Observatorio de Violencia Social, Género y Juventud

Figure 6.1. Cage Activity during severe cachexia. Cage activity was measured for three consecutive nights during the active cycle (1900-0700h). All animals were single housed. A) XY ambulatory count, the number of times a new beam is interrupted, and B) total Z counts were analyzed. T-test was run to test for significance. *significantly different from BL-6 p<0.001.

Figure 6.2. Metabolic gene response to a novel bout of low frequency stimulated contraction. mRNA levels of A) GLUT4 B) PPARγ C) PGC-1α and D) PGC-1β three hours after a novel bout of contraction in healthy wild type (BL-6) and cachectic ApcMin/+

(Min) mice *Significantly different from BL-6 control from pre-planned t-test. # main effect LoFS, & main effect genotype, † significantly different from all other

comparisons, p<0.05.

Figure 6.3. Expression of PGC-1α targets in response to a novel bout of low frequency stimulated contraction. mRNA levels for the PGC-1α target A) Nuclear Respiratory Factor-1 (NRF-1) and B) Mitochondrial transcription factor A (TFAM) were measured in the gastrocnemius muscle of wild type (BL-6) and cachectic ApcMin/+ (Min) mice. C) mitochondrial encoded cytochrome B mRNA was measured in the gastrocnemius, & main effect genotype, † significantly different from all other comparisons, p<0.05.

Figure 6.4. LoFS regulation of protein expression. A) The ratio of phosphorylated to total S6 in the gastrocnemius muscle was used as a marker of protein synthesis. The translational response of B) TFAM C) Cytochrome C and D) PGC-1α three hours after a novel bout of contraction in healthy wild type (BL-6) and cachectic ApcMin/+ (Min) mice. Lanes connected by a bar in the representative western blots represent the control (C) and LoFS (L) leg from the same animal. All comparisons were run on the same gel. # main effect LoFS, & main effect genotype, † significantly different from all other

comparisons, p<0.05.

Figure 6.5. Effect of Pyrrolidine dithiocarbamate on cachectic muscle response to LoFS. Inflammatory signaling was blocked 24h prior to stimulation by acute PDTC

administration A) The ratio of phosphorylation:total STAT3 and P65 were measured 3h after acute contraction. B) mRNA targets of PGC-1α, Nuclear Respiratory Factor-1 (NRF-1) and Mitochondrial transcription factor A (TFAM), and cytochrome B (Cyto B) were measured in mice administered PDTC. Data were normalized to the levels seen in the control leg of ApcMin/+ mice. The dotted line represents BL-6 control levels. C) The ratio of phosphorylated: total S6 was used as a marker of protein synthesis, and the protein levels of TFAM and cytochrome c (Cyto C) were measured by western blot analysis and normalized to Min controls run on the same gel. Lanes connected by a bar in the representative western blots represent the control (C) and LoFS (L) leg from the same animal. All comparisons were run on the same gel. * significantly different from Min based on pre-planned t-test. $ main effect PDTC, # main effect LoFS, & main effect genotype, ** significantly different from control leg within group, ^^ significantly

different from all other control groups, ∞ significantly different from all other LoFS comparisons, p<0.05.

Figure 6.6. LoFS mediated regulators of mTOR. Known activators and suppressors of mTOR were measured in gastrocnemius of BL-6 and Min mice 3 hours after LoFS. A) AMPK B) P-38 and C) Akt phosphorylation was measured and graphed as a ratio of phosphorylated to total protein. Lanes connected by a bar in the representative western blots represent the control (C) and LoFS (L) leg from the same animal. All comparisons were run on the same gel. # main effect LoFS, & main effect genotype, † significantly different from all other comparisons, p<0.05.

A)

B)

Figure 6.1. Cage Activity during severe cachexia. Cage activity was measured for three consecutive nights during the active cycle (1900-0700h). All animals were single housed. A) XY ambulatory count, the number of times a new beam is interrupted, and B) total Z counts were analyzed. T-test was run to test for significance. *significantly different from BL-6 p<0.001.

Figure 6.2.

A)

C)

D)

Figure 6.2. Metabolic gene response to a novel bout of low frequency stimulated contraction. mRNA levels of A) GLUT4 B) PPARγ C) PGC-1α and D) PGC-1β three hours after a novel bout of contraction in healthy wild

type (BL-6) and cachectic ApcMin/+(Min) mice *Significantly different from

BL-6 control from pre-planned t-test. # main effect LoFS, & main effect genotype, † significantly different from all other comparisons, p<0.05.

A)

B)

C)

Figure 6.3. Expression of PGC-1α targets in response to a novel bout of low frequency stimulated contraction. mRNA levels for the PGC-1α target A) Nuclear Respiratory Factor-1 (NRF-1) and B) Mitochondrial transcription factor A (TFAM) were measured in the gastrocnemius muscle of wild type

(BL-6) and cachectic ApcMin/+ (Min) mice. C) mitochondrial encoded

cytochrome B mRNA was measured in the gastrocnemius, & main effect genotype, † significantly different from all other comparisons, p<0.05.

A)

B)

D) C)

Figure 6.4. LoFS regulation of protein expression. A) The ratio of phosphorylated to total S6 in the gastrocnemius muscle was used as a marker of protein synthesis. The translational response of B) TFAM C) Cytochrome C and D) PGC-1α three hours after a novel bout of contraction in healthy wild type (BL-6) and cachectic ApcMin/+ (Min) mice. Lanes connected by a bar in the representative western blots represent the control (C) and LoFS (L) leg from the same animal. All comparisons were run on the same gel. # main effect LoFS, & main effect genotype, † significantly different from all other comparisons, p<0.05.

Figure 6.5.

A)

C) B)

Figure 6.5. Effect of Pyrrolidine dithiocarbamate on cachectic muscle response to LoFS.

Inflammatory signaling was blocked 24h prior to stimulation by acute PDTC administration A) The ratio of phosphorylation:total STAT3 and P65 were measured 3h after acute contraction. B) mRNA targets of PGC-1α, NRF-1, TFAM, and cytochrome B (Cyto B) were measured in

mice administered PDTC. Data were normalized to the levels seen in the control leg of ApcMin/+

mice. The dotted line represents BL-6 control levels. C) The ratio of phosphorylated: total S6 was used as a marker of protein synthesis, and the protein levels of TFAM and cytochrome c (Cyto C) were measured by western blot analysis and normalized to Min controls run on the same gel. Lanes connected by a bar represent the control (C) and LoFS (L) leg from the same animal. All comparisons were run on the same gel. * significantly different from Min based on pre-planned t-test. $ main effect PDTC, # main effect LoFS, & main effect genotype, ** significantly different from control leg within group, ^^ significantly different from all other control groups, ∞ significantly different from all other LoFS comparisons, p<0.05.

A)

B)

C)

Figure 6.6. LoFS mediated regulators of mTOR. Known activators and suppressors of mTOR were measured in gastrocnemius of BL-6 and Min mice 3 hours after LoFS. A) AMPK B) P-38 and C) Akt phosphorylation was measured and graphed as a ratio of phosphorylated to total protein. Lanes connected by a bar in the representative western blots represent the control (C) and LoFS (L) leg from the same animal. All comparisons were run on the same gel. # main effect LoFS, & main effect genotype, † significantly different from all other comparisons, p<0.05.

CHAPTER 7

Cachexia is defined as the unintentional loss of body weight including muscle mass with or without adipose tissue loss given an underlying condition (Batista et al., 2012; Bruera, 1997; Evans et al., 2008; Fearon et al., 2011). Cancer cachexia accounts for approximately 20% of all cancer related deaths and 40% of deaths related to colon cancer (Bruera, 1997; Tisdale, 2002). Given the complexity of the cachectic condition, it is not diagnosed until after significant muscle loss and there are currently no approved pharmaceutical interventions for the treatment of cachexia. While there are many models of cachexia there is an underlying increase in systemic inflammation that is associated with increased muscle protein degradation and functional decrements (Argiles et al., 2003; Argiles et al., 2012; Fearon et al., 2013; Fortunati et al., 2007; Strassmann et al., 1992; Strassmann and Kambayashi, 1995). Therefore the purpose of this dissertation was to determine the role of inflammatory signaling through gp130/STAT3 signaling on the regulation of muscle mass and mitochondrial content during cancer cachexia. We hypothesized that inhibition of systemic STAT3 signaling would attenuate muscle mass loss through inhibition of protein degradation and maintenance of mitochondria content and that inhibition of skeletal muscle gp130 signaling would be sufficient for muscle mass maintenance during cachexia.

The data presented support our hypothesis that inhibition of skeletal muscle gp130 signaling can attenuate skeletal muscle protein degradation and improve muscle oxidative capacity; however, muscle gp130 was unable to release cachexia-suppression of muscle protein synthesis or the induction of mitochondrial fission. Additionally, inhibition of trans IL-6 signaling attenuated cachexia and suppressed muscle inflammatory signaling and activation of protein degradation pathways. Trans IL-6

inhibition maintained muscle mitochondrial content and alleviated the increase of mitochondrial fission, but could not attenuate the suppression of mitochondrial fusion or the suppression of muscle protein synthesis. Similarly systemic inhibition of

STAT3/NFκB signaling inhibited muscle protein degradation and mitochondrial loss; however, STAT3/NFκB inhibition through PDTC administration increased muscle protein synthesis in cachectic mice. Inhibition of STAT3 alone was unable to rescue protein synthesis in LLC induced muscle atrophy suggesting that it is the systemic actions of PDTC that act to improve muscle protein synthesis. Finally cachectic muscle has an altered response to acute contraction. While contraction was able to induce signaling which should increase muscle mitochondrial biogenesis, increases in

mitochondrial biogenesis were blunted in cachectic muscle. Inhibition of inflammatory signaling attenuated the cachexia-block of contraction-induced mitochondrial biogenesis.

Regulation of mitochondrial biogenesis by inflammatory signaling during cachexia.

The loss of mitochondrial content and alterations in mitochondrial dynamics have been reported during cancer cachexia (Fermoselle et al., 2013; Julienne et al., 2012; Tisdale, 2002; Tzika et al., 2013; White et al., 2011a). Although cachexia is not associated with decrements in the ability of individual mitochondria to produce ATP (Julienne et al., 2012), the overall production is limited with the net loss of mitochondria. This decrease in ATP will inhibit energy using pathways such as protein synthesis and may ultimately lead to activation of energy saving/energy producing pathways such as protein degradation. We have previously demonstrated that inhibition of systemic IL-6 signaling can attenuate the progression of skeletal muscle loss after the initiation of cachexia and is associated with improved muscle mitochondrial content and dynamics

(White et al., 2013b; White et al., 2012c). Our initial studies blocked both classical and trans IL-6 signaling. We now show that trans IL-6 signaling can attenuate muscle

mitochondrial loss potentially through the suppression of mitochondrial fission; however, trans signaling was unable to rescue mitochondrial fusion. Muscle specific gp130

signaling inhibition also attenuated mitochondrial loss and was associated with increases in mitochondrial fusion, but did not block mitochondrial fission. These data suggest that mitochondrial fusion may be regulated through a gp130 independent pathway and may be more closely related to alterations in systemic inflammatory signaling than muscle

specific inflammatory signaling. Alternatively the suppression of mitochondrial fusion may be related to the disuse as activity generally declines prior to muscle loss in the Min mouse (Baltgalvis et al., 2010). Disuse of skeletal muscle as well as aging are associated with decreased mitochondrial fusion and increased fission (Iqbal et al., 2013). Long term decreases in mitochondrial fusion are detrimental to skeletal muscle as fusion is required for mitochondrial DNA stability and function and loss of MFN1/2 is associated with mitochondrial myopathies and muscle loss (Chen et al., 2010). Further work is necessary to understand the role of altered mitochondrial fusion on the progression of cachexia.

Increases in muscle mitochondrial fission are associated with inflammatory signaling (White et al., 2012b). Additionally mitochondrial fission can regulate the induction of protein degradation (Romanello et al., 2010; Romanello and Sandri, 2010) and the suppression of mitochondrial fission may be one mechanism through which repression of inflammation is acting to decrease protein degradation. While we see inhibition of IL-6 trans signaling suppressed mitochondrial fission, the suppression of systemic STAT3 and muscle gp130 signaling were unable to prevent FIS induction.

Interestingly inhibition of inflammatory signaling by all three methods resulted in attenuation of muscle degradation signaling. One possible explanation for the induction of fission and fusion with PDTC treatment and muscle gp130 inhibition is that there may be higher mitochondrial turnover with these treatments. Interestingly inhibition of

STAT3 and muscle gp130 were not sufficient to suppress mitochondrial fission, suggesting that the induction of mitochondrial fission may be independent of

inflammatory signaling and may not be regulating muscle protein breakdown during cancer cachexia. Further work is necessary to determine the mechanism by which PDTC administration and muscle gp130 inhibition attenuate protein degradation independently of increases in mitochondrial fission.

Interestingly, mice over-expressing PGC-1α are not protected from the rapid induction of muscle mass loss induced by LLC cachexia (Wang et al., 2012). A limitation to this study was that circulating IL-6 was increased in animals with muscle over-expression of PGC-1α, but not in wild type tumor bearing mice, and the suppression of muscle mitochondrial content with cachexia was not demonstrated. Over-expression of muscle PGC-1α4 can prevent LLC induced muscle wasting demonstrating the need for further research examining the role of mitochondrial content in muscle wasting (Ruas et al., 2012). We have shown that exercise training prior to development of cachexia can increase mitochondrial capacity and attenuate cachexia induced fission and suppression of fusion (White et al., 2012b). Acute contraction after the initiation of cachexia was unable to increase markers of mitochondrial biogenesis; however, when basal

inflammation was suppressed mitochondrial biogenesis was increased. Inflammatory signaling is a potent regulator of muscle mitochondrial content during cachexia, and

exercise has been shown to decrease inflammation. Although increased mitochondrial content is not sufficient for the prevention of cachexia, it is unknown if the mitochondria are functional. A buildup of dysfunctional mitochondria may be detrimental to the cachectic condition. Further work is necessary to determine if functional increases in muscle mitochondrial content can attenuate muscle loss associated with increased

inflammation and if exercise training after the initiation of cachexia can attenuate further mitochondrial and muscle mass loss.

Regulation of muscle protein turnover by inflammatory signaling during cachexia

Much of the focus of cachexia research has been aimed towards the suppression of cachexia induced muscle protein degradation. Inhibition of potential cachectic

mediators such as myostatin (Busquets et al., 2012; Murphy et al., 2011), FOXO (Reed et al.), C/EBPβ (Zhang et al., 2011), mitochondrial fission (Romanello et al., 2010;

Romanello and Sandri, 2010), and STAT3 (Bonetto et al., 2012) have been shown to attenuate muscle mass loss through suppression of protein catabolism pathways. We further this research demonstrating that inhibition of systemic STAT3/NFκB signaling, IL-6 trans signaling, and of muscle gp130 signaling can also attenuate the cachexia- induction of muscle protein degradation pathways. Skeletal muscle gp130 may be inhibiting degradation through a combination of STAT3 inhibition and inhibition of P38 which have both been implicated in muscle protein breakdown in models of cachexia (Bonetto et al., 2012; Zhang and Li, 2012). Inhibition of muscle gp130 was sufficient to inhibit muscle mass loss in the LLC model of cachexia, but not the Min model. The induction of atrogin was suppressed in both models, and the suppression of protein synthesis was unaltered by gp130 inhibition. Because the LLC is a rapid model of

cachexia, it is possible that the long term suppression of muscle protein synthesis in the Min model is responsible for the overall muscle mass loss, whereas in the LLC there was only a short term suppression of protein synthesis. Further work needs to be done to determine if the inhibition of muscle gp130 delays the onset of cachexia in the Min mouse. Many of the models currently used for cachexia research develop cachexia within a matter of days-weeks suggesting that the effectiveness of many of the treatments studied may only be beneficial for delaying the onset of cachexia in a slower model. Further research needs to be done to examine the effectiveness of long term inhibition on muscle protein degradation without improvements in synthesis for the prevention of muscle loss with cachexia.

Induction of muscle protein degradation is one mechanism inducing the loss of muscle mass with cachexia that has received much attention; however, cachexia is also associated with a suppression of muscle protein synthesis which could independently decrease muscle mass (Atherton et al., 2005; Dworzak et al., 1998; Goodman et al., 2011b; Risson et al., 2009; White et al., 2011b). To date no treatments aside from

exercise have been shown to release the cachexia-suppression of muscle protein synthesis and most studies have focused solely on protein breakdown. We have previously

demonstrated that exercise training prior to cachexia development can prevent IL-6 suppression of IGF-1 and mTOR signaling (White et al., 2013b). Our lab has also shown that high frequency stimulation, mimicking resistance exercise, in muscle after the initiation of cachexia can increase muscle protein synthesis (Sato, 2012).

One potential mechanism in which cachexia may be suppressing protein synthesis is through the up regulation of AMPK. Inflammation has been shown to