Effect of locally delivered IGF-1 on nerve regeneration during aging: an experimental study in rats

Abstract

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin-like growth factor 1 (IGF-1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF-1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF-1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF-1 significantly improved axon number, diameter, and density. IGF-1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF-1 treatment.

Keywords: aging, IGF-1, nerve injury, nerve regeneration, neuromuscular junction

Age is thought to be the most important predictor of neuromuscular recovery after peripheral nerve injury.¹ Of the 18 million extremity injuries recorded annually in the USA, >50% occur in patients 45 years of age or older.² Children often regain near-normal function after nerve injury,^3,4 but adults or aged patients have slow or absent neuromuscular recovery. Over half of patients >50 years of age do not achieve any functional recovery following nerve repair,¹ resulting in impaired activities of daily living. Aging is known to affect the structure and function of the peripheral nerve,^5,6 but the exact etiology of age-related impairment in regeneration is unknown, although delayed axonal regeneration^7,8 and instability of the neuromuscular junction (NMJ) have been implicated.⁹

Insulin-like growth factor 1 (IGF-1) is a single-chain, 70-amino-acid polypeptide, with a molecular mass of 7.6 kDa, similar in structure to pro-insulin, and is highly conserved across species.¹⁰ IGF-1 plays a significant role in neuronal development, ^11,12 recovery from neuronal injury,^13,14 neuronal survival,¹⁵ and neurite outgrowth following crush injury.¹⁶ In vitro studies have suggested that IGF-1 is produced locally by non-neuronal cells following injury and stimulates regeneration.¹⁷ Hansson et al. first showed that IGF-1 is secreted from Schwann cells in an autocrine fashion after peripheral nerve injury.¹³ IGF-1 has been shown to affect multiple facets of Schwann cell function in vitro, which likely contributes to improved nerve regeneration, ¹⁸ including proliferation,¹⁹ mobilization, myelination,²⁰ and Schwann cell–axon interaction. IGF-1 knockout mice have defects in neurologic development²¹ in addition to impaired recovery from neuronal injuries.²² Thus, there is substantial evidence that IGF-1 plays a key role in neuromuscular recovery following injury.

Age and developmental stage are the major determinants of IGF-1 levels. IGF-1 levels peak during puberty, initiating and maintaining the pubertal growth spurt.²³ Following the pubertal growth spurt, IGF-1 levels decline²⁴ at a rate of approximately 14% per decade.^25,26 With continued aging, IGF-1 concentrations fall to levels of 20–80% of values for young adults,²⁷ concordant with the decline in functional recovery following nerve transection and repair in aged adults.¹

Given the wide-ranging effects of IGF-1 on nerve regeneration and the known decline in IGF-1 with age, we sought to determine whether IGF-1 replacement would improve nerve regeneration in aged animals. Previously, we found that increasing systemic IGF-1 via pulsatile growth hormone injection did not improve nerve regeneration (unpublished data). Thus, the purpose of this study was to examine the effects of IGF-1 locally delivered to a nerve transection site in aged rats, and to evaluate neuromuscular recovery, axonal regeneration, myelination, Schwann cell activity, and the resulting changes at the NMJ. The hypothesis was that age-related decline in neuromuscular recovery following transection would be ameliorated by IGF-1 administered to the regenerating nerve.

METHODS

Animal Model

Thirty-two Fischer 344 × Brown Norway rats were obtained from the National Institute of Aging colony at the Harlan Sprague-Dawley facility (Indianapolis, Indiana). The animals were divided into two groups: young adult (8 months) and aged (24 months). Each age group was subdivided into saline and IGF-1 treatment groups. There were 6 young adult saline, 6 young adult IGF, 10 aged saline, and 10 aged IGF-1 animals. Animals underwent nerve transection and repair, as detailed in what follows, and were then killed after 12 weeks.

Surgical Procedure

Each animal was anesthetized with isoflurane, and an incision was made over the posterior aspect of the left thigh. Under the operating microscope, the sciatic nerve was exposed, and the tibial nerve was isolated and transected 1 cm from its insertion into the gastrocnemius. The nerve stumps were placed at opposing ends of a custom-made T-tube and the middle arm was attached to a minipump (Fig. 1). The minipump was buried subcutaneously under the skin of the back. The nerve conduit portion of the T-tube was made from 1016-µm internal diameter, semiporous tubing (Micro-Renathane; Braintree Scientific, Braintree, Massachusetts), and the T-arm was constructed of Silastic tubing (Dow Corning, Midland, Michigan). An Alzet 2004 mini-osmotic pump (Durect Corp., Cupertino, California) delivered either normal saline or IGF-1 at a rate of 0.25 µl/h. Half of the animals received recombinant human IGF-1 (Bachem AG, Torrance, California) at a 0.10-µg/µl (100 µg/ml) concentration. Thus, final IGF-1 delivery was 0.025 µg/h, modeled after previous studies of IGF-1 infusion.²⁸ The other animals received normal saline only. The subcutaneous pumps were replaced under anesthesia after 6 weeks. At 12 weeks after nerve transection and repair, tissue was harvested, and the animals were overdosed with isoflurane and perfused with fixative.

FIGURE 1 Diagram of experimental setup. The tibial nerve was transected 1 cm from the insertion into the gastrocnemius and allowed to relax to create a 7-mm gap. A custom-made T-tube device was interposed between the nerve ends and secured with a 9-0 nylon suture. (more …)

Animals were group-housed and allowed food and water ad libitum. All experiments were conducted under a protocol approved by the institutional animal care and use committee. The animals were housed in an appropriate facility in accordance with standards established by the Association for Assessment and Accreditation of Laboratory Animal Care.

Muscle and Tissue Harvest

At study termination, the animals were anesthetized with isoflurane, and the gastrocnemius was harvested. The muscle was cleaned, rinsed, flash frozen in liquid nitrogen, and stored at −80°C. Following removal of the gastrocnemius, the animals were transcardially perfused with warm saline followed by ice-cold 4% paraformaldehyde. The regenerated nerve was then removed and immersion-fixed in 2.5% glutaraldehyde.

Nerve Histomorphometry

The midpoint of the regenerated nerve was secondarily fixed in 1% osmium tetroxide, embedded in resin, sectioned to 1 µm, and stained with toluidine blue. All images were acquired microscopically (AxioImager M1; Carl Zeiss, Jena, Germany) using AxioVision version 4.5 software. Images were imported into ImageJ (National Institutes of Health, Bethesda, Maryland) for analysis. For the peripheral nerve, the following measurements were taken: nerve diameter; nerve cross-sectional area; axon number; axon density (axons per mm²); average axon diameter; and the average g-ratio (ratio of axon diameter to fiber diameter). Nerve diameter and nerve area were measured on 100× or 200× images. For axon number and axon density, two or three non-overlapping 400× fields were obtained from each of the four quadrants of the nerve. For average axon diameter and the average g-ratio, three or four non-overlapping 1000× fields were obtained from each of the four quadrants of the nerve. In each 400× high-power field, the myelinated axons were counted, and the area sampled was calculated, yielding axon density. For axon diameter and g-ratio, a minimum of 100 nerve fibers were analyzed. In order to focus analysis on motor neurons, axons <1 µm were not measured. For axon diameter, the measurement was from made from the inner table of the myelin sheath and did not include the myelin layer. The g-ratio was calculated by measuring axon diameter and total fiber diameter.

mRNA Expression of GAP43

The lateral gastrocnemius was pulverized, and total RNA was extracted with Trizol (Sigma-Aldrich, St. Louis, Missouri). Quantity and purity of the RNA was assessed using a spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, Delaware), and quality of RNA was assessed by microcapillary gel electrophoresis (Model 2100 Bioanalyzer; Agilent Technologies, Santa Clara, California) prior to downstream applications. The mRNA was transcribed to cDNA using oligo-dT primers (Invitrogen, Carlsbad, California) and Superscript II (Invitrogen, Carlsbad, California). Primers were obtained from Applied Biosystems for GAP43 (NM_017195.3). Relative quantitative reverse transcript–polymerase chain reaction (qRT-PCR) was then performed (Model 7900HT; Applied Biosystems) with a 384-well block. Muscle creatine kinase (MCK) was used as an endogenous control, as it has been shown to be stably expressed in skeletal muscle with age.²⁹

Histology of Postsynaptic NMJs

The medial gastrocnemius was pinned to resting length and immersed in isopentane cooled with dry ice. The muscle was sectioned on a cryotome longitudinally in 25-µm sections. The sections were permeabilized with 0.1% Triton X-100 for 20 minutes, and then treated with AlexaFluor 488–conjugated α-bungarotoxin (1:200; Invitrogen, Carlsbad, California) for 1 hour. Sections were rinsed and fixed in 1% paraformaldehyde for 1 hour. Confocal images were obtained on a laser scanning confocal microscope (Zeiss 510) with argon and helium–neon lasers. Slice thickness was set at ≤1.2 µm. Slices were then merged to create the final image. The NMJs were then qualitatively evaluated.

Statistical Analysis

All data were imported into SigmaStat version 3.11 (Systat Software, Inc., San Jose, California) for analysis. For each outcome measure, a two-way analysis of variance (ANOVA) model was established with age and treatment as the independent variables. Post hoc pairwise comparisons were made with the Student–Newman–Keuls test. Transformations to ranked sums were made when the data were nonparametric. Type 1 error was set at 0.05.

RESULTS

IGF-1 Increases Axon Number, Diameter, and Density in Regenerated Nerve of both Young and Aged Animals

Examination of the peripheral nerve revealed that, in aged saline-treated nerves, there were fewer total axons, fewer axons per mm², and less myelination than in young saline-treated nerves. IGF-1 had a marked effect on both young and aged animals, increasing axon number, diameter, and density, in addition to increasing myelination (Fig. 2). Quantitative data are presented in Figure 3. For the number of axons per nerve, IGF-1 increased total axon number in both young and aged animals; however, this was only statistically significant for aged animals. For axon density, IGF-1 increased axon density in both young and aged animals; however, this was only statistically significant for aged animals. For average axonal diameter, there was a significant increase in average axonal diameter in both young and aged animals when they were treated with IGF-1. There was no difference between the young IGF-1 group and aged IGF-1 group for number of axons per nerve, axon density, and average axonal diameter.

FIGURE 2 Cross-section of regenerated nerve segment. After 3 months, the regenerated nerve segment was removed, sectioned to 1 µm, stained with toluidine blue, and viewed at 1000×. (A) Young saline, (B) aged saline, (C) young IGF-1, and (D) aged (more …)

FIGURE 3 Effect of age and IGF-1 on axon number, density, and diameter. Quantification of histologic images reveals a significant effect of IGF-1 on (A) axons per nerve, (B) axon density (axons per mm2), and (C) average axonal diameter. Although there were differences (more …)

IGF-1 Increases Myelination and Schwann Cell Activity in Regenerated Nerve of both Young and Aged Animals

Three parameters of Schwann cell activity and myelination were examined: (1) myelin thickness; (2) axon/fiber ratio (g-ratio); and (3) GAP43 mRNA expression, a marker of Schwann cell activity. ^30,31 We found that IGF-1 significantly affected all measured aspects of Schwann cell activity and myelination (Fig. 4). For myelin thickness and g-ratio, this effect was not age-dependent. However, for GAP43 expression, aged animals had a greater response to IGF-1 than young animals (Fig. 4C).

FIGURE 4 Effect of age and IGF-1 on measures of myelination. Quantification of histologic images revealed a significant effect of IGF-1 on multiple facets of myelination and Schwann cell function. (A) Myelin thickness was significantly increased in IGF-1–treated (more …)

IGF-1 Preserves Morphology of Postsynaptic NMJs in Aged Animals

The postsynaptic NMJs were stained with fluorescent-labeled α-bungarotoxin and qualitatively examined using laser scanning confocal microscopy (Fig. 5). In saline-treated young animals, the postsynaptic NMJs were highly contiguous, complex, and had deep gutters, similar to uninjured control NMJs (images not shown). In young IGF-1 animals, the NMJs were very similar to those in the young saline group. In the aged saline group, numerous NMJs displayed a loss of complexity, with markedly more shallow gutters, and a loss of perimeter and endplate area. This is consistent with the morphology of denervated NMJs.^32,33 In contrast, in the aged IGF-1 animals, the complexity, gutter depth, and overall morphology more closely resembled that of young animals.

FIGURE 5 Response of the neuromuscular junction to saline or IGF-1 treatment. (A) Young saline, (B) aged saline, (C) young IGF-1, and (D) aged IGF-1. The postsynaptic NMJ was stained with fluorescent-labeled α-bungarotoxin and imaged under a confocal microscope. (more …)

•  Other Sections▼

DISCUSSION

In this study we sought to determine whether IGF-1 would improve neuromuscular recovery after nerve injury during aging. We found that IGF-1 improved nerve regeneration by acting on the axons and Schwann cells, and secondarily on the NMJs. These results suggest that there is no loss of sensitivity to IGF-1 with age and that IGF-1 can improve regeneration after nerve injury during aging.

Previous studies have examined the effect of systemically administered IGF-1 during neuromuscular recovery in neonatal rats. Vergani et al. found that systemic IGF-1 promoted neuronal survival following crush injury and improved muscle reinnervation.³⁴ Kanje et al. found that locally delivered IGF-1 following crush injury in juveniles led to improved sensory function.³⁵ Tiancgo et al. found that IGF-1 locally delivered to an end-to-side repair improved muscle function in young rats.³⁶ To the authors’ knowledge, the current study is the first to examine the effect of IGF-1 during nerve regeneration in aged animals.

Axonal outgrowth is central to nerve regeneration, and IGF-1 is a well-documented promoter of motor neuron survival,³⁷ axonal growth,³⁸ and axonal branching.¹⁸ In the regenerating nerve, IGF-1 is a key promoter of initial sprouting and subsequent elongation of axons³⁹ (for review, see Rabinovsky²). Although there has been extensive investigation of the effect of IGF-1 on neuron outgrowth and survival, nearly all of the studies were performed in vitro or in embryonic animals. Thus, it is not known how increased age affects the neuronal response to IGF-1. In this study we found that, for metrics of neuronal trophism, aged animals had a robust response to IGF-1 treatment, increasing axonal number, diameter, and density in the regenerated nerve segment. Thus, the neuronal response to IGF-1 of the regenerating axon does not appear to be limited due to age.

The Schwann cell is a key factor in promotion of nerve regeneration. The effect of IGF-1 on Schwann cells has also been studied. IGF-1 is known to promote Schwann cell survival,⁴⁰ motility,⁴¹ proliferation, ⁴² and myelination.⁴³ However, the physiology of Schwann cells from aged animals is largely unknown. This study has addressed some of the parameters of Schwann cell function in vivo, particularly myelination and GAP43 expression. GAP43 is upregulated in nearly all myelinating Schwann cells of the distal stump following axotomy.³¹ Expression of GAP43 is postulated to play a role in the interaction between the regenerating axon and Schwann cell.³⁰ In this study, we found that GAP43 expression was significantly upregulated in both young and aged animals with IGF-1 treatment. In addition, GAP43 expression was higher in aged animals treated with IGF-1 than in similarly treated young animals, suggesting that age increases sensitivity to IGF-1 for this aspect of Schwann cell function. However, due to pooling of the whole muscle mRNA, in this study we could differentiate between an increase in Schwann cell number and an increase in activity. Furthermore, because the sample was from muscle, the increase in GAP43 expression was due to intramuscular neurons, terminal Schwann cells, or both. GAP43 expression in the regenerated segment itself was not assayed. In addition to GAP43 activity, IGF-1 increased myelination in the regenerated nerve, independent of the increase in axon diameter. Thus, at the Schwann cell level, age does not impair the response to IGF-1 and results in an increased regenerative response.

Failure to achieve reinnervation of the muscle is thought to contribute to impaired neuromuscular recovery after nerve injury.⁴⁴ In this study we found by histologic examination that the motor endplate regained an innervated, pre-injury morphology in all but the aged saline group. This failure to reinnervate may have been due to delayed nerve regeneration or instability of the postsynaptic NMJ, both of which have been shown to occur with aging.⁹ In this study, treatment with IGF-1 may have accelerated axonal regeneration, leading to earlier reinnervation of the muscle, and thus preservational of the postsynaptic NMJ.

One limitation of our study is the lack of functional and electrical assessments. Functional recovery is the gold standard for clinical outcomes in human subjects, but, historically, functional recovery in rodent models has been difficult to assess.⁴⁵ In rats, there is a poor correlation between functional, electrophysiologic, and histologic outcomes. ⁴⁶ In this study, electrical measures of function were performed and showed a trend toward improvement with IGF-1 treatment, but this was not statistically significant (data not shown). Nonetheless, we were able to demonstrate that IGF-1 improved nerve regeneration in aged animals.

An important strength of our study is the in vivo comparison between young adult and aged rats after a complete nerve transection. Previous studies of IGF-1 on nerve regeneration utilized in vitro, neonatal, or juvenile animal models. In addition, many previous studies utilized crush injury models. Crush injuries in rodents typically have a near 100% rate of recovery and do not accurately model clinical scenarios wherein neuromuscular recovery is often very poor. Although previous studies have helped to answer many questions about the effects of IGF-1, this study is the first to examine in vivo the effects of age and IGF-1 on neuromuscular recovery following nerve transection.

Overall, our study has provided substantial evidence that locally delivered IGF-1 improves neuromuscular recovery after nerve injury in aged animals. We showed that, when local IGF-1 levels were equally supplemented in young and aged animals, nerve regeneration and NMJ preservation were similar, suggesting that IGF-1 may be a contributing factor in age-related impairment of neuromuscular recovery. However, our study has not conclusively demonstrated that IGF-1 deficiency is the immediate cause of age-related impairments in neuromuscular recovery after injury.

Myostatin inhibition increases muscle mass

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse

George Carlson C, Bruemmer K, Sesti J, Stefanski C, Curtis H, Ucran J, Lachey J, Seehra JS.

Source

Department of Physiology, Kirksville College Osteopathic Medicine, AT Still University, Kirksville, Missouri 63501, USA. ccarlson@atsu.edu

Abstract

INTRODUCTION:

In this study we investigated the action of RAP-031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF-κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice.

METHODS:

The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used.

RESULTS:

RAP-031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP-031 with UDCA produced increases in FPT that were associated with an increase in WBT.

CONCLUSIONS:

Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF-κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice.

Copyright © 2011 Wiley Periodicals, Inc.

PMID:

21462203

[PubMed – indexed for MEDLINE]

PMCID: PMC3075386

[Available on 2012/5/1]

Myostatin: a novel insight into its role in metabolism, signal pathways, and expression regulation.

Huang Z, Chen X, Chen D.

Source

Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China.

Abstract

Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a critical autocrine/paracrine inhibitor of skeletal muscle growth. Since the first observed double-muscling phenotype was reported in myostatin-null animals, a functional role of myostatin has been demonstrated in the control of skeletal muscle development. However, beyond the confines of its traditional role in muscle growth inhibition, myostatin has recently been shown to play an important role in metabolism. During the past several years, it has been well established that Smads are canonical mediators of signals for myostatin from the receptors to the nucleus. However, growing evidence supports the notion that Non-Smad signal pathways also participate in myostatin signaling. Myostatin expression is increased in muscle atrophy and metabolic disorders, suggesting that changes in endogenous expression of myostatin may provide therapeutic benefit for these diseases. MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate gene expression and recent evidence has accumulated supporting a role for miRNAs in the regulation of myostatin expression. This review highlights some of these areas in myostatin research: a novel role in metabolism, signal pathways, and miRNA-mediated expression regulation.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:

21609762

[PubMed – in process]

Myostatin propeptide (GDF-8) enhances the repair and regeneration of both muscle and bone in a model of deep penetrant musculoskeletal injury

BUY GDF-8 FROM US:  www.superhumangear.com

 

 

Recombinant myostatin (GDF-8) propeptide enhances the repair and regeneration of both muscle and bone in a model of deep penetrant musculoskeletal injury

Hamrick MW, Arounleut P, Kellum E, Cain M, Immel D, Liang LF.

Source

Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA. mhamrick@mail.mcg.edu

Abstract

BACKGROUND:

Myostatin (GDF-8) is known as a potent inhibitor of muscle growth and development, and myostatin is also expressed early in the fracture healing process. The purpose of this study was to test the hypothesis that a new myostatin inhibitor, a recombinant myostatin propeptide, can enhance the repair and regeneration of both muscle and bone in cases of deep penetrant injury.

METHODS:

We used a fibula osteotomy model with associated damage to lateral compartment muscles (fibularis longus and brevis) in mice to test the hypothesis that blocking active myostatin with systemic injections of a recombinant myostatin propeptide would improve muscle and bone repair. Mice were assigned to two treatment groups after undergoing a fibula osteotomy: those receiving either vehicle (saline) or recombinant myostatin propeptide (20 mg/kg). Mice received one injection on the day of surgery, another injection 5 days after surgery, and a third injection 10 days after surgery. Mice were killed 15 days after the osteotomy procedure. Bone repair was assessed using microcomputed tomography (micro-CT) and histologic evaluation of the fracture callus. Muscle healing was assessed using Masson trichrome staining of the injury site, and image analysis was used to quantify the degree of fibrosis and muscle regeneration.

RESULTS:

Three propeptide injections over a period of 15 days increased body mass by 7% and increased muscle mass by almost 20% (p < 0.001). Micro-CT analysis of the osteotomy site shows that by 15 days postosteotomy, bony callus tissue was observed bridging the osteotomy gap in 80% of the propeptide-treated mice but only 40% of the control (vehicle)-treated mice (p < 0.01). Micro-CT quantification shows that bone volume of the fracture callus was increased by ∼ 30% (p < 0.05) with propeptide treatment, and the increase in bone volume was accompanied by a significant increase in cartilage area (p = 0.01). Propeptide treatment significantly decreased the fraction of fibrous tissue in the wound site and increased the fraction of muscle relative to fibrous tissue by 20% (p < 0.01).

CONCLUSIONS:

Blocking myostatin signaling in the injured limb improves fracture healing and enhances muscle regeneration. These data suggest that myostatin inhibitors may be effective for improving wound repair in cases of orthopaedic trauma and extremity injury.

PMID:

20173658

[PubMed – indexed for MEDLINE]

>The wonderful L-arginine, a natural GH and NO booster, anabolic and wound healer

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20 ml 0,2g/ml L-arginine HCL infusion by Fresenius-Kabi $15 superhumangear@gmail.com

Investigation of the effects of oral supplementation of arginine in the increase of muscular strength and mass
Gerseli Angeli
1, Turibio Leite de Barros1, Daniel Furquim Leite de Barros2 and Marcelo Lima3

FULL PDF article DOWNLOAD

ABSTRACT
Introduction: Oral administration of arginine has been associated with physical performance improvement due to probable decrease of muscular fatigue derived from the vasodilatation factor of the nitric oxide over the skeletal muscles.
Objective: to evaluate the effects of oral administration of L-Arginine during an exercise program with weights. Methods: 20 male individuals, randomly divided in two groups: A and B, were submitted to eight weeks of training with weights (three times per week). Group A used 3 grams of L-Arginine + vitamin C during the eight weeks and group B used only vitamin C (control group).
Results: After eight weeks of training, group A presented body weight values and lean mass significantly higher (p < 0.05), body fat percentage significantly lower (p< 0.05), and strength of lower limbs significantly higher (p < 0.05), while group B did not present significant differences for the same period.
Conclusion: Oral administration of arginine associated with a training program with weights increased the stimuli of the exercise to the skeletal muscles level, enabling hence, increase of
muscular strength and mass.

>The role of insulin

>

Insulin physiology

It is often stated that the primary benefit of insulin in bodybuilding is that it increases the uptake of glucose into muscle and further that this movement of glucose is insulin dependent. But that is not exactly true. It may not be widely known but it is clearly established that insulin is NOT needed for glucose uptake and utilisation in man and therefore glucose uptake is NOT insulin dependent

There is a sufficient population of glucose transporters in all cell membranes at all times to ensure enough glucose uptake to satisfy the cell’s respiration, even in the absence of insulin. Insulin can and does increase the number of these transporters in some cells but glucose uptake is never truly insulin dependent.

Stimulatory & Inhibiting actions

Through stimulating the translocation or movement of ‘Glut 4’ glucose transporters from the cytoplasm of muscle and adipose tissue to the cell membrane insulin increases the rate of glucose uptake to values greater than the uptake that takes place in the basal state without insulin.

When insulin is administered to people with diabetes who are fasting, blood glucose concentration falls. It is generally assumed that this is because insulin increases glucose uptake into tissues, particularly muscle. In fact this is NOT the case and is another error arising from extrapolating from in vitro rat data. It has been shown quite unequivocally that insulin at concentrations that are within the normal physiological range lowers blood glucose through inhibiting hepatic glucose production without stimulating peripheral glucose uptake. As hepatic glucose output is ‘switched off’ by the inhibiting action of insulin, glucose concentration falls and glucose uptake actually decreases. Contrary to most textbooks and previous teaching, glucose uptake is therefore actually increased in uncontrolled diabetes and decreased by insulin administration.

When insulin is given to patients with uncontrolled diabetes it switches off a number of metabolic processes (lipolysis, proteolysis, ketogenesis and gluconeogenesis) by a similar inhibiting action. The result is that free fatty acid (FFA) concentrations fall effectively to zero within minutes and ketogenesis inevitably stops through lack of substrate. It takes a while for the ketones to clear from the circulation, as the ‘body load’ is massive as they are water and fat soluble and distribute within body water and body fat. Since both ketones and FFA compete with glucose as energy substrate at the point of entry of substrates into the Krebs cycle, glucose metabolism increases inevitably as FFA and ketone levels fall (despite the concomitant fall in plasma glucose concentration).

Thus insulin increases glucose metabolism more through reducing FFA and ketone levels than it does through recruiting more glucose transporters into the muscle cell membrane.

NOTE: The above was taken from:

Mechanism of action of insulin in diabetic patients: a dose-related effect on glucose production and utilisation, Brown P, Tompkins C, Juul S & Sonksen PH, British Medical Journal 1978 1239–1242.

Anabolic effect

Through facilitating glucose entry into cells in amounts greater than needed for cellular respiration insulin will stimulate glycogen formation.

It is possible to increase muscle bulk and performance not only through increasing muscle glycogen stores on a “chronic” basis but also to increase muscle bulk through inhibition of muscle protein breakdown. Just as insulin has an inhibiting action in inhibiting glucose breakdown in muscle glycogen, it also has an equally important inhibiting action in inhibiting protein breakdown.

The evidence now indicates that insulin does NOT stimulate protein synthesis directly (this process is under the control of growth hormone (GH) and insulin-like growth factor-I (IGF-I)). It has long been known that insulin-treated patients with diabetes have an increase in lean body mass when compared with matched controls. This results from insulin’s inhibition of protein breakdown in muscle tissue.

Growth Hormone Anabolic Actions

GH’s major action is to stimulate protein synthesis. It is at least as powerful as testosterone in this effect and, as they both operate through distinct pathways, their individual effects are additive or possibly even synergistic. In addition to stimulating protein synthesis, GH simultaneously mobilises fat by a direct lipolytic action. Together, these two effects are responsible for the ‘partitioning’ action of GH whereby it diverts nutritional calories to protein synthesis, possibly through using the energy derived from its lipolytic action. It most likely stimulates protein synthesis through mobilisation of amino acid transporters in a manner analogous to insulin and glucose transporters.

IGF-I also acts directly to stimulate protein synthesis but it has a weaker lipolytic action. GH, IGF-I and insulin thus act in concert to stimulate protein synthesis.

GH and IGF-I act in a promoting manner to stimulate protein synthesis while insulin acts in its characteristic inhibiting manner to inhibit protein breakdown. Thus they are synergistic in their powerful anabolic action.

Insulin is essential for the anabolic action of GH. GH administration in the absence of adequate insulin reserves (as during fasting or in Type 1 diabetes) is in fact catabolic and its lipolytic and ketogenic properties can induce diabetic ketoacidosis. Thus GH and insulin are closely linked in normal physiology and it is of great interest to see that athletes have discovered ways in which this normal physiological dependence can be exploited to enhance performance.

>Young skin and stronger tendons with stanozolol ("winny")

>

Turns out that stanozolol, the good old “Winny”, has unique collagen synthesis stimulating properties, that no other steroid has..

Stimulation of collagen synthesis by the anabolic steroid stanozolol

Researchers: Falanga V, Greenberg AS, Zhou L, Ochoa SM, Roberts AB, Falabella A, Yamaguchi Y; University of Miami School of Medicine, Department of Dermatology, Miami, Veterans Affairs Medical Center, Florida, USA.

Source: J Invest Dermatol 1998 Dec;111(6):1193-7

Summary: In this report, we measured the effect of the anabolic steroid stanozolol on cell replication and collagen synthesis in cultures of adult human dermal fibroblasts. Stanozolol (0.625-5 micrograms per ml) had no effect on fibroblast replication and cell viability but enhanced collagen synthesis in a dose-dependent manner. Stanozolol also increased (by 2-fold) the mRNA levels of alpha1 (I) and alpha1 (III) procollagen and, to a similar extent, upregulated transforming growth factor-beta1 (TGF-beta1) mRNA and peptide levels. There was no stimulation of collagen synthesis by testosterone. The stimulatory effects of stanozolol on collagen synthesis were blocked by a TGF-beta1 anti-sense oligonucleotide, by antibodies to TGF-beta, and in dermal fibroblast cultures derived from TGF-beta-1 knockout mice. We conclude that collagen synthesis is increased by the anabolic steroid stanozolol and that, for the most part, this effect is due to TGF-beta-1. These findings point to a novel mechanism of action of anabolic steroids.

Discussion: I must first acknowledge that the commonly held belief is that anabolic steroids predispose an athlete to tendon rupture. This conclusion is drawn from animal studies showing that some steroids produce a larger, stiffer tendon in rats and that these steroid-induced tendons “fail” before the tendons from the control animals. The term fail refers to the breaking point.

The interesting thing about the present study is that the steroid stanozolol (Winstrol) had a different effect than testosterone. If you are a regular reader of MESO-Rx you should be well aware that not all steroids act in the same manner. And that because of subtle differences in there molecular structure they are able to elicit different responses. For example, Deca seems to act primarily through the androgen receptor (AR) where as Dianabol has effects beyond those associated with the AR.

Because synthetic steroids have differ in their chemical properties it should not be surprising that testosterone did not have the same effect as Winstrol. Winstrol increased collagen synthesis as opposed to testosterone which did not in this study. Interpreting the results of this study are more difficult than simply describing them. Other researchers have suggested that steroids cause a rapid increase in protein synthesis within tendon fibroblasts which results in fibroids or fibrous nodules within the tendon (Michna,1988). These fibroids alter the mechanical properties of the tendon perhaps predisposing it to rupture. It is also noted that during short term use of steroids there is an alteration in the alignment of collagen fibers which may also lead to rupture. Interestingly these alterations in collagen metabolism are transient with markers of collagen turnover returning more or less to baseline after 3-4 weeks of steroid administration (Karpakka,1992). These same researchers noted that low dose anabolics effect primarily muscle collagenous tissue with tendon being effected only at higher doses (i.e. 5 times the therapeutic dose) which would more closely represent what is needed by bodybuilders to put on mass.

The question remains, dose this mean that Winstrol will actually help prevent tendon injury or will it lead to bigger yet stiffer tendons prone to injury? It is difficult to take animal research and extrapolate the results to humans. Stanozolol is used therapeutically in humans to treat a variety of connective tissue and vascular disorders and its clinical effects suggest that it can modulate connective tissue breakdown in people. Despite being labeled as “ineffective” by many bodybuilders it is very popular among athletes. As with most hormones, dosage plays a role in what effects are seen, be they positive or negative. Hopefully future studies will shed light on the therapeutic effects of different steroids on tendons in humans.

References:

Michna H Appearance and ultrastructure of intranuclear crystalloids in tendon fibroblasts induced by an anabolic steroid hormone in the mouse. Acta Anat (Basel) 1988;133(3):247-50

Karpakka JA, Pesola MK, Takala TE. The effects of anabolic steroids on collagen synthesis in rat skeletal muscle and tendon. A preliminary report. Am J Sports Med 1992 May-Jun;20(3):262-6

>Blocking DHT to avoid hair loss and prostate problems while using testosterone

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If you are using a DHT blocker such as finasteride, dutasteride or natural substances like green tea or saw palmetto, fortunately it doesn’t mean that you are blocking the anabolic properties of testosterone.

Inhibition of 5-reductase blocks prostate effects of testosterone without blocking anabolic effects

Stephen E. Borst,^1,2 Jun Hak Lee,¹ and Christine F. Conover²

¹Department of Applied Physiology & Kinesiology, University of Florida, Gainesville; and ²Geriatric Research, Education and Clinical Center, Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida

Submitted 12 July 2004 ; accepted in final form 26 August 2004

We studied the effect of the 5-reductase inhibitor MK-434 on responses to testosterone (T) in orchiectomized (ORX) male Brown Norway (BN) rats aged 13 mo. At 4 wk after ORX or sham surgery, a second surgery was performed to implant pellets delivering 1 mg T/day or placebo pellets. During the second 4 wk of the study, rats received injections of MK-434 (0.75 mg/day) or vehicle injections. Treatment with T elevated serum T to 75% above that for sham animals (P = 0.002) and did not affect serum dihydrotestosterone (DHT) or serum estradiol. T treatment also caused an elevation of prostate T and a marked elevation of prostate DHT. During the second half of the study, ORX rats lost an average of 18.86 ± 4.62 g body wt. T completely prevented weight loss, and the effect was not inhibited by MK-434 (P produced a nonsignificant trend toward a small (5%) decrease in the mass of the gastrocnemius muscle (P = 0.0819). This trend was also reversed by T, and the effect of T was not blocked by MK-434. T caused a significant 16% decrease in subcutaneous fat that was not blocked by MK-434 (P caused a 65% decrease in urine excretion of deoxypyridinoline, a marker of bone resorption, and again the effect was not blocked by MK-434 (P fivefold increase in prostate mass, and the effect was almost completely blocked by MK-434 (P that 5-reductase inhibitors may block the undesirable effects of T on the prostate, without blocking the desirable anabolic effects of T on muscle, bone, and fat.