Abstract

   Thrombospondin-1 (TSP1) is a matricellular protein with many important
   roles in mediating carcinogenesis, fibrosis, leukocyte recruitment, and
   metabolism. We have previously shown a role of diet in the absence of
   TSP1 in liver metabolism in the context of a colorectal cancer model.
   However, the metabolic implications of TSP1 regulation by diet in the
   liver metabolism are currently understudied. Therefore Discrete
   correlation summation (DCS) was used to re-interrogate data and
   determine the metabolic alterations of TSP1 deficiency in the liver,
   providing new insights into the role of TSP1 in liver injury and the
   progression of liver pathologies such as nonalcoholic fatty liver
   disease (NAFLD). DCS analysis provides a straightforward approach to
   rank covariance and data clustering when analyzing complex data sets.
   Using this approach, our previous liver metabolite data was re-analyzed
   by comparing wild-type (WT) and Thrombospondin-1 null (Thbs1^−/−) mice,
   identifying changes driven by genotype and diet. Principal component
   analysis showed clustering of animals by genotype regardless of diet,
   indicating that TSP1 deficiency alters metabolite handling in the
   liver. High-fat diet consumption significantly altered over 150
   metabolites in the Thbs1^−/− livers versus approximately 90 in the
   wild-type livers, most involved in amino acid metabolism. The absence
   of Thbs1 differentially regulated tryptophan and tricarboxylic acid
   cycle metabolites implicated in the progression of NAFLD. Overall, the
   lack of Thbs1 caused a significant shift in liver metabolism with
   potential implications for liver injury and the progression of NAFLD.

   Keywords: discrete correlation summation, thrombospondin, diet,
   non-alcoholic fatty liver disease, liver, oxidative stress

1. Summary

   Thrombospondin-1 (TSP1) is a matricellular protein that regulates
   cell-to-cell and cell-to-matrix interactions by modulating signaling in
   response to extracellular factors and through direct interaction with
   high-affinity cell surface receptors [[36]1]. TSP1 functions as an
   immunomodulatory molecule that is upregulated in inflammatory
   conditions. It activates latent transforming growth factor beta-1
   (TGF-β) and can lead to the recruitment of leukocytes to inflammatory
   sites [[37]2,[38]3,[39]4,[40]5,[41]6]. TSP1 is secreted from
   endothelial cells, monocytes, platelets, and adipocytes. TSP1 secreted
   from adipocytes functions as an adipokine, and elevated levels of TSP1
   are associated with obesity and insulin resistance [[42]7,[43]8].
   Therefore, it is not surprising that increased TSP1 levels are
   implicated in inflammation and metabolic syndrome. Still, the role of
   TSP1 in the regulation of cellular energetics is not entirely
   understood.

   TSP1 expression also limits carcinogenesis by inhibiting angiogenesis,
   preventing tumor neovascularization, and affecting tumor cell adhesion,
   invasion, and proliferation [[44]9]. Loss of TSP1 contributes to
   increased tumor multiplicity and decreased survival time in a murine
   model of colon carcinogenesis [[45]10]. We have previously demonstrated
   that the absence of TSP1 modulates liver metabolism in a
   C57BL/6J-ApcMin/j (Apc^Min) model of colorectal cancer [[46]10]. To
   determine the effects of TSP1 in this model, Apc^Min/+ mice were
   crossed with Thbs1^−/− mice to produce the Thbs1^+/−:Apc^Min/+ strain.
   These mice were then crossed with Thbs1^−/− mice to produce Thbs1^−/−
   Apc^Min/+ mice. Mice were pair-fed a 5% low fat or 21% high-fat diet
   starting at weaning [[47]10]. Thbs1^−/−:Apc^Min/+ mice had reduced
   survival times compared to Apc^Min/+ mice when fed a low-fat diet,
   whereas animals fed a high-fat diet had similar survival times
   regardless of TSP1 expression. Thbs1^−/− animals had an overall
   increase in tumor multiplicity, with the animals fed a high-fat diet
   having significantly more tumors than those fed a low-fat diet. The
   absence of TSP1 in the context of Apc^Min/+ also regulated amino acid
   and lipid metabolism and eicosanoids and ketone body formation in the
   liver [[48]10].

   The liver plays a significant role in regulating carbohydrate, lipid,
   and protein metabolism, with diet playing an essential role in the
   initiation and progression of nonalcoholic fatty liver disease (NAFLD)
   [[49]11]. NAFLD can progress to nonalcoholic steatohepatitis (NASH)
   when fat accumulation in the liver causes damage and the onset of
   inflammation [[50]12]. Lipid metabolism involves several pathways for
   the breakdown and generation of fatty acids. Complex lipids from the
   diet are broken down into non-esterified fatty acids, enter the cell by
   fatty acid transport proteins or diffusion across membranes, and are
   oxidized in the mitochondria or peroxisomes [[51]13]. In addition, de
   novo lipogenesis occurs in hepatocytes by converting carbohydrates into
   fatty acids, exported through lipoprotein production [[52]13]. Fatty
   acids and derivatives act as ligands and nuclear factors regulating
   gene transcription and driving liver metabolism by inducing changes in
   the activity of transcription factors in the peroxisome proliferator
   (PPAR) [[53]14,[54]15], liver x receptor (LXR) [[55]16,[56]17], hepatic
   nuclear factor 4 (HNF-4) [[57]18], and sterol regulatory
   element-binding protein (SREBP) families [[58]19,[59]20,[60]21].

   The mechanism of fatty acid regulation in the liver is relatively well
   understood, but no pharmaceuticals are currently approved to prevent or
   treat NAFLD/NASH [[61]22]. Preclinical NASH models implicate TSP1 in
   the modulation of NAFLD/NASH with evidence that Thbs1^−/− mice fed a
   choline-deficient L-amino acid-defined high-fat diet were protected
   from the liver damage associated with NASH [[62]12]. Furthermore,
   plasma TSP1 expression is increased in NAFLD human patients and reduced
   after fat-lowering lifestyle intervention, which is consistent with the
   ability of TSP1 to inhibit the uptake of free fatty acids via CD36 in
   endothelial cells [[63]23,[64]24]. In the same study, treatment with
   recombinant TSP1 or using a peptide mimetic resulted in the reduced
   accumulation of fat in hepatocytes mediated through CD36 [[65]23].

   Our previous studies examining liver metabolism in a
   Thbs1^−/−:Apc^Min/+ colorectal mouse model focused on the effects of
   diet and TSP1 in carcinogenesis [[66]10]. Given the paradoxical impact
   of TSP1 in liver fat accumulation, we reexamined our global
   metabolomics study focusing on WT and Thbs1^−/− mice fed a low-fat and
   a high-fat diet to further understand the role of this matricellular
   protein in liver metabolism and its potential implications in liver
   injury and diseases such as NAFLD/NASH.

2. Data Description

   Thbs1^−/− mice liver metabolites cluster differently than WT liver
   metabolites. To determine whether the lack of TSP1 gene expression
   results in metabolite variance, we reanalyzed metabolomics data from
   our previous study and compared WT and the Thbs1^−/− mice [[67]10].
   Mice were pair-fed a 5% low-fat diet or a 21% high-fat diet at weaning
   and maintained until 12 weeks of age [[68]10]. At the end of the study,
   mice were sacrificed, and liver tissue was subjected to metabolomics
   analysis [[69]10]. Following log transformation and imputation of
   missing values, ANOVA contrasts were used to identify biochemicals that
   differed significantly between experimental groups. Metabolite variance
   was examined using PCA plots ([70]Figure 1A,B); the leftmost points
   represent PCA variances for all metabolites with a p-value less than
   0.05, and the rightmost points represent all metabolites with a p-value
   less than 0.1. Discrete correlate summation analysis (DCS) was applied
   to all metabolites using unadjusted p-values. The unadjusted (p-values)
   were used due to the relative number of metabolites that were found to
   be significantly regulated in our data set. Still when considering
   standards for reporting metabolomics data [[71]25], we calculated
   adjusted (q values) which are found in the original dataset [[72]10]
   and in [73]Supplementary Tables S1–S4. The midpoints represent all
   metabolites with a p-value less than 0.05 plus metabolites clustered by
   DCS. PCA ([74]Figure 1C,D) was used on the midpoint metabolite sets
   with an overall (PC1 + PC2) percent variance increase, combined with an
   enrichment of key metabolites, which would not have been considered
   with the simple 0.05 p-value cut-off. Clustering of the matrices of
   metabolites from WT and Thbs1^−/− mice fed a low-fat diet ([75]Figure
   1A and [76]Appendix A [77]Figure A1) and mice fed a high-fat diet
   ([78]Figure 1B and [79]Appendix A [80]Figure A2) illustrates the
   aggregation of DCS metabolites. Principal component analysis indicated
   that liver metabolites of Thbs1^−/− mice fed a low-fat diet cluster
   differently than WT mice on the same diet ([81]Figure 1C). This
   indicates that some metabolic differences segregate mice by genotype.
   Similarly, analysis of liver metabolites from mice fed a high-fat diet
   revealed a clear clustering of metabolites between WT and Thbs1^−/−
   mice ([82]Figure 1D). This suggests that the absence of Thbs1 gene
   expression impacts metabolism during basal conditions and after feeding
   mice a high-fat diet.

Figure 1.

   [83]Figure 1
   [84]Open in a new tab

   Deficiency of TSP1 is associated with distinct liver metabolic
   signatures in low-fat and high-fat-diet-fed mice. Discrete correlate
   summation analysis was calculated to enrich metabolite sets together
   with those exhibiting a fold change p-value of < 0.05 from Thbs1^−/−
   vs. WT mice fed a low-fat (A) or a high-fat diet (B). (C) Principal
   component analysis of liver metabolites from Thbs1^−/− vs. WT animals
   fed a low-fat and (D) high-fat diet. N = 8/group.

   Liver metabolite alterations by consumption of a high-fat diet.
   Consumption of a high-fat diet in WT mice led to significant (p < 0.05)
   differential expression of 90 metabolites ([85]Figure 2A,B,
   [86]Supplemental Table S1). Forty-one metabolites were upregulated, and
   forty-nine were downregulated after feeding a high-fat diet to WT mice.
   The majority of regulated metabolites (52.2%) were lipid metabolites
   ([87]Figure 2B). In WT mice, a high-fat diet was associated with
   hepatic accumulation of various long-chain, saturated, monounsaturated,
   and polyunsaturated free fatty acids ([88]Figure 2C). Consistently,
   pathway enrichment analysis of WT HFD vs. WT LFD showed high enrichment
   of long-chain fatty acids; however, the highest enrichment was observed
   in ascorbate and aldarate metabolism, suggesting changes in the
   processing of vitamins and cofactors ([89]Figure 3A). Overall, these
   data suggest expected metabolic handling when feeding a high-fat diet.
   On the other hand, providing a high-fat diet to Thbs1^−/− mice resulted
   in broader alterations in 156 significantly regulated metabolites
   ([90]Figure 2D), suggesting altered handling of a high-fat diet between
   WT and Thbs1^−/− animals. As seen with WT, most of those altered (49%)
   are lipid metabolites ([91]Figure 2E). Pathway enrichment also
   suggested that the most-regulated pathways include sterol, medium-, and
   long-chain amino acids ([92]Figure 3B). Although Thbs1^−/− animals had
   more altered metabolites, the overall percentage of regulated
   metabolites is similar between groups. In addition to homeostasis and
   lipid metabolism, converting cholesterol to bile acids is a critical
   liver function, and NAFLD has been shown to alter bile acid production
   [[93]26]. WT mice fed a high-fat diet showed a significant reduction of
   the bile acid tauro-beta-muricholate relative to WT mice fed a low-fat
   diet, while Thbs1^−/− mice fed a high-fat diet had increased tauro-beta
   muricholate. This metabolite is known to regulate the Farnesoid X
   receptor (FXR) superfamily and plays a vital role in the action of bile
   acids on liver homeostasis [[94]27,[95]28]. Thbs1^−/− mice fed a
   high-fat diet showed increased beta-muricholate, implicated in liver
   steatoses [[96]29]. Thbs1^−/− mice fed a high-fat diet had increased
   levels of cholesterol, as well as the cholesterol precursor squalene
   and the primary bile acid precursor 7α-hydroxy-3-oxo-4-cholestenoate
   (7-Hoca) when compared to Thbs1^−/− mice fed a low-fat diet, suggesting
   increased bile acid synthesis in these animals ([97]Figure 2F and
   [98]Supplementary Table S2). The next most-regulated metabolic pathway
   was amino acid metabolism, with about 22.2% of metabolites regulated in
   WT livers belonging to the amino acid category vs. 28% in Thbs1^−/−
   livers ([99]Figure 2B,E). According to our analysis, tryptophan
   metabolism also played a crucial role in developing NAFLD and was one
   of the top enriched pathways ([100]Figure 3A,B). Serotonin is
   synthesized from tryptophan; inhibiting its synthesis protects mice
   from NAFLD by inhibiting obesity [[101]30]. One key difference is that
   indole acetate was the most elevated metabolite in the WT group
   ([102]Figure 2C) but was found at lower levels in Thbs1^−/− mice (2.89
   log2-fold in WT vs. 0.61 log2-fold in Thbs1^−/−). Indole acetate
   derived from dietary tryptophan is protective in NAFLD disease
   [[103]31], suggesting that altered tryptophan metabolism plays a role
   in disease pathogenesis.

Figure 2.

   [104]Figure 2
   [105]Open in a new tab

   Liver metabolism alterations by diet. (A) Volcano plot showing the
   number of metabolites regulated by diet in wild-type (WT) mice.
   Downregulated < −2-fold in green; upregulated > 2-fold in red;
   significantly altered between −2 and 2 in blue. (B) Pie chart depicting
   metabolites that exhibited significant diet-dependent changes in levels
   in WT mice. (C) Dot plot showing significantly regulated metabolites <
   −2- and > 2-fold in WT mice. (D) The volcano plot shows the number of
   metabolites regulated by diet in Thbs1^−/− mice. Downregulated <
   −2-fold in green; upregulated > 2-fold in red; significantly altered
   between −2 and 2 in blue. (E) Pie chart depicting metabolites that
   exhibited significant diet-dependent changes in levels in Thbs1^−/−
   mice. (F) Dot plot showing significantly regulated metabolites < −2-
   and > 2-fold in Thbs1^−/− mice. N = 8 in each group.

Figure 3.

   [106]Figure 3
   [107]Open in a new tab

   Metabolic pathway enrichment analysis. (A) Comparing metabolic pathways
   regulated by diet in wild-type (WT) mice. (B) Comparing metabolic
   pathways regulated by diet in thrombospondin 1-deficient (Thbs1^−/−)
   mice.

   A high-fat diet is associated with oxidative stress [[108]32], another
   component of the pathogenesis of NAFLD [[109]32,[110]33]. Glutathione
   is an important antioxidant highly concentrated in the liver [[111]34].
   Cysteine-glutathione disulfide, a rate-limiting precursor of
   glutathione synthesis, was significantly elevated in WT mice fed a
   high-fat diet compared to Thbs1^−/− mice fed a high-fat diet.
   Glutathione is oxidized to glutathione disulfide (GSSG) to compensate
   for oxidative stress. Our analysis shows that GSSG levels were
   decreased in Thbs1^−/− mice fed a high-fat diet. This could be mediated
   by the activity of glutathione reductase and glutathione peroxidase.
   Potentially due to an overall decrease in glutathione production or an
   increase in the reduced form. Another critical component of regulating
   redox homeostasis is the cysteine/cystine ratio, a lower percentage
   marker of oxidative stress in the clinic [[112]35]. Cystine, the
   oxidized form of cysteine, accumulated in Thbs1^−/− mice fed a high-fat
   diet. Increased cysteine levels have been shown to cause
   pro-inflammatory signaling in cardiovascular disease, and lower
   cysteine levels occur in patients with NAFLD [[113]35,[114]36].
   Methionine and S-methyl cysteine were elevated in Thbs1^−/− mice.
   Thbs1^−/− mice also had reduced levels of 4-hydroxy-nonenal-glutathione
   (HNE-GSH) when fed a high-fat diet compared to WT animals (−4.32
   log2-fold in WT vs. −6.64 log2-fold in Thbs1^−/−). Methionine is an
   essential amino acid, and methionine deficiency induces steatosis in
   animals [[115]37,[116]38]. Methionine is used to synthesize glutathione
   and metabolizes it to S-adenosylmethionine, an important methyl donor.
   Increased homocysteine levels are associated with increased oxidative
   stress, inflammation, and NAFLD [[117]39]. These changes in metabolites
   suggest TSP1-dependent regulation of redox stress responses.

   The progression of NAFLD is often associated with global and hepatic
   insulin resistance, which alters hepatic gluconeogenesis and glucose
   flux [[118]40]. Glycolytic metabolites, including lactate, were
   downregulated in WT mice fed a high-fat diet compared to those fed a
   low-fat diet, suggesting a shift in glucose metabolism due to fat
   consumption ([119]Figure 2C). Thbs1^−/− mice had increased pentose
   phosphate metabolites, including ribulose/xylulose 5-phosphate,
   6-phosphogluconate, and threitol, suggesting a potential shift in
   glucose metabolism toward the pentose phosphate pathway, which could
   perturb regulation of NADPH levels.

   Differential regulation of dietary metabolites by TSP1. To determine
   whether diet consumption is differentially regulated in WT and
   Thbs1^−/− mice, we compared liver metabolites from each genotype in
   high-fat and low-fat diet-fed mice. We observed 89 metabolites that
   differed in livers of Thbs1^−/− vs. WT mice fed a low-fat diet
   ([120]Figure 4A and [121]Supplementary Table S3). About 45% of these
   are amino acid metabolites, with 25 metabolites elevated in Thbs1^−/−
   compared to WT ([122]Figure 4A,B). Pathway enrichment analysis
   indicated that glutamate, alanine, and aspartate metabolic pathways are
   the most regulated when comparing Thbs1^−/− to WT. However, the most
   significantly upregulated metabolite was 5-hydroxy indole acetate, a
   tryptophan metabolite generated from serotonin ([123]Figure 4C and
   [124]Supplementary Table S3). Cholangiocytes, stellate liver cells, and
   microbes in the gut can produce serotonin from tryptophan
   [[125]30,[126]41]. Increases in serotonin metabolites, including
   5-hydroxy indole acetate, are associated with NAFLD development
   [[127]42]. The tryptophan metabolites indole acetate and kynurenine
   were also among the most elevated in Thbs1^−/− livers compared to WT
   livers, indicating a general TSP1-dependent regulation of tryptophan
   metabolism ([128]Figure 4C).

Figure 4.

   [129]Figure 4
   [130]Open in a new tab

   Lack of thrombospondin-1 (TSP1) alters liver metabolism. (A) Volcano
   plot showing the number of metabolites regulated by TSP1 deficiency in
   animals fed a low-fat diet (LFD). Downregulated < −2-fold in green;
   upregulated > 2-fold in red; significantly altered between −2 and 2 in
   blue. (B) Pie chart depicting metabolites that exhibited significant
   TSP1-dependent changes in mice fed an LFD. (C) Dot plot showing
   significantly regulated metabolites < −2- and > 2-fold in TSP1
   deficient mice fed an LFD. (D) Volcano plot shows the number of
   metabolites regulated by TSP1 deficiency in animals fed a high-fat diet
   (HFD). Downregulated < −2-fold in green. (E) Pie chart depicting
   metabolites that exhibited significant TSP1-dependent changes in mice
   fed an HFD. (F) Dot plot showing significantly regulated metabolites <
   −2- and > 2-fold in TSP1-deficient mice fed an HFD. N = 8/each group.

   Lipid metabolites account for about 26% of differentially regulated
   metabolites between Thbs1^−/− and WT mice fed a low-fat diet. The most
   significantly elevated metabolite was 1-linolenoyl
   glycerophosphocholine, a biomarker for developing insulin resistance,
   glucose intolerance, and type II diabetes [[131]43,[132]44]. Thbs1^−/−
   mice had decreased levels of the primary bile acids cholate and
   beta-muricholate as well as two secondary bile acids relative to WT
   mice, supporting a role for TSP1 in regulating cholesterol homeostasis
   [[133]45].

   Several TCA metabolites were elevated in Thbs1^−/− mice, most
   prominently α-ketoglutarate, with an over 3 log2-fold increase compared
   to WT mice fed the same low-fat diet. α-Ketoglutarate is a central
   intermediate metabolite of the TCA cycle, plays a vital role in
   regulating energy metabolism, and has been linked to obesity-associated
   liver pathology ([134]Supplementary Table S2)
   [[135]46,[136]47,[137]48].

   Approximately 9% of the metabolites regulated by genotype on a low-fat
   diet were nucleotide metabolites. All nucleotide metabolites were
   significantly depleted in Thbs1^−/− mice relative to WT. This may
   reflect decreased oxidative stress associated with a low-fat diet but
   may implicate TSP1 deficiency in the capacity to overcome liver injury.

   Comparing Thbs1^−/− to WT mice fed a high-fat diet, we observed 45
   differentially regulated metabolites, with the majority (60%) occurring
   in amino acid metabolites ([138]Figure 4D,E and [139]Supplementary
   Table S4). Similar to what was observed in the low-fat diet, 5-hydroxy
   indole acetate and indole acetate were also elevated in the Thbs1^−/−
   animals fed a high-fat diet ([140]Figure 4F). Pathway enrichment
   analysis indicated leucine, isoleucine, and valine metabolism
   regulation when comparing Thbs1^−/− to WT mice fed a high-fat diet,
   thus suggesting differences compared to low-fat diet-fed mice
   ([141]Figure 5A). Consistent with the animals fed a low-fat diet,
   α-ketoglutarate remained significantly elevated in Thbs1^−/− mice fed a
   high-fat diet, supporting the role of TSP1 in regulating this TCA cycle
   metabolite ([142]Figure 4F). As observed when feeding Thbs1^−/− a
   high-fat diet, we observed an over log2-fold reduction in
   4-hydroxy-nonenal-glutathione in Thbs1^−/− mice fed a high-fat diet
   compared to WT. Pathway enrichment analysis also shows that glutathione
   metabolism is significantly regulated in Thbs1^−/− mice ([143]Figure
   5B). These alterations suggest that Thbs1^−/− mice may handle oxidative
   stress associated with high-fat diet consumption differently from WT.

Figure 5.

   [144]Figure 5
   [145]Open in a new tab

   Metabolic pathway enrichment analysis. (A) Comparing TSP1-dependent
   metabolic pathways in mice fed a low-fat diet (LFD). (B) Comparing
   TSP1-dependent metabolic pathways in mice fed a high-fat diet (HFD).

   Results from this global biochemical profiling study revealed metabolic
   differences in liver tissue when comparing WT and Thbs1^−/− mice fed a
   low-fat or high-fat diet, with distinct clustering of metabolites based
   on genotype-specific handling of the diets. Exposure to a high-fat diet
   was associated with differential changes in lipid metabolism, TCA cycle
   intermediates, lipid-derived eicosanoids, amino acid, and protein
   metabolism compared to mice of the identical genotypes fed the low-fat
   diet. Notably, several of these pathways have been previously
   implicated in developing liver injury, including NAFLD. NAFLD is a
   spectrum of diseases in individuals who do not over-consume alcohol
   [[146]49]. NASH is an advanced presentation of the disease. It occurs
   when inflammation and hepatocellular ballooning with or without
   fibrosis in addition to the lipid accumulation and can advance to liver
   cirrhosis [[147]50]. Despite the relatively high prevalence of fatty
   liver disease with obesity, only a subset of the population progresses
   to inflammation and chronic liver disease [[148]51]. Histopathology is
   essential in evaluating NAFLD/NASH, which occurs clinically when at
   least 5% of hepatocytes display lipid accumulation [[149]49]. However,
   there is a need for designing non-invasive approaches and uncovering
   potential biomarkers to detect and stage NAFLD/NASH progression.

   Obesity, the pathological excess of body fat resulting from excess
   caloric intake, is a significant risk factor in developing NAFLD
   [[150]52]. Therefore, in order to determine whether TSP1 is implicated
   in regulating metabolism relevant to NAFLD, we performed a liver
   metabolomics analysis in WT and Thbs1^−/− animals fed low-fat or
   high-fat diets. Adipose tissue regulates energy balance and glucose
   homeostasis by secreting adipokines [[151]53]. Increased adiposity
   occurs during obesity and can lead to anatomic and functional
   abnormalities that manifest as altered adipokine production and other
   changes [[152]54,[153]55,[154]56]. A study on high-fat diet-induced
   obesity in a TSP1-deficient mouse model showed that TSP1 deficiency
   improved glucose–insulin homeostasis while decreasing adipose
   macrophage accumulation and inflammation [[155]57]. TSP1 mRNA
   expression and serum TSP1 were increased in rats fed high-fat diets.
   These animals also had lower insulin-stimulated glucose uptake and
   higher TSP1 expression in cultured adipocytes [[156]58].

   Furthermore, in a murine model of diet-induced NASH, Thbs1^−/− mice fed
   a choline-deficient L-amino acid-defined high-fat diet were protected
   against developing some of the characteristics of NASH including
   fibrosis, suggesting that TSP1 is critical for the development of NASH
   [[157]12]. In the same study, Thbs1^−/− mice exhibited decreased serum
   lipid levels and decreased mRNA levels of tumor necrosis factor-alpha
   (TNFα) and TGFβ1, which are associated with NAFLD progression
   [[158]12]. Serum TSP1 and TSP1 mRNA levels are increased in patients
   with NAFLD and other liver diseases and were positively associated with
   increasing severity of liver steatosis [[159]23,[160]59].
   Administration of recombinant TSP1 or the CD36-binding TSP1 mimetic
   ABT-526 inhibited liver steatosis in murine models of diet-induced
   obesity [[161]23]. These studies show the involvement of TSP1 in NAFLD
   and the potential role of the TSP1-CD36 signaling axis.

   When we compared the impact of consumption of a high-fat diet within
   each genotype, we observed a similar proportion of regulated
   metabolites associated with fatty acid metabolism. However, consuming a
   high-fat diet suppresses the number of genotype-dependent liver
   metabolites, whereas the number of high-fat diet-dependent metabolites
   is greater in the Thbs1^−/− mice. This is evidenced by the significant
   number of metabolites with an over 2-fold regulation in Thbs1^−/− mice
   and may further support studies suggesting no significant differences
   in lipid accumulation in the livers of Thbs1^−/− mice. Still, pathway
   enrichment analysis suggested high regulation of sterols in Thbs1^−/−
   mice, which may have implications for converting cholesterol to bile
   acids and liver homeostasis. Compared to WT mice fed a high-fat diet,
   Thbs1^−/− mice had increased tauro-beta-muricholate. This bile acid can
   act as an agonist of the Farnesoid X receptor (FXR) superfamily,
   regulating bile acid homeostasis and lipid metabolism, reducing bile
   acids, and contributing to hepatic steatosis [[162]27,[163]28]. When we
   compared the effect of diet between each genotype, we observed that the
   absence of TSP1 leads to the regulation of a larger proportion of amino
   acid metabolites. Previous studies examining transcriptional
   differences in murine models of NAFLD between WT and Thbs1^−/− mice
   suggested pathways related to amino acid metabolism as necessary in
   regulating NAFLD-mediated liver damage [[164]12]. Our analysis
   indicated differences in the regulation of tryptophan metabolism, which
   has been implicated in liver tissue integrity. Specifically, the
   microbiota-derived tryptophan metabolite indole acetate
   [[165]31,[166]60] was found to be upregulated in WT mice fed a high-fat
   diet compared to Thbs1^−/−. Administration of this metabolite in obese
   mice protects against NAFLD by reducing lipid accumulation,
   inflammation, and oxidative stress [[167]31]. Therefore, the relative
   reduction of indole acetate in Thbs1^−/− supports that the loss of TSP1
   may be detrimental to liver homeostasis during obesity-induced stress.
   Another metabolite associated with obesity severity that was
   upregulated in Thbs1^−/− regardless of diet was α-ketoglutarate; this
   TCA metabolite is linked to the severity of obesity and steatohepatitis
   [[168]46,[169]47]. We had previously reported the relative metabolite
   intensity of α-ketoglutarate in WT and Thbs1^−/− and had observed that
   it also remains elevated in the Thbs1^−/− Apc^Min/+, suggesting that
   TSP1 may be a key modulator of this metabolite. Loss of the TSP1
   receptor CD47 alters several metabolic pathways that consume or produce
   α-ketoglutarate [[170]61], suggesting that the observed alteration in
   Thbs1^−/− and Thbs1^−/− Apc^Min/+ mice may be mediated in part by the
   loss of CD47 signaling. This could be relevant to the pathogenesis of
   NASH because Cd47^−/− mice chronically fed a high-fat diet develop more
   severe hepatic steatosis and fibrosis [[171]62]. In contrast, Cd47^−/−
   mice fed a high-fat diet for 16 weeks were protected against hepatic
   lipid accumulation and had decreased inflammation [[172]63], suggesting
   a possible temporal implication of TSP1 signaling.

   Another pathway commonly regulated in Thbs1^−/− mice was glutathione
   metabolism, which is well known to be a regulator of oxidative stress
   [[173]64]. A critical metabolite in this pathway,
   4-hydroxy-nonenal-glutathione is reduced in the Thbs1^−/− mice;
   4-hydroxy-nonenal-glutathione is a hepatic metabolite formed by
   conjugating 4-hydroxynonenal (HNE) with glutathione and is a known
   marker of oxidative stress [[174]65]. It is a breakdown product of
   lipid peroxidation that reacts with DNA. Its conjugation with
   glutathione prevents the formation of DNA adducts, thus preventing DNA
   damage [[175]66]. This represents another mechanism by which the loss
   of TSP1 could mediate liver damage during oxidative stress and NAFLD
   progression [[176]67]. This is also consistent with other reports
   demonstrating that inhibition of TSP1 is associated with
   decreased/reduced glutathione activity in a model of
   acetaminophen-induced liver toxicity [[177]67]. This glutathione
   regulation was associated with lowering nuclear factor-erythroid
   2-related factor 2 (Nrf2), which is critical in mediating antioxidant
   signaling [[178]67]. Regulation of glutathione by TSP1 under conditions
   of redox stress could also be a CD47-dependent process, because
   CD47-deficient T cells were protected from the depletion of reduced
   glutathione induced by exposure to ionizing radiation and maintained
   higher levels of metabolites required for glutathione biosynthesis
   [[179]61].

   The mechanisms of liver pathology associated with NAFLD are complex.
   The current understanding of pathogenesis invokes a “multiple parallel
   hits” mechanism involving the gut, adipose tissue, immune system, and
   liver contributing to a cycle of inflammation and fibrosis and
   eventually damage [[180]68]. It is clear that many factors beyond fat
   accumulation in the liver drive the progression of NAFLD. Our data and
   others support the notion that TSP1 may be critical in regulating these
   processes. The paradoxical effects may be due to the spatiotemporal
   expression of this matricellular protein; while it may be increased in
   circulation in patients with NAFLD, the absence of TSP1 may also elicit
   harmful metabolic mechanisms that compromise liver tissue integrity.
   The difference may also be due to the source of TSP1;
   monocyte/macrophage populations release TSP1, contributing to insulin
   resistance and the progression of fatty liver and fibrosis, and
   macrophages of TSP1-deficient mice had reduced inflammatory phenotype
   macrophages and macrophage activation [[181]57,[182]60,[183]68]. The
   role of TSP1 in macrophage activation is partially mediated through
   TSP1 interacting with CD36, suppressing Smpdl3b expression, amplifying
   toll-like receptor 4, nuclear factor-κB, and TNF-α expression
   [[184]60,[185]69]. Therefore, the metabolic changes observed in liver
   tissue could be mediated by the effects of TSP1 release from
   macrophages on hepatocyte metabolism. Furthermore, the differential
   effects may be due to differences in microbiota; we observed the
   regulation of several secondary metabolites that depend on microbial
   processing. The role of TSP1 in modulating the microbiome remains to be
   defined and needs further study.

3. Methods

   Sample processing. The samples analyzed in this study were processed
   using the metabolon^® platform as reported previously [[186]10]. Scaled
   intensities were reported previously as a measurement of metabolite
   levels [[187]10]. Following log transformation and imputation of
   missing values, if any, with the minimum observed value for each
   compound, ANOVA contrasts were used to identify biochemicals that
   differed significantly between experimental groups. An estimate of the
   false discovery rate (q-value) is calculated both unadjusted and
   adjusted p-values, and scale intensities for each metabolite can be
   found in DOI: 10.1038/oncsis.2016.37(accessed 1 September 2022).

   Data set. The data set used in this study is based on the data
   published previously by Soto-Pantoja et al. in [[188]10] with the
   purpose of reanalyzing the data to determine genotype and diet effects
   between WT and Thbs1^−/− mice [[189]10]. Briefly, mice were fed an
   AIN-76A (5% fat) or a Western diet (#D12079B) with 21% fat beginning at
   weaning (Research Diets [190]https://researchdiets.com/) (accessed 1
   September 2022) [[191]10]. Livers of 12-week-old mice were harvested
   and subjected to tissue metabolomics using metabolon’s platform as
   described previously [[192]10].

   Discrete correlate summation and principal component analysis. One
   matrix of log correlation ratios (log[cr]) was calculated, as described
   previously [[193]70], for all metabolites in both conditions. Briefly,
   the correlation matrices were transformed into linear probability
   matrices using Student’s t-distribution for (n-2) degrees of freedom,
   returning two tails. A matrix of log[cr] was calculated (absolute value
   of the logarithm of the ratio of each comparison’s p-value) from the
   matrices of metabolites from WT and Thbs1^−/− mice fed a low-fat diet
   and mice fed a high-fat diet. The correlation matrix of this log[cr]
   matrix was calculated, and each column was totaled. The original
   log[cr]matrix was sorted in descending order based on these totals,
   establishing a clear clustering pattern. The upper cluster of this
   original matrix was determined by the totals of each variable column.
   The original matrix total array average was calculated right to left
   (RL-OriMaTArA). The last column in the contiguous block (LaCoCoB[25])
   reading right to left at which the RL-OriMaTArA is less than or equal
   to the 25th percentile [Q1, black sums and RL-OriMaTArA, ([194]Appendix
   A [195]Figure A1 and [196]Figure A2)] for the array was determined. If
   this point is in the rightmost 33% of the array (purple fraction, SF1
   and 2), clusters were defined below using Q1. If this point is in the
   center 33% of the array (green fraction, SF1 and 2), clusters were
   determined below the median (Q2, white sums and RL-OriMaTArA, SF1 and
   2). The value used as cut-off is Q*. Going left to right, the variables
   before the first value in the matrix total array less than Q* are in
   the DCS cluster. This distinction allows the discrete evaluation of
   clustering quality for the matrix, as is evident in SF1 and 2. For a
   matrix with a LaCoCoB[25] in the green fraction with little or diffuse
   clustering, the higher Q2 cut-off captures the smaller clustered
   segment. A matrix indicated by a purple fraction LaCoCoB[25], the lower
   Q1 cut-off allows for the more distinct and robust clustering.

   Volcano Plots: The volcano plots were calculated plotting the negative
   logarithm base 10 of the p-value on the y-axis and the logarithm of the
   fold-change of two conditions in the x-axis. The cut-off for
   significance is p < 0.05. The negative logarithm base 10 of adjusted
   q-values was also calculated and is included in the [197]Supplementary
   Tables S1–S4.

   Pathway enrichment analysis: We performed a pathway enrichment analysis
   ([198]https://portal.metabolon.com) using the formula (k/m)/((n − k)(N
   − m). Where K equals the number of significant metabolites; m equals
   the total number of detected metabolites in a pathway; n equals the
   total number of significant metabolites; and N equals the total number
   of metabolites.

Acknowledgments