**5. Strategies to Improve Metabolic Outcomes through Nutraceuticals**

#### *5.1. The Influence of Diet on Antioxidants*

Fresh fruit and vegetable intake represent the largest source of dietary antioxidants that are essential in maintaining health [142]. A great deal of research has evaluated fruit and vegetable intake as a means to counter the inflammation that has been attributed to nearly all chronic diseases of modern society, with many studies demonstrating a strong inverse correlation between fruit and vegetable intake and inflammatory markers [143,144]. Much of the research concerning dietary antioxidants and inflammation has centered on the Mediterranean diet due to its emphasis on fresh fruit and vegetable intake. Both cross-sectional and longitudinal trials have demonstrated a substantial lowering effect for the Mediterranean diet on a wide variety of inflammatory markers, including IL-6, IL-7, IL-19, CRP, and TNFα [145–147]. This has prompted the use of the Mediterranean diet in hopes of managing metabolic and vascular diseases, endocrine disorders, and some cancers [148–150]. Elucidation of specific antioxidants, and the benefits of dietary supplementation, have been a focus for many current research studies, giving rise to various nutraceuticals. Below, we describe the effects of such nutraceuticals and their beneficial actions on the HO-1 pathway and inflammation.

#### *5.2. The Benefits of Moderately Raising Plasma Bilirubin*

Natural substances that raise plasma bilirubin have been of interest for reducing adiposity [63]. One herbal method that is gaining interest in elevating plasma bilirubin is the use of the milk thistle plant (*Silybum marianum*) [63]. The plant contains a mix of polyphenols such as p-coumaric, vanillic acid, silybin, and α-tocopherol [151,152]. The primary compound in milk thistle that is considered the active component that increases plasma bilirubin is the silymarin flavonoids that suppress hepatic UGT1A1 [153]. Silymarin may protect against liver injury and hepatic fat accumulation [154–156]. However, how milk thistle or silymarin might function combined with exercise in reducing adiposity is unknown.

Bilirubin is a potent endogenous antioxidant that the body uses to support oxidative balance [17,41,42,119–122]. Plasma bilirubin has been empirically correlated with decreased risk for oxidative disorders such as coronary artery disease (CAD) [42,119]. The theory of action stemmed from individuals with Gilbert Syndrome, who have a mutation in the UGT1A1 gene, which causes defective processing of bilirubin [47,126]. Hence, lower hepatic UGT1A1 causes higher plasma unconjugated bilirubin [47]. Individuals with Gilbert Syndrome were found to have decreased incidence of CAD [157]. Previous studies postulate that unconjugated bilirubin is fluxing back into cells and acting as a scavenging agent of oxidative radicals. However, researchers have also hypothesized that elevated serum bilirubin acts as a marker that could predict greater expression or inducibility of intracellular HO-1, which will increase the intracellular concentration of bilirubin [132]. Using HPLC-TLS, these researchers detected bilirubin levels within vascular endothelial cells. They also showed that bilirubin within these vascular endothelial cells could effectively modulate HO-1 upregulation [158]. These findings suggest strong potential for developing pharmacotherapeutics that can target and upregulate this intrinsic antioxidant system within the vascular endothelium through the induction of HO-1 and help prevent or follow the progression of cardiovascular disease. This has the potential to lend more focused antioxidant and anti-inflammatory therapeutic approaches.

#### *5.3. Vitamin D Repletion*

Vitamin D is an important secosteroid in understanding metabolic disease [159]. Vitamin D deficiency (defined as a 25(OH)D level less than 20 ng/dL) is common and is associated with decreased muscle endurance, function, and strength [160–167]. Vitamin D deficiency is connected to muscle metabolic perturbations, including insulin resistance [168–170], and is linked to mitochondrial dysfunction [171] in both young and aged adults. Vitamin D deficiency is highly prevalent in obesity without vitamin D supplementation [172,173]. Obese adults are commonly prescribed a high-dose vitamin D repletion protocol to combat vitamin D deficiency and obesity-associated vitamin D resistance. Aggressive vitamin D repletion to correct the deficiency is linked to improved muscle mitochondrial function [171,174]. Increasing vitamin D status is consistently associated with skeletal muscle lipid deposition and distribution [175–178]. There is also evidence that vitamin D may improve hepatic steatosis with just 4-weeks of supplementation [179].

Calcitriol, the active form of vitamin D [1,25(OH)2D3], is the only form that can bind to the Vitamin D receptor (VDR). The VDR is a nuclear receptor transcription factor that controls gene expression changes that improve mitochondrial function in myotubes [180], insulin sensitivity, and myocellular lipid partitioning in high fat-treated SkM cells [181]. In humans, we found that vitamin D combined with aerobic exercise potentiated the metabolic benefit of training by producing the most intramyocellular lipid (IMCL) loss and increasing skeletal muscle tissue-level VO2 in older adults at risk for metabolic dysfunction [182]. These benefits were greater than when providing vitamin D repletion or exercising independently. These observations are consistent with reports that vitamin D coupled with exercise has positively affected muscle mitochondrial function [171,174]. In addition, VDR expression in SkM was increased by exercise [183]. Vitamin D supplementation has been associated with muscle regeneration and repair [123,145,146], suggesting an additive effect when combined with vitamin D repletion.

In addition to these findings, vitamin D has been described to have anti-inflammatory effects and is linked to insulin sensitivity and immuno-modulation [184–186]. Recent work has also highlighted a novel role of vitamin D in upregulating HO-1 expression in intestinal cells and reduced expression of macrophage HO-1 with an associated reduction in conjugated bilirubin [187]. Vitamin D has been shown to block the activation of M-1 macrophages, increase activation of M-2 macrophages, and impair monocyte/macrophage recruitment [187]. Collectively, these data suggest that vitamin D may ameliorate metabolic dysfunction by altering lipid availability for oxidation in response to exercise training and may help regulate inflammatory pathways. These observations require further exploration in obesity-inflammation studies.

Along with evidence that vitamin D repletion augments oxidative metabolism [171,174], we show in a muscle cell line that active calcitriol treatment altered total lipid, lipid species content, and increased gene expression of PLIN2, a lipid coatomer protein that facilitates IMCL availability for β-oxidation [188,189]. PLIN2-containing lipid droplets are also preferentially used during moderate-intensity exercise [190], suggesting that increased PLIN2 expression may increase lipolytic potential. In vitro findings from our group [180,181] indicate increased PLIN2-associated lipid accumulation and lipolysis after calcitriol treatment. These changes suggest an increased lipid flux—defined here as the rate at which lipids pass through SkM via IMCL accumulation and oxidation—and, by association, a decrease in lipid-mediated pathologies [191]. These cell culture results suggest that vitamin D is involved in muscle lipid packaging, partitioning, and mitochondrial lipid oxidation. Together with data showing that exercise improves muscle sensitivity to vitamin D storage and retention [192], evidence of muscle adaptations to the combination of vitamin D supplementation with exercise are tightly connected with improved mitochondrial function and may serve an integral role in delaying stagnant ectopic fat infiltration and metabolic dysfunction.

#### *5.4. Nitrate from Foods and Dietary Supplements*

Dietary nitrate is predominately found in green leafy vegetables and concentrated food sources (e.g., beetroot juice) and dietary supplements. Physically active individuals commonly use this EE to increase plasma nitrate concentrations and subsequently increase nitric oxide availability [193]. Increasing nitric oxide via the nitrate-nitrite-NO signaling pathway (with supplemental dietary nitrate) has been shown to decrease NADPH oxidase-derived oxidative stress via HO-1 induction and reduce p47phox expression [194]. Metabolically, nitrate has also been shown to reduce the oxygen cost of exercise [195] and improve exercise tolerance, economy, and performance. These benefits may also extend to those newly committed to exercise to lose fat and improve metabolic function. In addition to these observations, it seems reasonable that dietary nitrate may also work alongside exercise to preserve endothelial function [196]. Basaqr et al. recently found that four weeks of concentrated beetroot juice combined with vitamin C improved endothelial function and the lipid profile of overweight subjects with evidence of endothelial dysfunction [197]. The exact mechanism of action to explain these findings is unknown but is partially explained by the combined antioxidant effects of vitamin C and concentrated dietary nitrate supplements to decrease oxidative stress [198–201]. Improvements in blood lipids from others suggest that dietary nitrate supplementation with the addition of vitamin C (or other nutraceutical antioxidants) may be a valuable dietary approach alongside exercise to improve metabolic and cardiovascular health [60,61]. Future studies could determine how these impact exercise, inflammation, and metabolic outcomes.

#### *5.5. Vitamin E Supplementation*

Vitamin E (α/γ-tocopherol) is one of the most important dietary antioxidants that play a critical role as a radical savaging agent and mechanistic inducer [202]. Vitamin E acts as a potent antioxidant to neutralize free radicals and superoxide by using its free hydroxyl group to accept unpaired electrons [203]. Furthermore, unlike other dietary antioxidants (e.g., vitamin C, carotenoids, etc.), Vitamin E is uniquely connected with exercise-induced oxidative stress and insulin sensitivity. The regulation and distribution of Vitamin E are controlled by alpha-tocopherol transfer protein (α-TTP) in the liver. α-TTP secretes Vitamin E from the liver by releasing α-tocopherol into the circulation. However, this mechanism is still not clearly understood [204]. Data suggests that α-TTP is lower when the α-tocopherol levels are low and subjects with an α-TTP gene (TTPA) knockout presented with symptoms of vitamin E serum deficiency [202]. The α-TTP is also known to be induced by hypoxic states and stress-induced free radical production [205]. The administration of vitamin E in hypoxic states (similar to hypoxia observed with high-intensity exercise) has mitigated ROS- related biochemical changes in many tissues by preventing increases in malondialdehyde and myeloperoxidase and protecting against lipid peroxidation [206]. This hypoxia-based regulative mechanism has the potential to be evident during times of exercise; however, this hypothesis has yet to be tested. The rationale behind this hypothesis is that during exercise, hypoxia-induction of α-TTP will help increase serum α-tocopherol and protect the cells from free-radical damage during exercise-induced oxidative stress. This tocopherol can also provide non -antioxidant functions and induce mRNA levels of transcription factors, PPARγ, and the hormone, adiponectin [207,208]. Adiponectin and PPARγ are activated by vitamin E and are known to improve insulin sensitivity in diabetes. The vitamin E-induction of PPARγ is not through direct binding but through the increase in 15d-PGJ2, a commonly described ligand of PPARγ [208] that is also known to induce HO-1 through p38 MAP kinase and the Nrf-2 pathway [209]. This induction may further increase exercise-induced HO-1 activation and influence the BVRA-bilirubin-PPARαaxis. Both Vitamin E and PPARs are valuable targets in hepatic protection in non-alcoholic hepatic steatosis and fibrosis [210].

#### **6. Conclusions**

Exercise has clear benefits in reducing adiposity and inflammation while improving insulin sensitivity. A deeper understanding of the mechanisms of how exercise functions to improve these beneficial actions is needed. While bilirubin was once thought to be a harmful bile substance, current research argues otherwise and that slightly elevated levels have numerous health benefits against metabolic dysfunction. Studies on how exercise influences factors such as heme oxygenase, BVRA, and UGT1A1 that control bilirubin's turnover (half-life) are needed. Furthermore, nutraceuticals that activate and control these pathways might be beneficial in improving weight-loss regimens. Investigations in these areas might also benefit patients with inflammatory disorders as increasing plasma bilirubin has anti-inflammatory properties that probably originate from its antioxidant and hormone (as a ligand for PPARα) properties. Future work determining the interplay of exercise and nutraceuticals has many health benefits to help a broad spectrum of diseases.

**Author Contributions:** T.D.H.J. conceived the project. N.R.D. created graphic illustrations for Figures 1–3 and 6 using PowerPoint; D.E.S. developed Figure 4 using BioRender.com (accessed on 9 December 2021); T.D.H.J. generated Figure 5 using OmniGraffle. D.T.T., N.R.D., K.D.F., D.E.S. and T.D.H.J. wrote the manuscript and reviewed and revised the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases 1R01DK121797-01A1 (T.D.H.J.) and 1R01DK126884-01A1 (D.E.S.), the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.J.) and P01 HL05197-11 (D.E.S.), and the National Institute of General Medical Sciences P20GM104357-02 (D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

**Conflicts of Interest:** T.D.H.J. and D.E.S. have submitted patents on bilirubin and obesity-related disorders.

#### **References**

