*2.1. GH Stimulates Food Intake and Increases Body Weight in CKD Mice*

We empirically determined the optimal dose of GH treatments in our mouse model of CKD. Six-week-old c57BL/6J male mice were used for this study. Schematic representation of the experimental design is shown in Figure 1A. CKD in mice was induced by a two-stage subtotal nephrectomy, while a sham procedure was performed in control mice [13]. GH treatment was initiated in eight-week-old CKD or sham mice. CKD or sham mice were treated with recombinant human GH (5 mg/kg/day or 10 mg/kg/day, intraperitoneal) or vehicle for six weeks. During the treatment, all mice were housed in individual cage and fed ad libitum. Dietary intake as well as weight gain for each mouse was recorded weekly. Mice were sacrificed at the age of 14 weeks old. Serum and blood chemistry of CKD and sham mice are listed (Table 1). CKD mice were uremic, as CKD mice had a higher concentration of BUN and serum creatinine than control mice. Over the course of the six-week ad libitum experiment, GH stimulated food intake and improved weight gain in both CKD and sham mice. GH-treated CKD and GH-treated sham mice exhibited significantly more average daily energy intake and weight gain compared to vehicle-treated CKD and vehicle-treated sham mice, respectively (Figure 1B,C). More importantly, we found that CKD mice treated with 10 mg/kg/day demonstrated significantly improved food intake and weight gain relative to CKD mice treated with 5 mg/kg/day or vehicle. As a result, daily dosing of 10 mg/kg of GH for CKD mice was selected for the subsequent food-restrictive study.

**Table 1.** Serum and blood chemistry of mice from ad libitum study. Eight-week-old CKD and sham mice were treated with GH (5 mg/kg per day or 10 mg/kg per day) or normal saline as a vehicle for six weeks. All mice were fed ad libitum. Data are expressed as mean ± SEM. Results of all five groups of mice were compared to those of Sham + Vehicle mice, respectively. BUN, blood urea nitrogen. <sup>a</sup> *p* < 0.05, significantly higher than Sham + Vehicle mice.


**Figure 1.** GH attenuates cachexia in CKD mice. We performed two studies. For the first study, we used ad libitum dietary strategy (**A**). CKD and control mice were given GH (5 mg/kg/day or 10 mg/kg/day), or vehicle (normal saline), respectively, for six weeks. All mice were fed ad libitum. We calculated average daily caloric intake (**B**) and recorded final weight change in mice (**C**). Results of Sham + GH (5 mg/kg/day) and Sham + GH (10 mg/kg/day) mice were compared to those of Sham + Vehicle mice, while results of CKD + GH (5 mg/kg/day) and CKD + GH (10 mg/kg/day) mice were compared to those of CKD + Vehicle mice. In addition, results of CKD + GH (5 mg/kg/day) mice were compared to those of CKD + GH (10 mg/kg/day) mice. Furthermore, results of CKD + Vehicle, CKD + GH (5 mg/kg/day), and CKD + GH (10 mg/kg/day) mice were compared to those of Sham + Vehicle,

Sham + GH (5 mg/kg/day), and Sham + GH (10 mg/kg/day) mice, respectively. Data are expressed as mean ± SEM. For comparison of the means between two groups, data were analyzed by Student's 2-tailed *t*-test. Differences of the means for more than two groups containing two variables were analyzed using two-way ANOVA. Posthoc analysis was performed with Tukey's test. Specific *p*-values are shown above the bar. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. ns signifies not significant. For the second experiment, we employed a diet-restrictive strategy (**D**). CKD + Vehicle mice were given an ad libitum amount of food, whereas other groups of mice were given an equivalent amount of food (**E**). Weight gain, fat content, resting metabolic rate, lean content, gastrocnemius weight relative to length of tibia, and in vivo muscle function (rotarod and grip strength) were measured (**F**–**L**). Results of Sham + GH (10 mg/kg/day) mice were compared to those of Sham + Vehicle mice, while results of CKD + GH (10 mg/kg/day) mice were compared to those of CKD + Vehicle mice. Furthermore, results of CKD + Vehicle and CKD + GH (10 mg/kg/day) mice were compared to those of Sham + Vehicle mice, respectively. Data are expressed as mean ± SEM. For comparison of the means between two groups, data were analyzed by Student's 2-tailed *t*-test. Posthoc analysis was performed with Tukey's test. Specific *p*-values are shown above the bar. ns signifies not significant, \* *p* < 0.05, \*\* *p* < 0.01.
