Robust single-nucleus RNA sequencing reveals depot-specific cell population dynamics in adipose tissue remodeling during obesity

Classified as probably not a substantial claim.

scRNA-seq has been used to characterize dynamics of cell populations underlying adipose tissue remodeling, focusing on cell types within stromal vascular fractions, particularly preadipocytes.

“scRNA-seq has been used to characterize the dynamics of cell populations underlying adipose tissue remodeling, with a focus on the cell types within stromal vascular fractions, particularly preadipocytes (Schwalie et al., 2018; Burl et al., 2018; Hepler et al., 2018; Merrick et al., 2019).”

• cites Schwalie et al., 2018 sourceNot checked — no full text of the source
• cites Burl et al., 2018 sourceSupported
• cites Hepler et al., 2018 sourcePartly supported

  The Results section on single-cell RNA sequencing identifies distinct stromal populations in visceral adipose tissue, including committed preadipocytes. The Results section on high-fat-diet feeding reports changes in the frequencies of those populations during tissue remodeling, but those changes were assessed separately rather than by scRNA-seq. The source therefore supports the focus on precursor populations and remodeling, but not the claim that scRNA-seq itself characterized their dynamics. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Prolonged obesity leads to pathological changes and dysfunction in adipose tissue, characterized by disrupted lipid metabolism, impaired insulin signaling, inflammation, and cell death, which are critical precursors to type 2 diabetes and other metabolic diseases.

“However, prolonged obesity leads to pathological changes and dysfunction in adipose tissue, characterized by disrupted lipid metabolism, impaired insulin signaling, inflammation, and cell death. These changes are critical precursors to the onset of type 2 diabetes and other metabolic diseases (Longo et al., 2019).”

• cites Longo et al., 2019 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In obesity, adipose tissue undergoes significant remodeling, expanding substantially to enhance storage capacity, orchestrated by diverse cell types including adipocytes, preadipocytes, endothelial cells, and immune cells.

“In obesity, adipose tissue undergoes a significant remodeling process, expanding substantially to enhance its storage capacity. This process is orchestrated through coordinated actions among diverse cell types, including adipocytes, preadipocytes, endothelial cells, and various immune cells (Corvera, 2021).”

• cites Corvera, 2021 sourcePartly supported

  The Introduction identifies adipocytes as energy-storing cells, and the section on morphological and functional evidence describes white adipose tissue storing energy through adipocyte hypertrophy. The progenitor, endothelial-cell, and immune-cell sections document these cell types and changes associated with obesogenic diets. They do not establish that coordinated action among all four cell types drives substantial expansion specifically to increase storage capacity in obesity; page numbers are unavailable in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

snRNA-seq generates transcriptional profiles with high similarity to scRNA-seq while offering advantages: nuclei are promptly released during homogenization, minimizing cell type biases and artifactual transcriptional alteration; it is compatible with cryopreserved samples; and it can study cell types less amenable to scRNA-seq such as adipocytes and cardiomyocytes.

“While generating transcriptional profiles with high similarity to those by scRNA-seq (Grindberg et al., 2013; Habib et al., 2017), snRNA-seq offers advantages. First, this method utilizes nuclei that are promptly released from tissues during homogenization, obviating the need for an extended digestion process and thereby minimizing cell type biases and artifactual transcriptional alteration.”

• cites Grindberg et al., 2013 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Habib et al., 2017 sourceSupported

Classified as probably not a substantial claim.

More recently, snRNA-seq has revealed evidence of cellular heterogeneity within adipocyte populations.

“More recently, snRNA-seq has been employed, revealing evidence of the cellular heterogeneity within adipocyte populations (Sárvári et al., 2021; Emont et al., 2022; Holman et al., 2023).”

• cites Sárvári et al., 2021Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Emont et al., 2022 sourceSupported
• cites Holman et al., 2023 sourceNot checked — no full text of the source

May combine two claims.

scRNA-seq methods require fresh tissues, which can be difficult to obtain and lead to logistic complexities.

“First, these methods require the use of fresh tissues, which can be difficult to obtain and thus lead to logistic complexities (Alles et al., 2017; Attar et al., 2018; Wang et al., 2018; Lafzi et al., 2018).”

• cites Alles et al., 2017 sourcePartly supported

  The Background and Conclusions (pages not provided) describe difficulties preparing unperturbed cells, collecting rare cells, transporting clinical specimens, and coordinating experiments. But the paper demonstrates that fixed cells can be stored and used for droplet-based scRNA-seq, so it does not support the categorical claim that these methods require fresh tissues. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Attar et al., 2018 sourcePartly supported

  The Introduction (page unavailable) describes logistical barriers to processing cells when they become available, including distance, equipment scheduling, and difficulty storing intact cells. This supports the logistical point, but the paper discusses fresh cells or starting material rather than establishing that scRNA-seq methods require fresh tissues; it also describes preservation methods that can ease that constraint. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Wang et al., 2018 sourceSupported

Classified as probably not a substantial claim. May combine two claims.

The distribution of body fat in obesity is closely correlated with metabolic health; expansion of visceral fat depots increases risks for metabolic disorders, whereas subcutaneous fat depots pose lesser risks or can offer protection.

“The distribution of body fat in obesity is closely correlated with metabolic health. The expansion of visceral fat depots increases risks for metabolic disorders, whereas subcutaneous fat depots pose lesser risks or can even offer protection (Vishvanath and Gupta, 2019).”

• cites Vishvanath and Gupta, 2019 sourceSupported

May combine two claims.

Different adipose tissue depots respond differently to obesity, likely explaining the correlation between fat distribution and metabolic health.

“This is likely due to varying responses of different adipose tissue depots to obesity (Pellegrinelli et al., 2016).”

• cites Pellegrinelli et al., 2016 sourceSupported

May combine two claims.

Integration with Emont et al., 2022 and re-clustering all cells together consistently showed similar overall cellular composition with a substantially higher proportion of vascular cells in the authors' dataset.

“For a more robust comparison, we integrated our data with the previous study by Emont et al., 2022 and re-clustered all the cells together. The results consistently showed the similar overall cellular composition with a substantially higher proportion of vascular cells in our dataset (Figure 2—figure supplement 2C).”

• cites Emont et al., 2022 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

snRNA-seq has recently begun to reveal the heterogeneity of adipocytes within white adipose tissue.

“snRNA-seq has recently begun to reveal the heterogeneity of adipocytes within white adipose tissue (Sárvári et al., 2021; Emont et al., 2022; Holman et al., 2023).”

• cites Sárvári et al., 2021Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Emont et al., 2022 sourceSupported
• cites Holman et al., 2023 sourceNot checked — no full text of the source

May combine two claims.

APC2 exhibited higher expression of fibrotic genes such as Fn1 and Loxl1, resembling fibro-inflammatory progenitors.

“Notably, APC2 exhibited higher expression of fibrotic genes such as Fn1 and Loxl1 (Figure 3—figure supplement 1B), resembling ‘fibro-inflammatory progenitors’ (Hepler et al., 2018).”

• cites Hepler et al., 2018 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

The PC and SMC populations' respective association with capillaries and larger vessels is consistent with prior work.

“consistent with their respective association with capillaries and larger vessels (Rhodin, 1968; Yamazaki and Mukouyama, 2018).”

• cites Rhodin, 1968 sourceNot checked — no full text of the source
• cites Yamazaki and Mukouyama, 2018 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

eWAT appears to promote de novo adipogenesis through conversion of adipocyte progenitors to committed preadipocytes.

“eWAT appears to promote de novo adipogenesis through conversion of adipocyte progenitors to committed preadipocytes, as demonstrated in a prior study (Wang et al., 2013).”

• cites Wang et al., 2013 sourcePartly supported

  The Results (‘Cold-induced adipogenesis in epididymal adipose tissue’) and Discussion report new white adipocytes in eWAT after cold exposure or β3-agonist stimulation. The paper does not demonstrate the specific proposed step in which adipocyte progenitors convert to committed preadipocytes; it identifies committed-preadipocyte markers only as background in the Discussion. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

APC1 and APC2 corresponded to adipocyte progenitors, characterized by high expression of both Dpp4 and Pi16, while displaying distinct gene expression profiles from each other.

“APC1 and APC2 corresponded to ‘adipocyte progenitors’, characterized by high expression of both Dpp4 and Pi16 (Merrick et al., 2019Merrick et al., 2019), while displaying distinct gene expression profiles from each other (Figure 3—figure supplement 1A).”

• cites Merrick et al., 2019 sourcePartly supported

  In “Profiling and trajectory analysis of adipose progenitors” (page unavailable), the paper identifies a progenitor group expressing Dpp4 and Pi16. Its Discussion describes DPP4+ cells as adipose progenitors, but the paper does not identify the citing study’s APC1 and APC2 as two such populations. The claim that APC1 and APC2 have distinct expression profiles is attached to the citing study’s figure, not to this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Adipocytes undergo hypertrophy, an increase in cell size, during obesity, accompanied by significant cytoskeletal rearrangement.

“Adipocytes undergo hypertrophy, an increase in cell size, during obesity, accompanied by significant cytoskeletal rearrangement (Roh et al., 2020).”

• cites Roh et al., 2020 sourcePartly supported

  The Results section, “Adipocytes undergo remodeling of cytoskeletal organizations on an HFD” (page unavailable), reports substantially denser and more complex actin filaments in adipocytes from high-fat-diet mice, supporting cytoskeletal rearrangement. The Introduction describes adipose-tissue expansion during obesity, but the passages found do not establish that the adipocytes themselves increased in size; that hypertrophy component is not supported by our copy. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Recently, a few protocols have been introduced to enhance RNA quality by employing VRC or acidic buffers during nucleus isolation for snRNA-seq, particularly for kidney and pancreas samples.

“Recently, we encountered a few protocols that have been introduced to enhance RNA quality by employing VRC or acidic buffers during nucleus isolation for snRNA-seq, particularly for kidney (Soccio et al., 2014) and pancreas (Tosti et al., 2021) samples.”

• cites Soccio et al., 2014 sourceCould not verify

  The Basic Science section discusses ChIP-seq analysis of thiazolidinedione effects, not kidney nucleus-isolation protocols. Searches for VRC, acidic buffers, RNA quality, and kidney snRNA-seq found no passage in our copy addressing the claim attributed to Soccio et al.; the separate pancreas citation is not assessed here. Page numbers were not available in the indexed passages. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites Tosti et al., 2021 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Dead adipocytes are detected within CLS.

“As dead adipocytes are detected within CLS (Murano et al., 2008)”

• cites Murano et al., 2008 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

This observation aligns with a recent study that demonstrated the preservative properties of VRC, protecting tissue morphology, as well as RNA, protein, and genomic DNA quality.

“This observation aligns with a recent study that demonstrated the preservative properties of VRC, protecting tissue morphology, as well as RNA, protein, and genomic DNA quality (Shieh et al., 2018).”

• cites Shieh et al., 2018 sourceSupported

May combine two claims.

In integration with previous datasets, they observed a comparable adipocyte population identified in the previous study (mAd3 in Emont et al., 2022), with similar marker gene expression and lower transcript abundance.

“In the integration of our data with previous datasets, we observed a comparable adipocyte population identified in the previous study (mAd3 in Emont et al., 2022Emont et al., 2022; Figure 6—figure supplement 1A), with similar marker gene expression (Figure 6—figure supplement 1B) and lower transcript abundance (Figure 6—figure supplement 1C).”

• cites Emont et al., 2022 sourceSupported

May combine two claims.

The distinction between obesity-induced features occurring in the entire adipocyte population versus specific adipocyte subpopulations may explain the outstanding effectiveness of PPARγ agonists, such as thiazolidinediones, in improving adipose function and glucose homeostasis, surpassing the effects of other anti-inflammatory or anti-stress reagents.

“The distinctions between obesity-induced features occurring in the entire adipocyte population versus specific adipocyte subpopulations may explain the outstanding effectiveness of PPARγ agonists, such as thiazolidinediones, in improving adipose function and glucose homeostasis (Soccio et al., 2014), surpassing the effects of other anti-inflammatory or anti-stress reagents (Kraakman et al., 2015;”

• cites Soccio et al., 2014 sourcePartly supported

  Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Kraakman et al., 2015 sourceNot checked — no full text of the source
• cites Chen et al., 2016 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

DoubletFinder v2.0 was employed to identify and remove doublets, using an estimated multiplet rate derived from the total number of recovered nuclei.

“To identify and remove doublets, we employed DoubletFinder v2.0 (McGinnis et al., 2019) using an estimated multiplet rate derived from the total number of recovered nuclei.”

• cites McGinnis et al., 2019 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

This aligns with earlier findings, which demonstrated the failure of adipocytes to maintain their identity during obesity.

“This aligns with our earlier findings, which demonstrated the failure of adipocytes to maintain their identity during obesity (Roh et al., 2020).”

• cites Roh et al., 2020 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Ex vivo differentiated human adipocytes derived from various donors displayed distinct cellular phenotypes, consistent with the heterogeneity in adipocyte subpopulations identified by snRNA-seq.

“Interestingly, ex vivo differentiated human adipocytes derived from various donors displayed distinct cellular phenotypes, consistent with the heterogeneity in adipocyte subpopulations identified by snRNA-seq (Emont et al., 2022).”

• cites Emont et al., 2022 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

This finding may be consistent with previous studies demonstrating mosaic lipid transport activity among small white adipocytes from Rhesus Macaques monkeys.

“This finding may be consistent with previous studies demonstrating mosaic lipid transport activity among small white adipocytes from Rhesus Macaques monkeys (Varlamov et al., 2015).”

• cites Varlamov et al., 2015 sourceSupported

May combine two claims.

This finding contrasts with the heterogeneity of human adipocytes, where insulin sensitivity is shown to be a primary factor in adipocyte classification rather than cell size, as demonstrated by spatial transcriptomics mapping.

“This finding contrasts with the heterogeneity of human adipocytes, where insulin sensitivity is shown to be a primary factor in adipocyte classification rather than cell size, as demonstrated by spatial transcriptomics mapping (Massier et al., 2023).”

• cites Massier et al., 2023 sourcePartly supported

  The Results section, “Two clusters of cells are reciprocally associated with metabolic health” (page unavailable), reports that spatial-transcriptomic adipocyte markers have different associations with insulin resistance. But it does not show that insulin sensitivity is the primary basis for classifying adipocytes or compare its importance with cell size. The Discussion also cautions that adipocyte heterogeneity was less reproducible across studies. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Using the method, the authors generated snRNA-seq data of superior quality compared to existing datasets, offering novel and precise insights into specific changes within cell populations across different fat depots in response to obesity.

“Using this method, we have generated snRNA-seq data of superior quality compared to existing datasets, offering novel and precise insights into the specific changes within cell populations across different fat depots in response to obesity.”

May combine two claims.

The authors' robust nucleus isolation technique works across various tissue types and remarkably enhances snRNA-seq data quality.

“Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality.”

• Also stated in: § Introduction · plain

  “Here, we present a robust method for isolating nuclei for snRNA-seq, which enhances the quality of nuclear RNA and preserves nucleus integrity across various tissue types.”

• Also stated in: § Robust nucleus isolation protocols protect RNA quality and nucleus integrity · plain

  “Taken together, our optimized protocol, employing both VRC and recombinant RNase inhibitors, yields high-quality nuclei while preserving RNA integrity and nuclear structure for extended procedures up to 24 hr.”

May combine two claims.

Dysfunctional hypertrophic adipocytes have a key molecular feature of global shutdown in gene expression, along with elevated stress and inflammatory responses.

“Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses.”

May combine two claims.

Nuclei isolated with the optimized protocol had a smoother membrane surface and larger nucleus size compared to the standard protocol.

“Further analysis of nucleus morphology at a higher resolution revealed a smoother membrane surface and larger nucleus size when using the optimized protocol, as compared to the standard protocol (Figure 1F).”

May combine two claims.

Specifically in eWAT, there was an expansion of the APC4 proportion alongside a reduction in APC1 and APC2 populations during obesity.

“Specifically in eWAT, there was an expansion of the APC4 proportion alongside a reduction in APC1 and APC2 populations (Figure 3C) during obesity, suggesting distinct tissue expansion mechanisms in each adipose tissue depot.”

May combine two claims.

APC3 was identified as adipose regulatory cells, expressing F3 (encoding CD142) and showing strong expression of secretory signaling proteins, supporting its paracrine regulatory role.

“APC3 was identified as ‘adipose regulatory cells’, expressing F3 (encoding CD142) and showed strong expression of secretory signaling proteins (Figure 3A, Figure 3—figure supplement 1C), supporting its paracrine regulatory role (Schwalie et al., 2018).”

• cites Schwalie et al., 2018 sourceNot checked — no full text of the source

May combine two claims.

The authors' method produced substantially higher fractions of reads in nuclei, suggesting low ambient RNA contamination.

“Importantly, our method produced substantially higher fractions of reads in nuclei (Figure 2F), suggesting low ambient RNA contamination.”

May combine two claims.

HFD resulted in a relative reduction in adipocytes and an increase in macrophage/monocyte fractions in both eWAT and iWAT, with more prominent changes in eWAT.

“HFD resulted in a relative reduction in adipocytes and an increase in macrophage/monocyte fractions in both eWAT and iWAT, with more prominent changes in eWAT (Figure 2J, Figure 2—figure supplement 2E).”

Classified as probably not a substantial claim.

Capillary endothelial cells (CapEC) were further divided into arterial capillary endothelial cells (AcapEC) and venous capillary endothelial cells (VcapEC).

“CapEC was further divided into arterial capillary endothelial cells (AcapEC) and venous capillary endothelial cells (VcapEC) (Figure 3—figure supplement 3B)”

May combine two claims.

MC6 and MC7 resembled previously described regulatory macrophages (RegM) and collagen-expressing macrophages (CEM), respectively.

“We found two minor macrophage subpopulations, MC6 and MC7, which resembled previously described regulatory macrophages (RegM) and collagen-expressing macrophages (CEM), respectively (Figure 3D, Figure 3—figure supplement 2A).”

May combine two claims.

The most remarkable change in lymphocytes during HFD feeding was the emergence of a small B cell subpopulation, BC2, observed in eWAT, which highly expressed genes involved in B cell proliferation, such as Prkar2b and Mcc.

“The most remarkable change in lymphocytes during HFD feeding was the emergence of a small B cell subpopulation, BC2, observed in eWAT (Figure 3G), which highly expressed genes involved in B cell proliferation, such as Prkar2b and Mcc (Figure 3—figure supplement 2F).”

May combine two claims.

A total of 53,395 cells/nuclei were identified, distributed across 10 distinct cell types.

“After filtering nuclei based on the detected numbers of UMIs and genes, and subsequently removing doublets and ambient RNA, a total of 53,395 cells/nuclei were identified, distributed across 10 distinct cell types (Figure 2H).”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The paper's snRNA-seq data had higher recovery of vascular cells than prior studies.

“The higher recovery of vascular cells in our snRNA-seq data provided comprehensive insights into the vasculature of adipose tissues.”

May combine two claims.

In response to HFD feeding, only eWAT exhibited a marked relative reduction in MC1 and MC2 populations with a substantial increase in MC3 (LAMs).

“In response to HFD feeding, only eWAT exhibited a marked relative reduction in MC1 and MC2 populations with a substantial increase in MC3 (LAMs) (Figure 3F).”

May combine two claims.

The prevalence of MC6 and MC7 showed depot-dependent differences, with eWAT demonstrating a higher abundance of RegM and iWAT containing more CEM.

“Their prevalence showed depot-dependent differences, with eWAT demonstrating a higher abundance of RegM and iWAT containing more CEM (Figure 3F).”

May combine two claims.

Most minor cell populations exhibited minimal differences across conditions, except dendritic cells increased in eWAT in response to HFD, and mast cells were more abundant in iWAT than eWAT.

“Most of the minor cell populations exhibited minimal differences across the conditions, except for dendritic cells which increased in eWAT in response to HFD, and mast cells which were more abundant in iWAT than eWAT (Figure 2—figure supplement 2E).”

May combine two claims.

MC1 and MC2 cells corresponded to perivascular-like and non-perivascular-like macrophages, respectively.

“These MC1 and MC2 cells corresponded perivascular-like and non-perivascular-like macrophages, respectively, as described by Sárvári et al., 2021.”

• cites Sárvári et al., 2021Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

MC4 and MC5, accounting for approximately 20% of the myelocyte populations, were identified as classical monocytes and non-classical monocytes, respectively.

“MC4 and MC5, accounting for approximately 20% of the myelocyte populations, were identified as classical monocytes and non-classical monocytes, respectively (Figure 3D, Figure 3—figure supplement 2A).”

May combine two claims.

The distribution of APC subpopulations differed between adipose depots, with eWAT displaying a higher fraction of APC3 and APC4, while iWAT showed a larger fraction of APC1.

“The distribution of APC subpopulations differed between adipose depots, with eWAT displaying a higher fraction of APC3 and APC4, while iWAT showed a larger fraction of APC1 (Figure 3C).”

May combine two claims.

The distribution of T cell subpopulations differed between adipose depots: CD4+ and CD8+ T cells were more abundant in iWAT, whereas Treg were more prevalent in eWAT.

“The distribution of T cell subpopulations differed between adipose depots: CD4+ and CD8+ T cells were more abundant in iWAT, whereas Treg were more prevalent in eWAT (Figure 3I).”

May combine two claims.

Major cell types present in adipose tissues included adipocytes, adipocyte progenitor cells (APC), macrophages/monocytes, and endothelial cells; less abundant cell types included pericytes/smooth muscle cells, mesothelial cells, dendritic cells, mast cells, T cells, and B cells.

“The major cell types present in adipose tissues included adipocytes, adipocyte progenitor cells (APC), macrophages/monocytes, and endothelial cells. Additionally, we identified less abundant cell types, such as pericytes (PC)/smooth muscle cells (SMC), mesothelial cells, dendritic cells, mast cells, T cells, and B cells.”

May combine two claims.

The proportions of mitochondrial reads in the authors' datasets represented less than 1% (mean 0.37%), significantly lower than in other datasets (mean 38.7 and 2.31%).

“Also, the proportions of mitochondrial reads in our datasets represented less than 1% (mean 0.37%), which was significantly lower than in the other datasets (mean 38.7 and 2.31%; Figure 2E).”

May combine two claims.

The proportions of these cell types in the authors' data largely matched those reported in previous studies, except for notable improvement in recovering vascular cells (endothelial cells and PC/SMC).

“The proportions of these cell types in our data largely matched those reported in previous studies, except for the notable improvement in recovering vascular cells (endothelial cells and PC/SMC) (Figure 2—figure supplement 2B).”

May combine two claims.

Strong PCR amplification and highly specific expression of adipocyte markers in adipocyte nuclei and immune/endothelial cell markers in non-adipocyte nuclei were observed.

“We observed strong PCR amplification and highly specific expression of adipocyte markers in adipocyte nuclei and immune/endothelial cell markers in non-adipocyte nuclei (Figure 2-figure supplement 1A).”

May combine two claims.

Nuclear RNA isolated using the optimized protocol remained intact at 2 and 6 hr and maintained good quality even after 24 hr.

“In contrast, nuclear RNA isolated using the optimized protocol remained intact at 2 and 6 hr, maintaining good quality even after 24 hr (Figure 1G).”

May combine two claims.

APC fractions were smaller in eWAT compared to iWAT under chow diet, with marginal effects under HFD.

“APC fractions were smaller in eWAT compared to iWAT under chow diet, with marginal effects under HFD.”

May combine two claims.

APC4, the largest subpopulation, represented committed preadipocytes, expressing lipid metabolism-related genes such as Lpl, Hsd11b1, and Fabp4.

“APC4, the largest subpopulation, represented ‘committed preadipocytes’, expressing lipid metabolism-related genes such as Lpl, Hsd11b1, and Fabp4 (Figure 3A, Figure 3—figure supplement 1D).”

May combine two claims.

Gene cluster 1 represented a group of genes generally upregulated by HFD across all subpopulations, with the most significant induction observed in Ad3, followed by Ad4 and Ad2 to a lesser extent.

“Gene cluster 1 represented a group of genes generally upregulated by HFD across all subpopulations, with the most significant induction observed in Ad3, followed by Ad4 and Ad2 to a lesser extent.”

May combine two claims.

B2M was particularly found within crown-like structures (CLS), as well as in the cytoplasm of adipocytes neighboring these CLS.

“B2M was particularly found within crown-like structures (CLS), as well as in the cytoplasm of adipocytes neighboring these CLS (Figure 6C, D).”

May combine two claims.

Under HFD conditions, they observed markedly higher H2DCFDA fluorescence intensity in eWAT and a modest increase in iWAT, resulting in a significantly larger fraction of H2DCFDA-positive adipocytes, especially in eWAT.

“Under HFD conditions, we observed markedly higher H2DCFDA fluorescence intensity in eWAT and a modest increase in iWAT, resulting in a significantly larger fraction of H2DCFDA-positive adipocytes, especially in eWAT (Figure 6A, B).”

May combine two claims.

The population size of mAd3 was much smaller than Ad6 in their data and did not show consistent population changes during obesity.

“However, the population size of mAd3 was much smaller than Ad6 in our data and did not show consistent population changes during obesity (Figure 6—figure supplement 1D).”

May combine two claims.

In HFD-fed mice, eWAT predominantly contained larger adipocyte nuclei, while iWAT showed a heterogeneous size range from small to large nuclei.

“In HFD-fed mice, eWAT predominantly contained larger adipocyte nuclei, while iWAT showed a heterogeneous size range from small to large nuclei (Figure 4F, G).”

May combine two claims.

Differential gene expression analysis followed by gene ontology analysis revealed that large adipocytes promote processes involved in insulin response, vascularization and DNA repair, while inhibiting processes related to cell migration, metabolism and the cytoskeleton.

“Differential gene expression analysis followed by gene ontology analysis revealed that large adipocytes promote processes involved in insulin response, vascularization and DNA repair, while inhibiting processes related to cell migration, metabolism and the cytoskeleton (Figure 4—figure supplement 1G, H).”

May combine two claims.

eWAT and iWAT displayed contrasting preferences in adipocyte trajectories during HFD feeding: the dysfunctional trajectory leading to Ad6 was dominant in eWAT while the adaptive trajectory toward Ad3 remained more prevalent in iWAT.

“Importantly, eWAT and iWAT displayed contrasting preferences in adipocyte trajectories during HFD feeding: the dysfunctional trajectory leading to Ad6 was dominant in eWAT while the adaptive trajectory toward Ad3 remained more prevalent in iWAT (Figure 4C).”

May combine two claims.

Pseudotime trajectory analysis revealed two divergent trajectories: one moving toward Ad3 and Ad4, and the alternate progressing toward Ad5 and ultimately to Ad6.

“Two divergent trajectories were revealed: One trajectory moves toward Ad3 and Ad4, which may depict an ‘adaptive healthy transition’. Conversely, the alternate trajectory progresses toward Ad5 and ultimately to Ad6, indicating a potential ‘maladaptive dysfunctional transition’.”

May combine two claims.

While most GFP-labeled adipocyte nuclei robustly expressed PPARγ proteins under typical conditions, a significant proportion of adipocyte nuclei from eWAT in HFD-fed mice lacked PPARγ proteins.

“While most GFP-labeled adipocyte nuclei robustly expressed PPARγ proteins under typical conditions, a significant proportion of adipocyte nuclei from eWAT in HFD-fed mice lacked PPARγ proteins (Figure 6E, F)”

May combine two claims.

Their optimized protocol successfully preserved not only nuclear RNA quality in various tissue types, but also the integrity of the nuclear structure.

“In contrast, our optimized protocol successfully preserved not only nuclear RNA quality in various tissue types, but also the integrity of the nuclear structure.”

May combine two claims.

Gene clusters 2 and 3 comprised genes up- and down-regulated, respectively, with changes most pronounced in Ad6 and to a lesser extent in Ad5, during HFD feeding.

“On the other hand, Gene clusters 2 and 3 comprised genes up- and down-regulated, respectively, with changes most pronounced in Ad6 and to a lesser extent in Ad5, during HFD feeding.”

May combine two claims.

Certain genes differentiated between Ad3-Ad4 and Ad5-Ad6: Hdac9 was more enriched in Ad3, while Pde2a was more enriched in Ad5-Ad6.

“For example, Hdac9 was more enriched in Ad3, while Pde2a was in more enriched in Ad5–Ad6 (Figure 4I).”

May combine two claims.

The Ad6 proportion exhibited a substantial increase in eWAT and a moderate rise in iWAT.

“Notably, the Ad6 proportion exhibited a substantial increase in eWAT and a moderate rise in iWAT (Figure 4C, Figure 4—figure supplement 1D).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The automatically generated RNA integrity number (RIN) does not accurately reflect the quality of nuclear RNA due to the presence of a substantial amount of small RNA molecules.

“Since the automatically generated RNA integrity number provided by the system did not accurately reflect the quality of nuclear RNA due to the presence of a substantial amount of small RNA molecules, we opted for a qualitative analysis of the RNA quality.”

May combine two claims.

MUH adipocytes showed deficient expression of PPARγ as well as a substantial decline in metabolic gene expression.

“Interestingly, MUH adipocytes showed deficient expression of the master transcriptional regulator PPARγ as well as a substantial decline in metabolic gene expression.”

May combine two claims.

They validated the presence of MUH adipocytes, primarily around CLS in obese eWAT with oxidative stress and inflammation markers.

“We not only validated the presence of MUH adipocytes, primarily around CLS in obese eWAT with oxidative stress and inflammation markers”

May combine two claims.

During HFD feeding, adipocytes notably enlarged, particularly in eWAT, whereas iWAT exhibited a broader distribution with a mixture of small and large adipocytes.

“During HFD feeding, adipocytes notably enlarged, particularly in eWAT, whereas iWAT exhibited a broader distribution with a mixture of small and large adipocytes.”

May combine two claims.

This study introduces a robust nucleus isolation method for snRNA-seq, leading to enhanced data quality.

“In conclusion, this study introduces a robust nucleus isolation method for snRNA-seq, leading to enhanced data quality.”

May combine two claims.

They further noted two distinct types of hypertrophic adipocytes developed by HFD feeding: MHH adipocytes (Ad3) abundant in iWAT, and MUH adipocytes (Ad6) prevalent in eWAT.

“We further noted two distinct types of hypertrophic adipocytes developed by HFD feeding: MHH adipocytes (Ad3) abundant in iWAT, and MUH adipocytes (Ad6) prevalent in eWAT.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Nuclei isolated from in vivo tissues are typically more delicate than those from cultured cells, with potential variation depending on cell type.

“Nuclei isolated from in vivo tissues are typically more delicate than those from cells cultured in vitro, with potential variation depending on cell type.”

May combine two claims.

The study establishes a robust snRNA-seq method providing novel insights into biological processes involved in adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.

“Our study establishes a robust snRNA-seq method, providing novel insights into the biological processes involved in adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.”

Classified as probably not a substantial claim.

Acquiring high-quality snRNA-seq data from adipose tissues remains highly challenging, with technical difficulties posing a major hurdle to accuracy, sensitivity, and efficacy in understanding adipose tissue remodeling, especially regarding mature adipocyte populations during obesity.

“Despite this progress, acquiring high-quality snRNA-seq data from adipose tissues remains highly challenging. Technical difficulties have posed a major hurdle to achieving accuracy, sensitivity, and efficacy in understanding adipose tissue remodeling, especially regarding mature adipocyte populations during obesity.”

Classified as probably not a substantial claim.

Single-nucleus RNA sequencing (snRNA-seq) encounters technical challenges in obtaining high-quality nuclei and RNA, which hinder its applications.

“Single-nucleus RNA sequencing (snRNA-seq), an alternative to single-cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications.”

• Also stated in: § Introduction · plain

  “Nevertheless, snRNA-seq also poses challenges to be considered. Once released from cells, nuclei are highly fragile and susceptible to mechanical stress, resulting in leakage or aggregation. Moreover, their enclosed RNA becomes increasingly vulnerable to RNases, leading to rapid RNA degradation.”

May combine two claims.

Adipocyte subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot.

“These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot.”

May combine two claims.

Collectively, the high-quality data enabled accurate and detailed characterization of diverse immune cell populations in adipose tissue, revealing their dynamic changes during obesity in a depot-dependent manner.

“Collectively, our high-quality data enabled accurate and detailed characterization of diverse immune cell populations in adipose tissue, revealing their dynamic changes during obesity in a depot-dependent manner.”

May combine two claims.

The HFD-induced reduction of LEC, along with the decline in VcapEC, implies that metabolic drainage into lymphatic vessels and the venous circulation could be a key pathological feature of visceral adipose tissues during obesity.

“The HFD-induced reduction of LEC, along with the aforementioned decline in VcapEC, implies that metabolic drainage into lymphatic vessels and the venous circulation could be a key pathological feature of visceral adipose tissues during obesity.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Impaired metabolite transport into the venous circulation may potentially contribute to the pathology of visceral adipose tissue.

“which may potentially contribute to the pathology of visceral adipose tissue.”

May combine two claims.

The optimized protocol produces high-quality snRNA-seq data that outperforms other existing methods.

“Collectively, these findings suggest that our optimized protocol produces high-quality snRNA-seq data that outperforms other existing methods.”

Probably not a substantial claim: may not pass S4 (based).

The decrease in VcapEC indicates impaired metabolite transport into the venous circulation.

“indicating impaired metabolite transport into the venous circulation”

May combine two claims.

Their method addressed the issue of VRC inhibiting reverse transcriptase by reducing VRC from nucleus isolation buffer prior to loading onto microfluidic devices, as confirmed by robust qRT-PCR and snRNA-seq results.

“Our method addressed this issue by reducing VRC from nucleus isolation buffer prior to loading onto microfluidic devices, as confirmed by the robust qRT-PCR and snRNA-seq results we obtained.”

May combine two claims.

VRC functions as a transition state analog, binding to the active sites of RNases and inhibiting their enzymatic activity.

“VRC functions as a transition state analog, binding to the active sites of RNases and inhibiting their enzymatic activity (Gray, 1974; Berger and Birkenmeier, 1979).”

• cites Gray, 1974 sourceNot checked — no full text of the source
• cites Berger and Birkenmeier, 1979 sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

This discrepancy may be due to different technical robustness; the dysfunctional cellular state of this population, with severely compromised RNA contents, may have made it difficult to accurately capture using standard protocols in the previous study, while their protocol enabled robust and precise detection.

“This discrepancy may be due to different technical robustness; the dysfunctional cellular state of this population, with severely compromised RNA contents, may have made it difficult to accurately capture using standard protocols in the previous study, while our protocol enabled robust and precise detection.”

May combine two claims.

Ad3-Ad4 and Ad5-Ad6 may represent hypertrophic adipocytes.

“suggesting that they may represent hypertrophic adipocytes.”

May combine two claims.

The induction of genes in cluster 2, as well as the repression of genes in cluster 3, reflect stress response, inflammation, and cell death in adipose tissues, along with impaired machinery regulating gene expression during HFD-induced adipose pathology.

“The induction of genes in cluster 2, as well as the repression of genes in cluster 3, reflect stress response, inflammation, and cell death in adipose tissues, along with impaired machinery regulating gene expression (e.g., chromatin remodeling and mRNA metabolic process) during HFD-induced adipose pathology (Figure 5B).”

May combine two claims.

This subpopulation represented a dysfunctional state of adipocytes, characterized by elevated expression of stress response and inflammation genes, coupled with the loss of metabolic genes.

“This subpopulation represented a dysfunctional state of adipocytes, characterized by elevated expression of stress response and inflammation genes, coupled with the loss of metabolic genes.”

May combine two claims.

Taken together, Ad6 represents a subpopulation of stressed and dying adipocytes with transcriptional shutdown that arise during obesity.

“Taken together, Ad6 represents a subpopulation of stressed and dying adipocytes with transcriptional shutdown that arise during obesity.”

May combine two claims.

This implies that Ad3 represents 'metabolically healthy hypertrophic (MHH)' adipocytes, while Ad6 represents 'metabolically unhealthy hypertrophic (MUH)' adipocytes.

“This implies that Ad3 represents ‘metabolically healthy hypertrophic (MHH)’ adipocytes, while Ad6 represents ‘metabolically unhealthy hypertrophic (MUH)’ adipocytes.”

May combine two claims.

Their single-nucleus atlas of mouse white adipose tissues generally aligns with previous studies while offering deeper insights into depot specificity and cellular composition dynamics during obesity.

“Our single-nucleus atlas of mouse white adipose tissues generally aligns with previous studies while offering deeper insights into depot specificity and cellular composition dynamics during obesity.”

May combine two claims.

Their findings indicate that adipocytes may exhibit a unique pathological cellular state with significantly reduced RNA content, which may contribute to the poor consistency in adipocyte heterogeneity in prior studies with suboptimal RNA quality.

“Our findings indicate that adipocytes may exhibit a unique pathological cellular state with significantly reduced RNA content, which may contribute to the poor consistency in adipocyte heterogeneity in prior studies with suboptimal RNA quality.”

Classified as probably not a substantial claim.

Each sample was filtered prior to downstream analyses based on the number of UMIs (≥500) and genes (≥250), mitochondrial ratio (<15%), and the number of genes detected per UMI (>0.8).

“each sample was further filtered prior to downstream analyses based on the number of UMIs (≥500) and genes (≥250), mitochondrial ratio (<15%), and the number of genes detected per UMI (>0.8).”

Probably not a substantial claim: may not pass S2 (checkable).

Therefore, using a robust method to effectively preserve RNA quality may be critical for accurately characterizing adipocyte populations, especially in disease states.

“Therefore, using a robust method to effectively preserve RNA quality may be critical for accurately characterizing adipocyte populations, especially in disease states.”

Probably not a substantial claim: may not pass S3 (load-bearing).

During dissection of eWAT, epididymal ducts were carefully removed to minimize the risk of contamination.

“During the dissection of eWAT, we carefully removed epididymal ducts from the adipose tissues, minimizing the risk of contamination.”

Classified as probably not a substantial claim.

Gating based on forward scatter, side scatter, and Hoechst fluorescence was set to eliminate debris and nucleus doublets/multiplets.

“We set gating based on forward scatter, side scatter, and Hoechst fluorescence to eliminate debris and nucleus doublets/multiplets.”

May combine two claims.

They observed significant discrepancies in the size and changes of this population during obesity, likely due to differences in technical robustness.

“However, we observed significant discrepancies in the size and changes of this population during obesity, likely due to differences in technical robustness.”

Probably not a substantial claim: may not pass S3 (load-bearing).

CellBender v0.3.0 was applied to remove counts from ambient/background RNA.

“CellBender v0.3.0 (Fleming et al., 2023) was applied to remove counts from ambient/background RNA.”

• cites Fleming et al., 2023 sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

Significance was determined based on false discovery rate <0.05 and absolute log2 fold change >1.

“Significance was determined based on false discovery rate <0.05 and absolute log2 fold change >1.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

This adaptability has the potential to unlock a wide array of applications for snRNA-seq techniques.

“This adaptability has the potential to unlock a wide array of applications for snRNA-seq techniques.”

May combine two claims.

An important advancement in their dataset was the improved recovery of vascular cell populations, which were underrepresented in prior studies, likely due to their protocol's effective tissue homogenization and the high quality of RNA preserved in isolated nuclei.

“An important advancement in our dataset was the improved recovery of vascular cell populations, which were underrepresented in prior studies, likely due to our protocol’s effective tissue homogenization and the high quality of RNA preserved in isolated nuclei.”

Classified as probably not a substantial claim.

For iWAT, lymph nodes were removed to avoid over-representation of lymphocytes.

“For iWAT, the lymph nodes were removed to avoid over-representation of lymphocytes.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

This advancement holds the potential to greatly improve experimental logistics.

“This advancement holds the potential to greatly improve experimental logistics.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Gene counts were first normalized using SCTransform, and regressed on mitochondrial read ratio and cell cycle score by Seurat.

“Gene counts were first normalized using SCTransform, and regressed on mitochondrial read ratio and cell cycle score by Seurat.”

Probably not a substantial claim: may not pass S2 (checkable).

This advancement has enabled the discovery of novel insights into the dynamics of adipose tissue remodeling during obesity.

“This advancement has enabled the discovery of novel insights into the dynamics of adipose tissue remodeling during obesity.”

May combine two claims.

MUH adipocytes did not show significant expression of marker genes for APC, indicating that they are not in a state of dedifferentiation.

“However, MUH adipocytes did not show significant expression of marker genes for APC, indicating that they are not in a state of dedifferentiation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the clinical setting, this will enable patient biopsies to be collected for diagnostic tests and then be analyzed via snRNA-seq at a different facility.

“In the clinical setting, for example, this will enable patient biopsies to be collected for diagnostic tests and then be analyzed via snRNA-seq at a different facility.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

At the gene level, genes detected in less than 10 nuclei and mitochondrial genes were filtered.

“At the gene level, genes detected less than 10 nuclei and mitochondrial genes were filtered.”