**3. Results**

#### *3.1. E*ff*ect of Lactate on Skeletal Muscle Hypertrophy*

The body weight, TA muscle weight, and fiber CSA in response to oral lactate administration were shown in Figure 1. There was no significant di fference in body weight between groups. A significant increase in the absolute TA muscle weight and the muscle weight relative to body weight was observed in the lactate-administered L group (Figure 1A, *p* < 0.05). Similarly, the fiber CSA in the L group was significantly larger than that in the C group (Figure 1B, *p* < 0.05).

There was no significant change in the population number of Pax7-positive nuclei a fiber of TA muscle in the C group during the experimental period. In the L group, the population of Pax7-positive nuclei was significantly increased (200% and 138% at 1 and 2 weeks after lactate administration, respectively), compared with that in the C group (Figure 1C, *p* < 0.05).

**Figure 1.** (**A**) Effects of oral lactate administration on body weight (BW) and tibialis anterior (TA) weight in mice. Effect of oral lactate administration on the TA muscle fiber cross-sectional area (CSA) (**B**) and the number of Pax7-positive nuclei per muscle fiber (**C**). Representative images of histochemical and immuno-histochemical staining in TA muscle are shown. Arrowheads indicate the Pax7-positive nuclei. Scale bar = 50 μm. TA weight/BW: relative TA weight to BW. C: control group. L: lactate-administered group. 1W and 2W: 1 and 2 weeks of lactate administration, respectively. Values are means ± SEM. *n* = 6 per group. a: Significant main effect of lactate, *p* < 0.05. \*: *p* < 0.05.

#### *3.2. E*ff*ect of Lactate on Skeletal Muscle Regeneration*

Many regenerating fibers with centrally located nuclei in CTX-injected TA muscle were observed 1 and 2 weeks after the injection (Figure 2B). There was no significant change in body weight of not only CX but also LX groups during the experimental period (Figure 2A). Both TA muscle weight (24% and 29% of absolute and relative levels, respectively) and fiber CSA (44%) 2 weeks after CTX injection increased, compared with those 1 week after the injection. Two weeks after CTX injection, lactate administration-associated increase in the absolute TA muscle weight was observed (*p* < 0.05). A significant main effect of lactate administration was observed in the relative muscle weight as well as fiber CSA (*p* < 0.05).

**Figure 2.** (**A**) Effects of oral lactate administration on BW and TA weight in cardio-toxin (CTX)-injected mice. Effect of oral lactate administration on CTX-injected TA muscle fiber CSA (**B**) and the number of Pax7-positive nuclei per muscle fiber (**C**). Representative images of histochemical and immuno-histochemical staining in CTX-injected TA muscle are shown. Arrowheads indicate the Pax7-positive nuclei. Scale bar = 50 μm. CX: CTX-injected group. LX: lactate-administered after the CTX-injection group. 1W and 2W: 1 and 2 weeks after CTX injection, respectively. See Figure 1 for other abbreviations. Values are means ± SEM. *n* = 7 per group. a and b: Significant main effect of lactate and time, respectively. *p* < 0.05. \*: *p* < 0.05.

One week after CTX injection, the population of Pax7-positive nuclei was increased (0.08/myofiber), compared with that in the C group (0.02/myofiber). Furthermore, the additional increase in the population of Pax7-positive nuclei was observed in the lactate-administered LX group, when compared with the CX group, 1 week after CTX injection (*p* < 0.05).

#### *3.3. E*ff*ects of Lactate on Myotube Formation*

We examined the effects of lactate on myotube formation of C2C12 cells. The typical images of myotubes, on the 5th day of differentiation, with or without lactate in the medium were shown in Figure 3. In the lactate-administered cells, myotubes have a wider diameter, longer length, and more myonuclei compared with the control. A significant increase in the myotube diameter and muscular protein content was induced by lactate (Figure 3A,B, *p* < 0.05). Lactate administration also increased myotube length as well as myo-nuclei number (Figure 3C, *p* < 0.05).

**Figure 3.** Myo-tube diameter (**A**), muscular protein content (**B**), myo-tube length, and myonuclei number (**C**) of C2C12 cells in response to lactate. Scale bar = 300 μm. Values are means ± SEM. *n* = 6 wells per group. \*: *p* < 0.05.
