1. Evaluation Summary:

    With a series of elegant experiments, the authors have shown that the renin-angiotensin system (RAS) plays a critical role in thermogenesis. Involvement of brown fat and mitochondrial chain open new scenarios that may be helpful to define new target pathways for the treatment of obesity and diabetes.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Understanding the mechanisms of energy homeostasis is paramount to uncovering novel treatments for diseases such as obesity and diabetes. The authors found, consistent with previous reports (PMIDs: 29066463 and 31638856) that the renin-angiotensin system (RAS) - which normally controls blood pressure and which the authors studied previously in the context of diabetes - plays a critical role in thermogenesis (the process by which the fat stores in the body expends energy to produce heat in response to stimuli such as cold exposure). The authors find that Ace2 and Mas (the initiator and receptor of the RAS system, respectively) are upregulated in mouse brown adipose tissue (BAT; the fat stores responsible for thermogenesis) and are sensitive to cold-exposure.

    The authors then provide convincing evidence that mice lacking Ace2 or Mas activity have decreased energy expenditure, decreased body temperature when exposed to the cold, and functional BAT. The authors also show that Ace2 or Mas deficient mice express less PGC1-a than mice with competent RAS signaling. PCG1-a is a critical regulator of mitochondrial production and mitochondrial uncoupling, two processes required for BAT thermogenesis. This suggests in the absence of RAS there is an ability for BAT to produce necessary proteins for mitochondrial uncoupling and heat production. Further the authors provide evidence that Ace2 deficient mice have less mitochondrial activity, consistent with this inability to produce mitochondria as underlying the thermogenesis defect in RAS deficient mice. The authors also transplant Mas deficient BAT into otherwise normal mice and find that these animals have also have abnormal energy expenditure, suggesting RAS signaling specifically in the BAT is necessary for normal energy expenditure.

    The authors then determined if modulating RAS signaling could effect energy expenditure in diabetic mice that also harbor impairments in thermal regulation. The authors show that forced expression of Ace2 through adenoviral expression or treatment with angiotensin1-7 (the product of Ace2 activity) improves energy expenditure, and improves the ability to regulate body temperature when exposed to cold. White adipose tissue (WAT) which is normally not involved in thermogenesis can undergo "browning" and take on thermogenic properties in response to certain stimuli. The authors further find increased browning of WAT upon Ace2 re-expression measured by H&E staining, as well as increased expression of UCP1, PGC1a, and other BAT marker proteins.

    The authors then seek to determine how RAS signaling leads to increased thermogenesis in BAT. Based on previous literature and transcriptomics of BAT upon RAS inhibition, the authors hypothesize that AKT and cAMP-PKA signaling could be critical in relaying the RAS signaling to elicit thermogenesis. The authors first find that RAS signaling modulates activating phosphorylation of AKT and PKA, and subsequently show that treatment of isolated adipocytes with AKT and PKA inhibitors reverse expression of mitochondrial and thermogenesis genes that are increased upon RAS signaling. Further, these inhibitors also block increased mitochondrial function upon triggering RAS signaling with angiontensin1-7 in isolated adipocytes. Collectively, these experiments suggest AKT and PKA signaling are critical for RAS regulation of thermogenesis but mechanistic elements remain to be elucidated about how RAS engages AKT and PKA signaling.

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  3. Reviewer #2 (Public Review):

    Previous reports, including studies from the Yang group, had identified ACE2 as an important mediator of glucose and lipid homeostasis in the context of obesity. These studies hinted that ACE2 and other factors in the RAS system may affect adipocyte biology, but rigorous characterization of the physiological effects of the ACE2 pathway on adipose tissue - particularly that of brown adipose tissue - had not been completed. This manuscript by Cao, X, Shi, T, and Zhang C et al. fills these gaps in knowledge by demonstrating that multiple components of the ACE2 pathway impact thermogenesis and BAT function. The authors utilize a variety of methods, including six independent mouse models as well as isolated primary cells to test the hypothesis that the ACE2 pathway regulates thermogenesis and energy metabolism. Within the abstract, the authors make the following claims, which are supported by the evidence that follows:

    1. "that ACE2 is highly expressed in brown adipose tissue (BAT) and that cold stimulation increases ACE2 and Ang-(1-7) levels in BAT and serum."

    The authors show that ACE2 is expressed BAT at the protein (Figure 1A) and RNA (Figure 1B) levels. The authors demonstrate that ACE2 is induced by cold exposure for 6 hours (Figure 1C) and 24 hours of cold exposure (Figures 1D and E). The authors also demonstrate increased levels of ACE2 and Ang-(1-7), a cleaved product of ACE2, in serum after 2 or 4 days of cold exposure (Figures 1F and G).

    2. "ACE2 knockout mice (ACE2-/y), Mas knockout mice (Mas-/-) and mice transplanted with brown adipose tissue from Mas-/- mice displayed impaired thermogenesis."

    The authors show that ACE2-/y mice have decreased body temperature at 22C and 4C for eight hours (Figure 2F) and acutely after transition to 4C (Supplemental figure 1G). They show that Mas-/- knockout mice show similar phenotypes - decreased body temperature at 22C and 4C for eight hours (Supplemental figure 4J) and acutely after transition to 4C (Supplemental figure 4L). The authors then show that wild type C57BL/6 mice transplanted with BAT from Mas-/- mice show metabolic impairments consistent with ACE2-/y and Mas-/- mice, but do not show data regarding their body temperature.

    3. "...impaired thermogenesis of db/db obese diabetic mice and high-fat diet-induced obese mice were ameliorated by overexpression of ACE2 or continuous fusion of Ang-(1-7)."

    The authors show that db/db obese mice have decreased body temperature at 22C and 4C for eight hours which is rescued (or perhaps partially rescued) by adenoviral expression of ACE2 (Figure 3F). The authors additionally show that subcutaneous infusion of Ang-(1-7) elevates body temperature in db/db obese mice in response to cold challenges at 22C and 4C for eight hours (Figure 4F). The authors also show that infusion of Ang-(1-7) can increase body temperature acutely after cold challenge in mice fed a high fat diet (HFD) (Figure 4H), but they do not test the effects of overexpression of ACE2 on body temperature in the HFD model.

    4. "Activation of {the} ACE2 pathway was associated with improvement of metabolic parameters, including blood glucose, lipids, and energy expenditure in multiple animal models."

    The authors show that adenoviral overexpression of ACE2 in db/db mice increases their energy expenditure (Figure 3E) and improves glucose tolerance (Supplemental Figure 2B) and serum triglyceride levels (Supplemental figure 2C) relative to control mice. The authors show similar phenotypes for their model infusing Ang-(1-7) into the db/db mouse model, showing increased energy expenditure (Figure 4E), improved glucose tolerance (Supplemental figure 3B), and decreased serum triglycerides (Supplemental Figure 3C).

    5. "...[the] ACE2 pathway activated Akt/FOXO1 and PKA pathway, leading to induction of UCP1 and activation of mitochondrial function."

    The authors show that primary brown adipocytes have increased basal oxygen consumption and spare respiratory capacity when treated with Ang-(1-7), and that these effects are negated by treatment with an AKT inhibitor (MK2206, Figure 6E) or a PKA inhibitor (HA, Figure 6J), strongly implicating these signaling pathways in increased mitochondrial function (as assayed by oxygen consumption). The authors also show that Ang-(1-7) induces UCP1 expression and that this is blunted by an AKT inhibitor (Figure 1C) or a PKA inhibitor (Figure 6I).

    Strengths

    This is a strong paper addressing the role of the angiotensin II pathway in obesity-related metabolic parameters and thermogenesis. The authors use multiple orthogonal methods to rigorously characterize the physiology of the RAS pathway in BAT. First, the authors use six independent mouse models to test the effects of the ACE2 pathway on metabolic homeostasis and thermogenesis. This includes three distinct loss of function models, including ACE2-/y mice, Mas-/- mice, and wild type C57BL/6 mice with subcutaneously transplanted Mas-/- BAT mice. In each of these loss of function models the authors found that loss of the ACE2 pathway leads to worsened metabolic parameters, and in most of these models, decreased thermogenesis and BAT function. Collectively, these data strongly suggest that intact ACE2 pathway signaling is required for BAT function, thermogenesis, and subsequent metabolic health in mice. Additionally, the authors characterize three distinct gain of function models, including an adenoviral ACE2 overexpression system in db/db mice, and two continuous infusion models of Ang-(1-7) in db/db mice or mice fed a high fat diet. In each of these models, increasing function of the ACE2 pathway is associated with improvements in systemic metabolism, and, in most models, improved thermogenesis. Finally, the authors utilize primary cells to test the pathways involved in Ang-(1-7) function to identify the AKT and PKA signaling pathways as mediators of the physiological response seen. The authors show that both AKT and PKA signaling are required to elevate mitochondrial function (as assayed by oxygen consumption) in response to Ang-(1-7) treatment. While the precise molecular mechanisms of how the ACE2 pathway activates these signaling pathways and downstream mitochondrial are not fully worked out, these details are well beyond the scope of this study.

    In summary, this paper identifies the ACE2 pathway as critical for maintaining thermogenesis and energy expenditure in mice. The authors demonstrate this through multiple independent mouse models as well as primary cells. Their data give important insights on the ACE2 pathway in mediating brown adipose tissue function in metabolism and thermogenesis in obesity.

    Weaknesses

    There are few major weaknesses in this study. Most noted weaknesses in the manuscript can be resolved without the need for further experimentation (e.g., by clarifying and/or reanalyzing select data or figure panels). One note is that some of the claims put forth in the abstract that have not been directly addressed (as noted above). This can be addressed by either performing experiments to address the claims or by modifying the claims to reflect the current experimentation.

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