Altered Skeletal Muscle Fiber Composition and Size Precede Whole ...

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The Journal of Clinical Endocrinology & Metabolism 92(4):1530 –1534 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-2360

Altered Skeletal Muscle Fiber Composition and Size Precede Whole-Body Insulin Resistance in Young Men with Low Birth Weight Christine B. Jensen, Heidi Storgaard, Sten Madsbad, Erik A. Richter, and Allan A. Vaag Steno Diabetes Center (C.B.J., H.S., A.A.V.), 2820 Gentofte, Denmark; Department of Human Physiology (E.A.R.), Copenhagen Muscle Research Centre, Institute of Exercise and Sport Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark; and Department of Endocrinology (S.M.), Hvidovre University Hospital, 2630 Hvidovre, Denmark Context: Low birth weight (LBW), a surrogate marker of an adverse fetal milieu, is linked to muscle insulin resistance, impaired insulinstimulated glycolysis, and future risk of type 2 diabetes. Skeletal muscle mass, fiber composition, and capillary density are important determinants of muscle function and metabolism, and alterations have been implicated in the pathogenesis of insulin resistance. Objective: The aim of this study was to investigate whether an adverse fetal environment (LBW) induces permanent changes in skeletal muscle morphology, which may contribute to the dysmetabolic phenotype associated with LBW. Design and Subjects: Vastus lateralis muscle was obtained by percutaneous biopsy from 20 healthy 19-yr-old men with birth weights at 10th percentile or lower for gestational age (LBW) and 20 normal birth weight controls, matched for body fat, physical fitness, and

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ETAL PROGRAMING IS the phenomenon whereby alterations in fetal growth and development in response to the prenatal environment may have long-term or permanent effects (1). Low birth weight (LBW), a surrogate marker of an adverse fetal environment, is linked to muscle insulin resistance and increased future risk of type 2 diabetes (T2D) (2). It has been speculated that early influences on the growth and development of muscle fibers may underlie this relationship (3). Evidence for fetal programming of adult body composition and musculoskeletal development in humans is derived mostly from epidemiological studies, associating LBW and poor prenatal nutrition with increased fat mass (4), central distribution of fat (5), and reduced muscle mass (6). In addition to muscle mass per se, alterations in skeletal muscle morphology may also play a role in the pathogenesis of insulin resistance. In obese and T2D individuals, the distribution of muscle fiber types is shifted toward faster, more glycolytic fibers (7–11), a finding that is associated with a lower oxidative capacity (7, 11) and reduced GLUT4 content (12, 13). In parallel with fiber type alterations, insulin-resistant or glucose-intolerant subjects may have reduced capilFirst Published Online February 6, 2007 Abbreviations: DEXA, Dual-energy x-ray absorptiometry; LBW, low birth weight; NBW, normal birth weight; T2D, type 2 diabetes; VO2max, maximal aerobic capacity. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

whole-body glucose disposal. Myofibrillar ATPase staining was used to classify muscle fibers as type I, IIa, and IIx (formerly type IIb), and double immunostaining was performed to stain capillaries (LBW, n ⫽ 8; normal birth weight, n ⫽ 12). Results: LBW was associated with increased proportion of type IIx fibers (⫹66%; P ⫽ 0.03), at the expense of decreased type IIa fibers (⫺22%; P ⫽ 0.003). No significant change was observed in proportion of type I fibers (⫹16%; P ⫽ 0.11). In addition, mean area of type IIa fibers was increased (⫹29%; P ⫽ 0.01) and tended to be increased for type I fibers as well (⫹17%; P ⫽ 0.08). Capillary density was not significantly different between groups. Conclusion: Alterations in fiber composition and size may contribute to development of type 2 diabetes in individuals with LBW. (J Clin Endocrinol Metab 92: 1530 –1534, 2007)

lary density (14) and abnormalities of capillary structure, which could reduce the rate of diffusion of insulin to its receptor, thereby also contributing to insulin resistance. It is still a matter of debate whether these changes are a cause rather than a consequence of obesity, insulin resistance, and/or lower physical fitness. We hypothesized that an adverse fetal environment induces primary changes in muscle histology, and that these changes are at least partly responsible for the observed link between birth weight, insulin resistance, and subsequently T2D in adult life. To that end, we investigated the relation between LBW (birth weight ⱕ 10th percentile for gestational age), muscle fiber composition, and capillary density in a cohort of 19-yr-old lean men who had normal glucose tolerance, normal whole-body insulin sensitivity, and were matched for physical fitness. Subjects and Methods Subjects (Table 1) Forty singleton men born at term (39 – 41 wk) in 1980 in Copenhagen County were identified and recruited from the Danish Medical Birth Registry according to birth weight, as previously described (15). Twenty men had had birth weights below the 10th percentile (LBW, 2702 ⫾ 202 g), and 20 men had had birth weights in the upper normal range [normal birth weight (NBW), 50 –75th percentile] (3801 ⫾ 101 g). Information on postnatal weight gain was not available. None of the participants had a family history of diabetes (parents, grandparents), hypertension, or ischemic heart disease. All participants had normal glucose tolerance after a standard 75-g oral glucose tolerance test, according to the World Health Organization criteria. The participants provided written informed consent before participation. The protocol

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Jensen et al. • Muscle Fibers after Low Birth Weight

J Clin Endocrinol Metab, April 2007, 92(4):1530 –1534

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TABLE 1. Subject characteristics Variable

Birth weight (g) Height (cm) Weight (kg) Body mass index (kg/m2) Waist-to-hip ratio Total lean body massDEXA (kg) Total fat massDEXA (kg) Abdominal fat massDEXA (kg) VO2max (liters/min) Fasting plasma glucose (mmol/liter) Fasting plasma insulin (pmol/liter) Rd40mU (mg/kg FFM/min) GF40mU (mg/kg FFM/min) Forearm glucose uptakeINS (␮mol/100 ml)a

LBW (n ⫽ 20)

NBW (n ⫽ 20)

P value

Mean

SD

Mean

SD

2702 178.5 73.6 23.1 0.82 54.9 15.5 33.1 3.4 5.6 56.1 11.1 3.9 0.6

202 4.0 8.5 2.7 0.04 4.3 6.8 18.1 0.4 0.4 39.6 3.1 2.6 0.0

3801 181.7 74.7 22.6 0.81 56.9 15.6 30.7 3.5 5.4 48.6 11.9 5.4 1.0

101 4.8 13.1 3.6 0.06 7.3 6.9 21.9 0.7 0.4 22.2 3.0 2.6 0.2

⬍0.0001 0.03 NS NS NS NS NS NS NS 0.05 NS NS 0.04 0.04

Rd, Whole-body glucose uptake during euglycemic hyperinsulinemic clamp; GF, whole-body glycolysis during euglycemic hyperinsulinemic clamp; NS, not significant. a Done in a subset of this population (LBW, n ⫽ 14; NBW, n ⫽ 16). was approved by the regional ethical committee, and all procedures were performed according to the principles of the Helsinki Declaration. Briefly, all subjects were subjected to a dual-energy x-ray absorptiometry (DEXA) scan, underwent a 6-min submaximal exercise test with continuous monitoring of heart rate on a ergometer bicycle for calculation of the maximal aerobic capacity (VO2max) (16), and on a separate day, a two-step 4-h hyperinsulinemic euglycemic clamp (2 h at 10 mU/ m2䡠min, 2 h at 40 mU/m2䡠min) in combination with tritiated glucose tracer, indirect calorimetry, and a baseline muscle biopsy. In addition, assessments of insulin-stimulated endothelial function and glucose uptake were performed in a subgroup of this cohort in a forearm model (blood flow measured by venous occlusion plethysmography during dose-response studies of acetylcholine and sodium nitroprusside, glucose uptake measured by arteriovenous difference after acute intraarterial insulin infusion). Detailed description of the demographic and metabolic data (5, 15), forearm blood flow, and glucose uptake (17), as well as skeletal muscle expression of proximal insulin signaling proteins (18), has been provided previously. Briefly, these studies revealed that 19-yr-old men with LBW were slightly shorter, had comparable current body weight, body mass index, waist-to-hip ratio, VO2max, and total lean and fat mass, but had a tendency toward more central fat [abdominal fat mass (%), P ⫽ 0.12, abdominal fat mass/total fat mass, P ⫽ 0.03]. In addition, LBW was associated with slightly but significantly higher fasting plasma glucose, reduced insulin-stimulated whole-body glycolysis and reduced glucose uptake in the forearm, in the face of normal blood flow, whole-body glucose disposal, glucose oxidation, and nonoxidative glucose metabolism. Finally, LBW was associated with lower protein expression of the regulatory (p85␣) and catalytic (p110␤) subunits of PI3-kinase, protein kinase C␨, and the glucose transporter GLUT4 in skeletal muscle, whereas the expression of IR␤, protein kinase B␣, GSK3␣, GSK3␤, and PGC1␣ was unaltered.

Muscle biopsies Percutaneous muscle biopsies (n ⫽ 40) were obtained from the vastus lateralis muscle during local anesthesia using a Bergstro¨m needle. Samples were blotted free of blood, connective tissue, and visible fat, embedded in Tissue Tek (Miles Diagnostics Division, Elkhart, IN), and frozen in liquid nitrogen. Samples were stored at ⫺80 C until further analysis. Serial cross-sections (10 ␮m) were cut and stained for myofibrillar ATPase to identify type I, IIa, and IIx (formerly type IIb) muscle fibers (19). On two additional cross-sections, respectively, capillaries were stained using the method of Qu et al. (20). Stained sections were examined using a Zeiss Axiolab light microscope (Broch & Michelsen A/S, Birkerød, Denmark), coupled to a Sanyo VCC-2972 color CCD camera. Muscle fiber composition and capillary density were analyzed in all subjects by the same blinded observer using the TEMA image analysis software (CheckVision, Støvring, Denmark). The mean number of fibers and capillaries per biopsy analyzed were

153 and 471, respectively. Due to technical reasons, fiber determinations were only obtained in 19 LBW and 18 NBW subjects, and capillary density in 8 LBW and 12 NBW subjects.

Statistical analysis Statistical analysis was performed using SigmaStat 3.0 statistical software. The data were normally distributed, and there was no significant difference in the variances of the two groups. The significance of any difference between groups was examined by the Student’s t test, and a P value of less than 0.05 was considered statistically significant. Results are given as mean ⫾ sem, except for anthropometrical and metabolic data presented in Table 1 (mean ⫾ sd). Correlations between muscle morphology and various metabolic, demographic, and protein data were assessed by Spearman rank order test.

Results Muscle fiber type and size

Table 2 shows data on muscle fiber type, fiber size, and capillary density. Fiber type distribution expressed as relative number (% no.) of fibers or relative fiber area (% area) differed as a function of birth weight, such that LBW was associated with shift in distribution of type II fibers with a relative increase in type IIx fibers (absolute area, ⫹66%, P ⫽ 0.028; % area, ⫹50%, P ⫽ 0.059), at the expense of decreased type IIa fibers (% no., ⫺27%, P ⬍ 0.001; % area, ⫺22%, P ⫽ 0.003). Type I fibers (% no., ⫹16%; P ⫽ 0.11) were not significantly different between groups. In addition, type IIa fibers were larger (mean area, ⫹29%; P ⫽ 0.014) in LBW, and type I fibers tended to be larger in LBW as well (mean area, ⫹17%; P ⫽ 0.078). Capillary density

Due to the low number of eligible samples (LBW, n ⫽ 8; NBW, n ⫽ 12), caution should be exerted when interpreting differences in capillary density. Overall, the number of capillaries per fiber and capillaries per mm2 was similar between groups (P ⫽ 0.158, P ⫽ 0.380), whereas the number of capillaries per type IIa (⫹16%; P ⫽ 0.106) and type I fibers (⫹14%; P ⫽ 0.127) tended to be higher in LBW subjects.

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TABLE 2. Fiber composition and capillary density LBW Mean

Type I

NBW SEM

Mean

19

P value SEM

n Absolute no. Percent no. Absolute area (␮m2) Percent area Mean area (␮m2) No. of cap. to each fiber

71.6 49.7 391,066 44.4 5,660 6.8

7.2 2.7 41,024 2.5 345 0.5

69.4 43.0 345,107 40.2 4,848 5.9

18 7.8 3.1 44,739 2.7 279 0.2

0.838 0.110 0.453 0.260 0.077 0.127

Type IIa

Absolute no. Percent no. Absolute area (␮m2) Percent area Mean area (␮m2) No. of cap. to each fiber

43.4 30.7 313,301 35.9 7,444 7.3

4.5 2.1 30,498 2.5 503 0.6

63.2 42.2 360,889 46.4 5,754 6.3

4.5 2.3 34,941 2.2 411 0.3

0.004 0.001 0.310 0.003 0.014 0.106

Type IIx

Absolute no. Percent no. Absolute area (␮m2) Percent area Mean area (␮m2) No. of cap. to each fiber

25.9 18.6 159,446 18.7 5,389 6.1

3.9 2.7 19,930 2.6 525 0.4

21.3 13.7 96,224 12.5 4,898 5.7

4.1 2.0 18,374 1.8 409 0.3

0.414 0.160 0.028 0.059 0.473 0.444

Capillaries

n Total no. of cap Cap./fiber Cap./mm2

68.3 0.4 25.1

483.1 2.8 476.9

38.0 0.1 22.2

0.686 0.158 0.380

8 453.4 3.2 448.3

12

cap., Capillary.

Correlations

Discussion

We found significant correlations between absolute type I fiber area and VO2max (all: R ⫽ 0.43, P ⫽ 0.008) (Fig. 1), and between type I fiber area (%) and abdominal fat mass (%) (all: R ⫽ ⫺0.46, P ⫽ 0.004) (Fig. 2). Type IIa fiber number (%) correlated weakly with insulin-stimulated glycolysis assessed during hyperinsulinemic euglycemic clamp (all: R ⫽ 0.40, P ⫽ 0.01) (Fig. 3), and with fasting plasma glucose (all: R ⫽ ⫺0.36, P ⫽ 0.03) (Fig. 4). Furthermore, absolute type IIx fiber area was weakly negatively correlated with insulinstimulated glycolysis (all: R ⫽ ⫺0.35, P ⫽ 0.04), and with fasting plasma glucose (LBW fasting plasma glucose, R ⫽ 0.48, P ⫽ 0.04) and GLUT4 protein (LBW, R ⫽ ⫺0.47, P ⫽ 0.05) in the LBW group. We found no consistent trends for associations between capillary density and clinical, metabolic, or protein expression data.

This study is the first in humans to demonstrate an effect of the early environment on muscle fiber composition and fiber size. Given that these were young healthy individuals with normal glucose tolerance, normal total whole-body glucose disposal, and VO2max, we speculate that these are primary changes programmed in utero, contributing to development of muscle insulin resistance and T2D in LBW. Muscle fibers are classified in terms of their contractile and metabolic properties. In untrained individuals, the proportion of slow twitch oxidative fibers (type I) in the vastus lateralis muscle (the largest of the quadriceps muscles and the most commonly studied muscle in humans) is typically approximately 55%, with fast twitch type IIa fibers being twice as common as fast twitch type IIx fibers (21). The close coupling between muscle fiber type and associated morphological, metabolic, and functional properties applies to both

FIG. 1. Relation between type I fiber area (square micrometers) and VO2max (liters per min). Filled symbols represent LBW subjects, and open symbols represent NBW subjects. Spearman correlation coefficient R ⫽ 0.48, P ⫽ 0.008.

FIG. 2. Relation between type I fiber area (%) and abdominal fat mass (%). Filled symbols represent LBW subjects, and open symbols represent NBW subjects. Spearman correlation coefficient R ⫽ ⫺0.46, P ⫽ 0.004.


FIG. 3. Relation between type IIa fiber number (%) and insulin-stimulated glycolysis (mg/kg FFM/min). Filled symbols represent LBW subjects, and open symbols represent NBW subjects. FFM, Fat free mass. Spearman correlation coefficient R ⫽ 0.40, P ⫽ 0.01.

physical and metabolic performance (22). Insulin sensitivity correlates with the proportion of type I fibers (14). Specifically, insulin-stimulated glucose transport is greater in skeletal muscle enriched in type I fibers (23), possibly related to a higher GLUT4 content (13, 24). A shift in fiber distribution, from type I to type II, alters activity of key oxidative and glycolytic enzymes (25), and the ratio between glycolytic and oxidative enzyme activities in skeletal muscle of T2D or obese individuals is closely related to insulin resistance (26, 27). T2D patients (9 –11,14), their insulin-resistant offspring (28), and individuals with impaired glucose tolerance (29) have a relative increase in glycolytic type IIx fibers, of similar magnitude to these LBW subjects. Using simple correlation analysis, we found weak but significant associations between type IIx fiber area, fasting plasma glucose, and GLUT4 protein expression in LBW, suggesting that increased type IIx fiber area may indeed influence whole-body and muscle glucose metabolism in these individuals. Similar to insulinresistant first-degree relatives of T2D patients (28) and glucose-intolerant subjects (29, 30), LBW subjects did not have fewer type I fibers. On the contrary, LBW subjects tended to have more type I fibers (P ⫽ 0.11), despite increased abdominal adiposity and the strong negative correlation between abdominal fat and type I fibers. Interestingly, newborn piglets exposed to intrauterine growth restriction have accelerated development of skeletal muscle with precocious type II to type I conversion and, as a consequence, increased proportion and maturation of type I fibers (31, 32). Future studies

FIG. 4. Relation between type IIa fiber area (%) and fasting plasma glucose (millimoles per liter). Filled symbols represent LBW subjects, and open symbols represent NBW subjects. Spearman correlation coefficient R ⫽ ⫺0.36, P ⫽ 0.03.


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must address whether LBW subjects have alterations in muscle development. Alterations in type IIa fibers have not previously been found in insulin-resistant states or diabetes. Interestingly, patients with severe chronic obstructive pulmonary disease and stable chronic hypoxemia had fewer type IIa and more type IIx fibers in brachial biceps muscle (33). Exposure of young rats to hypoxia during postnatal development induced slow-to-fast transition in major histocompatibility complex expression (i.e. fewer type I/IIa fibers and more type IIx) (34). Moreover, the muscle-specific hypoxia-inducible factor-1␣ knockout mouse, characterized by impaired glycolysis and compensatory upregulation of mitochondrial oxidative pathways, had fewer type IIa fibers in soleus muscle (35). Thus, we speculate that hypoxia and hypoxia-induced adaptational responses may contribute to muscle fiber type alterations in LBW. Two previous studies examined the effect of fetal growth restriction on capillary density and function in humans. In a group of insulin-resistant women, thinness at birth (ponderal index ⬍ 23 kg/m3) was not related to differences in fiber composition, capillary density or blood flow (36). However, women with a low ponderal index had faster reoxygenation rates after exercise. More recently, birth weight was positively associated with capillary recruitment during postocclusive reactive hyperemia in prepubertal children, independently of blood pressure and insulin sensitivity (37), suggesting that LBW is associated with an impaired capillary hypoxic response. Combined with more capillaries in newborn piglets with intrauterine growth restriction (31), it is therefore of interest that our LBW men tended to have more capillaries per type I/IIa fibers. However, due to risk of type I error, our findings should be reproduced in larger studies. Finally, our LBW subjects had larger type I and IIa fibers. Obesity correlates positively with muscle fiber area (14). Increased size of type I (29) and IIa fibers (30) was previously described in glucose-intolerant subjects, and insulin-resistant women with Turner syndrome have larger type IIa fibers (38). Increased fiber size thus appears to track with insulin resistance and precedes onset of clinical disease in insulinresistant people. We speculate that increased fiber size is an early marker of insulin resistance in this population. In conclusion, our study is the first in humans to demonstrate an effect of the early environment on skeletal muscle development. Given that the individuals were young and lean with normal glucose tolerance, whole-body glucose disposal, and VO2max, we speculate that these are primary changes, involved in development of muscle insulin resistance in individuals with LBW. Although we do not yet understand the molecular basis for these findings, it is interesting that myogenesis and fiber type switching are regulated by a complex network of transcription factors (22), including the transcriptional coactivator PGC1␣, also implicated in the pathogenesis of T2D. We previously found no differences in protein expression of PGC1␣ in this population (18). Thus, understanding mechanisms by which an adverse fetal milieu may affect muscle development and fiber type specification will be an important goal of future studies.

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Acknowledgments The authors thank Betina Bolmgren for expert technical assistance in preparing and staining the muscle sections. Furthermore, Dr. Susan E. Ozanne kindly provided access to raw data on protein expression for correlation analyses. Received October 27, 2006. Accepted January 25, 2007. Address all correspondence and requests for reprints to: Christine Bjørn Jensen, M.D., Ph.D., Steno Diabetes Center, Niels Steensens Vej 2, 2820 Gentofte, Denmark. E-mail: [email protected]. This work was assisted by funding from The Danish Agency for Science, Technology, and Innovation, The Danish Diabetes Association, The Danish Medical Association Research Fund, The AP Moller Foundation for the Advancement of Medical Science, and Integrated Project (LSHM-CT-2004-005272) funded by the European Union and the Danish Medical Research Council and the Lundbeck Foundation. C.B.J. was supported by a postdoctoral fellowship from The Agency for Science, Technology, and Innovation (DK). C.B.J., H.S., S.M., and E.A.R have nothing to declare. A.A.V. is currently employed by, has equity interests in, and received lecture fees from Novo Nordisk A/S.

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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.