Empagliflozin mitigates type 2 diabetes-associated peripheral neuropathy: a glucose-independent effect through AMPK signaling

Abstract

Diabetic peripheral neuropathy (DPN) represents a severe microvascular condition that dramatically affects diabetic patients despite adequate glycemic control, resulting in high morbidity. Thus, recently, anti-diabetic drugs that possess glucose-independent mechanisms attracted attention. This work aims to explore the potentiality of the selective sodium-glucose cotransporter-2 inhibitor, empagliflozin (EMPA), to ameliorate streptozotocin-induced DPN in rats with insight into its precise signaling mechanism. Rats were allocated into four groups, where control animals received vehicle daily for 2 weeks. In the remaining groups, DPN was elicited by single intraperitoneal injections of freshly prepared streptozotocin and nicotinamide (52.5 and 50 mg/kg, respectively). Then EMPA (3 mg/kg/p.o.) was given to two groups either alone or accompanied with the AMPK inhibitor dorsomorphin (0.2 mg/kg/i.p.). Despite the non-significant anti-hyperglycemic effect, EMPA improved sciatic nerve histopathological alterations, scoring, myelination, nerve fibers' count, and nerve conduction velocity. Moreover, EMPA alleviated responses to different nociceptive stimuli along with improved motor coordination. EMPA modulated ATP/AMP ratio, upregulated p-AMPK while reducing p-p38 MAPK expression, p-ERK1/2 and consequently p-NF-κB p65 as well as its downstream mediators (TNF-α and IL-1β), besides enhancing SOD activity and lowering MDA content. Moreover, EMPA downregulated mTOR and stimulated ULK1 as well as beclin-1. Likewise, EMPA reduced miR-21 that enhanced RECK, reducing MMP-2 and -9 contents. EMPA's beneficial effects were almost abolished by dorsomorphin administration. In conclusion, EMPA displayed a protective effect against DPN independently from its anti-hyperglycemic effect, probably via modulating the AMPK pathway to modulate oxidative and inflammatory burden, extracellular matrix remodeling, and autophagy.

Keywords: AMPK; Diabetic peripheral neuropathy; Dorsomorphin; Empagliflozin; mTOR; p38 MAPK.

Fig. 1

Experimental design

Fig. 2

Effect of EMPA on body weight and blood glucose level in STZ-induced DPN in rats. Panels represent (a) % change in body weight and (b) fasting blood glucose level. Every bar with a vertical line displays the mean ± S.D (n = 10). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; STZ: streptozotocin

Fig. 3

Effect of EMPA on motor and sensory performance in STZ-induced DPN in rats. Panels represent: (a) rotarod fall-off latency, (b) Randell-Selitto mechanical withdrawal threshold, (c) cold allodynia hind paw withdrawal latency, and (d) hot plate reaction latency. Every bar with a vertical line displays the mean ± SD (n = 8-10). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; STZ: streptozotocin

Fig. 4

Effect of EMPA on the histopathological alterations of the sciatic nerves in STZ-induced DPN in rats. (a) Sections of sciatic nerves stained with hematoxylin and eosin. CONT section showing myelinated nerve fibers (arrow) and scattered Schwann cells (arrowhead). STZ section showing myelin sheath loss (dashed arrows), Schwann cells loss (arrowhead), and inflammatory cells (red arrow). EMPA section showing myelinated nerve fibers (arrow) and scattered Schwann cells (arrowhead). DORS+EMPA section showing edema (dashed arrows) and mild loss of Schwann cells (arrowhead) (Scale bar is 50 μm). (b) Pathological scoring. Every bar with a vertical line displays the median ± range (n= 4). (c) Sections of sciatic nerves stained with toluidine blue (Scale bar is 50 μm). (d) Nerve fibers' count. Every bar with a vertical line displays the mean ± SD (n=3). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; STZ: streptozotocin

Fig. 5

Effect of EMPA on the micromorphological alterations of the sciatic nerves in STZ-induced DPN in rats. (a) Sections of sciatic nerves stained with uranyl acetate and lead citrate. CONT section showing intact well-formed myelin sheath (arrow). STZ section showing deformed nerve fibers, axonal atrophy, and delamination of myelin lamellae (arrow). EMPA section showing almost well-formed axon with an intact myelin sheath. DORS+EMPA section showed axonal damage with areas of myelin loss (arrow) (Scale bar is 2 μm). (b) The ratio of the axon to myelin sheath areas. CONT: control; DORS: dorsomorphin; EMPA: empagliflozin; STZ: streptozotocin

Fig. 6

Effect of EMPA on ATP/AMP ratio and AMPK expression in the sciatic nerves in STZ-induced DPN in rats. Panels represent: (a) ATP/AMP ratio, (b) protein expression of p-AMPK (Thr172), and (c) corresponding p-AMPK western blotting bands. Results are displayed as mean ± SD (n= 3-6). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. AMP: adenosine monophosphate; AMPK: adenosine monophosphate kinase; ATP: adenosine triphosphate; CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; STZ: streptozotocin

Fig. 7

Effect of EMPA on NF-κB p65, p38 MAPK, ERK1/2, RECK, and miR-21 expressions in the sciatic nerves in STZ-induced DPN in rats. Panels represent protein expression of (a) p-NF-κB p65/total NF-κB p65, (b) p-p38 MAPK/total p38 MAPK, (c) p-ERK1/2/total ERK1/2, (d) RECK, and (e) corresponding western blotting bands along with relative expression of (f) miR-21. Results are displayed as mean ± SD (n = 3-4). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; ERK: extracellular signal-regulated kinases; MAPK: mitogen-activated protein kinase; miR: micro-RNA; NF-κB: nuclear factor kappa-B; RECK: reversion-inducing cysteine-rich protein with Kazal motifs; STZ: streptozotocin

Fig. 8

Effect of EMPA on MMP-2 and MMP-9 expression in the sciatic nerves in STZ-induced DPN in rats.Representative photomicrographs depicting (a) MMP-2 and (b) MMP-9 immunohistochemical staining in sciatic nerves (Scale bar is 50 μm). Panels represent the corresponding area % of (c) MMP-2 and (d) MMP-9 immunoexpression. Results are displayed as mean ± SD (n = 4). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; MMP: metalloproteinase; STZ: streptozotocin

Fig. 9

Effect of EMPA on mTOR and ULK1 expressions and beclin-1 content in the sciatic nerve in STZ-induced DPN in rats. Panels represent protein expression of (a) mTOR and (b) ULK1 (Ser317), (c) corresponding mTOR and ULK1 western blotting bands, and (d) beclin-1 content. Results are displayed as mean ± SD (n = 3-6). (*) vs CONT, (@) vs STZ, (#) vs EMPA; P < 0.05. Beclin-1: mammalian orthologue of yeast Atg6; CONT: control; DPN: diabetic peripheral neuropathy; DORS: dorsomorphin; EMPA: empagliflozin; mTOR: mammalian target of rapamycin; STZ: streptozotocin; ULK1: Unc-51 like autophagy activating kinase 1