Hindlimb immobilization induces insulin resistance and elevates mitochondrial ROS production in the hippocampus of female rats

Abstract

Alzheimer's disease (AD) is the fifth leading cause of death in older adults, and treatment options are severely lacking. Recent findings demonstrate a strong relationship between skeletal muscle and cognitive function, with evidence supporting that muscle quality and cognitive function are positively correlated in older adults. Conversely, decreased muscle function is associated with a threefold increased risk of cognitive decline. Based on these observations, the purpose of this study was to investigate the negative effects of muscle disuse [via a model of hindlimb immobilization (HLI)] on hippocampal insulin sensitivity and mitochondrial function and identify the potential mechanisms involved. HLI for 10 days in 4-mo-old female Wistar rats resulted in the following novel findings: 1) hippocampal insulin resistance and deficits in whole body glucose homeostasis, 2) dramatically increased mitochondrial reactive oxygen species (ROS) production in the hippocampus, 3) elevated markers for amyloidogenic cleavage of amyloid precursor protein (APP) and tau protein in the hippocampus, 4) and reduced brain-derived neurotrophic factor (BDNF) expression. These findings were associated with global changes in iron homeostasis, with muscle disuse producing muscle iron accumulation in association with decreased serum and whole brain iron levels. We report the novel finding that muscle disuse alters brain iron homeostasis and reveal a strong negative correlation between muscle and brain iron content. Overall, HLI-induced muscle disuse has robust negative effects on hippocampal insulin sensitivity and ROS production in association with altered brain iron homeostasis. This work provides potential novel mechanisms that may help explain how loss of muscle function contributes to cognitive decline and AD risk.NEW & NOTEWORTHY Muscle disuse via hindlimb immobilization increased oxidative stress and insulin resistance in the hippocampus. These findings were in association with muscle iron overload in connection with iron dysregulation in the brain. Overall, our work identifies muscle disuse as a contributor to hippocampal dysfunction, potentially through an iron-based muscle-brain axis, highlighting iron dysregulation as a potential novel mechanism in the relationship between muscle health, cognitive function, and Alzheimer's disease risk.

Keywords: brain insulin resistance; hippocampus; iron overload; muscle disuse; muscle-brain axis.

Graphical abstract

Figure 1.

Hindlimb immobilization (HLI) induces loss of body weight and severe muscle atrophy without altering stress markers. A: body weights before and after the 10-day HLI intervention in Ctrl (n = 12), Ctrl Cast (n = 12), and HLI (n = 12) groups. Muscle wet weight of the soleus (B), plantaris (C), gastrocnemius (D), quadriceps (Quad; E), tibialis anterior (TA; F), and extensor digitorum longus (EDL; G) normalized to body weight following HLI. H: adrenal wet weights normalized to body weight following HLI. I: serum corticosterone levels following HLI (n = 6/group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2.

Muscle excitability, neuromuscular junction (NMJ) transmission, and mitochondrial function of hindlimb muscles are greatly reduced following 10 days of muscle disuse. Electrophysiological measurements of compound muscle action potential (CMAP; A) and repetitive nerve stimulation (RNS; B) pre- and post-hindlimb immobilization (HLI) (n = 8, RNS pre-HLI n = 5). C: Western blot images of the mitochondrial complexes in the soleus muscle of Ctrl Cast and HLI rats. D: quantification of the mitochondrial complexes expressed as fold change relative to Ctrl Cast (n = 6). E: Pgc1α levels in Ctrl Cast and HLI groups (n = 6). *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 3.

Muscle disuse disrupts systemic glucose homeostasis and produces insulin resistance in the hippocampus. A: glucose tolerance test (GTT) on day 7 of hindlimb immobilization (HLI). B: AUC GTT between Ctrl Cast and HLI groups. C: detection of membrane insulin receptor expression in the soleus via radioligand binding following 10 days of HLI. D: measuring membrane insulin receptor expression in the hippocampus via radioligand binding following 10 days of HLI. E: Western blot images of Akt and p-Akt. F–H: quantification of Akt, p-Akt (Ser 473), and p-Akt:Akt ratio protein levels. *P < 0.05.

Figure 4.

Hippocampal mitochondrial reactive oxygen species (ROS) production is significantly increased following 10 days of muscle disuse. A: high-resolution respirometry across various states of respiration in mitochondria isolated from the hippocampus. B: H₂O₂ emissions under basal respiration conditions. C: H₂O₂ emissions during State 2 respiration. D and E: expression of Sod1 (D) and Nrf2 (E) in the DG region of the hippocampus. F: representative Western blot images of GPX4 and catalase. G and H: quantification of GPX4 (G) and catalase (H) protein levels. n = 6/group for all measurements. *P < 0.05; **P < 0.01.

Figure 5.

Hindlimb immobilization induced pathological APP cleavage, elevated Tau, reductions in brain-derived neurotrophic factor (BDNF), and dysregulated AMPA receptor function. A: Western blot images of Alzheimer’s disease (AD)-related proteins. B: mRNA expression for the APP-processing protein, BACE1. Protein levels of APP (C) and its cleavage product, AICD (D). Protein levels of Tau (E) and pTau (F). G: transcript levels of the coding exon for BDNF, exon IX. GluA1 protein levels (H) and its phosphorylation status at S831 (I). J: the ratio of total GluA1 to phosphorylated GluA1. K: GluA2 protein levels. n = 6/group for all measurements. *P < 0.05; **P < 0.01.

Figure 6.

Hindlimb immobilization (HLI) for 10 days induced signs of iron overload in the soleus and iron dysregulation in the serum and brain. A: Western blot images for proteins involved in iron storage [ferritin heavy chain 1 (FTH1)], uptake [transferrin receptor 1 (TFR1)], and export [ferroportin 1 (FPN1)] in the soleus. B: fold change protein levels of FTH1, TFR1, and FPN1 in the soleus (n = 6/group). C: fold change transcript levels of Fpn1 in the soleus (n = 6/group). D and E: total iron concentrations using inductively coupled plasma optical emission spectroscopy (ICP-OES) in the soleus and serum, respectively (n = 8/group). F: Western blot images for FTH1, TFR1, and FPN1 proteins in the dentate gyrus (DG). G: fold change protein levels of FTH1, TFR1, and FPN1 in the DG (n = 6/group). H: fold change transcript levels of Fpn1 in the DG (n = 6/group). I: whole brain total iron concentrations using ICP-OES (n = 8/group). J: correlation between soleus and brain iron concentrations. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 7.

Muscle disuse for 5 days induced iron accumulation in the soleus prior to significant muscle atrophy. A and B: body weight and soleus wet weight following 5 days of hindlimb immobilization (HLI) (n = 8/group). C: representative Western blot images of FTH1, TFR1, and FPN1 protein expression in the soleus. D: fold change protein levels of ferritin heavy chain 1 (FTH1), transferrin receptor 1 (TFR1), and ferroportin 1 (FPN1) in the soleus in response to 5 days of HLI (n = 8/group). ****P < 0.0001.