Amyloidogenic proteins are a class of polypeptides that tend to misfold and self-assemble into oligomeric species called amyloid fibrils (Chisholm and Hunter, 2024, Ke et al., 2020). A number of diseases, including neurodegenerative diseases like Alzheimer’s and Parkinson’s and metabolic diseases like Type 2 Diabetes Mellitus (T2DM), have been associated with the aggregation and deposition of different amyloidogenic proteins (Almeida and Brito, 2020). One such amyloidogenic protein is the human Islet Amyloid polypeptide (hIAPP) or amylin, which has been associated with pancreatic β-cell cytotoxicity, inflammation, and dysfunction in T2DM – the major form of diabetes (Hsu et al., 2024, Roham et al., 2022). T2DM is characterized by defects in insulin secretion by pancreatic β-cells and the development of insulin resistance (IR) due to which insulin-responsive tissues, like adipose tissue, liver and skeletal muscles do not respond appropriately to insulin, leading to the development of hyperglycemia (Galicia-Garcia et al., 2020). Moreover, the formation of these amyloidogenic hIAPP aggregates has also been associated with the development of hyperglycemia (Chaari and Ladjimi, 2019, Dubey et al., 2017, Roham et al., 2022).
hIAPP, a hormone involved in maintaining glucose homeostasis, is normally co-synthesized and co-secreted by the pancreatic β-cells along with insulin (Roham et al., 2022). It is a 37 amino acid long peptide that is soluble in its monomeric state where it exists as a random coil (Marzban et al., 2003). However, various regions in this protein, including the amino acid residues from position 20–29 have been found to be amyloidogenic (Akter et al., 2016, Jaikaran and Clark, 2001). Therefore, hIAPP can undergo a conformational change to β-sheet structure from the unfolded state (Brender et al., 2012, Marzban et al., 2003) and aggregate into cross β-fibrils in a sequential manner following sigmoidal kinetics (Caillon et al., 2016). It is important to note that the C-terminus of the mature peptide is amidated; however, studies have shown that the variants with a free C-terminus are also cytotoxic and form amyloids (Dubey et al., 2017). The cellular cues for the conversion of hIAPP monomers into toxic oligomers in the pathogenesis of T2DM are not clearly understood (Hsu et al., 2024). Both insulin and pro-insulin are known to inhibit hIAPP aggregation and therefore it has been proposed that insulin-resistant conditions could stimulate hIAPP aggregation (Alrouji et al., 2023, Brender et al., 2011). Previous studies have shown that the presence of zinc in the secretary granules could also prevent hIAPP fibril formation (Khemtemourian et al., 2021). On the other hand, the exposure to high glucose and fatty acid (glucolipotoxic conditions) associated with T2DM tends to activate the hIAPP promoter increasing its expression and glycation, while decreasing insulin expression. Together these conditions stimulate the aggregation of hIAPP (Jaikaran and Clark, 2001, Roham et al., 2022). Plasma components, including LDL and sugars also interact with hIAPP, forming further toxic oligomers (Rodriguez Camargo et al., 2018). In addition, defects in the cellular processing of hIAPP by the proteasome and autophagy machinery also contribute positively towards the accumulation of hIAPP aggregates (Milardi et al., 2021).
Skeletal muscles are the major sites for glucose utilization, accounting for ∼ 60–70 % of whole-body glucose uptake during the post-prandial period, and are therefore important players in the development of insulin resistance (Karlsson and Zierath, 2007). hIAPP deposits have also been reported in tissues like RBCs (Verma et al., 2020), kidneys (Gong et al., 2007) and the brain (Jackson et al., 2013) in addition to pancreatic islets and are also known to co-aggregate with amyloid β (Leibold and Despa, 2024). Also, in vitro and in vivo studies show that hIAPP induces the development of insulin resistance in peripheral tissues like the liver (Molina et al., 1990, Ohsawa et al., 1989); stimulates glycogenolysis and gluconeogenesis with no significant effect on glycogen synthesis in the liver (Ciaraldi et al., 1992); inhibits insulin-stimulated glycogen synthesis in rat soleus muscles (Leighton and Cooper, 1988); and reduces glucose uptake and muscle glycogen synthesis (Frontoni et al., 1991). However, there are limited studies evaluating the possible role of hIAPP in inducing the development of insulin resistance in skeletal muscles. To the best of our knowledge, no studies to date (September, 2024) have successfully identified the events that are associated with the development of insulin resistance in skeletal muscles due to hIAPP aggregation.
The incidence of T2DM is on the rise and has been associated with changes in lifestyle, over-nutrition, stress, and lack of physical activity, to name a few (Pradeepa and Mohan, 2021). The situation has been further complicated due to the lack of early and specific biomarkers to detect the same (Zimmet et al., 2001). Metabolomics is the youngest omics platform after genomics, transcriptomics and proteomics, which involves the detection of small molecular weight metabolites from body fluids, cells and their extracts, tissues, etc. (Trifonova et al., 2023). These metabolites are the end products of different biochemical processes that are highly dynamic and get affected by both genetic and environmental changes. Hence, these can reveal the changes that occur in response to pathophysiological conditions, thereby serving as biomarkers for different diseases (Clish, 2015, Gowda et al., 2008). Indeed, metabolomics investigations done in the past have identified elevated levels of branched-chain amino acids (BCAAs) in T2DM (Giesbertz et al., 2015) and have proposed elevated glutamine, BCAAs (Klén et al., 2020), C18:0 sphingolipids (Tonks et al., 2016) and dimethylarginine (Lee et al., 2018) levels as biomarkers of insulin resistance in skeletal muscles.
In the present study, using different cellular and biochemical assays, we have established that exposure to hIAPP induces insulin resistance in L6 myotubes that is associated with cytotoxicity and build-up of oxidative stress. We then attempted to identify the metabolic perturbations associated with the development of insulin resistance and identified metabolites, including o-phosphocholine, sn-glycero-3-phosphocholine, and dimethylamine, that were significantly perturbed upon hIAPP exposure. We propose that the results obtained from this study would help understand the major metabolic pathways associated with protein misfolding. Thus these pathways can be used to identify early biomarkers to predict the onset of insulin resistance and T2DM.