Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis

Reviewer #1 (Public Review):

The authors put forth the hypothesis that hepatocyte and/or non-parenchymal liver MCT1 may be responsible for physiologic effects (lower body weight gain and less hepatic steatosis) in MCT1 global heterozygote mice. They generate multiple tools to test this hypothesis, which they combine with mouse diets that induce fatty liver, steatohepatitis and fibrosis. Novel findings include that deletion of hepatocyte MCT1 does not change liver lipid content, but increases liver fibrosis. Deletion of hepatic stellate cell (HSC) MCT1 does not substantially affect any liver parameter, but concomitant HSC MCT1 deletion does reverse fibrosis seen with hepatocyte MCT1 knockout or knockdown. In both models, plasma lactate levels do not change, suggesting that liver MCT1 does not substantially affect systemic lactate. In general, the data match the conclusions of the manuscript, and the studies are well-conducted and well-described. Further work would be necessary to dissect mechanism of fibrosis with hepatocyte MCT1, and whether this is due to changes in local lactate (as speculated by the authors) or another MCT1 substrate. This would be important to understand this novel potential cross-talk between hepatocytes and HSCs.

A parallel and perhaps more important advance is the generation of new methodology to target HSC in mice, using modified siRNA and by transduction of AAV9-Lrat-Cre. Both methods would reduce the need to cross floxed mice with the Lrat-Cre allele, saving time and resources. These tools were validated to an extent by the authors, but not sufficiently to ensure that there is no cross-reactivity with other liver cell types. For example, AAV9-Lrat-Cre-transduced MCT1 floxed mice show compelling HSC but not hepatocyte Mct1 knockdown, but other liver cell types should be assessed to ensure specificity. This is particularly important as overall liver Mct1 decreased by ~30% in AAV9-Lrat-Cre-transduced mice, which may exceed HSC content of these mice, especially when considering a 60-70% knockdown efficiency. This same issue also affects Chol-MCT1-siRNA, which the authors demonstrate to affect hepatocytes and HSC, but likely affects other cell types not tested. As this is a new and potentially valuable tool, it would be important to assess Mct1 expression across more non-parenchymal cells (i.e. endothelial, cholangiocytes, immune cells) to determine penetration and efficacy.

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1. The significance of the findings is moderately impacted due to the following considerations:

a) Fibrosis in human NAFLD is a significant problem as a predictor of liver related mortality and is associated with type 1 and type 3 collagen. However, the reduction in COL1 in stellate cells did not amount to a reduction in liver fibrosis even in cell specific KO (in Fig 7E, there is no indication of whether Sirius red staining was different between HSC KO and control mice- the authors mention a downward trend in the text). The authors postulate that type 1 COL may not be the more predominant form of fibrosis in the model. This does not seem likely, since the same ob/ob mouse model was used to determine that fibrosis was enhanced with hepatocyte specific MCT-1 KO and decreased with Chol MCT-1KO. Measurements of different types of collagens in their model and the effect of MCT-1 on different types could be more informative. In particular, although collagens are the structural building blocks for hepatic fibrosis, fibrosis can also be controlled by matrix remodeling factors such as Timp1, Serpine 1, PAI-1 and Lox.

b) The authors use multiple animal models including cell specific KO to conclude that stellate cell MCT-1 inhibition decreases COL-1. However, the mechanisms behind this reduced expression of COL-1 are not discussed or explored, making it descriptive.

c) Different types of diets are used in this study which could impact lactate availability. Choline deficiency diets are reported to cause weight loss, and importantly have none of the metabolic features of human NASH. Therefore, their utility is doubtful, especially for this study which proposes to investigate if metabolic dysregulation and substrate availability could be a tool for therapy.

d) Hepatocyte specific MCT-1 KO mice seem to have increased COL-1 production, despite no noticeable difference in hepatocyte steatosis. The reasons for this are not discussed. Fibrosis in NASH is thought to be from stellate cell activation secondary to signals from hepatocellular damage. There is no evidence that there was a difference in either of these parameters in the mouse models used.

e) The authors report that serum lactate levels did not rise after MCT-1 silencing, but the reasons behind this are unclear. There is insufficient data about lactate production and utilization in this model, which would be useful to interpret data regarding steatosis and fibrosis development. For example, does the MCT-1 KO prevent hepatocyte and stellate cell net import or export of lactate? What is the downstream metabolic consequence in terms of pyruvate, acetylCoA and the NAD/NADH levels. Does the KO have downstream effects on mitochondrial TCA cycling?

f) MCT-1 protein expression is measured only in the in vitro assay. Similar quantitation through western blot is not shown in the animal models.

Reviewer #3 (Public Review):

A major finding of this work is that loss of monocarboxylate transporter 1 (MCT1), specifically in stellate cells, can decrease fibrosis in the liver. However, the underlying mechanism whereby MCT1 influences stellate cells is not addressed. It is unclear if upstream/downstream metabolic flux within different cell types leads to fibrotic outcomes. Ultimately, the paper opens more questions than it answers: why does decreasing MCT1 expression in hepatocytes exacerbate disease, while silencing MCT1 in fibroblasts seems to alleviate collagen deposition? Mechanistic studies in isolated hepatocytes and stellate cells could enhance the work further to show the disparate pathways that mediate these opposing effects. The work highlights the complexity of cellular behavior and metabolism within a disease environment but does little to mechanistically explain it.

The observations presented are compelling and rigorous, but their impact is limited by the nearly complete lack of mechanistic insight presented in the manuscript. As also mentioned elsewhere, it is important to know whether lactate import or export (or the transport of another molecule-like ketone bodies, for example) is the decisive role of MCT1 for this phenotype. Beyond that, it would be interesting, albeit more difficult, to determine how that metabolic change leads to these fibrotic effects.

Kuppfer cells are initially analyzed and targeted. These cells may play a major role in fibrotic response. It will be interesting to determine the effects of lactate metabolism in other cells within the microenvironment, like Kuppfer cells, to gain a complete understanding of how metabolism is altered during fibrotic change.

The timing of MCT1 depletion raises concern, as this is a largely prophylactic experiment, and it remains unclear if altering MCT1 would aid in the regression of established fibrosis. Given the proposal for translation to clinical practice, this will be an important question to answer.