On-target action of anti-tropomyosin drugs regulates glucose metabolism

Figures

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  Figure 1. ATM-1001 targets Tpm3.1-containing actin filaments. (A) Structural diagram of the predicted molecular interactions of ATM-1001 and the C-terminus (last 37 amino acids) of the two Tpm3.1 helices. (B) ATM-1001 impact on Tpm3.1 filaments was quantitated in MEF cells using a High Content Imaging assay in combination with the CSIRO linear feature detection algorithm. Tpm3.1 filaments/cell area were quantitated using >300 cells (n = 3 independent experiments; ****p < 0.0001, T-test). (C) Depolymerization time course of actin filaments (35% pyrene labeled) with or without saturating amounts of Tpm3.1. Data: mean ± SEM, 4–6 replicates (***p < 0.001, T-test). (D) Tpm3.1 was pre-incubated with 50 μM ATM-1001 or 1% (v/v) DMSO prior to mixing with F-actin. Fluorescence data normalized to the T0 fluorescence and initial rates of depolymerisation (V0), calculated from the first 600 s, for F-actin alone or Tpm3.1/F-actin in the presence of DMSO or ATM-1001.

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  Figure 2. Anti-tropomyosin drugs have on-target impact on glucose clearance. (A) Experimental design to examine acute (single dose) impact of ATM drugs on glucose metabolism. (B) Glucose tolerance test (GTT; 1.5 g glucose/kg BW, i.p.) and (C) area-under-the-curve for the GTT, 1 h after TR100 (40 mg/kg BW, i.p.) or vehicle injection in wild-type (WT) and Tpm3.1 knock-out (KO) mice (n = 5/group). (D) Glucose tolerance test and (E) area-under-the-curve for the GTT, 1 h after ATM-1001 (40 mg/kg BW, i.p.) or vehicle injection in wild-type and Tpm3.1 KO mice (n = 6–7/group). Results are shown as mean ± SEM and the results of statistical analysis (ANOVA with post-hoc Tukey’s multiple comparison test) are indicated: ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 3. ATM drugs do not impact on whole body insulin sensitivity, but inhibit insulin-stimulated glucose uptake in skeletal muscle ex vivo. (A) Insulin tolerance test (ITT; 2U insulin/kg BW, i.p.) and (B) area-above-the-curve for the ITT, 1 h after TR100 (40 mg/kg BW, i.p.) or vehicle injection in wild-type (WT) and Tpm3.1 knock-out (KO) mice. (C) Insulin tolerance test and (D) area-above-the-curve for the ITT, 1 h after ATM-1001 (40 mg/kg BW, i.p.) or vehicle injection in WT and Tpm3.1 KO mice (n = 5–6/group). (E,F) Impact of TR100 (50 μM) or vehicle on insulin-stimulated glucose uptake in WT and Tpm3.1 KO (E) extensor digitorum longus (EDL) and (F) soleus muscle. (G,H) Impact of ATM-1001 (50 μM) or vehicle on insulin-stimulated glucose uptake in WT and Tpm3.1 KO (G) EDL and (H) soleus muscle. Results are shown as mean ± SEM (n = 6–8/group) and the results of statistical analysis (ANOVA with post-hoc Tukey’s multiple comparison test) are indicated: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 4. ATM compounds do not impact on insulin-stimulated Akt phosphorylation. Impact of 50 μM TR100 (A,B) and 50 μM ATM-1001 (C,D) on ex vivo insulin-stimulated phosphorylation of Akt (Ser473) in soleus (A,C) and extensor digitorum longus (EDL) muscle (B,D) from wild-type (WT) and Tpm3.1 knock-out (KO) mice. Representative blots of P-Akt and the house-keeping loading control protein, 14-3-3 are shown (same samples run on separate gels for Akt and 14-3-3) and as well as the quantitation of P-Akt for n = 5–7 mice per treatment group. Data are represented as mean ± SEM. Densitometry values on each gel were corrected for loading (pan-14-3-3) and then results are expressed as a ratio to an internal standard (insulin-stimulated muscle) run on each gel. There was a similar effect of insulin on Akt phosphorylation in both muscles from WT and Tpm3.1 KO mice with and without ATM compound (1-way ANOVA). (E) Western blots of total AKT in WT and Tpm3.1 KO Soleus and EDL muscle. Total AKT levels were similar in WT and Tpm3.1 KO soleus and EDL muscle. The blots for Akt and 14-3-3 were from the same samples run on separate gels.

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  Figure 5. ATM drugs impact on insulin secretion. (A and B) Changes in insulin levels during a glucose tolerance test (2 g glucose/kg BW, i.p.) in wild-type (WT) and Tpm3.1 knock-out (KO) mice (n = 5/group), 1 h after (A) TR100, (B) ATM-1001 (40 mg/kg BW, i.p.) or vehicle administration. Results of statistical analysis (ANOVA with post-hoc Tukey’s multiple comparison test) are indicated by: (1) *P < 0.05; **P < 0.01; ***P < 0.001, for drug- versus vehicle-treated mice of the same genotype; and (2) +P < 0.05; ++P < 0.01, for drug-treated WT versus drug-treated KO mice. (C and D) Insulin secretion in pancreatic islets isolated from WT and Tpm3.1 KO mice treated with increasing amounts of ATM-1001 (1 h treatment). There was no impact of ATM-1001 on (C) basal (5 mM glucose) secretion, but there was a dose-dependent suppression of (D) glucose-stimulated (20 mM glucose) insulin secretion with increasing concentration of drug in WT but not KO islets. Results are mean ± SEM from n = 5 islets/group. The results of statistical analysis (ANOVA with post-hoc Tukey’s multiple comparison test) are indicated: *P < 0.05; **P < 0.01, ***P < 0.001.

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  Figure 6. Tpm3.1 is at the cell cortex with actin and ATM drugs impact on insulin secretion by disrupting cortical actin filaments in β cells. (A) Representative immunofluorescent confocal images of Tpm3.1 (γ/9d antibody) and insulin and (B) Tpm3.1 and filamentous actin (F-actin; Phalloidin stain) in wild-type mouse pancreas. Tpm3.1 is concentrated at the cell cortex in β cells (upper images) colocalized with F-actin (magnified insets in B). Scale bars = 20 μm. (C) Representative confocal images of MIN-6 cells at the cell centre, treated with vehicle, TR100 or ATM-1001 (1 h) in the presence of with basal (2.8 mM) or high (25 mM) glucose and stained for F-actin (Phalloidin) after 20 min glucose incubation. In vehicle-treated cells, there was a loss of F-actin at the cell cortex in cells in high versus basal glucose. TR100 and ATM-1001 disrupted actin at the cell cortex in both basal and high glucose conditions. (D) Insulin secretion in MIN6 β cells treated with increasing amounts of TR100 and ATM-1001. Cells were incubated with basal (2.8 mM) or high (25 mM) glucose in the presence of vehicle or ATM drug and insulin secretion measured over a 1 h period. There was a significant increase in…