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. 2023 Dec;14(6):2866-2881.
doi: 10.1002/jcsm.13350. Epub 2023 Nov 8.

Improved endurance capacity of diabetic mice during SGLT2 inhibition: Role of AICARP, an AMPK activator in the soleus

Shintaro Nakamura  1 , Yasutaka Miyachi  1 , Akihito Shinjo  1 , Hisashi Yokomizo  1 , Masatomo Takahashi  2 , Kohta Nakatani  2 , Yoshihiro Izumi  2 , Hiroko Otsuka  1 , Naoichi Sato  1 , Ryuichi Sakamoto  1 , Takashi Miyazawa  1 , Takeshi Bamba  2 , Yoshihiro Ogawa  1
Affiliations

Improved endurance capacity of diabetic mice during SGLT2 inhibition: Role of AICARP, an AMPK activator in the soleus

Shintaro Nakamura et al. J Cachexia Sarcopenia Muscle. 2023 Dec.

Abstract

Background: Diabetes is associated with an increased risk of deleterious changes in muscle mass and function or sarcopenia, leading to physical inactivity and worsening glycaemic control. Given the negative energy balance during sodium-glucose cotransporter-2 (SGLT2) inhibition, whether SGLT2 inhibitors affect skeletal muscle mass and function is a matter of concern. However, how SGLT2 inhibition affects the skeletal muscle function in patients with diabetes remains insufficiently explored. We aimed to explore the effects of canagliflozin (CANA), an SGLT2 inhibitor, on skeletal muscles in genetically diabetic db/db mice focusing on the differential responses of oxidative and glycolytic muscles.

Methods: Db/db mice were treated with CANA for 4 weeks. We measured running distance and handgrip strength to assess skeletal muscle function during CANA treatment. At the end of the experiment, we performed a targeted metabolome analysis of the skeletal muscles.

Results: CANA treatment improved the reduced endurance capacity, as revealed by running distance in db/db mice (414.9 ± 52.8 vs. 88.7 ± 22.7 m, P < 0.05). Targeted metabolome analysis revealed that 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (AICARP), a naturally occurring AMP-activated protein kinase (AMPK) activator, increased in the oxidative soleus muscle (P < 0.05), but not in the glycolytic extensor digitorum longus muscle (P = 0.4376), with increased levels of AMPK phosphorylation (P < 0.01).

Conclusions: This study highlights the potential role of the AICARP/AMPK pathway in oxidative rather than glycolytic skeletal muscles during SGLT2 inhibition, providing novel insights into the mechanism by which SGLT2 inhibitors improve endurance capacity in patients with type 2 diabetes.

Keywords: AICARP; AMPK; SGLT2 inhibitor; endurance exercise; fatty acid oxidation; skeletal muscle.

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Conflict of interest statement

Yoshihiro Ogawa received research grants from Mitsubishi Tanabe Pharma Corporation, Fujifilm Holdings Corporation, Cosmic Corporation, AstraZeneca K.K. and Nippon Boehringer Ingelheim Corporation. The remaining authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Changes in body weight and glucose metabolism during sodium–glucose cotransporter‐2 (SGLT2) inhibition. (A) Animal experimental protocol. Male 8‐week‐old db/+ and db/db mice were fed with normal chow (NC) or canagliflozin (CANA)‐containing diet for 4 weeks. Changes in random blood glucose levels (B) and body weight (C) during CANA treatment (n = 7). (D) Average daily food intake at 4 weeks of CANA treatment (n = 7). (E) Intraperitoneal glucose tolerance test (ipGTT) (1 g/kg) at 3 weeks of CANA treatment (n = 3). Area under the curve (AUC) of ipGTT. (F) Insulin tolerance test (ITT) (0.5 U/kg) at 3 weeks of CANA treatment and AUC of ITT (n = 4). Y axis indicates the percentage changes in blood glucose from baseline (0 min). Data are presented as the mean ± SEM. Group difference was assessed using one‐way analysis of variance (ANOVA) in (D) and (E) (AUC of ipGTT) or two‐way ANOVA in (B), (C) and (E) (ipGTT) and (F) (ITT). *P < 0.05, **P < 0.01 and ***P < 0.001 versus CANA‐treated db/db mice. ns, not significant.
Figure 2
Figure 2
Effect of sodium–glucose cotransporter‐2 (SGLT2) inhibition on skeletal muscle weight and function. Lower limb muscle weight (upper left), cross‐sectional area (upper right) and representative wheat germ agglutinin staining of muscle fibres (lower) in soleus (SOL) (A) and extensor digitorum longus (EDL) muscles (B). Scale bar = 100 μm. (C–F) Running distance assessed by treadmill exercise test and grip strength. Changes in distance (C) and grip strength (E) during canagliflozin (CANA) treatment. Distance (D) and grip strength (F) at 4 weeks of CANA treatment. Data are expressed as the mean ± SEM. Group difference was assessed using one‐way analysis of variance (ANOVA) in (D) and (F) or two‐way ANOVA in (C) and (E). *P < 0.05, **P < 0.01 and ***P < 0.001. ††† P < 0.001 versus control db/+ mice. ns, not significant.
Figure 3
Figure 3
Metabolome analysis of skeletal muscles. (A) Soleus (SOL) and extensor digitorum longus (EDL) from db/+, db/db and canagliflozin (CANA)‐treated db/db mouse groups (n = 4, each group) were clustered based on metabolomics data (rows). Clustering results shown as a heatmap (distance measure using Euclidean and clustering algorithm using ward.D). Each column represents the mean hydrophilic metabolite from four mice, where red indicates high metabolite level and blue indicates low metabolite level within the three groups. The scale bar indicates z‐score values, which are calculated by subtracting the mean peak intensity of metabolites across all samples from the individual peak intensity of a specific metabolite and then dividing the result by the standard deviation. (B) Top 25 enriched metabolic pathways affected by CANA treatment in SOL and EDL. (C) Volcano plot representation of hydrophilic metabolites that were significantly changed in SOL and EDL from CANA‐treated db/db mice. The dotted horizontal line represents the significance threshold (Student's t test, P < 0.05 vs. control db/db mice, n = 4).
Figure 4
Figure 4
Glycolysis and tricarboxylic acid (TCA) cycle in skeletal muscles. Ratio of metabolites in canagliflozin (CANA)‐treated db/db mice to control db/db mice in soleus (SOL) (A) and extensor digitorum longus (EDL) (B), calculated as fold change. Dark red indicates metabolites with fold change ≥1.5, and dark blue indicates those with fold change ≤0.6. Grey indicates those with no difference between the CANA‐treated and control db/db mice. White indicates those undetected. Relative metabolite changes in SOL (C) and EDL (D) (n = 4). Data are expressed as the mean ± SEM. *P < 0.05 versus control db/db mice (Student's t test).
Figure 5
Figure 5
Assessment of metabolites and key enzyme related to fatty acid oxidation in skeletal muscles. (A–D) Heatmap of relative acyl‐CoA and acyl‐carnitine levels in skeletal muscles from db/+, db/db and canagliflozin (CANA)‐treated db/db mice. Each column represents the mean metabolite from four mice, where red indicates high metabolite level and blue indicates low metabolite level within the three mouse groups. Relative acyl‐CoA (A) and acyl‐carnitine (B) levels in soleus (SOL). Relative acyl‐CoA (C) and acyl‐carnitine (D) levels in extensor digitorum longus (EDL). The scale bar indicates z‐score values, which are calculated by subtracting the mean peak intensity of metabolites across all samples from the individual peak intensity of a specific metabolite and then dividing the result by the standard deviation. (E) Schematic of key enzymatic steps in fatty acid oxidation. (F) Representative immunoblot images and quantification of carnitine palmitoyltransferase 1 (CPT1) expression in SOL and EDL. Data are expressed as the mean ± SEM. *P < 0.05 (analysis of variance [ANOVA], n = 4). ns, not significant.
Figure 6
Figure 6
Changes in amino acid levels in skeletal muscles and serum. Heatmap of relative amino acid levels in soleus (SOL) (A) and extensor digitorum longus (EDL) (B) muscles and serum (C) from db/+, db/db and canagliflozin (CANA)‐treated db/db mice (n = 4). The scale bar indicates z‐score values, which are calculated by subtracting the mean peak intensity of metabolites across all samples from the individual peak intensity of a specific metabolite and then dividing the result by the standard deviation. Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.
Figure 7
Figure 7
Changes in 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranosyl 5′‐monophosphate (AICARP) levels and AMP‐activated protein kinase (AMPK) phosphorylation in skeletal muscles. AICARP peak areas (A) and relative AMP/ATP ratios (B) in soleus (SOL) and extensor digitorum longus (EDL) after canagliflozin (CANA) treatment (n = 4). Representative immunoblot images and quantification for the expression of p‐AMPK and t‐AMPK in SOL (C) and EDL (D) (n = 7). Data are expressed as the mean ± SEM. Group difference was assessed using Student's t test in (A) and (B) or one‐way analysis of variance (ANOVA) in (C) and (D). *P < 0.05 and **P < 0.01. arb. unit, arbitrary unit; ns, not significant.
Figure 8
Figure 8
Schematic of major metabolites and 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranosyl 5′‐monophosphate (AICARP)/AMP‐activated protein kinase (AMPK) pathway in soleus muscle from db/db mice. The colours of metabolites are described in Figures 3 and 4 . Long‐chain acyl‐CoAs (C12–C18) accumulated in soleus muscle from db/db mice, partly because of reduced AMPK activation. Impaired fatty oxidation reduced energy production by the mitochondria and caused muscle dysfunction, such as reduced endurance. Canagliflozin (CANA) treatment induced AICARP accumulation in soleus muscle from db/db mice, leading to AMPK activation. AMPK activation enhanced fatty acid β‐oxidation in soleus muscle from db/db mice, which consequently improved their endurance capacity.
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