Claire E. Stewart

Tumor necrosis factor-?-induced apoptosis is associated with suppression of insulin-like growth factor binding protein-5 secretion in differentiating murine skeletal myoblasts

Wasting of muscle and fat during cachexia exceeds that explained by reduced food intake alone. This wasting may result from an imbalanced cytokine environment, which could lead to increased protein catabolism. Supporting this, tumor necrosis factor‐α (TNF‐α) is raised in several animal models of cachectic muscle wasting. Therefore, we assessed the effects of TNF‐α and its second messenger, ceramide, on the proliferation, differentiation, and survival of murine C2 skeletal myoblasts. Because insulin‐like growth factor binding protein‐5 (IGFBP‐5) and insulin‐like growth factor‐II (IGF‐II) are potent regulators of myoblast proliferation and differentiation, we monitored the ability of exogenous TNF‐α to manipulate this system. Fibroblast growth factor (FGF) ceramide, or TNF‐α suppressed differentiation of C2 cells compared with controls. All treatments suppressed IGF‐II production but only TNF‐α blocked IGFBP‐5 secretion. TNF‐α increased apoptotic cell death, which otherwise remained basal (low serum differentiation medium (LSM), FGF) or low (ceramide). Suppression of both IGFBP‐5 and IGF‐II secretion may explain why of all triggers tested, only TNF‐α not only blocked differentiation, but also promoted cell death. This suggests a fundamental role of IGFBP‐5 for maintaining muscle survival. Supporting this hypothesis, no increase in apoptosis was seen in IGFBP‐5 cDNA tranfected C2 cells after TNF‐α treatment. In summary, the IGF system is essential for maintaining skeletal muscle cell survival and differentiation, and its suppression by TNF‐α is fundamental regarding muscle wasting, and may be associated in vivo with cancer cachexia. J. Cell. Physiol. 183:330–337, 2000.

Multifaceted roles of TNF-α in myoblast destruction: A multitude of signal transduction pathways

In catabolic conditions, such as cancer cachexia, a balance favouring a cytokine environment culminates in muscle destruction. Utilising an in vitro model to mimic muscle wasting, we elucidate here the multifaceted roles that one such cytokine, TNF‐α, invokes in the degeneration process. Treatment of C2 skeletal myoblasts with TNF‐α not only suppresses morphological and biochemical differentiation, but following an initial wave of proliferation, and of survival (24 h), induces apoptosis. Investigating the mechanisms underlying these diverse actions of TNF‐α, we demonstrate that cell replication is dependent on rapid and sustained activation of MAP kinase. Map kinase is not, however, central to the death process, which is associated with a progressive rise in caspase‐8 activity, and is accompanied by sustained activation of JNK1 and transient activation of JNK2. Caspase inhibition caused a dose responsive reduction in cell death, while inhibition of the JNKs caused a significant increase in apoptosis. We further report that PI3 kinase is not involved in conferring early protection against TNF‐α‐induced death. By contrast, inhibition of NF‐κB in the presence of TNF‐α culminates in increased cell cycle progression, decreased gadd45β expression and significant and precociously increased cell death, when compared with TNF‐α alone. Our results begin to characterise the mechanisms underlying the acute mitogenic and anti‐apoptotic roles of TNF‐α, which appear to be defined by a balance between MAP kinase, Jun kinase (JNK), NF‐κB and gadd45β. They establish that inhibition of any one of these molecules, as may occur following caspase activation, could eliminate vital stem cells required for skeletal muscle regeneration during chronic catabolic conditions. J. Cell. Physiol. 198: 237–247, 2004© 2003 Wiley‐Liss, Inc.

Powerful signals for weak muscles.

The aim of the present review is to summarise, evaluate and critique the different mechanisms involved in anabolic growth of skeletal muscle and the catabolic processes involved in cancer cachexia and sarcopenia of ageing. This is highly relevant, since they represent targets for future promising clinical investigations. Sarcopenia is an inevitable process associated with a gradual reduction in muscle mass and strength, associated with a reduction in motor unit number and atrophy of muscle fibres, especially the fast type IIa fibres. The loss of muscle mass with ageing is clinically important because it leads to diminished functional ability and associated complications. Cachexia is widely recognised as severe and rapid wasting accompanying disease states such as cancer or immunodeficiency disease. One of the main characteristics of cancer cachexia is asthenia or lack of strength, which is directly related to the muscle loss. Indeed, apart from the speed of loss, muscle wasting during cancer and ageing share many common metabolic pathways and mediators. In healthy young individuals, muscles maintain their mass and function because of a balance between protein synthesis and protein degradation associated with rates of anabolic and catabolic processes, respectively. Muscles grow (hypertrophy) when protein synthesis exceeds protein degradation. Conversely, muscles shrink (atrophy) when protein degradation dominates. These processes are not occurring independently of each other, but are finely coordinated by a web of intricate signalling networks. Such signalling networks are in charge of executing environmental and cellular cues that ultimately determine whether muscle proteins are synthesised or degraded. Increasing our understanding for the pathways involved in hypertrophy and atrophy and particularly the interaction of these pathways is essential in designing therapeutic strategies for both prevention and treatment of muscle wasting conditions with age and with disease.

Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation

Previous studies have shown that exogenous insulin‐like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF‐II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF‐II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF‐II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF‐II overproduction. Similar to observations with other muscle cell lines, IGF‐II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low‐serum differentiation medium. Accelerated differentiation of IGF‐II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24–48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle‐specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein‐5 when compared with controls. These results confirm a role for IGF‐II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways

ABSTRACT In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways.

Fatty acid‐induced defects in insulin signalling, in myotubes derived from children, are related to ceramide production from palmitate rather than the accumulation of intramyocellular lipid

The elevation of free fatty acids (FFAs), observed in childhood obesity results in intramyocellular lipid (IMCL) accumulation with consequent insulin resistance. Using in vitro differentiated myotubes from normal weight pre‐pubertal children (n = 8), we examined the effects of saturated (palmitate) and unsaturated (oleate) FFAs on insulin‐stimulated AKT phosphorylation (pAKT) and IMCL accumulation. Palmitate decreased pAKT (Mean [SEM] % change pAKT with palmitate 750 µM vs. control; pThr308 site −50.5% [28.7] and pSer473 site −38.7% [11.7]; P < 0.001) with no effect on IMCL formation. Equimolar bromopalmitate did not effect pAKT and blocking ceramide production abolished the palmitate‐induced reduction in signalling, suggesting that ceramide synthesis is critical for palmitates actions. Oleate did not effect pAKT (1,000 µM oleate; pSer473 site −3.4% [11.4]; P = NS) but increased IMCL accumulation (+32.3% [7.1%]; P < 0.001). Co‐administration of oleate diminished the reduction in pAKT seen with palmitate (+36.4% [23.6] vs. −13.3% [13.6]; P = 0.28), with similar IMCL levels to oleate alone. Co‐administration also caused a significant reduction in 14C‐ceramide synthesis from 14C‐palmitate (101.6 [21.6] vs. 371.5 [122.4] DPM/mg protein; P < 0.001). In summary, palmitate appears to cause insulin resistance in childrens myotubes via its metabolism to ceramide, and this process appears unrelated to IMCL formation and is ameliorated by oleate. J. Cell. Physiol. 211: 244–252, 2007.

Characterization of differentiated subcutaneous and visceral adipose tissue from children: the influences of TNF-alpha and IGF-I

The relationship between subcutaneous and visceral adipocyte metabolism and development has been extensively studied in adult but not in pediatric tissue. Our aim was to isolate, develop, characterize, and compare primary cell cultures of subcutaneous and visceral preadipocytes from 16 normal prepubertal children (10 male and 6 female). Subculture techniques were developed to increase cell number and allow differentiation using a chemically defined serum-free medium. Removal of insulin from the differentiation medium prevented adipogenesis in both subcutaneous and visceral preadipocytes, whereas coincubation with rosiglitazone markedly enhanced glycerol-3-phosphate dehydrogenase activity, peroxisome proliferator-activated receptor γ expression, and triglyceride accumulation in cells from both fat depots. Adiponectin secretion increased with differentiation from undetectable levels at day 0. Histological analyses demonstrated significant differences in lipid droplet number and size, with subcutaneous cells having fewer but larger vesicles compared with visceral cells. Downregulation and reorganization of the cytoskeleton appeared comparable. We further demonstrate regional differences in adipogenesis manipulation. Tumor necrosis factor-α was more effective at inhibiting differentiation in subcutaneous cells, whereas insulin-like growth factor-I stimulated differentiation more effectively in visceral cells. Insulin-like growth factor binding protein-3 enhanced differentiation equally. These observations may have important physiological and pharmacological implications for the development of obesity in later life.

Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake.

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio‐economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin‐like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS‐1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.

Insulin-like growth factors (IGF-I and IGF-II) inhibit C2 skeletal myoblast differentiation and enhance TNF alpha-induced apoptosis.

IGF‐I and IGF‐II are thought to be unique in their ability to promote muscle cell differentiation. Murine C2 myoblasts differentiate when placed into low serum media (LSM), accompanied by increased IGF‐II and IGF binding protein‐5 (IGFBP‐5) production. Addition of 20 ng/ml TNFα on transfer into LSM blocked differentiation, IGF‐II and IGFBP‐5 secretion and induced apoptosis. We, therefore, wished to assess whether IGFs could protect against the effects of TNFα. Neither inhibition of differentiation or induction of apoptosis was rescued by co‐incubation with IGF‐I or IGF‐II. A lower dose of TNFα (1 ng/ml) while not inducing apoptosis still inhibited myoblast differentiation by 56% ± 12, (P < 0.001), indicating that induction of apoptosis is not the sole mechanism by which TNFα inhibits myoblast differentiation. Addition of IGF‐I or IGF‐II alone reduced differentiation by 49% ± 15 and 33% ± 20, respectively, (P < 0.001), although neither induced apoptosis. For muscle cells to differentiate, they must arrest in G0. We established that addition of IGF‐I, IGF‐II or TNFα to the myoblasts promoted proliferation. The myoblasts could not exit the cell cycle as efficiently as controls and differentiation was thus reduced. Unexpectedly, co‐incubation of IGF‐I or IGF‐II with 1 ng/ml TNFα enhanced the inhibition of differentiation and induced apoptosis. In the absence of apoptosis we show an association between IGF‐induced inhibition of differentiation and increased IGFBP‐5 secretion. These results indicate that the effects of the IGFs on muscle may depend on the cytokine environment. In the absence of TNFα, the IGFs delay differentiation and promote myoblast proliferation whereas in the presence of TNFα the IGFs induce apoptosis.

Age-dependent alteration in muscle regeneration: the critical role of tissue niche

Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury and to modify its contractile and metabolic properties in response to changing demands. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic precursor cells that reside between the basal lamina and plasmalemma and that are rapidly activated in response to appropriate stimuli. However, in pathologic conditions or during aging, the complete regenerative program can be precluded by fibrotic tissue formation and resulting in functional impairment of the skeletal muscle. Our study, along with other studies, demonstrated that although the regenerative program can also be impaired by the limited proliferative capacity of satellite cells, this limit is not reached during normal aging, and it is more likely that the restricted muscle repair program in aging is presumably due to missing signals that usually render the damaged muscle a permissive environment for regenerative activity.