Annette Karen Serup,1 Thomas Junker Alsted,1,2 Andreas Børsting Jordy,1

Peter Schjerling,3 Cecilia Holm,4 and Bente Kiens1

Partial Disruption of Lipolysis IncreasesPostexercise Insulin Sensitivityin Skeletal Muscle DespiteAccumulation of DAGDiabetes 2016;65:2932–2942 | DOI: 10.2337/db16-0655

Type 2 diabetes and skeletal muscle insulin resistancehave been linked to accumulation of the intramyocellularlipid-intermediate diacylglycerol (DAG). However, recentanimal and human studies have questioned such an as-sociation. Given that DAG appears in different stereoiso-mers and has different reactivity in vitro, we investigatedwhether the described function of DAGs as mediatorsof lipid-induced insulin resistance was dependent on thedifferent DAG isomers. We measured insulin-stimulatedglucose uptake in hormone-sensitive lipase (HSL) knock-out (KO) mice after treadmill exercise to stimulate theaccumulation of DAGs in skeletal muscle. We found that,despite an increased DAG content in muscle after exer-cise in HSL KO mice, the HSL KO mice showed a higherinsulin-stimulated glucose uptake postexercise comparedwith wild-typemice. Further analysis of the chemical struc-ture and cellular localization of DAG in skeletal musclerevealed that HSL KO mice accumulated sn-1,3 DAG andnot sn-1,2 DAG. Accordingly, these results highlight theimportance of taking the chemical structure and cellularlocalization of DAG into account when evaluating the roleof DAG in lipid-induced insulin resistance in skeletal mus-cle and that the accumulation of sn-1,3 DAG originatingfrom lipolysis does not inhibit insulin-stimulated glucoseuptake.

Skeletal muscle accounts for a large part of insulin-stimulatedglucose disposal and is an important tissue in regard to

peripheral glucose uptake (1,2). The general perceptionhas been that insulin resistance can be attributed to theaccumulation of diacylglycerol (DAG) in liver and skeletalmuscle (3–5). The mechanism by which DAG is believed tocause insulin resistance is by the activation of proteinkinase C (PKC) e and PKC u leading to phosphorylationand inhibition of insulin receptor substrate 1 and theinsulin signaling cascade in both liver (6,7) and skeletalmuscle (8–10). However, not all studies have been able toshow these associations among the accumulation of DAG,impaired insulin signaling, and insulin resistance in skel-etal muscle (11–13).

In adipose tissue and skeletal muscle, adipose triglyceridelipase (ATGL) has been found to be the major triacylglycerol(TAG)-lipase and hormone-sensitive lipase (HSL), the majorDAG lipase (14–16). Consequently, a partial disruption oflipolysis by knocking out the HSL gene leads to the accumu-lation of DAG (14). DAGs are, in skeletal muscle, generatedfrom the following three different pathways: from hydro-lysis of TAG by ATGL, from esterification of fatty acids (FAs)to the phosphoglycerol backbone, and from hydrolysis ofphospholipids (17). In addition, DAG can occur in threedifferent stereoisomers, each with unique biological proper-ties, and their structure and localization differ depend-ing on their origin. In COS7 cells, it was shown that ATGLhas a preference for hydrolyzing TAG in the sn-2 position,producing primarily sn-1,3 DAGs located in the lipid drop-let membrane, whereas re-esterification of plasma FA and

1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports,Faculty of Science, University of Copenhagen, Copenhagen, Denmark2Clamp Competency Center, Novo Nordisk A/S, Måløv, Denmark3Institute of Sports Medicine, Department of Orthopedic Surgery M, BispebjergHospital and Center for Healthy Aging, Faculty of Health Sciences, University ofCopenhagen, Copenhagen, Denmark4Department of Experimental Medical Science, Lund University Biomedical Cen-tre, Lund, Sweden

Corresponding author: Bente Kiens, bkiens@nexs.ku.dk.

Received 23 May 2016 and accepted 28 July 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0655/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

2932 Diabetes Volume 65, October 2016

METABOLISM

the hydrolysis of phospholipids primarily generates sn-1,2DAG located in the endoplasmic reticulum membrane andat the plasma membrane, respectively (18). Furthermore,in rats fed a high-fat diet it was shown that elevatedDAG production from hydrolysis of TAG by lysosomallipase (LIPA) in the liver was located in two differentlocalizations: in the dense and the light lysosomes and,to a larger extent, in the light lysosomes (19); however,whether lipase has the same preference as ATGL for hydro-lyzing TAG in the sn-2 position and producing sn-1,3 DAGsis not known. It could therefore be speculated that the roleof DAG in lipid-induced insulin resistance may be depen-dent on cellular localization, stereo chemical structure, andcell type. No studies have evaluated the effect of the differ-ent cellular localization and stereochemical structure ofDAG on insulin resistance and insulin-stimulated glucoseuptake in skeletal muscle. Therefore, the aim of the currentstudy was to test the hypothesis that the described functionof DAGs as mediators of lipid-induced insulin resistance isdependent on the origin and presence of the different DAGisomers. To test this hypothesis, we studied the HSL knock-out (KO) mice and their wild-type (WT) littermates. Therehave been conflicting findings in the literature as towhether mice lacking HSL are insulin resistant. Earlier find-ings (20,21) suggested that lack of HSL led to impairedinsulin sensitivity in skeletal muscle. In contrast, when ahyperinsulinemic-euglycemic clamp combined with D-3-H-glucose were applied, no significant differences were ob-served in insulin-mediated whole-body glucose uptakebetween HSL KO and WT mice (22). In addition, HSL KOmice actually are protected from high-fat diet–induced in-sulin resistance in skeletal muscle (23). Because of theseconflicting findings we investigated insulin-stimulated glu-cose uptake in skeletal muscle in the basal resting state andduring the recovery period after an in vivo exercise boutand took advantage of the fact that skeletal muscle sub-jected to exercise displays increased insulin sensitivity upto 48 h postexercise (POST EX) (2,24,25) and that HSL KOmice accumulate DAG POST EX (26). We combined this andmeasured insulin-stimulated glucose uptake ex vivo usingradiolabeled glucose 90 min after treadmill running in HSLKO mice and their WT littermates.

RESEARCH DESIGN AND METHODS

AnimalsHSL KO mice were generated by targeted disruption ofthe HSL gene as previously described (20,27). Female HSLKO mice and WT littermates on a mixed genetic back-ground (C57BL/6 and SV129) were generated by hetero-zygous breeding and used for experiment at 16–25 weeksof age. The animals were kept in standard laboratorycages on a 12-h light/dark cycle at room temperature,and received standard chow diet (Brogården Aps, Lynge,Denmark) and water ad libitum. All experiments wereapproved by the Danish Animal Experimental Inspector-ate and complied with the “European Convention for theProtection of Vertebrate Animals Used for Experiments

and Other Scientific Purposes” (Council of Europe no.123, Strasbourg, France, 1985).

Running ProtocolAll animals were acclimatized to treadmill running (TSETreadmill; TSE Systems, Bad Homburg, Germany) before allexperimental testing. During acclimatization, the mice wereallowed to rest at a still treadmill for 5 min, and then wereset to run for 5 min at 7 m/min followed by 5 min at14 m/min, and the speed was increased over a 2-minperiod. All running was performed at 0% incline. The inten-sities used for acclimatization were low to avoid trainingadaptation. A shocker at the bottom of the treadmill wasused to encourage running if necessary. On the third day,the mice were subjected to a maximal running capacity testin which the mice ran at 10 m/min for 2 min; thereafter,the speed was increased by 2 m/min every second minuteuntil fatigue. Fatigue was defined as spending .10 s at thelower part of the treadmill despite manual encouragement.Three days later, the mice were subjected to an endurancerunning test at 70% of the individual maximal runningspeed until fatigue. The main experiment was performed3 days after the endurance test. On the basis of the resultsfrom the maximal running capacity and endurance tests,the mice were set to run for 40 min at 65% of their indi-vidual maximal running speed during the experiment. Dur-ing the main experiment, mice in the basal group weretransferred to a still treadmill for 40 min. All the micewere fasted 4 h prior to the experiment.

Muscle Incubations

Glucose UptakeBasal and insulin-stimulated glucose uptake at rest and90-min POST EX were measured ex vivo. After the com-pletion of treadmill running, mice were anesthetized byan intraperitoneal injection of sodium pentobarbital(6 mg/100 g body wt). The soleus (SOL) muscle andextensor digitorum longus (EDL) muscle were dissectedfree, and silk sutures were tied around the intact tendonsand transferred to incubation chambers (Multi MyographSystem 610M; Danish Myo Technology A/S, Aarhus,Denmark) and suspended at resting tension. SOL andEDL were preincubated in Krebs–Henseleit–Ringer buffer(pH 7.4) containing 7 mmol/L mannitol, 2 mmol/L pyruvate,and 0.1% BSA at 30°C and continuously gassed with amixture of 5% CO2 and 95% O2 for 75 min. SOL andEDL muscles were insulin stimulated 75 min after comple-tion of exercise for 15 min in preincubation buffer contain-ing 100, 200, or 10,000 mU/mL insulin (Actrapid; NovoNordisk, Bagsværd, Denmark). The SOL and EDL musclesfrom the contralateral leg were used to evaluate basal glu-cose uptake without insulin.

Basal and insulin-stimulated glucose uptake were mea-sured ex vivo 90 min after treadmill running by changingthe incubation media to the preincubation buffer containingthe desired insulin concentration, 1 mmol/L 2-deoxyglucose,0.056 MBq/mL 2-[3H]deoxyglucose, and 0.0167 MBq/mL[14C] mannitol (PerkinElmer, Buckinghamshire, U.K.) for

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10 min. After tracer incubation, the muscles were washed inKrebs–Henseleit–Ringer buffer, frozen in liquid nitrogen,and stored at 280°C until further analysis.

Glucose uptake was measured on muscle lysate as theaccumulation of 2-[3H]deoxyglucose, and [14C] mannitolwas used as a marker of the extracellular space, as de-scribed previously (28).

FA OxidationFA metabolism experiments were conducted in incubationreservoirs (Radnoti, Monrovia, CA) using procedures thathave been described previously (29,30). Isolated SOL mus-cles were incubated at 30°C in Krebs–Henseleit–Ringerbuffer, pH 7.4, containing 2 mmol/L pyruvate, 2% FA-freeBSA, and 0.5 mmol/L palmitic acid. After incubation atresting tension (4–5 mN) for 20 min, the incubation bufferwas refreshed and supplemented with 0.5 mCi/mL [1-14C]palmitate (Amersham BioScience, Buckinghamshire, U.K.).For resting experiments, FA metabolism was measured for25 min. Contracting experiments of SOL muscle (50 Hz,350 ms pulse duration, 6 tetani/min) was performed for25 min. Oxidation of [1-14C] palmitate was measured inresting and contracting SOL muscles from gaseous 14CO2

produced from the exogenous oxidation of [1-14C] palmi-tate, as previously described (31). The incorporation of ex-ogenous FAs into DAG and intracellular TAG was measuredby thin-layer chromatography (TLC), as described below.

Body CompositionBody composition was measured 3 days prior to the ex-periment on unanesthetized mice using quantitative MRIby EchoMRI (Echo Medical Systems, Houston, TX).

DAG and TAG in Skeletal MuscleThe quadriceps muscle was freeze dried and dissected freeof visible adipocytes under a microscope, as describedpreviously (32), and muscle DAG was measured by TLC.Initially, the lipids were extracted in chloroform-methanol(2:1) using the method of Folch et al. (33) and were dis-solved in chloroform. The lipids were separated by TLCusing two different separate mobile phases consisting ofchloroform-methanol-acetic acid-water (50:50:5:5) followedby petroleum ether-diethyl ether-acetic acid (120:25:1.5).Butylated hydroxytoluene (50 mg/L) was added to bothof the mobile phases. The lipids were developed by a10% copper sulfate pentahydrate and 8% phosphoric acidsolution at 120°C for 15 min, and subsequently were visu-alized and analyzed using a Kodak image station (2000MM;Kodak, Glostrup, Denmark). Specific standards were usedto identify sn-1,2 DAG (catalog #D9135; Sigma-Aldrich)and sn-1,3 DAG (catalog #D1639; Sigma-Aldrich).

Intramyocellular TAG (IMTG) was measured by abiochemical method on 1 mg of freeze-dried and dissectedquadriceps muscle, as described previously (34,35).

Muscle GlycogenMuscle glycogen was measured as glycosyl units after acidhydrolysis on 30 mg w/w quadriceps muscle, as describedpreviously (36).

Muscle Homogenization, SDS-PAGE, and WesternBlottingEntire SOL and EDL muscles were homogenized andsubjected to SDS-PAGE and Western blotting analysis, asdescribed previously (37,38).

AntibodiesA detailed list of the primary antibodies used to deter-mine phosphorylation status and total protein expressionis presented in Supplementary Table 1.

StatisticsAll data are expressed as the mean 6 SEM. Statisti-cal analysis was performed using IBM SPSS Statistics20.0 (SPSS, Chicago, IL) and Sigma Plot 12.3 (SystatSoftware Inc., Erkrath, Germany). A three-way ANOVAwith repeated measures was used for comparison of in-sulin, 90-min POST EX, and genotype. Two-way ANOVAwith repeated measures was used for comparison of90-min POST EX and genotype. Two-way ANOVA wasused for comparison of exercise/contraction and geno-type in Fig. 3A–F and Fig. 7A–F. Finally, a t test was used

Figure 1—Glucose uptake in the presence of no insulin and during100 mU/mL and 10,000 mU/mL insulin stimulation in SOL muscle(m. soleus) (A) and in EDL muscle (m. extensor digitorum longus) (B)in WT and HSL KO mice. Measured between the ages of 16 and 25weeks (n = 3–4 in each group). ww, wet weight. Data are expressedas the mean 6 SEM. **Significant difference from no insulin (P <0.01) within WT and HSL KO mice, respectively; ΔΔsignificantdifference from 100 mU/mL insulin within WT and HSL KO mice,respectively. There are no differences between WT and HSL KOmiceat either insulin concentration.

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for comparison of body composition, and running differ-ences between groups were considered statistically signifi-cant at P , 0.05.

RESULTS

Body Composition and Running CapacityThere were no differences in body weight (WT 24.7 60.3 g; HSL KO mice 25.5 6 0.4 g), fat mass (WT 2.6 60.2 g; HSL KO mice 2.9 6 0.1 g), or lean body mass(WT 19.8 6 0.2 g; HSL KO mice 20.6 6 0.2 g) betweenWT and HSL KO mice. In addition, maximal runningcapacity (WT 33.4 6 0.6 m/min; HSL KO mice 33.6 60.7 m/min), running endurance at 70% of maximalrunning speed (WT 3,018 6 106 s; HSL KO mice3,079 6 97 s), and experimental running speed (WT21.05 6 0.53 m/min; HSL KO mice 20.26 6 0.62 m/min)measured on a treadmill at 0% incline were not differentbetween the genotypes.

Insulin Dose ResponseInsulin was found to dose-dependently increase glucoseuptake in both SOL and EDL muscles, and in both genotypes(Fig. 1A and B). There were no significant differences inglucose uptake between genotypes at basal conditions withno insulin in the media or at 100 or 10,000 mU/mL insulin.

POST EX Insulin-Stimulated Glucose Uptake Is Higherin HSL KO MiceGlucose uptake without insulin exposure, measured 90-minPOST EX, was similar to that of resting muscles in bothgenotypes and in both SOL and EDL muscles (Fig. 2A and C).When stimulated with insulin (100 mU/mL), muscle glu-cose uptake was significantly higher compared with no in-sulinat rest, and POST EX in both SOL and EDL musclesin both WT and HSL KO mice (P , 0.01). Furthermore,insulin-stimulated glucose uptake was higher in pre-exercisedcompared with nonexercised SOL and EDL muscles in both

Figure 2—Glucose uptake in the presence of no insulin and during 100 mU/mL insulin stimulation at rest (white bars) and 90-min POST EX(black bars) in SOL muscle (m. soleus) (A) and EDL muscle (m. extensor digitorum longus) (C ) in WT and HSL KO mice. DGlucose uptakecalculated by subtracting glucose uptake for the muscle without insulin from glucose uptake for the contralateral muscle during 100 mU/mLinsulin stimulation in SOL muscle (B) and EDL muscle (D) in WT and HSL KO mice, measured between ages 16 and 25 weeks (n = 7–9in each group). ww, wet weight. Data are expressed as the mean 6 SEM. **Significant main effect of 100 mU/mL insulin (P < 0.01);‡significant interaction between 100 mU/mL insulin and 90-min POST EX (P < 0.05); §significant interaction among 100 mU/mL insulin,genotype, and 90-min POST EX (P < 0.05); #significant effect of 90-min POST EX (P < 0.05); ##significant effect of 90-min POST EX(P < 0.01); †significant effect of genotype (P < 0.05); ††significant effect of genotype (P < 0.01).

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WT and HSL KO mice (P , 0.05) (Fig. 2A and C). In addi-tion, in pre-exercised muscle, insulin-stimulated glucose up-take as well as delta glucose uptake was significantly higherin HSL KO mice compared with WT mice both in SOL andEDL muscles (P , 0.01) (Fig. 2A–D).

Muscle DAG Was Higher in HSL KO After ExerciseTotal muscle DAG content at rest was not different be-tween genotypes. After exercise, DAG content remainedunchanged compared with at rest in WT mice, whereas anincreased DAG content was observed in HSL KO mice(P , 0.05) (Fig. 3A). The majority of the DAG content inthe muscle samples was of the sn-1,3 DAG isomer originboth in resting and pre-exercised muscle, and sn-1,3 DAGcontent was more than 17-fold higher than sn-1,2 DAGcontent in both WT and HSL KO mice (Fig. 3B). To fur-ther visualize the changes within sn-1,3 DAG and sn-1,2DAG, the data are displayed separately in Fig. 3C and D,respectively. In the resting, nonexercised muscle, sn-1,3DAG content was similar between genotypes. In the ex-ercised muscle, sn-1,3 DAG content was similar to theresting muscle in WT, whereas an increase in sn-1,3 DAGwas observed in exercised muscle from HSL KO mice(P , 0.05) (Fig. 3C). In addition, muscle sn-1,3 DAGcontent was 43% higher in HSL KO mice compared withWT after exercise (P, 0.01). Muscle sn-1,2 DAG contentwas significantly higher in HSL KO mice at rest and afterexercise (P , 0.05) and was reduced by exercise in bothgenotypes (P , 0.05) (Fig. 3D).

IMTG and Glycogen Content Were Reduced WithTreadmill RunningIMTG content was similar in resting muscle between ge-notypes. A similar reduction in IMTG content (P , 0.01)was observed after exercise in WT and HSL KOmice (Fig. 3E).

Muscle glycogen levels at rest were similar betweengenotypes, and were 29% and 46% reduced after exercisein WT and HSL KO mice, respectively, compared with at rest(P, 0.01). There was no difference in glycogen use betweengenotypes during in vivo treadmill running (Fig. 3F).

Insulin Signaling Was Similar 90-Minute POST EXPhosphorylation of AKT thr308 and AKT ser473 was in-creased by insulin stimulation to the same extent in thenonexercised and pre-exercised muscles in both genotypes(Fig. 4A, B, D, and E). Total AKT2 protein expression wasnot affected by either of the interventions (90-min POSTEX and insulin) or by genotype (data not shown).

Insulin stimulation increased AS-160 thr642 phosphor-ylation by ;139% and 67% in SOL and EDL muscle, re-spectively, in both WT and HSL KO mice (Fig. 4C and F)(P , 0.05) with no effect of the intervention in eitherSOL or EDL muscle, and with no differences between thegenotypes.

Phosphorylation of AMPK on thr172 was not affectedby the interventions either in SOL and EDL muscles or ingenotypes (Fig. 5A and C). Acetyl-CoA carboxylase (ACC)ser212 phosphorylation, a downstream target of AMPK,confirmed that AMPK was not activated in either of the

Figure 3—Energy stores in quadriceps muscles at rest (white bars) and after treadmill exercise (black bars) in WT and HSL KO mice.Content of total muscle DAG (A), muscle sn-1,3 DAG and sn-1,2 DAG muscle (B), sn-1,3 DAG (C), muscle sn-1,2 DAG (D), IMTG (E),and muscle glycogen (F). Measured between age 16 and 25 weeks (n = 7–9 in each group). dw, dry weight. Data are expressed as themean 6 SEM. ##Effect of exercise (P < 0.01); †significant effect of genotype (P < 0.05); ††a significant effect of genotype (P < 0.01);¶¶significant effect of sn-1,2 DAG (P < 0.01).

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interventions in both SOL and EDL muscles in both ge-notypes (Fig. 5B and D).

GLUT4 Protein Expression Was Upregulated inHSL KO MiceGLUT4 protein expression was ;24% (P , 0.05) and;32% (P, 0.01) higher in HSL KO mice compared withWT mice in SOL and EDL muscles, respectively (Fig. 6Aand D). Both hexokinase (Fig. 6B and E) and ATGL (Fig.6C and F) protein expression were similar in both ge-notypes, and were unaffected by insulin and exercise.

Similar Uptake and Oxidation of Exogenous FAs inHSL KO and WT MiceExogenous palmitate uptake and oxidation in SOL muscle atrest were similar in WT and HSL KO mice (Fig. 7A and B).During muscle contraction, exogenous palmitate uptakeand oxidation increased in both genotypes. Exogenouspalmitate incorporation into muscle DAG and TAG wassimilar between genotypes, at rest and with contractions(Fig. 7C and D). The ratio between palmitate esterification

and oxidation was similar between genotypes at rest andwith muscle contractions, but the ratio was significantlylower with contractions than at rest, indicating that theexogenous FAs are directed toward oxidation rather thanesterification during muscle contractions (Fig. 7E).

DISCUSSION

It is well accepted that in skeletal muscle ATGL is themajor TAG lipase (15,16), and HSL is mainly respon-sible and rate limiting for the hydrolysis of DAG tomonoacylglycerol (14). Accordingly, activating lipolysisin the absence of HSL activity is expected to lead to anaccumulation of DAGs in skeletal muscle, which was pre-viously found in overnight fasted and exercised HSL KOmice (14,26). In the current study, we demonstrated thatinsulin-stimulated glucose uptake in skeletal muscle wasincreased after exercise in both mice lacking HSL miceand their WT littermates. However, this increase wassignificantly higher in muscles from HSL KO mice thanWT mice despite an increased content of DAG in HSL KO

Figure 4—AKT thr308 phosphorylation related to total AKT2 protein in SOL muscle (m. soleus) (A) and EDL muscle (m. extensor digitorumlongus) (D) and AKT ser472 phosphorylation related to total AKT2 protein in SOL muscle (B) and EDL muscle (E). AS-160 phosphorylation inSOL muscle (C ) and EDL muscle (F ), in the presence of no insulin and during 100 mU/mL insulin stimulation at rest (white bars) and 90-minPOST EX (PEX) (black bars) in WT and HSL KO mice. A representative blot is shown above each graph, measured between 16 and25 weeks of age (n = 7–9 in each group). Data are expressed as the mean 6 SEM. **Significant effect of 100 mU/mL insulin (P < 0.01).

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muscles. These findings may seem counterintuitive giventhe proposed inhibitory effect of intracellular DAG oninsulin sensitivity and signaling (3). However, a furtheranalysis of the DAG isomer content in the musclesrevealed that only the sn-1,3 DAG isomer was increasedby exercise and only in the HSL KO muscles. Together,these findings suggest that the increased DAG contentoriginating from ATGL-mediated lipolysis does not im-pair insulin signaling or effect.

In the current study, total DAG content, measured inthe quadriceps muscle was ;40% higher in HSL KO miceafter exercise compared with WT mice. In the gastrocne-mius muscle, an even higher total DAG content (;2.5-fold)was obtained after exercise by Fernandez et al. (26), com-pared with the present finding, which probably is due to amore energy-demanding exercise protocol in the study byFernandez et al. (26) or to muscle-specific differences.

Given that DAGs exist in different stereoisomers,depending on how they are generated (18), we examinedthe two most abundant DAG isomers, sn-1,2 DAG andsn-1,3 DAG, in skeletal muscle to determine their relativecontribution to the increased DAG content after exercisein skeletal muscle. We found that the majority of DAGpresent in skeletal muscle samples were of the sn-1,3DAG stereoisomers and that the overall level of sn-1,2DAG represents only a minor fraction of the total DAGpool. Even though not quantified in the study by Fernandezet al. (26), their data suggest a higher sn-1,2 DAG contentin the gastrocnemius muscle of HSL KO mice comparedwith the present findings, resulting in a more equitabledistribution between sn-1,2 DAG to sn-1,3 DAG contentthan in the current study. The reason for this could bedue to differences in the metabolic state of the ani-mals and the muscle type being analyzed, as mentioned

Figure 5—AMPK thr172 phosphorylation related to total AMPKa2 in SOL muscle (m. soleus) (A) and EDL muscle (m. extensor digitorumlongus) (C). ACC ser212 phosphorylation related to total ACCb in SOL muscle (B) and EDL muscle (D) in the presence of no insulin andduring 100 mU/mL insulin stimulation at rest (white bars) and 90-min POST EX (PEX) (black bars) in WT and HSL KO mice. A representativeblot is shown above each graph, measured between 16 and 25 weeks of age (n = 7–9 in each group). Data are expressed as the mean 6SEM.

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previously. Only the sn-1,3 DAG levels in skeletal musclewere increased after exercise in HSL KO mice in the cur-rent study, whereas the sn-1,3 DAG level was unaltered byexercise in WT mice. This finding is in line with a recentstudy by Eichmann et al. (18), who showed that ATGL inCOS7 cells had a strong preference for the hydrolysis ofFA esters at the sn-2 position of the glycerol backbone,resulting in the formation of sn-1,3 DAG.

The increased content of the sn-1,3 DAG isomers ap-parently had no inhibitory effect on insulin signaling andglucose uptake in skeletal muscle. Support for this notionare the previous in vitro findings showing that only sn-1,2DAG is able to activate conventional and novel PKCs,whereas the others, sn-1,3 DAG and sn-2,3 DAG, lackthis bioactivity (39,40). We observed that the muscle con-tent of sn-1,2 DAG represented only a very small part ofthe total DAG content in skeletal muscle in the presentsituation. Nonetheless, sn-1,2 DAG content in skeletalmuscle was reduced in both genotypes after exercise andwas significantly higher in HSL KO mice both in the basalstate and POST EX. Nevertheless, insulin-stimulated glucose

uptake was similar in the resting state and higher POST EXin HSL KO mice compared with WT mice, respectively. Thisindicates that the minor sn-1,2 DAG content in skeletalmuscle did not play a role in determining insulin sensitivityin the basal state or after exercise.

The exercise effect on insulin-stimulated glucose uptakein skeletal muscle could be attributed to AMPK activation, asit was shown that activation of AMPK by AICAR or hypoxiaincreased insulin-stimulated glucose uptake ex vivo in therat skeletal muscle 3.5 h after removal of the stimulus (41).In addition, recent findings by Kjøbsted et al. (38), demon-strating that insulin-stimulated glucose uptake in skeletalmuscle was increased 4 h after AICAR stimulation in WTmice, but not in mice where AMPK activity was blunted,further supporting the notion that AMPK activation is suf-ficient to increase skeletal muscle insulin sensitivity. Sincewe observed that AMPK and ACC phosphorylation were sim-ilar between genotypes, both at basal and POST EX and withinsulin stimulation in both SOL and EDL muscles, AMPKactivation cannot account for the higher insulin-stimulatedglucose transport in the skeletal muscle of HSL KO

Figure 6—GLUT4 protein expression in SOL muscle (m. soleus) (A) and EDL muscle (m. extensor digitorum longus) (D), hexokinase IIprotein expression in SOL muscle (B) and EDL muscle (E), and ATGL protein expression in SOL muscle (C) and EDL muscle (F ) in thepresence of no insulin and during 100 mU/mL insulin stimulation at rest (white bars) and 90-min POST EX (PEX) (black bars) in WT and HSLKO mice. A representative blot is shown above each graph. Measured between 16 and 25 weeks of age (n = 7–9 in each group), data areexpressed as the mean 6 SEM. †Significant effect of genotype (P < 0.05); ††significant effect of genotype (P < 0.01).

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mice. Part of insulin sensitization after exercise stems froman effect on GLUT4-mediated glucose transport (42,43). In-terestingly, we observed a significantly higher GLUT4 pro-tein expression in HSL KO mice compared with WT mice inboth SOL and EDL muscles, which may explain the higherglucose uptake POST EX in HSL KO mice than in WT mice.On the other hand, higher GLUT4 levels have previouslybeen connected to higher insulin responsiveness (44), whichdid not differ between genotypes in the current study. Themechanisms for the higher GLUT4 protein content in thesemice are unknown, as this, to our knowledge, is the firststudy to measure GLUT4 protein expression in HSL KOmice in skeletal muscle. An upregulation of genes involvedin carbohydrate metabolism was previously found in SOLmuscles of HSL KO mice, compared with WT mice (26). Itcould be speculated that the higher GLUT4 expression is dueto a compensatory upregulation of GLUT4 protein due tothe partial disruption of lipolysis in HSL KO mice in adiposetissue (45) and skeletal muscle (15,26), thereby decreasingthe availability of circulating FA and FA generated locallyfrom lipolysis in the muscle, for oxidation, and hence in-creasing the reliance on carbohydrate as a substrate.

Randle et al. (46) were the first to propose that reducedinsulin sensitivity was caused by elevated fat oxidation; how-ever, studies from the last 2 decades reveal that reduced in-sulin sensitivity is associated with reduced fat oxidativecapacity (47). It was previously demonstrated that HSL KOmice during exercise have reduced plasma FA levels and de-creased lipid metabolism (27,44). Together with this, the re-sults of the current study suggest that reduced FA availability

for mitochondrial oxidation plays a role in the observed in-crease in POST EX insulin sensitivity in HSL KO mice.One could argue that the reduced lipolysis, and thus thedecreased FA oxidation, may be due to a reduced capacityof mitochondrial FA metabolism in HSL KO mice. Wemeasured exogenous FA uptake and oxidation in skeletalmuscle at rest and with muscle contractions in HSL KOmice and their WT littermates. The data clearly dem-onstrated a similar handling of exogenous FAs in bothgenotypes, which is in line with previous findings (44).

It could be hypothesized that decreased lipid availabil-ity should be preferential in increasing insulin sensitiv-ity rather than increasing lipid use. This hypothesis issupported by studies in rodents (48), patients with type2 diabetes (49,50), and healthy male subjects (51). Ourfindings suggest that partial disruption of lipolysis inwhite adipose tissue and skeletal muscle could be benefi-cial in increasing insulin sensitivity and glucose uptake byincreasing the reliance on carbohydrate to maintain themetabolic demand of the cell.

In conclusion, we show that HSL KO mice accumulateDAG in skeletal muscle after exercise and that insulin-stimulated glucose uptake in muscle POST EX was higher inHSL KO mice compared with WT. The sn-1,3 DAG isomerwas the DAG isoform with the highest concentration inskeletal muscle and was higher in HSL KO mice after exer-cise than in WT mice. The sn-1,2 DAG isomer was onlyfound at low levels in both genotypes. Accordingly, theseresults highlight the importance of taking the chemical struc-ture and cellular localization of DAG into account when

Figure 7—Palmitate uptake (A), palmitate oxidation (B), palmitate incorporation into DAG (C) and TAG (D), and palmitate esterification/oxidation (E ) in SOL muscle at rest (white bars) and during muscle contractions (black bars) in WT and HSL KO mice. Measured between16 and 25 weeks of age (n = 8–9 in each group). The data are expressed as the mean6 SEM. ##Significant effect of contraction (P< 0.01).

2940 Disruption of Lipolysis and Insulin Sensitivity Diabetes Volume 65, October 2016

evaluating the role of DAG in lipid-induced insulin resistancein skeletal muscle and that the accumulation of sn-1,3 DAGoriginating from lipolysis does not inhibit insulin-stimulatedglucose uptake. This increase in insulin-stimulated glucoseuptake could be associated with an upregulation of GLUT4 protein in HSL KO mice, increasing the capacity to take upglucose upon insulin stimulation. The data suggest that areduction in FA availability and oxidation should be favoredin order to increase insulin sensitivity in skeletal muscle.

Acknowledgments. The authors thank Irene B. Nielsen and BetinaBolmgren (Department of Nutrition, Exercise and Sports, University of Copenha-gen) for their skilled technical assistance. The authors also thank Jørgen F.P.Wojtaszewski (Department of Nutrition, Exercise and Sports, University of Copen-hagen) and Jonas Thue Treebak (The Novo Nordisk Foundation Center for BasicMetabolic Research, Section of Integrative Physiology, University of Copenhagen)for helpful insight into the experimental protocol.Funding. This study was financially supported by the Novo Nordisk Foundation(grant 10807) and the UNIK Project Food, Fitness & Pharma for Health and Disease(see www.foodfitnesspharma.ku.dk), which was supported by the Danish Ministryof Science, Technology and Innovation; the Swedish Research Council (project11284 to C.H.); and the Novo Nordisk Foundation Center for Basic MetabolicResearch. The Novo Nordisk Foundation Center for Basic Metabolic Researchis an independent Research Center at the University of Copenhagen that ispartially funded by an unrestricted donation from the Novo Nordisk Foundation(www.metabol.ku.dk).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. A.K.S. and T.J.A. conducted the laboratoryexperiments, contributed to the analysis of the data, and wrote the first versionof the manuscript. T.J.A. and B.K. designed the study. A.B.J. conducted thelaboratory experiments and contributed to the analysis of the data. P.S.contributed to the analysis of the data. C.H. donated the hormone-sensitivelipase knockout mice. B.K. wrote the manuscript. All authors contributed to theinterpretation of the results, revised the manuscript, and approved the finalversion of the manuscript. B.K. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.

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