distribution patterns of the glucose transporters glut4 and glut1 in skeletal muscles of rats...

gy, Part A 146 (2007) 274–282www.elsevier.com/locate/cbpa

Comparative Biochemistry and Physiolo

Distribution patterns of the glucose transporters GLUT4 and GLUT1 inskeletal muscles of rats (Rattus norvegicus), pigs (Sus scrofa),

cows (Bos taurus), adult goats, goat kids (Capra hircus),and camels (Camelus dromedarius)

R. Duehlmeier a,⁎, K. Sammet d, A. Widdel b, W. von Engelhardt c,U. Wernery e, J. Kinne e, H.-P. Sallmann b

a Clinic for Pigs, Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover,Foundation, Bischofsholer Damm 15, D-30173 Hannover, Germany

b Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Foundation, Germanyc Department of Physiology, University of Veterinary Medicine Hannover, Foundation, Germany

d Centre for Food Science, Institute for Food Quality and Safety, University of Veterinary Medicine Hannover, Foundation, Germanye Central Veterinary Research Laboratory, Dubai, United Arab Emirates

Received 29 March 2006; received in revised form 25 September 2006; accepted 19 October 2006Available online 27 October 2006

Abstract

Earlier studies demonstrated that forestomach herbivores are less insulin sensitive than monogastric omnivores. The present study was carriedout to determine if different distribution patterns of the glucose transporters GLUT1 and GLUT4 may contribute to these different insulinsensitivities. Western blotting was used to measure GLUT1 and GLUT4 protein contents in oxidative (masseter, diaphragm) and glycolytic(longissimus lumborum, semitendinosus) skeletal muscle membranes of monogastric omnivores (rats and pigs), and of forestomach herbivores(cows, adult goats, goat kids, and camels). Muscles were characterized biochemically. Comparing red and white muscles, the isocitratedehydrogenase (ICDH) activity was 1.5–15-times higher in oxidative muscles of all species, whereas lactate dehydrogenase (LDH) activity was1.4–4.4-times higher in glycolytic muscles except in adult goats. GLUT4 levels were 1.5–6.3-times higher in oxidative muscles. GLUT1 levelswere 2.2–8.3-times higher in glycolytic muscles in forestomach herbivores but not in monogastric animals. We conclude that GLUT1 may be thepredominant glucose transporter in glycolytic muscles of ruminating animals. The GLUT1 distribution patterns were identical in adult and pre-ruminant goats, indicating that GLUT1 expression among these muscles is determined genetically. The high blood glucose levels of camels citedin literature may be due to an “NIDDM-like” impaired GLUT4 activity in skeletal muscle.© 2006 Elsevier Inc. All rights reserved.

Keywords: Camel; Cow; GLUT1; GLUT4; Goat; Pig; Rat; Skeletal muscles

1. Introduction

Glucose is themain energy source in themammalian organism.There are great differences in the strategies for maintaining theglucose demands of the metabolism in monogastric omnivoresand in animals with forestomachs. One main difference between

⁎ Corresponding author. Tel.: +49 511 8567263; fax: +49 511 8567684.E-mail address: [email protected] (R. Duehlmeier).

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representatives of these two nutrition strategies is the glucosesource. In monogastrics glucose is mainly absorbed in thesmall intestine by a sodium-dependent transport protein (SGLT1).Adult ruminants for the most part utilize glucose produced byendogenous gluconeogenesis, with propionate as the majorprecursor (Reynolds, 1992). Probably due to these differentsources of glucose, basal plasma glucose levels are significantlyhigher in monogastrics than in adult ruminants, but not in camels(Elmahdi et al., 1997). Unlike adult ruminants, suckling forest-omach animals meet their glucose demands by enteral absorption

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Table 1Experimental animals

Species Breed Sex Age Body mass Number

Rats Spraque–Dawley Male 259±9 g 6Pigs Pic Male, castrated ca.

7 months121±6 kg 6

Cows Holstein–Frisian Female,nonpregnant,lactating

3–4years

580±81 kg 6

Goats Weiβe DeutscheEdelziege (WDE)

Female,nonpregnant,nonlactating

ca.1 year

ca. 50 kg 4

Goatkids

Female 4 weeks ca. 5 kg 5

Camels Racing camel Female,nonpregnant,nonlactating

5.5±1.9 years

ca. 350 kg 5

275R. Duehlmeier et al. / Comparative Biochemistry and Physiology, Part A 146 (2007) 274–282

as do monogastric omnivores, which leads to plasma glucoseconcentrations and a SGLT1 expression in the small intestinecomparable to those inmonogastrics (Shirazi-Beechy et al., 1995).The second important difference between ruminant and mono-gastric animals in glucose metabolism concerns the insulin-dependent glucose transport into peripheral tissues. Insulin is themost important endocrine factor controlling glucose homeostasis.In hyperglycemic states, insulin is secreted from the endocrinepancreas and stimulates the glucose uptake into skeletal musclesand adipose tissue. However, hyperinsulinemic, euglycemicclamp tests have shown that the insulin-stimulated whole bodyglucose utilization is significantly lower in adult ruminants than inmonogastric omnivores. This indicates a lower insulin sensitivityof peripheral tissues in ruminating foregut fermenters than inmonogastric animals (Chandrasena et al., 1984;Kaske et al., 2001;Sano et al., 1999). Camels are exceptional with regard to theircarbohydrate metabolism. On the one hand, plasma glucose levelsin camels are in the range or of those of monogastrics or evenhigher (Abdel-Fattah et al., 1999; Al-Ali et al., 1988), althoughcamels ferment carbohydrates to short-chain fatty acids on thesame scale as sheep and lactating cows (Höller et al., 1989) andlike ruminants, they also meet their glucose demands byendogenous gluconeogenesis (Shirazi-Beechy et al., 1995). Onthe other hand, thewhole body insulin sensitivity of camels is evenlower than that of adult ruminants (Kaske et al., 2001). High basalplasma glucose levels and simultaneously low insulin sensitivitiesalso occur in humans suffering from non-insulin-dependentdiabetes mellitus (NIDDM) (Zierath et al., 1998), so the insulinsensitivity of camels may be similar to that of NIDDM patients.

The absorption and distribution of glucose is mediated by afamily of specific facilitative transport proteins, the glucosetransporters. Presently 14 of those transporters are known, 11 ofwhich are involved in sugar transport (Scheepers et al., 2004), butonly glucose transporter 4 (GLUT4) is insulin-dependent. Inmyocytes and adipocytes, GLUT4 is associated with membranestructures and recycles between the plasma membrane and anintracellular tubulovesicular pool. Under hyperglycemic condi-tions, insulin is secreted from the pancreas and increases thetranslocation of cytoplasmatic GLUT4 vesicles towards and intothe plasma membrane, thus stimulating the transport of glucoseinto the cells (Kahn, 1996). In addition to GLUT4, muscle andadipose cells also contain a second glucose transport protein,insulin-independent GLUT1, which is recognized to be respon-sible for the basal glucose supply. GLUT4 and GLUT1 areglycoproteins of between 45 and 50 kDs (Mueckler, 1994) and arehighly conserved among mammalian species, especially withregard to their C-terminal domain, which is responsible forantigenic attitudes. The 38-amino acid C-terminal regions ofGLUT4 in rats, pigs, goats, and bovines exhibit a single aminoacid difference: the asparagine508 in bovines is replaced byhistidine in porcines, rats, and goats (Abe et al., 1998). There areno differences between rats (Birnbaum et al., 1986), pigs (Weiler-Guttler et al., 1989), and bovines (Borado and Pardridge, 1991) inthe corresponding GLUT1 sequences.

Since skeletal muscle is the main mammalian tissue ofglucose utilization and therefore the major site of differencesconcerning the insulin sensitivity (Zierath, 1995), we aimed to

determine if it is likely that the lower in vivo insulin sensitivityof adult ruminants is mediated by a different distribution patternof GLUT1 and GLUT4 in the crude membranes of skeletalmuscles of forestomach herbivores and monogastric omnivores.The second objective was to clarify whether the impairedinsulin sensitivity of camels could be due to the absence ofGLUT4 in the skeletal muscle.

2. Materials and methods

2.1. Animals and tissue sampling

GLUT4 and GLUT1 levels were estimated in oxidative andglycolytic muscles of rats, pigs, cows, goat kids, adult goats,and adult camels. Table 1 shows breed, sex, ages, and bodyweights of the animals used in this experiment. Rats wereobtained from the Harlan Winkelmann Corporation (Borchen,Germany). They were kept on sawdust in groups of three inboxes for laboratory rodents and fed a common rodent chow(Altromin Corporation, Lage, Germany). Pigs were housed instable boxes and received a diet composed of barley (50%),wheat (28.5%), soybean meal (18%), soybean oil (1%), andmineral premix (2.5%). Cows were tethered in stalls and fed tosatisfy the nutrient requirements of maintenance and dailyproduction of 30 kg milk. The diet consisted of maize (47%)and grass silage (22%), concentrate (15%), soybean meal (2%),molasses (7%), tapioca (6%), and mineral–vitamin premix(1%). Goat kids were housed in a single group on a flat deckstable and were exclusively fed with milk exchanger. Adultgoats were housed in a stable box on straw and received a dietcomposed of hay and mineral premix. Camels were keptextensively in the desert of Dubai and were given supplemen-tary alfalfa and dates. All animals had free access to waterthroughout the entire experiment. Pigs, cows and goats wereslaughtered by stunning and exsanguination. Cows wereslaughtered within six weeks after parturition. Rats were killedby decapitation. Camels were slaughtered by exsanguinationaccording to traditional Islamic rules in Dubai. Immediatelyafter slaughter or killing, tissue samples of about 5 g wereremoved from the masseter, diaphragm, longissimus lumborum,

276 R. Duehlmeier et al. / Comparative Biochemistry and Physiology, Part A 146 (2007) 274–282

and semitendinosus muscles. Muscle samples were frozen inliquid nitrogen and stored at −80 °C until analysis of GLUTcontents and enzyme activities.

2.2. Metabolic classification of muscles

The muscles were classified as oxidative or glycolyticaccording to ICDH and LDH metabolic activity, respectively.Specific activities (IU/mg) of both enzymes were measuredspectrophotometrically as described previously (Briand et al.,1981; Goldberg and Ellis, 1983). One IU was defined as theoxidation of 1 μmol isocitrate (ICDH) and lactate (LDH) perminute at 37 °C and at pH 7.4 and at pH 6.5, respectively.

2.3. Determination of GLUT1 and GLUT4 levels in musclecrude membranes

To evaluate total muscle cell GLUT contents, GLUT levelswere determined in myocyte crude membranes reflectingplasma membrane GLUT concentrations as well as GLUTproteins associated with cytoplasmatic membranes (Balageet al., 1997; Bonnet et al., 2004; Dühlmeier et al., 2005;Hocquette et al., 2006; Lacombe et al., 2003; Lemosquet et al.,2002; McCutcheon et al., 2002; Zhao et al., 1996).

2.3.1. Preparation of myocyte crude membranesMuscle crude membranes were prepared according to the

method of Gumà et al. (1995). To isolate muscle crude mem-branes, about 100 mg of muscle tissue were homogenizedusing an Ultra-Turrax for 2×5 s in 2.5 mL ice-cold homog-enization buffer (20 mM HEPES, 250 mM sucrose, 5 mMsodium azide [NaN3], 1 μM leupeptin, 1 μM antipain, 2 μg/mLaprotinin, and 100 mM phenylmethylsulfonyl fluoride[PMSF], pH 7.4). The homogenate was centrifuged at1200 ×g and 4 °C for 10 min and the supernatant (SN1)retained. The pellet (P1) was resuspended in 2.5 mL of ho-mogenization buffer, re-homogenized, and centrifuged in thesame way. The new pellet (P2) was discarded and the super-natant (SN2) pooled with SN1 and centrifuged at 9000 ×g and4 °C for 10 min to remove mitochondria. The pellet (P3) wasdiscarded and the supernatant (SN3) centrifuged at 190,000 ×gand 4 °C in an ultracentrifuge. The resulting pellet P4 (crudemembranes) was collected and resuspended in 500 μL of ice-cold homogenization buffer. Protein concentrations of thecrude membrane preparations were determined using theBradford protein assay with BSA as standard (Bradford,1976). The protein yield in muscle cell crude membranesranged between approximately 120 and 550 μg/100 mg musclewet mass. Since muscle dry matter was about 20% of the wetweight, protein recovery was 0.6–2.7% of muscle drysubstance. Crude membrane proteins were precipitated with10 mM trichloroacetic acid (TCA) for 30 min at 4 °C. Thencrude membrane proteins were resuspended in Laemmli buffercontaining 63 mM tris(hydroxymethyl)amino methane hydro-chloride (Tris/HCl), pH 6.8, 10% (v/v) glycerol, 2% (w/v)SDS, 5% (v/v) 2-mercaptoethanol, and 30 mM bromophenolblue (Laemmli, 1970) to a final dilution of 30 μg/15 μL.

2.3.2. Electrophoresis and immunoblotting of membranesCrude membrane proteins were separated by SDS-polyacryl-

amide gel electrophoresis. Proteins were transferred to a Hybondnitrocellulose membrane in buffer consisting of 25 mM Tris,192 mM glycine, and 10% methanol (v/v), pH 8.6. After thetransfer, the membranes were blocked with 10% (w/v) non-fatdry milk in Tris-buffered saline solution (20 mM Tris base,137 mM NaCl, 0.05% [w/v] Tween 20, pH 7.6) for 1 h at roomtemperature and then incubated overnight at 4 °Cwith antibodiesdirected against 12-amino acid carboxy terminus of rat GLUT4,and rat whole GLUT1 protein. Primary antibodies were dilutedaccording to the manufacturers' recommendations (GLUT4:1:750; GLUT1: 1:200) in Tris-buffered saline solution asdescribed above containing 5% (w/v) non-fat dry milk and0.05% NaN3. The immune complex was detected using anenhanced chemiluminescence (ECL) system. The resultingautoradiograms were quantified by scanning densitometry(Scion Image, Scion Company, Frederick, MD, USA). Theresulting optical densities (OD) of the GLUT-specific bandswere used to calculate the distribution of GLUT1 and GLUT4 inpercent among the four muscles of each animal. The ODs of theGLUT-specific bands in the four muscles of each animal weresummarized and equated with 100% and the percentage ofGLUT1 and GLUT4 calculated in all four muscles. Thisprocedure made it possible to compare GLUT1 and GLUT4distribution patterns in the different species.

To test the specificity of the GLUT4 antibody used throughoutthis experiment, blots of rat, pig, cow, goat, goat kid, horse, andcamel masseter muscles were simultaneously incubated with theGLUT4 antibody and with the corresponding synthetic carboxyterminal sequence of bovine GLUT4 (synthesized by BiogenesCorporation, Berlin, Germany). As shown in Fig. 1A, GLUT4appeared as two distinct bands in Western blot audiograms.Comparable results were received in many studies dealing withtotal skeletal and heart muscle cell GLUT4 contents. On the otherhand, in partly purified plasma membranes of porcine and bovinesemitendinosus muscles only one single GLUT4 specific bandwas observed (Dühlmeier et al., 2005). Thus, it might be possiblethat GLUT4 proteins either in the intracellular or in the plasmamembrane pool slightly differ with regard to their molecular massaccording to different glycolsylations as described in Xenopuslaevis oocytes (Marshall et al., 1993). However, both bandscorresponding toGLUT4 (Fig. 1A)were blunted in all animals bypreincubation of the primary antibody with the bovine peptide.Therefore, both bands were estimated for calculation of musclecell GLUT4 contents. A third series of blots was incubated onlywith the secondary antibody to check the unspecific binding of thesecondary antibody (horseradish peroxidase-labeled anti-rabbitIgG at a final dilution of 1:10,000). No binding signal wasdetected in the corresponding autoradiograms (Fig. 1C).We couldnot test the specificity of the primary GLUT1 antibody since itwas raised against whole rat GLUT1 and the amino acidsequences of goat and camel GLUT1 have not been determinedyet. Nevertheless, inWestern blots of GLUT1, only a single bandcorresponding to a molecular mass of about 50 kDa (Fig. 1D) andno unspecific binding of the secondary antibody (data not shown)were observed.

Table 2Isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities(IU/mg) of rat, pig, cow, goat, and goat kid muscles (mean±SEM; n=4)

Species Enzyme[IU/mg]

M. masseter Diaphragm M. long.lumb.

M. semitend.

Rats ICDH 1.49±0.13B 3.67±0.47A 1.06±0.14B 0.86±0.06B

LDH 58.8±7.70b,c 47.6±4.0c 84.0±7.3a,b 112.0±6.2a

Pigs ICDH 1.80±0.23A 2.75±0.25A 0.31±0.04B 0.66±0.04B

LDH 86.7±8.4b 78.1±15.4b 209.2±15.7a 160.6±29.9a

Cows ICDH 3.22±0.56A 1.43±0.33B 0.26±0.02C 0.22±0.02C

LDH 35.9±4.6b 55.5±5.5b 126.3±17.8a 137.1±7.1a

Goats ICDH 3.53±0.54A 1.07±0.09A 0.69±0.17B 0.37±0.06B

LDH 34.1±4.7a 43.6±3.1a 80.0±23.5a 74.4±10.3a

Goatkids

ICDH 3.83±0.35A 1.90±0.21B 1.13±0.19C 0.93±0.07C

LDH 30.6±2.7b 55.8±5.1b 133.9±12.1a 118.7±10.5a

Different indices (ICDH: A, B, and C; LDH a, b, and c) indicate significant( pb0.05) differences for values in the same line.

Fig. 1. Specificity of heterologous antibodies against GLUT4 and GLUT1 ofrats, pigs, cows, goats, goat kids, horses, and camels. Concerning GLUT4, crudemembrane preparations of masseter muscle from each species (and age group ofgoats) were analyzed in three experiments. Autoradiograms show immunore-active bands after incubation with the primary antibodies alone (A), in thepresence of the synthetic GLUT4 carboxy terminus 12-amino acid peptide (B),and after incubation only with the secondary antibody (C). The positions ofimmunoreactive bands were compared with molecular mass standards. D showsrepresentative autoradiograms of GLUT1 in masseter, diaphragm, longissimuslumborum, and semitendinosus muscles of rats, pigs, cows, goats, and camels.One single strong band corresponding to a molecular mass of approximately50 kDa was detected in these autoradiograms.


2.4. Reagents

Most commonly used chemicals and the protease inhibitorsantipain, leupeptin, and aprotinin were from Sigma-AldrichCompany (St. Louis, MO, USA). All chemicals were of thehighest purity grade available. Western blotting reagents andmaterials (Hybond© nitrocellulose membrane, Hyperfilm©,ECL© reagents, and secondary horseradish-peroxidase-linkedanti-rabbit antibody) were from Amersham Biosciences EuropeGmbH (Freiburg, Germany). Bradford reagent was from Bio-Rad (Hercules, CA, USA). Polyclonal antibodies against the 12C-terminal amino acid residues of rat GLUT4 and whole ratGLUT1 raised in rabbit were from Biotrend Chemicals GmbH(Cologne, Germany).

3. Data analysis

Data are expressed as means±SE. Statistical analysis wasconducted using Sigma Stat® 3.0 (SPSS Inc., Chicago, IL,USA). Normal distribution of data was confirmed by the

Kolmogoroff–Smirnoff test. To evaluate significant differencesin GLUT1 and GLUT4 levels and in LDH and ICDH activitiesof the four muscles, a one-way ANOVA for repeated measureswas carried out for each species and each age group of goats. Ifsignificant differences in main effects were detected by theANOVA procedure, means were compared with a Tukey test.Correlations between GLUT contents and metabolic enzymeactivities were calculated using the linear regression model. AnANOVA on ranks was performed when data were not distributednormally. Significance was set at p=0.05 in all statisticalanalyses.

4. Results

4.1. LDH and ICDH activities of oxidative and glycolyticmuscles

Specific ICDH activities were generally significantly higherin red muscles (masseter and diaphragm), except in the masseterin rats. Specific LDH activities were mostly significantly higherin white muscles (longissimus lumborum and semitendinosusmuscles), except in the masseter in rats and in all four muscles inadult goats where differences were not significant. However, inadult goats, specific LDH activities tended ( p=0.089) to behigher in longissimus lumborum and semitendinosus than inmasseter and diaphragm muscle (Table 2).

Unfortunately, it was not possible to characterize these fourmuscles in camels in the laboratory in Dubai.

4.2. GLUT4 contents of oxidative and glycolytic muscles

In general, GLUT4 specific bands and the correspondingpercentage GLUT4 contents were higher in oxidative than inglycolytic muscles of all species investigated throughout thisexperiment (Fig. 2). Nevertheless, some gradual differenceswere observed with regard to the GLUT4 distribution patterns.In rats, the estimated percentages of GLUT4 were significantly( pb0.05) higher in the masseter than in both glycolyticmuscles, whereas the percentages of GLUT4 in rat diaphragmdid not differ significantly from those in the masseter and the

Fig. 2. GLUT4 levels in skeletal muscles of rats , pigs, cows, adult goats, goat kids, and camels (mean±SE; n=4–6). The figure shows representative Western blots ofGLUT4 in the masseter (M), diaphragm (D), longissimus lumborum (LL), and semitendinosus (ST) muscles. The corresponding GLUT4 contents shown in the tablesbelow the blots are expressed as percentage contents of the total OD of GLUT4 in all four muscles of each species. Values with different superscripts indicatesignificant ( pb0.05) differences among the muscles.


white muscles. In pigs and adult goats, GLUT4 levels weresignificantly ( pb0.05) higher in the masseter as well as in thediaphragm muscles than in the two glycolytic muscles. In cows

Fig. 3. GLUT1 levels in skeletal muscles of rats, pigs, cows, adult goats, goat kids, anGLUT1 in the masseter (M), diaphragm (D), longissimus lumborum (LL), and semitebelow the blots are expressed as percentage contents of the total OD of GLUT1 isignificant ( pb0.05) differences among the muscles.

and in goat kids, significantly highest ( pb0.05) GLUT4 levelsoccurred in the masseter, followed by the diaphragm and thelongissimus lumborum and the semitendinosus muscles. There

d camels (mean±SE; n=4–6). The figure shows representative Western blots ofndinosus (ST) muscles. The corresponding GLUT1 contents shown in the tablesn all four muscles of each species. Values with different superscripts indicate

Fig. 4. Linear regression between GLUT1 and GLUT4 levels and LDH activitiesin porcine and bovine muscles (n=16). Skeletal muscle GLUT1 (A) and GLUT4(B) levels of pigs (open symbols) and cows (closed symbols) are expressed independency of LDH activities; r = coefficient of correlation.


were no significant differences in the GLUT4 contents of the lasttwo muscles in both species. The concentrations of GLUT4 inthe camel masseter were significantly ( pb0.05) higher than inall other muscles; and there were no significant differences in theGLUT4 concentrations in the diaphragm, the longissimuslumborum, and the semitendinosus muscles. The differencesbetween the GLUT4 levels in oxidative and glycolytic muscleswere considerably greater in forestomach herbivores than inmonogastric animals. In goat kids, the GLUT4 levels were 6.3times higher in the masseter than in the semitendinosus muscle.This enrichment was 4.5-fold in camels, 3.4-fold in cows, 3.2-fold in adult goats, 2.7-fold in pigs, and 1.5-fold in rats.

4.3. GLUT1 contents of oxidative and glycolytic muscles

GLUT1 distribution in forestomach herbivores differed clearlyfrom that in monogastric animals (Fig. 3). In rats and in pigs, theGLUT1-specific bands were comparable in strength in the fourmuscle sites. Thus, there were no significant differences in thecorresponding percentages of GLUT1. In adult forestomachomnivores and in camels, the glycolytic longissimus lumborumand semitendinosus muscles contained significantly (pb0.05)higher GLUT1 protein levels than the oxidative masseter anddiaphragm muscles. In goat kids, the highest GLUT1 concentra-tion occurred in the longissimus lumborum, where a significantly(pb0.05) higher GLUT1 percentage was determined than in themasseter, but not in the diaphragm and the semitendinosusmuscles. There was no significant difference in GLUT1 levels inthe last three muscles in goat kids.

4.4. Relation between glucose transporter levels and metabolicmuscle activity

In pigs, cows, adult goats, and goat kids the muscle GLUT4levels correlated significantly ( pb0.05) positively with thespecific ICDH activity and negatively with the specific LDHactivity (Table 3). In rats, GLUT4 levels tended to be correlatednegatively ( p=0.05) with the specific LDH activity. There wasno statistically proven relation between muscle GLUT4 levelsand the specific ICDH activity in rats. In adult ruminants (cows,goats) there was a significantly ( pb0.05) negative correlationbetween GLUT1 levels and specific ICDH activity, and asignificantly positive correlation between muscle GLUT1 levelsand specific LDH activity. No such relationship was detected in

Table 3Relation between metabolic activity and glucose transporter content (n=16)

Species GLUT1–LDH GLUT1–ICDH GLUT4–LDH GLUT4–ICDH

Rat r=0.299 r=−0.199 r=−0.498 r=0.211Pig r=0.101 r=−0.128 r=−0.800⁎ r=0.801⁎

Cow r=0.898⁎ r=−0.759⁎ r=−0.924⁎ r=0.844⁎

Goat r=0.625⁎ r=−0.723⁎ r=−0.570⁎ r=0.682⁎

Goat kids r=0.153 r=−0.463 r=−0.713⁎ r=0.744⁎

Coefficients of correlation (r) between the glucose transporters and themetabolic enzyme activities in the muscles of rats, pigs, cows, goats, and goatkids. An asterisk marks a significant ( pb0.05) positive or negative correlationbetween the activity of an enzyme and glucose transporter levels in the fourmuscles of a species.

the monogastric rats and pigs. In pre-ruminant goats, GLUT1levels tended to be correlated negatively with the specificICDH activity ( p=0.071), but there was no significant relationbetween muscle GLUT1 levels and specific LDH activity. Fig. 4shows the different relationships between muscle cell GLUT1levels and marker enzyme activities in cows and pigs.

5. Discussion

5.1. Biochemical muscle classification

Recent investigations in humans and animals confirmed arelation between muscle fiber composition and metabolicactivity on the one hand and muscle GLUT4 and GLUT1expression on the other hand (Daugaard and Richter, 2001). Redoxidative muscles are enriched in slow-twitch type-I and fast-twitch type-IIa myosin heavy chains (MHC) and in aerobicglycolysis enzymes, for example the intramitochondrial ICDH(Goldberg and Ellis, 1983). White glycolytic muscles mainlycontain fast-twitch type-IIb and type-IIx (human) MHCs andanaerobic glycolysis enzymes (Spangenburg and Booth, 2003)like LDH (Briand et al., 1981). Thus, muscles can becharacterized by determination of muscle fiber composition orby measuring the activities of marker enzymes of anaerobic andaerobic metabolism. We used the biochemical method of muscleclassification since several studies have confirmed a closercorrelation between the distribution of muscle glucose trans-porters and the metabolic muscle activity than betweentransporter expression and fiber composition (Daugaard andRichter, 2001). The ICDH and LDH enzyme activities wedetermined in the muscles of pigs, cows, goat kids and withregard to the ICDH in adult goats (Table 2) were in goodagreement with the findings for calves and goats (Hocquetteet al., 1995). Our findings for LDH activity in pigs agree withthose in fattened pigs (Sepponen et al., 2003). Our results alsoconfirmed the classification of the masseter and the diaphragmas oxidative muscles and the longissimus lumborum and thesemitendinosus as glycolytic muscles. However, in rats ICDHand LDH activities differed to a considerably lesser extentbetween oxidative and glycolytic muscles than in most otherspecies, indicating a more uniform metabolic activity of the four


muscles in rats. We could not verify significant differences ofmuscle LDH activities in adult goats. Due to the tendency ofhigher LDH activities in longissimus lumborum and semitendi-nosus muscles and the significantly higher ICDH activities inmasseter and diaphragm muscles, we assume that the muscleclassification described above also applies for adult goats.Unfortunately, we could not characterize the four muscles of thecamels. Previous investigations indicated some particularities ofcamel muscles compared with other species: The muscles weredominated by type I and type IIa fibers and there was no firmrelationship between fiber type composition and glycolytic oroxidative enzyme activity (Saltin et al., 1994). Nevertheless,also in camels the semitendinosus muscle has been identifiedas a glycolytic muscle with regard to fiber composition andenzyme activities (Saltin et al., 1994). Thus, we expect that thesame classification as observed in the other species would alsoapply to the camel muscles.

5.2. Distribution of GLUT4 and GLUT1 in red and whiteskeletal muscles

In rodents, red oxidative muscles were shown to containsignificantly higher amounts of GLUT4 than white glycolyticmuscles (Goodyear et al., 1991; Henriksen et al., 1990; Kernet al., 1990; Marette et al., 1992; Megeney et al., 1993;Richardson et al., 1991). Our data correspond to these findings.GLUT4 levels were significantly higher in the masseter and indiaphragm muscles than in the longissimus lumborum andsemitendinosus muscles in all species investigated, except in thediaphragm of camels and rats. However, in rats, the GLUT4enrichment in oxidative muscles was lower than described inearlier investigations (Goodyear et al., 1991; Henriksen et al.,1990; Kern et al., 1990; Marette et al., 1992; Megeney et al.,1993; Richardson et al., 1991), probably due to the fact that notexactly the same muscles were investigated by the latter authorsand by us. The findings of earlier studies on GLUT4 levels inruminants are inconsistent. Hocquette et al. (1995) foundsignificantly higher GLUT4 protein levels in the semitendino-sus muscle than in the masseter and diaphragm of calves andadult goats, but higher levels of GLUT4 mRNA in oxidativethan in glycolytic muscles (Hocquette et al., 1996). Thoseauthors assumed that a post-translational degradation of GLUT4precursors in oxidative muscles might be responsible for theseconflicting results. Abe et al. (1994, 1997) estimated adistribution of GLUT4 protein and mRNA levels in musclehomogenates comparable to that in humans and rodents, andthis corresponds well with our findings.

In camels, insulin does not develop a short-acting effect onglucose utilization (Elmahdi et al., 1997; Kaske et al., 2001)supporting the hypothesis of the absence of GLUT4. It wastherefore surprising that GLUT4 protein was detected in theskeletal muscles of the camels. In patients suffering from non-insulin-dependent diabetes mellitus (NIDDM), skeletal muscleGLUT4 protein is expressed as in healthy persons, but theinsulin-stimulated GLUT4 translocation into the myocyteplasma membrane is interrupted (Zierath et al., 1998). Theimpaired insulin-dependent pathway is partly compensated by

the insulin-independent glucose transport into myocytes, prob-ably via GLUT1 in these patients (Zierath et al., 1998). Theglucose uptake by GLUT1 is a facilitated diffusion along theglucose concentration gradient and takes place only under con-ditions of mild hyperglycemia reflected by slightly increasedbasal plasma glucose concentrations in NIDDM patients (Kahn,1996; Mueckler, 1994). Camels too exhibit relatively high basalplasma glucose levels of between 5.5 and 7.0 mmol/L (Al-Aliet al., 1988; Chandrasena et al., 1979). Summarizing these factsand our findings, camels show particularities of an “NIDDM-like” insulin–glucose metabolism: GLUT4 is expressed inskeletal muscles but the insulin-stimulated glucose utilization isreduced and may be compensated by the insulin-independentglucose uptake via GLUT1. In this context, the high basalplasma glucose levels in camels may be a regulatory necessity tomaintain glucose requirements of muscle cells. Further studiesare needed to verify this hypothesis.

Little is known about GLUT1 expression in red and whiteskeletal muscles of livestock animals. Even the findings ofearlier investigations on rodents are inconsistent. Kern et al.(1990) detected similar GLUT1 protein and mRNA levels in redand white portions of the quadriceps and gastrocnemius musclesof Sprague–Dawley rats, whereas other investigators foundhigher GLUT1 levels in oxidative than in glycolytic parts ofthese muscles (Goodyear et al., 1991; Marette et al., 1992). Wefound no significant differences in GLUT1 levels in red andwhite muscles of rats and porcines, thus our findings agree withthose of Kern et al. (1990). More striking was the finding thatGLUT1 expression was significantly higher in the glycolyticthan in the oxidative muscles of herbivores with forestomachs.This different GLUT1 distribution in red and white muscles offorestomach herbivores and of monogastric omnivores corre-sponds to the fact that GLUT1 contents correlated positivelywith LDH activities and negatively with ICDH activities inbovines and goats, but not in the monogastric omnivores.

Although GLUT4 distribution among the four musclesanalyzed in our study was qualitatively similar in all species,there were quantitative differences. The GLUT4 enrichments inoxidative muscles were considerably greater in forestomachherbivores than in monogastric animals and in contrast to all otherspecies in rats GLUT4 levels were not correlated significantlywith muscle marker enzyme activities. The quantitative discre-pancies concerning themuscle GLUT4 expression in rats and pigswere unexpected because in vivo studies displayed largelyidentical whole body glucose tolerances and insulin sensitivitiesin rats (Clément et al., 2002) and in pigs (Guan et al., 2000).Concerning the GLUT1 expression, we observed qualitative dif-ferences between monogastric animals and forestomach herbi-vores. The species investigated show some metabolic differenceswhich may contribute to the skeletal muscle GLUTexpression. Inruminants, early lactation is characterized by distinctivemetabolicchanges including the development of insulin resistance in skeletalmuscles and adipose tissue (Bell and Bauman, 1997) to maintainmammary gland lactose production. This insulin resistance israther due to a decreased GLUT4 expression than a down-regulation of insulin receptors. In goat myocytes GLUT4 contents(Balage et al., 1997) and in cow muscle cells GLUT4 mRNA


levels (Zhao et al., 1996) are decreased during early lactation. Sofar we have no knowledge about the influence of metabolic adap-tations to lactation onGLUT1 expression inmuscle cells. Physicaltraining increased GLUT4 expression in horses (Lacombe et al.,2003) aswell as in humans (Goodyear andKahn, 1998; Hjeltnes etal., 1998). However, findings on the influence of training onGLUT1 expression are contradictory: GLUT1 levels rose signifi-cantly in patients suffering from complete spinal cord lesions andparalysis of the vastus lateralis muscle after a period of electricalmuscle stimulation (Chilibeck et al., 1999), but not in healthyhumans and laboratory rodents after physical training (Gasteret al., 2000; Rodnick et al., 1992). Furthermore, in camels it hasbeen shown that muscle adaptation to physical conditioningappears only after quite intense training and ismost consistent withregard to enzyme activities, whereas alterations in muscle fibercomposition had been altered inconsistently (Cluer et al., 1994).Another environmental factor is the diet composition. It is knownthat fat-enriched diets reduce the insulin-stimulated glucosetransport into myocytes in rats. This decreased insulin sensitivityis accompanied by an increment of muscle GLUT4 but not ofGLUT1 protein (Han et al., 1995). The goat kids in our studywerefed a milk exchanger, they therefore received a fat-enriched diet,and this may have influenced the GLUT expression. In lactatingcows, nonlactating adult goats, suckling goats and physicallytrained camels, we detected slightly different GLUT4 but identicalGLUT1 distribution patterns, indicating that muscle cell GLUT4contents may have been influenced by environmental factors. ButGLUT1 distribution patterns observed in goats, cows and camelsappear to be a particularity of forestomach fermenters and areprobably defined genetically.

5.3. Conclusions

The present study indicates that GLUT1 is the predominantglucose transporter in cows, goats and camels, at least inglycolytic muscles. GLUT1 may be of much greater impor-tance in the whole body glucose utilization in herbivores withforestomachs than in monogastric omnivores. We recentlyobserved similar glucose transport rates in the semitendinosusmuscle of cows and pigs, although the translocation of GLUT4into plasma membrane of the semitendinosus muscle wassignificantly lower in the cows (Dühlmeier et al., 2005). Inmonogastric omnivores, glucose is absorbed from the smallintestine, whereas in foregut fermenters it is synthesizedendogenously by hepatic gluconeogenesis from short-chainedfatty acids generated microbiologically in the forestomach(Reynolds, 1992). In ruminants, this nutrient strategy isassociated to lower basal plasma glucose levels as well as tosmaller postprandial increments in glucose and insulin levelsthan in monogastric omnivores (Jenny and Polan, 1975; Yanget al., 2000). To maintain glucose levels in large locomotormuscles of forestomach fermenters these smaller fluctuationsof plasma insulin and glucose levels seem to require a glucosetransport pathway partly independent from an insulin-stimu-lated uptake. Further studies on glucose transport capacities ofGLUT1 in foregut herbivores and monogastric omnivores arerequired to confirm this hypothesis.

Acknowledgements

The authors wish to thank Prof. Dr. K.-H. Waldmann andDr. A. von Altrock, Clinic for Pigs, Small Ruminants, ForensicMedicine and Ambulatory Service, University of VeterinaryMedicine Hannover, Foundation, Hannover, Germany; Prof. Dr.G. Breves, Department of Physiology, University of VeterinaryMedicine Hannover, Foundation, Hannover, Germany andDr. J. Voigt, Department of Physiological Nutrition “OskarKellner”, Research Institute for the Biology of Farm Animals,Dummerstorf, Germany, for providing access to the experi-mental animals. Special thanks go to H.H. General SheikhMohammed Bin Rashid Al Maktoum, who enabled us toperform the investigations in camels at the Central VeterinaryResearch Laboratory, Dubai, UAE. We are also grateful to Dr. J.McAlister-Hermann for careful editing of the English manu-script. This work was supported by the German ResearchFoundation (DFG) grant SA 160/17-2.

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distribution patterns of the glucose transporters glut4 and glut1 in skeletal muscles of rats...

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