabolism in myotubes from lean, obese and type 2 diabetic individuals. Diabetologia 50, 2171–2180 Guillet-Deniau, I., Pichard, A. L., Kone, A., Esnous, C., Nieruchalski, M., Girard, J. and Prip-Buus, C. (2004) Glucose induces de novo lipogenesis in rat muscle satellite cells through a sterol-regulatory-element-binding-protein-1c-dependent pathway. J. Cell Sci. 117, 1937–1944 Mandarino, L. J., Printz, R. L., Cusi, K. A., Kinchington, P., O’Doherty, R. M., Osawa, H., Sewell, C., Consoli, A., Granner, D. K. and DeFronzo, R. A. (1995) Regulation of hexokinase II and glycogen synthase mRNA, protein, and activity in human muscle. Am. J. Physiol. 269, E701–E708 Osawa, H., Sutherland, C., Robey, R. B., Printz, R. L. and Granner, D. K. (1996) Analysis of the signaling pathway involved in the regulation of hexokinase II gene transcription by insulin. J. Biol Chem. 271, 16690–16694 Mitro, N., Mak, P. A., Vargas, L., Godio, C., Hampton, E., Molteni, V., Kreusch, A. and Saez, E. (2007) The nuclear receptor LXR is a glucose sensor. Nature 445, 219–223 Iizuka, K. and Horikawa, Y. (2008) ChREBP: a glucose-activated transcription factor involved in the development of metabolic syndrome. Endocrine J. 55, 617–624 Denechaud, P. D., Bossard, P., Lobaccaro, J. M., Millatt, L., Staels, B., Girard, J. and Postic, C. (2008) ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver. J. Clin. Invest. 118, 956–964 Kawaguchi, T., Takenoshita, M., Kabashima, T. and Uyeda, K. (2001) Glucose and cAMP regulate the L-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein. Proc. Natl. Acad. Sci. USA. 98, 13710–13715 Aas, V., Hessvik, N. P., Wettergreen, M., Hvammen, A. W., Hallen, S., Thoresen, G. H. and Rustan, A. C. (2011) Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes. Biochim. Biophys. Acta 1812, 94–105 Cersosimo, E., Coppack, S. and Jensen, M. (1993) Lack of effect of hyperglycemia on lipolysis in humans. Am. J. Physiol. 265, E821–E824 Carlson, M. G., Snead, W. L., Hill, J. O., Nurjhan, N. and Campbell, P. J. (1991) Glucose regulation of lipid metabolism in humans. Am. J. Physiol. 261, E815–E820 Jensen, M. D., Caruso, M., Heiling, V. and Miles, J. M. (1989) Insulin regulation of lipolysis in nondiabetic and IDDM subjects. Diabetes 38, 1595–1601 Nahle, Z., Hsieh, M., Pietka, T., Coburn, C. T., Grimaldi, P. A., Zhang, M. Q., Das, D. and Abumrad, N. A. (2008) CD36-dependent regulation of muscle FoxO1 and PDK4 in the PPARδ/β-mediated adaptation to metabolic stress. J. Biol. Chem. 283, 14317–14326 Haeusler, R. A., Han, S. and Accili, D. (2010) Hepatic FoxO1 ablation exacerbates lipid abnormalities during hyperglycemia. J. Biol. Chem. 285, 26861–26868 Frescas, D., Valenti, L. and Accili, D. (2005) Nuclear trapping of the forkhead transcription factor FoxO1 via Sirt-dependent deacetylation promotes expression of glucogenetic genes. J. Biol. Chem. 280, 20589–20595 Canto, C., Jiang, L. Q., Deshmukh, A. S., Mataki, C., Coste, A., Lagouge, M., Zierath, J. R. and Auwerx, J. (2010) Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle. Cell Metab. 11, 213–219

Received 6 September 2012/9 January 2013; accepted 15 January 2013 Published as Immediate Publication 15 January 2013, doi: 10.1042/CS20120481

684


Clinical Science (2013) 124, 675–684 (Printed in Great Britain) doi: 10.1042/CS20120481

SUPPLEMENTARY ONLINE DATA

Independent and combined effects of acute physiological hyperglycaemia and hyperinsulinaemia on metabolic gene expression in human skeletal muscle Kostas TSINTZAS*, Luke NORTON†, Kamal CHOKKALINGAM*, Nusrat NIZAMANI*, Scott COOPER*, Francis STEPHENS*, Rudolf BILLETER* and Andrew BENNETT*‡ *School of Biomedical Sciences, Nottingham University Medical School, Queens Medical Centre, Nottingham NG7 2UH, U.K. †Division of Diabetes, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, U.S.A. ‡FRAME Alternatives Laboratory, Nottingham University Medical School, Queens Medical Centre, Nottingham NG7 2UH, U.K.

See the following page for Supplementary Table S1.

Correspondence: Dr Kostas Tsintzas (email [email protected]).

www.clinsci.org


Table S1

Names and symbols of gene expression assay targets that were pre-loaded on to the microfluidic cards

Number

Gene name (human skeletal muscle)

Symbol

1

α-Actin

ACTA1

2

18S ribosomal RNA

18S

3

Hydroxymethylbilane synthase

HMBS

4

Phosphorylase, glycogen, muscle

PYGM

5

Glycogenin

GYG1

6

Protein phosphatase 1, catalytic subunit, gamma

PPP1G (PPP1CC)

7

Solute carrier family 2, facilitated glucose transporter, member 4 (GLUT4)

SLC2A4

8

Hexokinase 2

HK2

9

Sterol regulatory element binding protein 1c (00100529.1)

SREBP1c (SREBF1)

10

Fatty acid synthase

FASN

11

Acetyl-CoA carboxylase β

ACACB

12

Phosphofructokinase (muscle)

PFKM

13

Aldolase A

ALDOA

14

Pyruvate kinase, muscle

PKM2

15

Lactate dehydrogenase B

LDHB

16

Pyruvate dehydrogenase kinase, isoenzyme 2

PDK2

17

Pyruvate dehydrogenase kinase, isoenzyme 4

PDK4

18

Pyruvate dehydrogenase phosphatase, isoenzyme 1 (PPM2C)

PDP1 (PPM2C)

19

Pyruvate dehydrogenase phosphatase, isoenzyme 2

PDP2

20

Glucose-6-phosphate dehydrogenase

G6PD

21

Transaldolase 1

TALDO1

22

Transketolase, muscle

TKT

23

CD36 antigen (collagen type I receptor/fatty acid transporter)

CD36

24

Adipose differentiation-related protein

ADRP (ADFP)

25

Carnitine palmitoyltransferase I (muscle)

CPT 1B

26

Acyl-CoA dehydrogenase, long chain (ACADL)

LCAD (ACADL)

27

Diacylglycerol kinase, isoenzyme α

DGKα

28

Diacylglycerol kinase, isoenzyme δ

DGKδ

29

Diacylglycerol acyltransferase, isoenzyme 1

DGAT1

30

ATP citrate lyase

ACLY

31

Fatty acid transport protein 1

FATP1 (SLC27A1)

32

Carbohydrate response element binding protein (MLXIPL)

ChREBP (MLXIPL)

33

Peroxisome proliferator-activated receptor gamma coactivator-1α

PGC1α

34

Forkhead transcription factor 1A, also called FKHR (forkhead in rhabdomyosarcoma)

FOXO1A

35

Forkhead transcription factor 3, also called FKHRL1 (FKHR-like 1)

FOXO3

36

Insulin receptor substrate-1

IRS1

37

Insulin receptor substrate-2

IRS2

38

Phosphatidylinositol 3-kinase, regulatory 1 (p85α)

PIK3R1

39

Protein kinase B/Akt, isoform 2

Akt2

40

Liver X receptor, isotype α

NR1H3 (LXRα)

41

Liver X receptor, isotype β

NR1H2 (LXRβ)

42

ATP binding cassette transporter, subfamily A, member 1

ABCA1

43

Stearoyl-CoA desaturase 1 (SCD1 9 desaturase)

SCD-1

44

Apolipoprotein E

apoE

45

NADH dehydrogenase (ubiquinone) Fe-S protein4

NDUFS4

46

Cytochrome c oxidase subunit VIIa polypeptide 2

COX7A2

47

Succinate dehydrogenase complex, subunit B

SDHB

48

Surfeit 1

SURF1

Received 6 September 2012/9 January 2013; accepted 15 January 2013 Published as Immediate Publication 15 January 2013, doi: 10.1042/CS20120481
