Whole body glucoregulation and tissue-specific glucose uptake in a novel Akt substrate of 160 kDa knockout rat model

Abstract

Akt substrate of 160 kDa (also called AS160 or TBC1D4) is a Rab GTPase activating protein and key regulator of insulin-stimulated glucose uptake which is expressed by multiple tissues, including skeletal muscle, white adipose tissue (WAT) and the heart. This study introduces a novel rat AS160-knockout (AS160-KO) model that was created using CRISPR/Cas9 technology. We compared male AS160-KO versus wildtype (WT) rats for numerous metabolism-related endpoints. Body mass, body composition, energy expenditure and physical activity did not differ between genotypes. Oral glucose intolerance was detected in AS160-KO versus WT rats (P<0.005). A hyperinsulinemic-euglycemic clamp (HEC) revealed insulin resistance for glucose infusion rate (P<0.05) with unaltered hepatic glucose production in AS160-KO versus WT rats. Genotype-effects on glucose uptake during the HEC: 1) was significantly lower in epitrochlearis (P<0.01) and extensor digitorum longus (P<0.05) of AS160-KO versus WT rats, and tended to be lower for AS160-KO versus WT rats in the soleus (P<0.06) and gastrocnemius (P<0.08); 2) tended to be greater for AS160-KO versus WT rats in white adipose tissue (P = 0.09); and 3) was significantly greater in the heart (P<0.005) of AS160-KO versus WT rats. GLUT4 protein abundance was significantly lower for AS160-KO versus WT rats in each tissue analyzed (P<0.01-0.001) except the gastrocnemius. Ex vivo insulin-stimulated glucose uptake was significantly lower (P<0.001) for AS160-KO versus WT rats in isolated epitrochlearis or soleus. Insulin-stimulated Akt phosphorylation (in vivo or ex vivo) did not differ between genotypes for any tissue tested. Ex vivo AICAR-stimulated glucose uptake by isolated epitrochlearis was significantly lower for AS160-KO versus WT rats (P<0.01) without genotype-induced alteration in AMP-activated protein phosphorylation. This unique AS160-KO rat model, which elucidated striking genotype-related modifications in glucoregulation, will enable future research aimed at understanding AS160's roles in numerous physiological processes in response to various interventions (e.g., diet and/or exercise).

Fig 1. Total AS160 abundance and AS160 phosphorylation (pAS160 Thr⁶⁴²) in tissues collected immediately after the hyperinsulinemic-euglycemic clamp performed in WT and KO rats.

EDL, extensor digitorum longus; WAT, white adipose tissue. Images of representative immunoblots and loading controls (Memcode protein stain) are provided for each group. Neither total AS160 nor pAS160 Thr⁶⁴² was detectable in any of the tissues from KO rats. N = 5–6 rats per group.

Fig 2. Oral glucose tolerance test (OGTT) and glucose infusion rate (GIR during the hyperinsulinemic-euglycemic clamp; HEC) in wildtype (WT; open bars and open circles) and AS160-KO (KO; filled bars and filled circles) rats.

Area under the curve (AUC; inset) was calculated by the trapezoidal method for (A) plasma glucose and (B) plasma insulin. Data were analyzed by Student’s t-test. *P<0.05, ^†P<0.01 and ^‡P<0.005 for WT versus KO rats. Values are means ± SEM. N = 6 rats per group. (C) GIR in rats undergoing HEC. AUC for GIR was calculated by the trapezoidal method (inset). Data were analyzed by Student’s t-test. *P<0.05 and ^†P<0.005 for WT versus KO rats. Values are means ± SEM. N = 5–6 rats per group.

Fig 3. In vivo tissue 2-deoxyglucose (2-DG) uptake in wildtype (WT; open bars) and AS160-KO (KO; filled bars) rats from the hyperinsulinemic-euglycemic clamp experiment.

Extensor digitorum longus (EDL); White adipose tissue (WAT). Data were analyzed by Student’s t-test. *P<0.05 and ^‡P<0.005 for WT versus KO rats. Values are means ± SEM. N = 5–6 rats per group.

Fig 4. Phosphorylation of Akt on Ser⁴⁷³ (pAkt Ser⁴⁷³) and Thr³⁰⁸ (pAkt Thr³⁰⁸) in tissues collected immediately after the hyperinsulinemic-euglycemic clamp performed in WT (open bars) and KO (filled bars) rats.

EDL, extensor digitorum longus; WAT, white adipose tissue. Bar graphs represent the ratio of the values for the immunoblots and their respective loading controls (Memcode stain). Because no genotype differences were detected for loading controls, images of the loading controls are not included to improve the clarity of these figures, as well as the other immunoblotting figures that follow. *P<0.05, for WT versus KO rats. Values are means ± SEM. N = 5–6 rats per group.

Fig 5. Total abundance TBC1D1 in tissues collected immediately after the hyperinsulinemic-euglycemic clamp performed in WT (open bars) and KO (filled bars) rats.

EDL, extensor digitorum longus; WAT, white adipose tissue. Bar graphs represent the ratio of the values for the immunoblots and their respective loading controls (Memcode stain). Values are means ± SEM. N = 5–6 rats per group.

Fig 6. Total abundance of GLUT4 in tissues collected immediately after the hyperinsulinemic-euglycemic clamp performed in WT (open bars) and KO (filled bars) rats.

EDL, extensor digitorum longus; WAT, white adipose tissue. Bar graphs represent the ratio of the values for the immunoblots and their respective loading controls (Memcode stain). *P<0.05 and ^#P<0.001 for WT versus KO rats. Values are means ± SEM. N = 5–6 rats per group.

Fig 7. Akt2 phosphorylation on Ser⁴⁷⁴ (pAkt2^Ser474) and abundance of SGLT1, SERCA2, and CD36 in heart collected immediately after the hyperinsulinemic-euglycemic clamp performed in WT (open bars) and KO (filled bars) rats.

Bar graphs represent the ratio of the values for the immunoblots and their respective loading controls (Memcode stain). Values are means ± SEM. N = 5–6 rats per group.

Fig 8. 2-Deoxyglucose (2-DG) uptake by ex vivo incubated skeletal muscles (epitrochlearis and soleus) from WT and AS160-KO rats.

Paired muscles were incubated without (open bars) or with (filled bars) 500 μU/ml insulin. Data were analyzed by two-way ANOVA, and Holm-Sidak post hoc analysis was used to identify the source of significant variance. *P<0.001 (WT) and ^#P<0.05 (KO), no insulin versus insulin within the same genotype; ^‡P<0.001, WT versus KO with the same insulin concentration. A paired t-test revealed significantly greater glucose uptake with insulin versus without insulin in the KO soleus (P<0.05). Values are means ± SEM. N = 12–21 rats per group.

Fig 9. AS160 phosphorylation (pAS160 Thr⁶⁴²) and Akt phosphorylation (pAkt Ser⁴⁷³ and pAkt Thr³⁰⁸) in ex vivo incubated skeletal muscles (epitrochlearis and soleus) from WT and AS160-KO rats.

Paired muscles were incubated without (open bars) or with (filled bars) 500 μU/ml insulin. Bar graphs represent the ratio of the values for the immunoblots and their respective loading controls (Memcode stain). Fig 9 (A) (B), AS160 pThr⁶⁴² was undetectable in any of the tissues from KO rats. Data were analyzed by t-test. *P<0.05 and ^‡P<0.005, no insulin versus insulin in WT rats. Values are means ± SEM. N = 3–7 rats per group. Fig 9 (C) (D) (E) (F), *P<0.001, ^†P<0.05, no insulin versus insulin in rats with same genotype. Values are means ± SEM. N = 3 rats per group.

Fig 10. 2-Deoxyglucose (2-DG) uptake and phosphorylation of AMP-activated protein kinase (AMPK) on Thr¹⁷² (pAMPK Thr¹⁷²) in ex vivo incubated epitrochlearis from WT and AS160-KO rats.

Paired muscles were incubated without (open bars) or with (filled bars) 2 mM AICAR. Data were analyzed by two-way ANOVA, and Holm-Sidak post hoc analysis was used to identify the source of significant variance. (A) *P<0.01 for no AICAR versus AICAR within the same genotype; ^†P<0.01 for WT versus KO with the same AICAR concentration. Values are means ± SEM. N = 5–9 rats per group. (B) Bar graphs represent the ratio of the values for the immunoblots and their respective loading controls (Memcode stain). ^†P<0.01 (in WT) for no AICAR versus AICAR; ^‡P<0.005 for no AICAR versus AICAR in KO. Values are means ± SEM. N = 5–9 rats per group.