Fig 2: Effect of cadmium and/or linagliptin administration on testicular AMPK/mTOR in rats. The co-treatment with linagliptin stimulated the testicular pro-autophagic AMPK/mTOR pathway that was suppressed by cadmium metal. This was demonstrated by increased phosphorylation of the AMP-activated protein kinase (AMPK; Ser487) to the total AMPK protein (A) together with the diminished phosphorylation of the mammalian target of rapamycin mTOR (Ser2448) to the total mTOR protein (B). * p < 0.05, and *** p < 0.001 show statistical significance compared to the group of control rats; ## p < 0.01 show statistical significance compared to the group of cadmium-intoxicated rats. Values are stated as the mean ± standard error of the mean for 6 animals per group. LIN, linagliptin; Cd, cadmium chloride.

Fig 3: Effect of cadmium and/or topiramate on AMPK/mTOR pathway in testes of rats. The co-treatment with topiramate activated the testicular pro-autophagy AMPK/mTOR pathway that was inhibited by cadmium administration. This was manifested by increased p-AMPK (Ser487)/total AMPK ratio [H (3, 20) = 5.71, p = 0.0054] (A) and lowered p-mTOR (Ser2448)/total mTOR ratio [H (3, 20) = 18.83, p < 0.0001] (B). Results are demonstrated as the mean ± standard error of the mean (SEM) for six rats in each experimental group. * p < 0.05, ** p < 0.01, **** p < 0.0001 signifies statistical significance versus the control group; # p < 0.05, ## p < 0.01 signifies statistical significance versus cadmium chloride-treated group. AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin. TPM, topiramate; Cd, cadmium chloride.

Fig 4: An outline of the mechanisms that intercede linagliptin’s amelioration of cadmium-evoked testicular impairment. According to the current findings, the proposed mechanisms are: (A,A′) Curbing the inflammatory responses, including HMGB1/TLR4/NF-κB and NLRP3/caspase-1/IL-1β pathways in the testes of cadmium-intoxicated rats. (B) Stimulation of the autophagy response with activation of AMPK/mTOR pathway in the testes of cadmium-intoxicated rats. (C) Interference with the pro-apoptotic responses, including Bax downregulation, Bcl2 upregulation, and caspase 3 inhibition in the testes of cadmium-intoxicated rats. Of note, the beneficial outcomes of linagliptin are mainly due to DPP-4 inhibition, which has been linked to marked anti-inflammatory/immune-modulatory actions [21,22]. DPP-4 inhibition triggers several downstream targets, including the upregulated protein expression of SDF-1α, which prompts intensified testicular repair and regeneration [21,33]. Moreover, the beheld activation of autophagy may suppress the pro-inflammatory responses/NLRP3 inflammasome activation [12,20] and limit the proapoptotic responses [12,17,19] in the testes of cadmium-intoxicated rats. Activation is represented by solid arrows, whereas inhibition is depicted by blunt arrows. The question marks denote unknown mechanisms.

Fig 5: Dapagliflozin rescued impaired hippocampal autophagy events in cadmium-intoxicated rats. (A) The protein expression of sequestosome-1/protein 62 (SQSTM-1/p62) was reduced by dapagliflozin. (B) The protein expression of Beclin 1 was enhanced by dapagliflozin. Moreover, dapagliflozin elevated the phosphorylated adenosine-monophosphate-activated protein kinase (p-AMPK)/total AMPK (C) and diminished the phosphorylated mechanistic target of rapamycin (p-mTOR)/total mTOR in the hippocampus tissue (D), indicating that AMPK/mTOR pathway was stimulated. The mean ± standard error (SE) was presented in the graph (n = 6). Statistical comparisons versus the control group were demonstrated by **** p < 0.0001, *** p < 0.001, or ** p < 0.01. Statistical comparisons versus the Cd group were demonstrated by ### p < 0.001, ## p < 0.01, or # p < 0.05 (Bonferroni’s test and one-way ANOVA). Cd, cadmium chloride (5 mg/kg/day, via gavage for 2 months); DPG, dapagliflozin (1 mg/kg/day, by gavage for 2 months).