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miR ‐ 34a − / − mice are susceptible to diet ‐ induced obesity

View Article: PubMed Central - PubMed

ABSTRACT

Objective: MicroRNA (miR)−34a regulates inflammatory pathways, and increased transcripts have been observed in serum and subcutaneous adipose of subjects who have obesity and type 2 diabetes. Therefore, the role of miR‐34a in adipose tissue inflammation and lipid metabolism in murine diet‐induced obesity was investigated.

Methods: Wild‐type (WT) and miR‐34a−/− mice were fed chow or high‐fat diet (HFD) for 24 weeks. WT and miR‐34a−/− bone marrow‐derived macrophages were cultured in vitro with macrophage colony‐stimulating factor (M‐CSF). Brown and white preadipocytes were cultured from the stromal vascular fraction (SVF) of intrascapular brown and epididymal white adipose tissue (eWAT), with rosiglitazone.

Results: HFD‐fed miR‐34a−/− mice were significantly heavier with a greater increase in eWAT weight than WT. miR‐34a−/− eWAT had a smaller adipocyte area, which significantly increased with HFD. miR‐34a−/− eWAT showed basal increases in Cd36, Hmgcr, Lxrα, Pgc1α, and Fasn. miR‐34a−/− intrascapular brown adipose tissue had basal reductions in c/ebpα and c/ebpβ, with in vitro miR‐34a−/− white adipocytes showing increased lipid content. An F4/80high macrophage population was present in HFD miR‐34a−/− eWAT, with increased IL‐10 transcripts and serum IL‐5 protein. Finally, miR‐34a−/− bone marrow‐derived macrophages showed an ablated CXCL1 response to tumor necrosis factor‐α.

Conclusions: These findings suggest a multifactorial role of miR‐34a in controlling susceptibility to obesity, by regulating inflammatory and metabolic pathways.

No MeSH data available.


Related in: MedlinePlus

High‐fat diet (HFD) ‐fed miR‐34a−/− mice show an F4/80high phenotype, with increased type 2 cytokines and a reduction in splenic neutrophils. (A) Representative dot‐plots showing the gating strategy used to identify macrophages in the SVF from murine epididymal (e)WAT at week 24 in WT and miR‐34a−/− (KO) mice on chow vs. HFD. (B) Representative dot‐plots showing the F4/80high macrophage phenotype in KO eWAT after HFD feeding, from the same study as panel A. (C) Quantification of panel B, showing median fluorescence intensity (MFI) values representing surface expression and percentage F4/80+ cells in the macrophage FSC‐SSC gate; n = 9 for all groups, except n = 10 for KO chow. (D) RT‐qPCR data of interleukin (IL)‐10 gene expression in eWAT from same study as panel A, normalized to 18s rRNA; n = 6 for all groups, except n = 5 for KO HFD and WT chow. (E) Significant changes from cytokine Luminex data of serum from mice fasted for 16 to 18 h from the same study as panel A; n = 6 for KO chow and WT HFD groups and n = 5 for WT chow and KO HFD groups. (F) RT‐qPCR gene expression and supernatant, cytokine Luminex protein data for CXCL1, from WT and KO in vitro bone marrow‐derived macrophages (BMDM) ± 45.45 ng/mL tumor necrosis factor (TNF)‐α for 24 h; n = 3. RT‐qPCR data normalized to 18s rRNA. (G) FACS quantification of the percentage of neutrophils (CD45+ CD11b+ F4/80− CD11c−Ly6c‐Ly6g+) in the FSC‐SSC population from spleens of mice in the same study as panel A. Gating strategy is shown in Supporting Information Figure S4D; n = 8 for all groups, except n = 4 for WT chow and n = 6 for WT HFD. (H) RT‐qPCR quantification of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and c/ebpα transcripts in the same samples as panel F; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as relative quantification (RQ) with RQmin − RQmax values. *P < 0.05, **P < 0.01, ***P < 0.001, one‐way ANOVA, with Bonferroni's multiple comparisons post‐test.
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oby21561-fig-0004: High‐fat diet (HFD) ‐fed miR‐34a−/− mice show an F4/80high phenotype, with increased type 2 cytokines and a reduction in splenic neutrophils. (A) Representative dot‐plots showing the gating strategy used to identify macrophages in the SVF from murine epididymal (e)WAT at week 24 in WT and miR‐34a−/− (KO) mice on chow vs. HFD. (B) Representative dot‐plots showing the F4/80high macrophage phenotype in KO eWAT after HFD feeding, from the same study as panel A. (C) Quantification of panel B, showing median fluorescence intensity (MFI) values representing surface expression and percentage F4/80+ cells in the macrophage FSC‐SSC gate; n = 9 for all groups, except n = 10 for KO chow. (D) RT‐qPCR data of interleukin (IL)‐10 gene expression in eWAT from same study as panel A, normalized to 18s rRNA; n = 6 for all groups, except n = 5 for KO HFD and WT chow. (E) Significant changes from cytokine Luminex data of serum from mice fasted for 16 to 18 h from the same study as panel A; n = 6 for KO chow and WT HFD groups and n = 5 for WT chow and KO HFD groups. (F) RT‐qPCR gene expression and supernatant, cytokine Luminex protein data for CXCL1, from WT and KO in vitro bone marrow‐derived macrophages (BMDM) ± 45.45 ng/mL tumor necrosis factor (TNF)‐α for 24 h; n = 3. RT‐qPCR data normalized to 18s rRNA. (G) FACS quantification of the percentage of neutrophils (CD45+ CD11b+ F4/80− CD11c−Ly6c‐Ly6g+) in the FSC‐SSC population from spleens of mice in the same study as panel A. Gating strategy is shown in Supporting Information Figure S4D; n = 8 for all groups, except n = 4 for WT chow and n = 6 for WT HFD. (H) RT‐qPCR quantification of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and c/ebpα transcripts in the same samples as panel F; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as relative quantification (RQ) with RQmin − RQmax values. *P < 0.05, **P < 0.01, ***P < 0.001, one‐way ANOVA, with Bonferroni's multiple comparisons post‐test.

Mentions: Given that miR‐34a transcripts were increased in WT BMDMs stimulated with TNF‐α, they may contribute to the in vivo metabolic profile. Therefore, using FACS we gated on the larger, more granular F4/80+ cells from WT and 34a−/− eWAT to identify macrophages, after 24 weeks on chow or HFD (Figure 4A). These cells were CD45+, CD11b+, MHCII+, and CD86+. Further analysis revealed a population of F4/80high cells within the miR‐34a−/− HFD group not observed in WT (Figure 4B). We observed a significant increase in F4/80 surface expression on miR‐34a−/− cells, when fed HFD (P = 0.0497), and a lower F4/80+ macrophage content within the eWAT of miR‐34a−/− chow mice, compared with WT chow (P = 0.0078) (Figure 4C). There were no changes in the other macrophage M1/M2 surface markers examined (Supporting Information Figure S4A), or M1/M2 genes within the eWAT, except a basal increase in miR‐34a−/− chow eWAT Nos2 (P = 0.0500) and decreased HFD‐fed miR‐34a−/− eWAT Retnla (P = 0.0006) transcripts, over WT diet controls (Supporting Information Figure S4B). To check whether an increase in cytosolic lipid could contribute to the F4/80high macrophage phenotype in miR‐34a−/− HFD eWAT, we back‐gated on this population for side‐scatter, a measurement of internal complexity, but observed no difference in side‐scatter MFI between any of the groups (Supporting Information Figure S4C).


miR ‐ 34a − / − mice are susceptible to diet ‐ induced obesity
High‐fat diet (HFD) ‐fed miR‐34a−/− mice show an F4/80high phenotype, with increased type 2 cytokines and a reduction in splenic neutrophils. (A) Representative dot‐plots showing the gating strategy used to identify macrophages in the SVF from murine epididymal (e)WAT at week 24 in WT and miR‐34a−/− (KO) mice on chow vs. HFD. (B) Representative dot‐plots showing the F4/80high macrophage phenotype in KO eWAT after HFD feeding, from the same study as panel A. (C) Quantification of panel B, showing median fluorescence intensity (MFI) values representing surface expression and percentage F4/80+ cells in the macrophage FSC‐SSC gate; n = 9 for all groups, except n = 10 for KO chow. (D) RT‐qPCR data of interleukin (IL)‐10 gene expression in eWAT from same study as panel A, normalized to 18s rRNA; n = 6 for all groups, except n = 5 for KO HFD and WT chow. (E) Significant changes from cytokine Luminex data of serum from mice fasted for 16 to 18 h from the same study as panel A; n = 6 for KO chow and WT HFD groups and n = 5 for WT chow and KO HFD groups. (F) RT‐qPCR gene expression and supernatant, cytokine Luminex protein data for CXCL1, from WT and KO in vitro bone marrow‐derived macrophages (BMDM) ± 45.45 ng/mL tumor necrosis factor (TNF)‐α for 24 h; n = 3. RT‐qPCR data normalized to 18s rRNA. (G) FACS quantification of the percentage of neutrophils (CD45+ CD11b+ F4/80− CD11c−Ly6c‐Ly6g+) in the FSC‐SSC population from spleens of mice in the same study as panel A. Gating strategy is shown in Supporting Information Figure S4D; n = 8 for all groups, except n = 4 for WT chow and n = 6 for WT HFD. (H) RT‐qPCR quantification of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and c/ebpα transcripts in the same samples as panel F; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as relative quantification (RQ) with RQmin − RQmax values. *P < 0.05, **P < 0.01, ***P < 0.001, one‐way ANOVA, with Bonferroni's multiple comparisons post‐test.
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oby21561-fig-0004: High‐fat diet (HFD) ‐fed miR‐34a−/− mice show an F4/80high phenotype, with increased type 2 cytokines and a reduction in splenic neutrophils. (A) Representative dot‐plots showing the gating strategy used to identify macrophages in the SVF from murine epididymal (e)WAT at week 24 in WT and miR‐34a−/− (KO) mice on chow vs. HFD. (B) Representative dot‐plots showing the F4/80high macrophage phenotype in KO eWAT after HFD feeding, from the same study as panel A. (C) Quantification of panel B, showing median fluorescence intensity (MFI) values representing surface expression and percentage F4/80+ cells in the macrophage FSC‐SSC gate; n = 9 for all groups, except n = 10 for KO chow. (D) RT‐qPCR data of interleukin (IL)‐10 gene expression in eWAT from same study as panel A, normalized to 18s rRNA; n = 6 for all groups, except n = 5 for KO HFD and WT chow. (E) Significant changes from cytokine Luminex data of serum from mice fasted for 16 to 18 h from the same study as panel A; n = 6 for KO chow and WT HFD groups and n = 5 for WT chow and KO HFD groups. (F) RT‐qPCR gene expression and supernatant, cytokine Luminex protein data for CXCL1, from WT and KO in vitro bone marrow‐derived macrophages (BMDM) ± 45.45 ng/mL tumor necrosis factor (TNF)‐α for 24 h; n = 3. RT‐qPCR data normalized to 18s rRNA. (G) FACS quantification of the percentage of neutrophils (CD45+ CD11b+ F4/80− CD11c−Ly6c‐Ly6g+) in the FSC‐SSC population from spleens of mice in the same study as panel A. Gating strategy is shown in Supporting Information Figure S4D; n = 8 for all groups, except n = 4 for WT chow and n = 6 for WT HFD. (H) RT‐qPCR quantification of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and c/ebpα transcripts in the same samples as panel F; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as relative quantification (RQ) with RQmin − RQmax values. *P < 0.05, **P < 0.01, ***P < 0.001, one‐way ANOVA, with Bonferroni's multiple comparisons post‐test.
Mentions: Given that miR‐34a transcripts were increased in WT BMDMs stimulated with TNF‐α, they may contribute to the in vivo metabolic profile. Therefore, using FACS we gated on the larger, more granular F4/80+ cells from WT and 34a−/− eWAT to identify macrophages, after 24 weeks on chow or HFD (Figure 4A). These cells were CD45+, CD11b+, MHCII+, and CD86+. Further analysis revealed a population of F4/80high cells within the miR‐34a−/− HFD group not observed in WT (Figure 4B). We observed a significant increase in F4/80 surface expression on miR‐34a−/− cells, when fed HFD (P = 0.0497), and a lower F4/80+ macrophage content within the eWAT of miR‐34a−/− chow mice, compared with WT chow (P = 0.0078) (Figure 4C). There were no changes in the other macrophage M1/M2 surface markers examined (Supporting Information Figure S4A), or M1/M2 genes within the eWAT, except a basal increase in miR‐34a−/− chow eWAT Nos2 (P = 0.0500) and decreased HFD‐fed miR‐34a−/− eWAT Retnla (P = 0.0006) transcripts, over WT diet controls (Supporting Information Figure S4B). To check whether an increase in cytosolic lipid could contribute to the F4/80high macrophage phenotype in miR‐34a−/− HFD eWAT, we back‐gated on this population for side‐scatter, a measurement of internal complexity, but observed no difference in side‐scatter MFI between any of the groups (Supporting Information Figure S4C).

View Article: PubMed Central - PubMed

ABSTRACT

Objective: MicroRNA (miR)&minus;34a regulates inflammatory pathways, and increased transcripts have been observed in serum and subcutaneous adipose of subjects who have obesity and type 2 diabetes. Therefore, the role of miR&#8208;34a in adipose tissue inflammation and lipid metabolism in murine diet&#8208;induced obesity was investigated.

Methods: Wild&#8208;type (WT) and miR&#8208;34a&minus;/&minus; mice were fed chow or high&#8208;fat diet (HFD) for 24 weeks. WT and miR&#8208;34a&minus;/&minus; bone marrow&#8208;derived macrophages were cultured in vitro with macrophage colony&#8208;stimulating factor (M&#8208;CSF). Brown and white preadipocytes were cultured from the stromal vascular fraction (SVF) of intrascapular brown and epididymal white adipose tissue (eWAT), with rosiglitazone.

Results: HFD&#8208;fed miR&#8208;34a&minus;/&minus; mice were significantly heavier with a greater increase in eWAT weight than WT. miR&#8208;34a&minus;/&minus; eWAT had a smaller adipocyte area, which significantly increased with HFD. miR&#8208;34a&minus;/&minus; eWAT showed basal increases in Cd36, Hmgcr, Lxr&alpha;, Pgc1&alpha;, and Fasn. miR&#8208;34a&minus;/&minus; intrascapular brown adipose tissue had basal reductions in c/ebp&alpha; and c/ebp&beta;, with in vitro miR&#8208;34a&minus;/&minus; white adipocytes showing increased lipid content. An F4/80high macrophage population was present in HFD miR&#8208;34a&minus;/&minus; eWAT, with increased IL&#8208;10 transcripts and serum IL&#8208;5 protein. Finally, miR&#8208;34a&minus;/&minus; bone marrow&#8208;derived macrophages showed an ablated CXCL1 response to tumor necrosis factor&#8208;&alpha;.

Conclusions: These findings suggest a multifactorial role of miR&#8208;34a in controlling susceptibility to obesity, by regulating inflammatory and metabolic pathways.

No MeSH data available.


Related in: MedlinePlus