It is well known that CVD and NAFLD are major public health concerns globally with high morbidity and mortality. Both have been associated with elevated circulating levels of Cho and Hcy, an intermediate in Met metabolism. Though endogenous as well as dietary Cho sources contribute to circulating levels of Cho, non-pharmaceutical management (i.e., dietary approaches) is well-known for lowering Cho levels. With the emerging controversy about the role of Cho in CVD, it remains evident that elevated blood Cho can greatly affect liver function, as the liver is a main processing center for Cho [41]. Also, there are reports that point to the liver being affected by HChol more than the heart and that chronic liver disease can have a direct impact on heart function [42].
Our experimental approach was to feed a dietary excess of Cho and Met independently, but moreso in combination since studies on the combined effects are not as numerous. We also aimed to investigate the cardioprotective potential of sitagliptin, which is documented as improving cardiac function and ejection fraction. Sitagliptin is used in pharmacotherapy of glucose management in type II diabetics and has displayed positive effects (e.g., weight lowering, reduction of inflammation / oxidative stress / fibrotic responses) independent of glucose-lowering [43, 44, 45].
Adverse biochemical events occurring in the heart can result in conditions like arrhythmias, myocardial infarction, and heart failure eventually [46, 47, 48]. Diagnosis of such events requires a concerted effort that usually commences with cardiac function tests. This involves imaging techniques (e.g., echocardiograms, magnetic resonance imaging scans, computed tomography scans, nuclear cardiac stress test, coronary angiogram or left heart catheterization, X-rays, etc.), biopsies, and/or serological assays [49, 50, 51, 52, 53]. As it relates to a clinical diagnosis of acute myocardial infraction, elevated blood cTn-I protein levels are what typically aids that determination. It serves as indicator of cardiac structural damage [53, 54]. In our animal study, we assessed cardiac function primarily by histopathological evaluation and quantification of cTn-I protein in heart tissue. Cho feeding resulted in a significant loss of the protein, an effect that was exacerbated with sitagliptin – a novel finding contrary to our central hypothesis, which was to anticipate such an effect in MC-feeding and without sitagliptin administration. Similarly, Han et al. (2018) saw a reduction of cTn-I protein in heart tissue as a result of feeding male SD rats a high-fat, high Cho diet for 14 and 28 days. Interestingly, we observed an approximate 350% increase to cTn-I gene expression in HChol (+ sitagliptin), an effect that is possibly due to a compensatory response, as demonstrated by Sasse et al. (1993). Studies by Packer (2018) also show a positive correlation between DPP-4 inhibitor use and adverse cardiac events, citing their ability to cause and/or worsen heart failure. Rouse et al. (2014) and Shahbaz et al. (2018) correlate sitagliptin use with pancreatic injury and acute hepatitis, respectively.
Cho feeding in our study was shown to increase collagen deposition surrounding blood vessels of the heart, as well as within the interstitial spaces. Sitagliptin appears to exacerbate the effect to some degree. Notably, cardiac fibrosis is classified as either endomyocardial fibrosis, infiltrative & reactive interstitial fibrosis, or replacement fibrosis [61]. HChol (+/- sitagliptin) seems to have resulted in a form cardiac perivascular fibrosis, which is characterized by collagen accumulation around blood vessels [62, 63]. This is known to precede or coincide with reactive interstitial fibrosis - collagen accumulation that causes expansion of cardiac interstitial spaces with minor cardiomyocyte loss [62, 63]. Although the increase in collagen deposition by Cho may not seem unique, as this was demonstrated by Han et al. (2018), the seemingly sitagliptin exacerbation is interesting. A reason for such an observation could be due to sitagliptin’s interaction with Cho that affects some factor in Tgfβ signaling. Three isotypes of Tgfβ have been identified in mammals (Tgfβ1, Tgfβ2, Tgfβ3) and many animals studies identify type 1 as the “master regulator” that promotes fibrotic development in several tissues [64, 65, 66]. Tgfβ1 utilizes several signaling pathways to elicit a variety of actions (e.g., autophagy, differentiation, apoptosis, and cellular proliferation). However, it is the Smad-dependent (canonical) pathway that is most noted as resulting in fibrosis [64, 65, 66]. Sitagliptin could possibly stimulate the canonical pathway in some way, but this remains to be proven. Similar to cardiac smooth muscle, myofibroblasts in heart tissue express αSMA and are abundantly located in the thick myocardial layer. Myofibroblasts help to regulate various functions such as matrix deposition & degradation and growth-factor secretion [67]. Expression of αSMA and Tgfβ1 (gene and/or protein) was increased in HChol as well and exacerbated with sitagliptin.
Insight into the underlying molecular mechanisms by which adverse structural responses were seen in HChol, with and with sitagliptin administration, was investigated in our study. Biochemical changes are those precede structural changes in all cell types and are related to processes like oxidative stress and inflammation [68, 69]. Such changes could, in fact, be sex-specific, as Marques et al. (2018) discovered an association between increased IL-6 and C-reactive protein expression and the development of interstitial myocardial fibrosis in men. Additional literature also points to Tnfα and IL-1/6 being key mediators for myocardial alterations [71]. Serum Cho was increased approximately 100% in our rats due to Cho feeding - sitagliptin had no added effect on either decreasing or increasing serum Cho. This, however, resulted in a substantial increase in hepatic gene and protein expression of several biomarkers related to inflammation and oxidative stress, when rats were administered sitagliptin; Pathak et al. (2019), Kumar et al. (2020). Even though we observed significant increases to pro-inflammatory, pro-fibrotic, and Cho/fatty acid transport biomarkers in the heart (+ sitagliptin), they are far outweighed by those in the liver. This could be due to the liver’s increased exposure to compounds in the blood, since it primarily functions to metabolize, transport, and filter compounds that are absorbed and placed into circulation [72]. In either case of the liver or heart, sitagliptin was shown to enhance the adverse biochemical responses seen in HChol.
The addition of Met to the Cho diet did not produce an added adverse effect as originally hypothesized by our group. In fact, it proved beneficial by way of attenuating all adverse cardiac responses in HChol, bringing them closer to normal levels. This was interesting because Met restriction is outlined in literature as being beneficial, however, our results were on the contrary. We did not notice any obvious disruptions to Met metabolism, as Hcy levels and gene expression of the Met-metabolizing enzymes were unaffected. Unexpectedly, Met and MC feeding led to increased serum taurine levels. Taurine is a compound with anti-oxidative and anti-inflammatory effects, both of which could have contributed to the beneficial responses we observed [73]. In addition to taurine, there are other intermediates in Met metabolism that are documented to elicit multiple health benefits, i.e., anti-oxidation & -inflammation, vasodilation [73, 75]. Additional studies are needed to better understand this.
In summary, our study provides insight into the effects of DPP4-inhibitor use and atherogenic diets on the biochemical and structural changes of the heart. We demonstrated that sitagliptin administration exacerbates adverse cardiac responses seen in HChol, while also revealing the beneficial potential of high dietary Met to attenuate such effects. For a better understanding of this diet-drug relationship, additional studies are needed. The beneficial aspect of high dietary Met observed in our study merit mechanistic understanding for exploring future therapeutic options considering the public health relevance of CVD and are thus translational.