Dysregulation of Metabolic Peptides Precedes Hyperinsulinemia and Inflammation Following Exposure to Rotenone in Rats

Abstract

Rotenone, a naturally occurring compound derived from the roots of tropical plants, is used as a broad-spectrum insecticide, piscicide, and pesticide. It is a classical, high-affinity mitochondrial complex I inhibitor that causes not only oxidative stress, α-synuclein phosphorylation, DJ-1 (Parkinson's disease protein 7) modifications, and inhibition of the ubiquitin-proteasome system but it is also widely considered an environmental contributor to Parkinson's disease (PD). While prodromal symptoms, such as loss of smell, constipation, sleep disorder, anxiety/depression, and the loss of dopaminergic neurons in the substantia nigra of rotenone-treated animals, have been reported, alterations of metabolic hormones and hyperinsulinemia remain largely unknown and need to be investigated. Whether rotenone and its effect on metabolic peptides could be utilized as a biomarker for its toxic metabolic effects, which can cause long-term detrimental effects and ultimately lead to obesity, hyperinsulinemia, inflammation, and possibly gut-brain axis dysfunction, remains unclear. Here, we show that rotenone disrupts metabolic homeostasis, altering hormonal peptides and promoting infiltration of inflammatory T cells. Specifically, our results indicate a significant decrease in glucagon-like peptide-1 (GLP-1), C-peptide, and amylin. Interestingly, levels of several hormonal peptides related to hyperinsulinemia, such as insulin, leptin, pancreatic peptide (PP), peptide YY (PYY), and gastric inhibitory polypeptide (GIP), were significantly upregulated. Administration of rotenone to rats also increased body weight and activated macrophages and inflammatory T cells. These data strongly suggest that rotenone disrupts metabolic homeostasis, leading to obesity and hyperinsulinemia. The potential implications of these findings are vast, given that monitoring these markers in the blood could not only provide a crucial tool for assessing the extent of exposure and its relevance to obesity and inflammation but could also open new avenues for future research and potential therapeutic strategies.

Keywords: diabetes; hormonal peptides; hyperinsulinemia; inflammation; obesity; rotenone.

Figure 1

Schematic representation of the experimental design. Young adult Lewis rats received vehicle or rotenone (2 mg/kg body weight) in respective treatment groups. Rotenone treatment groups received 10 sub-cutaneous injections of rotenone (total 20 mg/kg body weight). The change in body weight was measured before the treatment (C-pre-treatment, and R-pre-treatment) and one month post-treatment (C-post-treatment and R-post-treatment). Rats were sacrificed one month post-treatment, and samples were collected.

Figure 2

Alterations of hormonal peptides GIP and GLP-1 in rats following rotenone administration. Plasma samples from control and rotenone-treated rats were analyzed by Metabolic Hormone 10-Plex Discovery Assay. (A) GIP level shows a highly significant increase (p = 0.0003) in the plasma of rotenone-treated rats compared to the control rats. (B) Analysis of GLP-1 showed a significant reduction of this metabolic peptide in the plasma of rotenone-treated rats (p = 0.0045) compared to the control group. N = 5–8.

Figure 3

Rotenone administration elevated anorectic gut hormones, pancreatic polypeptide and peptide YY, in rats. Both pancreatic polypeptide (A) and peptide YY (B) hormonal peptides were significantly increased (p < 0.0001) in the plasma of the rotenone-treated group compared to the control group. N = 6–9.

Figure 4

Effects of rotenone on incretin levels in rat plasma one month post-treatment. (A) Metabolic profile in the plasma demonstrates a highly significant increase (p < 0.0001) in the insulin level following rotenone treatment compared to the control group. (B) Meanwhile, the C-peptide level in rotenone-treated rats was significantly decreased (p < 0.05). (C) Amylin levels were also decreased significantly (p < 0.0008) in the rotenone group compared to the control group. (D) However, the plasma level of leptin hormone was increased significantly (p < 0.001) in rotenone-treated rats compared to the control group. These data suggest that rotenone disrupts incretin levels, promoting inflammation and insulinemia. N = 6–9.

Figure 5

Rotenone treatment increased rat body weight. The change in body weight was measured before the treatment (C-pre-treatment and R-pre-treatment) and one month post-treatment (C-post-treatment and R-post-treatment). The data showed that the rats in the rotenone post-treatment (R-post-treatment) group gained substantial body weight, and it was significantly increased (p < 0.0001) compared to before starting the rotenone treatment (R-pre-treatment) and to the post-vehicle control (C-post-treatment) group (p < 0.005). Body weights of the control pre-treatment and the rotenone pre-treatment group are not significantly different (p < 0.05) at the beginning of the experiment. Moreover, no significant difference (ns) in body weights was detected in the control groups, C-pre-treatment and C-post-treatment. N = 3–7.

Figure 6

Administration of rotenone promoted activation of macrophages in rats. (A) Immunostaining of CD68 in cryosections of spleens from control and rotenone treatment groups. There was a distinct upregulation of CD68-positive cells in the rat spleen’s red pulp (RP) area following rotenone treatment compared to the vehicle treatment control group. (B) Counting of CD68-stained cells by ImageJ software showed a significant increase (p < 0.05) in CD68-positive cells in the rotenone treatment group compared to the control. N = 4.

Figure 7

Rotenone treatment promoted activation of inflammatory CD4⁺ T cells in rats. (A) Immunostaining for the presence of CD4 and TNF-α in spleens from control and rotenone treatment groups counterstained with DAPI. Expansion of CD4⁺ T cells in the spleen following rotenone treatment was noticeable compared to control. These CD4⁺ T cells also expressed TNF-α, indicating inflammatory changes in the rotenone-treated animals. (B,C) Counting of cells expressing CD4 (B) and CD4/TNF-α markers (C). The rotenone treatment group showed a significant increase (p < 0.01) in the CD4⁺ T cell population in the spleen compared to the spleens from the control group. Moreover, these CD4⁺ T cells also co-expressed increased TNF-α in rotenone-treated rats compared to the control group (p < 0.005). N = 4.

Figure 8

Administration of rotenone disrupts metabolic hormones and influences gut–brain axis and hyperinsulinemia. The observed low levels of GLP-1 and amylin indicate a potential dysregulation of appetite and blood sugar levels. This dysregulation may be associated with increased body weight and hyperinsulinemia. Furthermore, the hyperinsulinemia observed in the rotenone-treated rats indicates that the peripheral toxicity caused by rotenone may have disrupted regulation of insulin secretion mediated by dopamine. Red arrows indicate levels and pathways affected by rotenone treatment. Black arrows indicate the cell types and the metabolic hormones they secret.