*4.7. DGA Regimen Hinders Excessive ROS Generation under Metabolic Stress in Hepatocytes and Astrocytes*

The DGA regimen effectively hindered excessive ROS generation (Supplementary Materials) [46]. The exact reason why the two-day DGA priming and acute DGA dose with fresh cell culture media 2 h earlier is able reduce net ROS generation remains elusive, but it is likely that the activation on the HO-1 pathway plays an important role in both hepatocytes [9] and astrocytes [7].

#### **5. Conclusions**

The inducible heme oxygenase pathway was activated transiently after each DGA dose during the 21-day study period. Simultaneously, blood bilirubin level was strongly upregulated towards Day21 under the DGA regimen. The positive associations of blood bilirubin and systemic inflammation (GlycA and IL-6) and blood lipids (TGs) were observed. Also, HIF-1α mRNA expression was shown to be strongly activated in WBCs by the 3-week

DGA regimen. All these activations likely relate to the mitochondrial energy metabolic activation by the DGA [27].

During the first four days under the DGA regimen, the reduction in blood TGs in the LC subgroup may be partially linked to the hepatic inflow of bilirubin with plasma albumin. Simultaneously, subclinical inflammation was markedly improved in the LC subgroup, i.e., in the participants with somewhat elevated systemic inflammation before the study. Fast improvement of the anti-inflammatory status likely reduced HO-1 pathway activity in four days in the LC subgroup.

It seems that the DGA regimen is able to both up- and downregulate the HO-1 pathway in humans. This possibly materializes via temporary activation of ROS generation by OXPHOS after each DGA dose, and a more permanent reduction of oxidative stress inter alia via HO-1 pathway activation. In vitro studies with hepatocytes and astrocytes showed that the DGA regimen efficiently reduces ROS generation in metabolic stress.

All in all, the DGA regimen seems to be able to activate whole-body aerobic energy metabolism without excessive ROS generation. Benefits materialize in the whole body, but the liver is activated the most due to its central role in maintaining glucose homeostasis and its other vital homeostatic tasks. Increased plasma bilirubin and beta-hydroxybutyrate, and the activation of the HIF-1α pathway are beneficial also for neurodegenerative diseases. The therapeutic combination activated by the DGA regimen poses benefits for chronic diseases related to elevated systemic inflammation.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/antiox11122319/s1, Supplementary S1. Human primary hepatocyte ROS study model and results; Supplementary S2. Astrocyte ROS study, study outline, main results and materials and methods [46].

**Author Contributions:** O.P.H.: conceptualization, methodology, formal analyses, writing the original draft. H.K. (Heikki Kyröläinen): supervision, review and editing, validation. H.K. (Heikki Kainulainen): supervision, review and editing, validation. M.L.: review and editing, validation. All authors have read and agreed to the published version of the manuscript.

**Funding:** University of Jyväskylä (JyU) provided the study site and equipment without cost. Replicon Health Oy paid the salary for the corresponding author.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical committee of the Central Finland Health Care District (Dnro 1U/2019, Approval date 4 June 2019).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The original data contributions presented in the study are included in the graphs of the article. Anonymous data on presented biomarkers is available from the corresponding author upon reasonable request. Additional anonymous data can be found in Hirvonen et al., (2021) [27] Supplement A.

**Conflicts of Interest:** Replicon Health Oy develops DGA as a health beneficial agent. Corresponding Author is an owner of this company and H.K. (Heikki Kainulainen) owns shares in it. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **References**

