**3. Discussion**

Patients with CKD frequently have cachexia, which has been linked to higher morbidity and mortality rates [1]. We looked into how GH affected cachexia in CKD mice. First, we showed that intraperitoneal administration of GH significantly increased caloric intake and weight growth in CKD mice (Figure 1B,C). We also demonstrated that the beneficial metabolic benefits of GH go beyond appetite stimulation. GH improves organismal metabolism (Figure 1F–L) as well as specific tissue energy balance (skeletal muscle and adipose tissue) in CKD mice (Figure 2). The findings of this study are consistent with those of other, earlier studies. In hemodialysis patients, GH enhances nutrition intake and

increases lean body mass [8–10]. With the help of GH, the body's anabolism is stimulated, and protein accretion happens in the muscles and extramuscular tissues [6,7,24].

By increasing muscle mass and improving energy efficiency, GH may improve muscle strength. Anaerobic and aerobic energy sources comprise the continuum of energy needed to fuel muscular function. Anaerobic energy systems are stimulated by GH, which suppresses the aerobic energy system. This increases muscle strength. After six months of GH therapy, healthy males showed a considerable improvement in their lower body muscle strength [5]. By "uncoupling" ATP synthesis, UCPs regulate energy homeostasis by dissipating the mitochondrial proton gradient for ATP synthesis and producing heat [25,26]. UCP3 is expressed in skeletal muscle, and upregulation of UCP3 has been reported in various conditions characterized by skeletal muscle atrophy, including denervation, diabetes, cancer, and sepsis [27]. Gastrocnemius UCP3 protein content along with ATP content was normalized in GH-treated CKD mice (Figure 2A,B). Putative functions of UCP3 are controversial. Interesting evidence for and against UCP3 involvement in thermogenesis has been published [27,28]. Furthermore, increased muscle expression of UCP3 has been postulated to modulate oxidative stress and lipotoxicity in a rat model of cachexic sepsis [29]. GH therapy reduced abnormal UCP1 and ATP content in WAT and BAT in CKD mice (Figure 2C–F). However, the precise role of UCP1 in disease-associated cachexia in humans is still a topic of debate. Several studies have described UCP1 expression, a biomarker of WAT browning, as a critical component of WAT dysfunction in cancer cachexia [17,18,30]. Results also suggested that a UCP-1 independent cascade could also regulate adipocyte homeostasis and influence tumor-induced WAT wasting [31]. Moreover, activation of BAT has been associated with hypermetabolism in cachexia, but information from human studies is scarce. A recent study investigated the relationship between activation of BAT and hypermetabolism in patients with emphysematous COPD (chronic obstructive pulmonary disease). BAT activity and gene expression of beige markers of BAT in WAT (Tmem26, Cidea, CD137, Shox2, and Tnfrsf9) were not different between COPD patients versus controls [32]. Medications may influence the sympathetic nervous system and BAT metabolism. Adrenergic receptor blockers and calcium channel blockers are commonly used by COPD patients. Involvement of β-adrenergic receptor signaling in BAT metabolism was reported in humans and rodents [33,34]. Data also indicated that calcium channel blockers regulated adipogenesis and BAT browning [35,36].

CKD-associated cachexia is a progressive, multifactorial metabolic syndrome that results in significant loss of adipose tissue and skeletal muscle mass. Fat loss from adipose tissue in CKD-associated cachexia may be due to the increased rate of lipolysis. Recent longitudinal studies found that the magnitude of adipose tissue wasting predicts poorer survival in cancer patients [37–39]. The bulk of lipid mobilization from adipose tissue is mediated through lipolysis. In canonical adipose tissue lipolysis, triglycerides stored in lipid droplets are hydrolyzed by ATGL and HSL to produce free glycerol and fatty acids and fuel peripheral tissue metabolism [40]. ATGL is the rate-limiting lipase and hydrolyzes triacylglycerol in lipid droplets to diacylglycerol. GH treatment attenuated inguinal WAT mRNA expression and protein content of ATGL in CKD mice (Figure 3A,B). Previous studies have shown increased ATGL expression in the adipose tissue of cancerassociated cachectic animals and humans [21,41,42]. Inguinal WAT gene expression and protein content of HSL was not different among groups of mice (Figure 3C,D). However, phosphorylated HSL Ser552 protein content in inguinal WAT, a surrogate marker for protein kinase A-activated lipolysis [43], was significantly increased in CKD mice (Figure 3E). Importantly, GH attenuated inguinal WAT protein content of phosphorylated HSL in CKD mice. Evidence of enhanced protein kinase A-activated lipolysis correlated with elevated whole-organism energy expenditure and increased adipose tissue thermogenesis, and increased expression of biomarkers of adipose tissue browning in WAT was reported in a mouse model of cancer cachexia [21]. Moreover, increased WAT protein content of phosphorylated HSL and protein Kinase A was also shown in a mouse model of CKD [44].

Browning of adipose tissue is associated with a hypermetabolic state and cachexia. Adipose tissue browning is evident in animal models of CKD-associated cachexia and cancer as well as in cachectic cancer patients [17,18,30]. We demonstrated that in CKD mice, GH reduced the browning of adipose tissue. The expression of biomarkers of beige adipocyte in WAT (CD137, Tbx-1, Tmem26, Prdm16, Pgc1a, and Cidea) was attenuated in CKD mice treated with GH (Figure 4A–F). Cox2/Pgf2 and inflammatory Tlr2, MyD88, and Traf6 signaling pathways have been associated with the biogenesis of browning [22]. GH treatment restored the expression of inflammatory molecules (Tlr2, MyD88, and Trap6) in the inguinal WAT of CKD mice treated with GH (Figure 4G–K). GH influences the metabolism of adipose tissue by binding to the GH receptor (GHR). Disrupted GH/GHR in mice results in multiple metabolic disorders. Global or adipose-specific GHR-deficient mice fail to demonstrate metabolic adaptability when challenged with a high-fat diet or cold temperature [45].

We looked at how GH affected the expression of molecules that control skeletal muscle metabolism in CKD mice. GH increases the expression of promyogenic factors (MyoD, Myogenin, and Pax-7) while decreasing or normalizing the expression of negative regulators of skeletal muscle mass (Atrogin-1, Murf-1, Myostatin, and Soc2, and inflammatory cytokines IL-1β, IL-6, and TNFα) (Figure 5A–J). Recent research indicates that the immune system and the GH/IGF-I axis interact in complicated and bidirectional ways. For example, the GH/IGF-I axis may be suppressed by inflammatory cytokines such as IL-1, IL-6, and TNFα, while GH/IGF-I may also influence systemic inflammation [46]. In cancer cachectic mice, IL-6 causes a decrease in fat content and stimulates adipose tissue browning [47]. In children with GH deficiency, GH has been found to reduce serum concentrations of IL-1β and TNFα [48]. In addition, GH lowers the serum concentrations of TNFα in adult hemodialysis patients [8]. Skeletal muscle growth and repair are influenced by the transcription factors Pax-3 and Pax-7. Pax-3 and Pax-7 regulate MyoD and myogenin [49]. MyoD and Myogenic Factor 5 (Myf5) are required to promote myogenic precursors. A downstream target of MyoD, myogenin controls the differentiation of myoblasts into myocytes and myotubes [49,50].

Because GH and IGF-I are powerful anabolic hormones that stimulate muscle mass increase and are crucial for maintaining skeletal mass, muscle loss in CKD has been linked to disruptions in the GH/IGF axis. As a result, IGF-I resistance may be a factor in the wasting of muscle in CKD [4]. In fact, GH therapy improved muscle mass compared to height in children with CKD [51]. Patients receiving continuous hemodialysis experienced an increase in blood IGF-I concentration following GH therapy [52–54]. The IGF-I signaling pathway, which promotes the proliferation and differentiation of satellite cells into myoblasts and the development of new myofibers, is one of the mechanisms by which GH affects skeletal muscle metabolism [55]. After a prolonged denervation injury, GH enhances muscle reinnervation, nerve regeneration, and functional outcomes [56]. Furthermore, Gautsch et al. have demonstrated that GH stimulates endocrine IGF-I-stimulated protein accretion, enhancing somatic and skeletal muscle growth in malnourished rats [57]. Interesting findings also point to a possible IGF-I-independent mechanism by which GH may exert anabolic effects in muscle [58]. Muscle wasting associated with CKD is brought on by an impaired JAK-STAT signal [14,15]. We have verified that recombinant human GH treatments resulted in high circulating concentration of human GH in CKD and control mice (Table 2). Muscle expression of IGF-I was decreased in CKD mice, and GH treatments normalized muscle IGF-I expression as well as restored the phosphorylated JAK2 and STAT5 muscle protein levels to normal in CKD mice (Figure 5K–M). Previous studies also showed that GH treatment increased muscle mRNA expression of IGF-I and attenuated JAK-STAT signaling in rodent models of CKD [14].

The GH dose administered to the mice in this study was about 200-fold higher than the dose typically used in humans. The recommended dose approved for treatment of growth failure in children with CKD is 0.35 mg/kg per week [59], whereas we used 10 mg/kg/day in mice for this study. However, our dose was comparable to those commonly used in

rodent studies [60], and the observation of increased muscle mRNA expression of IGF-I as well as JAK/STAT phosphorylation after GH treatment in our CKD mice (Figure 5K–M) argues against any effect of GHR saturation.

IGF-I is the most important downstream mediator of GH. Thus, IGF-I is generally considered to be the most important biomarker of GH action, as reflected in the inclusion of serum concentrations of IGF-I in the current guidelines for diagnosis and treatment of GH disorders in humans [61]. We have not measured serum IGF-I concentration in this study, as recent studies suggested that concentration of serum IGF-I is not a reliable marker for exogenous growth hormone activity in mice [60]. Male and female mice from four different strains of mice, including the 57BL/6J mouse strain used in this study, were treated with recombinant human GH (500 ug/day, intraperitoneally, for a period of 14 days). The total amount of GH administrated to mouse is ~7 mg in their study [60], which is comparable to the dose we used for the diet-restrictive study (total amount of GH is ~9.2 mg, presumably 22 g of body weight for CKD mice). In agreement to our observation (serum concentration of human GH in Table 2), GH treatment resulted in high circulating concentrations of human GH in all four strains of mice, whereas no human GH was detectable in control mice receiving isotonic 0.9% NaCl as vehicle. Two weeks of daily GH treatment significantly increased body and organ weight in male and female mice of all four inbred mouse strains when compared with controls. GH treatment failed to affect circulating (total) IGF-I concentrations in all strains and in both sexes. The liver is the main source of circulating IGF-I [62]. Hepatic expression of IGF-I mRNA did not show any difference between GH-treated mice versus control mice in any of these four strains of mice and sexes [60]. List et al. investigated the effects of GH in a mouse model of diet-induced diabetes [63]. Male c57BL/6J mice were fed a high-fat diet to induce obesity and type 2 diabetes. Subsequently, obese and diabetic mice were treated with various doses of GH for a period of six weeks. Comparable to our findings in CKD mice (Figure 1C,F,I), their highest dose of GH (215 μg/day/mouse for their study versus 220 μg/day/mouse in our study, presumable 22 g of body weight for CKD mice) resulted in a significant increment of total body mass and lean mass content. However, in contrast to the findings that the treatment of GH did not influence serum concentration of IGF-I in mice [60], GH treatment led to a significant increase in serum IGF-I in diabetic mice [63]. The mice used by List et al. were obese and hyperinsulinemic and showed impaired glucose tolerance. These factors may account for the difference in the results. Serum concentration of insulin, especially insulin concentration of portal vein, is an important regulator of hepatic GHR expression in rodents [64,65].

In this study, eight-week-old male CKD or sham mice on c57BL/6J background were given GH or vehicle for 6 weeks, and all mice were sacrificed at the age of 14 weeks old. We showed that GH administration elicited beneficial metabolic effects in CKD mice. c57BL/6J mice are the most widely used inbred strain for biomedical research. For c57BL/6J mice, many developmental processes such as T-cell and B-cell immunity, as well as the central nervous system, are still ongoing until 26 weeks of life [66–68]. Furthermore, growth patterns and body composition were evaluated in c57BL/6J mice. Data suggested that cortical bone property and peak bone mass on male c57BL/6J mice are not reached until around 26 weeks of age [69–72]. Thus, the results of our present study are of immerse importance, as multiple disturbances in the GH/IGF-I axis have been observed in children with CKD.

We recognize the limitations of this study. Firstly, according to our restrictive study design, vehicle-treated CKD mice were fed ad libitum, whereas other mouse groups received an energy intake amount equal to that of vehicle-treated CKD mice. However, we observed that pair-fed mice consumed their restricted amount of the rodent diet within a short period of time. These pair-fed mice were in an overnight fasting state. Mice, as nocturnal creatures, are active mainly during the dark phase. Circadian rhythm affects adipose tissue metabolism [73–75]. Disruption of circadian regulation has been implicated in cancer-induced WAT wasting [42]. Secondly, our work was performed in male c57BJ/6J

mice. Results generated from male mice cannot be unambiguously extrapolated to female mice. Sex hormones influence regional adipose tissue fatty acid storage and BAT function in animals and humans. Disruption of estrogen signaling such as by performing ovariectomy resulted in reduced energy expenditure, gain of fat mass, and loss of BAT activity, and these metabolic phenotypes can be reversed by subsequent estrogen replacement in ovariectomized rodents [76]. The reduction of circulating concentration of estradiol is associated with central obesity and decreased metabolism in menopause [77]. Murine and human brown adipocytes express estrogen receptor α [78,79]. Intracerebral administration of estrogen increased BAT activity in mice [80]. On the other hand, follicle-stimulating hormones, which are elevated with estrogen deficiency, downregulated in vivo BAT function in mice [81]. Currently, there are no published data on the effect of estrogen or estrogen deficiency on in vivo BAT function in humans. Dieudonne et al. investigated the effects of sex hormones on adipogenesis in preadipocytes from male versus female rats. They found that androgens and estrogens did not affect adipogenesis in cultured preadipocytes from male rats. However, opposite effects of androgens and estrogens on adipogenesis have been demonstrated in cultured preadipocytes from female rats. Estrogens increased adipogenesis, while androgens acted as negative effectors of terminal differentiation on rat preadipocytes. Subsequent studies suggest that these opposite effects could be related to differential expression of IGF-IR and Pparγ2 on those cultured preadipocytes [82]. Thirdly, uncertainty remains about the precise role of BAT metabolic responses in the pathogenesis of cachexia, and this is partly due to the lack of BAT-specific pharmacological agents. Currently, there is no convincing evidence to suggest that BAT activity can be selectively modulated by any pharmacological agents without influencing WAT metabolism along with cardiac chronotropic side-effects [83,84]. Moreover, BAT activity is mostly driven by the sympathetic signal mediated by β-adrenergic receptors, namely, ADRB3 in mice and ADRB1/ADRB2 in humans. However, the in vivo BAT metabolic activity is the result of the interaction between sympathetic output signal to BAT and other concomitant signaling processes such as α-adrenergic receptors and adenosine receptors as well as postsignaling modulation of these signaling processes [84]. The complexity and redundancy of the endogenous sympathetic regulation of BAT metabolic activity may explain the lack of an optimal pharmacological approach to modulate BAT in vivo.

Previously, we performed RNAseq analysis in the gastrocnemius muscle in CKD and control mice and identified the top 12 differentially expressed genes that have been associated with energy metabolism, skeletal and muscular system development and function, nervous system development and function, as well as organismal injury and abnormalities [23]. We evaluated the effects of GH treatment on muscle transcriptome in this study. A total of 7 of the 12 muscle genes with variable expression in CKD mice were normalized or reduced by GH (Figure 6). These seven muscle genes—Atp2a2, Cyfip2, Fhl1, Tnnc1, Atf3, Fos, and Itpr1—had aberrant expression patterns that have been linked to enhanced tissue thermogenesis, compromised mechanical muscle properties, poor muscle regeneration, and diminished muscle-neuron regeneration capacity [23].

In conclusion, our findings imply that GH might be a useful treatment for adipose tissue browning and muscular atrophy in CKD-associated cachexia.
