Dysregulated homeostatic pathways in sarcopenia among frail older adults

Abstract Sarcopenia, a core feature of the physical frailty syndrome, is characterized by multisystem physiological dysregulation. No study has explored qualitatively the hierarchical network of relationships among different dysregulated pathways involved in the pathogenesis of sarcopenia. We used 40 blood biomarkers belonging to community‐dwelling prefrail and frail older persons to derive measures of multiple physiological pathways, and structural equation modeling to generate path network models of the multisystem physiological dysregulations associated with muscle mass and function (MMF). Insulin–leptin signaling and energy regulation, anabolic sex steroid regulation (testosterone, leptin), and tissue oxygenation (hemoglobin, red cell count) appear to be primary mediating factors exerting direct influences on MMF. There was additionally secondary mediatory involvement of myocyte‐ and adipocyte‐derived cytokines, hypothalamic pituitary adrenal (HPA) stress hormones (cortisol, DHEAS), glomerular function, and immune cell regulatory and inflammatory cytokines and glycoproteins. We conclude that within a hierarchical network of multisystem physiological dysregulations in sarcopenia, dysregulated anabolic and catabolic pathways via sex steroids and insulin–leptin dual signaling and tissue hypoxemia are primary physiological dysregulations responsible for sarcopenia and frailty.


| INTRODUCTION
Studies suggest that multiple cellular, hormonal, metabolic, and molecular mechanisms underlie the development of sarcopenia in animal models, including impaired insulin signaling, imbalanced anabolic and catabolic energy metabolism, upregulation of catabolic cytokine expression, immune dysfunction, systemic inflammation, and increased oxidative stress (A Sayer, Stewart, Patel, & Cooper, 2010).
Testosterone is well known to increase muscle mass by increasing the rate of muscle protein synthesis partly via stimulating IGF-1 expression (Mudali & Dobs, 2004). Insulin appears to play a crucial role in maintaining the rate of muscle protein synthesis and breakdown, both in health and disease (Abdulla, Smith, Atherton, & Idris, 2016).
Although the exact mechanism is not fully understood, as a key anabolic hormone, insulin stimulates muscle growth via secreting IGF-1 followed by activation of the PI3K and Akt (also known as protein kinase B) signaling pathways via mammalian target of rapamycin (mTOR) that controls protein synthesis and the forkhead box O (FOXO) that controls protein degradation. Current evidence suggests that the anabolic effect of insulin is driven by attenuating muscle protein breakdown rather than increasing muscle protein synthesis.  et al., 2011), insulin-like growth factor-1 (IGF-1; Gielen et al., 2015), and dehydroepiandrosterone sulfate (DHEAS; Tajar et al. (2011)) have been interrogated in a majority of studies mostly in isolation and with conflicting results. In contrast, several studies, such as the InCHIANTI Study, the WHAS I and II, and the MSSA Gruenewald, Seeman, Karlamangla, & Sarkisian, 2009;Maggio et al., 2010), have found that an increased number of multiple hormonal biomarker abnormalities strongly predict physical frailty when individual biomarker abnormalities did not. Determining the effect of a single physiological derangement in sarcopenia is complicated by the effects of intermediary changes in the complex network of physiological pathways that are deranged in sarcopenia.
Because sarcopenia involves physiological dysregulation within and across multiple systems, its pathophysiology could be better understood when combinations of biomarkers and physiological impairments are investigated together rather than singly. Thus far, no study has explored the qualitative hierarchy of relationships within a network of different dysregulated pathways involved in the pathogenesis of sarcopenia.
In this study, we used archival baseline blood specimens collected in a previously reported study of prefrail and frail older persons in the Frailty Intervention Trial in Singapore (Ng et al., 2015), to analyze known and potential blood biomarkers of sarcopenia and frailty. We chose 40 analytes of hormones, cytokines, metabolites, and other biomarkers that were identified from literature search to be involved with homeostatic processes associated with muscle mass and function and dysregulated in sarcopenia and frailty. The study subjects' mean age was 69.3 (SD: 4.2) years; they included 34 males and 63 females and 18 with known history of diabetes. The mean appendicular mean mass, knee strength, and gait speed are shown in Supporting information Table S1. Using a guided supervision approach to principal component analyses of the 40 blood biomarker measurements, we derived various measures of physiological and molecular activity across multiple important regulatory systems (Table 1).
We analyzed the inter-relationships of these blood biomarker suggests that these are primary processes involved in muscle growth and function that are dysregulated in sarcopenia.

1.
Insulin signaling and energy metabolism (ISEM: denoted by insulin, C-peptide, leptin). Based on present evidence, it remains unclear whether increased insulin resistance or reduced endogenous insulin secretion (or both) is underlying mechanisms in sarcopenia and frailty (Cleasby et al., 2016;Tanaka, Kanazawa, & Sugimoto, 2015). Our result shows that low fasting plasma insulin and C-peptide (a better measure of portal insulin secretion) levels are associated with low muscle mass and function and suggests that in this Asian group, endogenous reduction of insulin level and dysregulated insulin signaling activity in older individuals (with and without diabetes) diminish the anabolic capacity of insulin to alleviate muscle protein breakdown in skeletal muscles. As expected, basal plasma leptin is increased together with increased insulin, and higher concentrations of both hormones appear to be directly related to greater muscle mass and function. Conversely, in physically frail individuals with low body mass, the presence of decreased levels of leptin (known to be associated with weight loss) may reflect a state of persistent energy deprivation that characterizes physical frailty.

2.
Anabolic sex steroid regulation (ASSR: free testosterone, leptin). Next to insulin-leptin signaling and energy metabolism, ASSR appears to occupy a co-pivotal role in the multiple hormonal dysregulation in sarcopenia. ASSR is inversely linked to ISEM, reflecting what is known about their complementary actions in protein synthesis and muscle accretion (Mudali & Dobs, 2004).
Taken together, dysregulated anabolic and catabolic pathways via insulin signaling and energy metabolism and anabolic sex steroid regulation appear to be primary pathophysiological mechanisms directly related to the development of sarcopenia. (hemoglobin, rbc count). This index of tissue oxygenation is also directly and indirectly (via its link to ASSR) associated with muscle mass and function. Reduced tissue oxygen delivery with anemia, hypoxia, or ischemia in skeletal muscle has been shown to impair muscle strength and performance (Dodd, Powers, Brooks, & Crawford, 1993). In line with this, there are reports of the association of anemia with physical frailty (Hirani et al., 2016;Roy, 2011), which may be due to chronic inflammation deficiency.

Oxygen transport and delivery
There appears to be a second tier of five intermediary physiological processes that are indirectly linked to muscle mass and function via their positive or inverse associations with ISEM or ASSR.

Myocyte protein signaling (irisin and myostatin). Its positive link to
ISEM is consistent with their known effects on activation of the PI3K-Akt-mTOR pathway and the FOXO pathway mentioned above. Myostatin, a negative regulator of skeletal muscle growth, is known to negatively regulate the activity of the Akt pathways, which promotes protein synthesis and increases the activity of the ubiquitin-proteasome system to induce atrophy (Rodriguez et al., 2014). On the other hand, the negative relationship between myocyte signaling proteins and ASSR is consistent with studies showing androgens to have strong negative impact on myostatin expression (Mendler, Baka, Kovács-Simon, & Dux, 2007). F I G U R E 1 Path analysis of blood biomarkers and muscle mass and function 5. Immune cell and inflammation homeostasis CRP,sgp130). Age-associated decline in immune cell function (such as neutrophil migration) and chronic inflammation and dysregulation of the phosphoinositide 3-kinase (PI3K)-Akt pathway in neutrophils are strongly believed to contribute pathogenically to sarcopenia and frailty, but a direct causative role is not well established (Wilson, Jackson, Sapey, & Lord, 2017). In particular, it showed a positive association with ASR in this study, but this is not consonant with prior experimental findings that IL-6 and TNF-α inhibit testosterone secretion via central hypothalamic-pituitary and peripheral gonadal influences, and observations in men that serum levels of testosterone are inversely related to soluble IL-6 receptor (sIL-6R) and the effect of testosterone supplementation in reducing inflammatory markers in hypogonadal men (Maggio et al., 2005).

Adipocyte protein signaling (adiponectin and obestatin). This is neg
In connection to the above, the findings also reveal an associated series of innate immune system response processes represented almost entirely of positive or negative acute phase proteins (plasma transport, coagulation, and other inflammatory glycoproteins), which have also been reported in human observational studies to be associated with sarcopenia and frailty (Darvin et al., 2014;Reiner et al., 2009;Shamsi et al., 2012).

ACKNOWLEDGMENTS
The study was supported by research grants from the National Med-

CONFLI CT OF INTEREST
None reported.

AUTHOR CONTRI BUTION
TPN formulated the hypothesis, performed literature review, designed the study, analyzed and reviewed the data, and drafted and reviewed the manuscript. RWMC performed the statistical analyses.
CTYT and EWHM performed the laboratory analyses. YL, CTYT, MSZN, QG, EWHM, and AL contributed to the review of the literature, study design, and data collection, and review of the data and manuscript drafts.