Title: Effect of pemafibrate in reducing intestinal long-chain fatty acid absorption and hepatic
fibrosis in metabolic dysfunction-associated steatohepatitis rats
Version: 0 Date: 05 Mar 2025
Reviewer's report:
The manuscript by Okada et al., examines the role of pemafibrate on intestinal lipid metabolism
in the context of a rat model of MASH.  The study design is straightforward and well controlled.
The data support reduced lipid accumulation in the intestinal epithelium and a modest reduction
in fat absorption that is associated with a reduction in mRNA levels of lipid metabolizing and
transport enzymes in the intestine.  However, there are a number of limitations that reduce
enthusiasm for acceptance of the manuscript.
Major concerns:
1) The analysis is limited to a measure of fat absorption, intestinal and liver histology and rtPCR
of a handful of genes implicated in intestinal lipid handling and liver phenotypes.
2) Gene expression of markers expected to increase with PPAR alpha agonism in both liver and
intestine would give reviewers confidence that the drug is affecting this transcriptional pathway
as previously published (i.e fatty acid oxidation enzymes).  As it is all target genes are either
reduced or unchanged in fibrate-treated rats.
3) Confirmation of changes in lipid metabolizing enzymes at the protein level, particularly
CD36.
4) The effects of pemafibrate on liver collagen deposition and a-SMA levels are expected and
may or may not have any connection to the observed phenotypes in the intestine, which is the
focus of the manuscript as outlined in the background and title.
5) The reduction in fat absorption as determined by mass measurements is ~1%.  It is difficult
to know if such a difference is biologically relevant in the setting of a diet composed of 27%
palm oil and the development of MASH phenotypes.
6) The measurement of fat absorption is not state-of-the-art and would be strengthened by the
use of a non-absorbable marker.
7) The accumulation of lipid in Oil Red O positive areas in the intestinal epithelium is interpreted
as evidence of reduced lipid absorption.  Firstly, the reduction in staining appears to be far greater
than the reduction in fat absorption (1%).  Secondly, the reduction could reflect more rapid
chylomicron assembly and secretion or increased fat oxidation within the intestinal epithelium
following fibrate treatment.
8) The suppression of intestinal CD36 by pemafibrate is somewhat puzzling, but not necessarily
incorrect.  A host of transcription factors have been implicated in the upregulation of CD36,
including PPAR alpha.  Admittedly, CD36 has been more heavily studied in other cell types
which may not reflect regulation in the intestinal epithelium.  In the present study, mice are fed a
diet containing palm oil, cholesterol, and cholate.  All of which may influence CD36 expression
via PPARs, LXR, and NF-KB.
Minor Concerns:
1) The diet should be better described and percent lipid expressed in kCal.
Title: Effect of pemafibrate in reducing intestinal long-chain fatty acid absorption and hepatic
fibrosis in metabolic dysfunction-associated steatohepatitis rats
Version: 0 Date: 05 Mar 2025
Reviewer's report:
The manuscript by Okada et al., examines the role of pemafibrate on intestinal lipid metabolism
in the context of a rat model of MASH.  The study design is straightforward and well controlled.
The data support reduced lipid accumulation in the intestinal epithelium and a modest reduction
in fat absorption that is associated with a reduction in mRNA levels of lipid metabolizing and
transport enzymes in the intestine.  However, there are a number of limitations that reduce
enthusiasm for acceptance of the manuscript.
Major concerns:
1) The analysis is limited to a measure of fat absorption, intestinal and liver histology and rtPCR
of a handful of genes implicated in intestinal lipid handling and liver phenotypes.
2) Gene expression of markers expected to increase with PPAR alpha agonism in both liver and
intestine would give reviewers confidence that the drug is affecting this transcriptional pathway
as previously published (i.e fatty acid oxidation enzymes).  As it is all target genes are either
reduced or unchanged in fibrate-treated rats.
3) Confirmation of changes in lipid metabolizing enzymes at the protein level, particularly
CD36.
4) The effects of pemafibrate on liver collagen deposition and a-SMA levels are expected and
may or may not have any connection to the observed phenotypes in the intestine, which is the
focus of the manuscript as outlined in the background and title.
5) The reduction in fat absorption as determined by mass measurements is ~1%.  It is difficult
to know if such a difference is biologically relevant in the setting of a diet composed of 27%
palm oil and the development of MASH phenotypes.
6) The measurement of fat absorption is not state-of-the-art and would be strengthened by the
use of a non-absorbable marker.
7) The accumulation of lipid in Oil Red O positive areas in the intestinal epithelium is interpreted
as evidence of reduced lipid absorption.  Firstly, the reduction in staining appears to be far greater
than the reduction in fat absorption (1%).  Secondly, the reduction could reflect more rapid
chylomicron assembly and secretion or increased fat oxidation within the intestinal epithelium
following fibrate treatment.
8) The suppression of intestinal CD36 by pemafibrate is somewhat puzzling, but not necessarily
incorrect.  A host of transcription factors have been implicated in the upregulation of CD36,
including PPAR alpha.  Admittedly, CD36 has been more heavily studied in other cell types
which may not reflect regulation in the intestinal epithelium.  In the present study, mice are fed a
diet containing palm oil, cholesterol, and cholate.  All of which may influence CD36 expression
via PPARs, LXR, and NF-KB.
Minor Concerns:
1) The diet should be better described and percent lipid expressed in kCal.
Please review this paper.
user
Title: Effect of pemafibrate in reducing intestinal long-chain fatty acid absorption and hepatic
fibrosis in metabolic dysfunction-associated steatohepatitis rats
Version: 0 Date: 05 Mar 2025
Reviewer's report:
The manuscript by Okada et al., examines the role of pemafibrate on intestinal lipid metabolism
in the context of a rat model of MASH.  The study design is straightforward and well controlled.
The data support reduced lipid accumulation in the intestinal epithelium and a modest reduction
in fat absorption that is associated with a reduction in mRNA levels of lipid metabolizing and
transport enzymes in the intestine.  However, there are a number of limitations that reduce
enthusiasm for acceptance of the manuscript.
Major concerns:
1) The analysis is limited to a measure of fat absorption, intestinal and liver histology and rtPCR
of a handful of genes implicated in intestinal lipid handling and liver phenotypes.
2) Gene expression of markers expected to increase with PPAR alpha agonism in both liver and
intestine would give reviewers confidence that the drug is affecting this transcriptional pathway
as previously published (i.e fatty acid oxidation enzymes).  As it is all target genes are either
reduced or unchanged in fibrate-treated rats.
3) Confirmation of changes in lipid metabolizing enzymes at the protein level, particularly
CD36.
4) The effects of pemafibrate on liver collagen deposition and a-SMA levels are expected and
may or may not have any connection to the observed phenotypes in the intestine, which is the
focus of the manuscript as outlined in the background and title.
5) The reduction in fat absorption as determined by mass measurements is ~1%.  It is difficult
to know if such a difference is biologically relevant in the setting of a diet composed of 27%
palm oil and the development of MASH phenotypes.
6) The measurement of fat absorption is not state-of-the-art and would be strengthened by the
use of a non-absorbable marker.
7) The accumulation of lipid in Oil Red O positive areas in the intestinal epithelium is interpreted
as evidence of reduced lipid absorption.  Firstly, the reduction in staining appears to be far greater
than the reduction in fat absorption (1%).  Secondly, the reduction could reflect more rapid
chylomicron assembly and secretion or increased fat oxidation within the intestinal epithelium
following fibrate treatment.
8) The suppression of intestinal CD36 by pemafibrate is somewhat puzzling, but not necessarily
incorrect.  A host of transcription factors have been implicated in the upregulation of CD36,
including PPAR alpha.  Admittedly, CD36 has been more heavily studied in other cell types
which may not reflect regulation in the intestinal epithelium.  In the present study, mice are fed a
diet containing palm oil, cholesterol, and cholate.  All of which may influence CD36 expression
via PPARs, LXR, and NF-KB.
Minor Concerns:
1) The diet should be better described and percent lipid expressed in kCal.
assistant