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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

In vitro miR‐34a−/− brown adipocytes show an increase in percentage lipid+ cells but no change in mitochondrial markers. (A,B) RT‐qPCR quantification of miR‐34a, 34b*, and 34c* over in vitro WT brown adipocyte differentiation (day 0–8) from intrascapular (i)BAT SVF precursors, normalized to RNU6B; three mice were pooled together for each replicate, n = 3–4. (C,D) RT‐qPCR gene expression at day 8 of in vitro differentiation of SVF brown preadipocytes from miR‐34a−/− (KO) and WT murine iBAT, after a 4‐h stimulation with 0.1 µM noradrenaline (NA) or PBS control, normalized to 18s rRNA; three mice were pooled together for each replicate, n = 4. (E,F) Quantification of mitochondria in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 4 h, by FACS and MitroTracker Deep Red, shown as geometric mean fluorescence intensity (MFI) values representing content, and percentage positive cells; n = 3. (G,H) Quantification of lipid in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 20 h, by FACS and BODIPY, shown as geometric MFI values representing content, and percentage positive cells; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as 1/ΔCt. Statistics were calculated using a one‐way ANOVA, with Bonferroni's multiple comparisons post‐test, or an unpaired Student's t‐test for FACS measurements. *P < 0.05, **P < 0.01, ***P < 0.001. (I) A diagrammatic representation of the theoretical mechanism in miR‐34a−/− (KO) mice, predisposing them to diet‐induced obesity. The imbalance in WAT metabolic genes caused by chronic overexpression of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and mitochondrial dysfunction promotes lipid uptake/storage and weight gain when stressed with a high‐fat diet (HFD). As the tissue increases in size there is an increase in inflammatory cytokines, such as tumor necrosis factor (TNF)‐α, which polarize macrophages to an M1 phenotype. However, the KO macrophages are unresponsive to TNF‐α induction of CXCL1 and possibly other pro‐M1 genes, through overexpression of PGC1α inhibiting the NF‐κB subunit p65, suggesting an M2 phenotype. This inhibits the recruitment of neutrophils which could promote an M1 phenotype and inhibit lipogenic processes. The M2 phenotype is further promoted by interleukin (IL)‐5, which can induce eosinophils to produce pro‐M2 IL‐4. IL‐10 likely produced by macrophages can then stimulate adipocytes to be more insulin sensitive and dampen inflammatory processes, reducing pro‐obesity processes. Inhibition of key thermogenic genes in BAT predisposes the mice to obesity development, with overcompensation by the induction of PGC1α during HFD feeding.
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oby21561-fig-0006: In vitro miR‐34a−/− brown adipocytes show an increase in percentage lipid+ cells but no change in mitochondrial markers. (A,B) RT‐qPCR quantification of miR‐34a, 34b*, and 34c* over in vitro WT brown adipocyte differentiation (day 0–8) from intrascapular (i)BAT SVF precursors, normalized to RNU6B; three mice were pooled together for each replicate, n = 3–4. (C,D) RT‐qPCR gene expression at day 8 of in vitro differentiation of SVF brown preadipocytes from miR‐34a−/− (KO) and WT murine iBAT, after a 4‐h stimulation with 0.1 µM noradrenaline (NA) or PBS control, normalized to 18s rRNA; three mice were pooled together for each replicate, n = 4. (E,F) Quantification of mitochondria in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 4 h, by FACS and MitroTracker Deep Red, shown as geometric mean fluorescence intensity (MFI) values representing content, and percentage positive cells; n = 3. (G,H) Quantification of lipid in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 20 h, by FACS and BODIPY, shown as geometric MFI values representing content, and percentage positive cells; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as 1/ΔCt. Statistics were calculated using a one‐way ANOVA, with Bonferroni's multiple comparisons post‐test, or an unpaired Student's t‐test for FACS measurements. *P < 0.05, **P < 0.01, ***P < 0.001. (I) A diagrammatic representation of the theoretical mechanism in miR‐34a−/− (KO) mice, predisposing them to diet‐induced obesity. The imbalance in WAT metabolic genes caused by chronic overexpression of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and mitochondrial dysfunction promotes lipid uptake/storage and weight gain when stressed with a high‐fat diet (HFD). As the tissue increases in size there is an increase in inflammatory cytokines, such as tumor necrosis factor (TNF)‐α, which polarize macrophages to an M1 phenotype. However, the KO macrophages are unresponsive to TNF‐α induction of CXCL1 and possibly other pro‐M1 genes, through overexpression of PGC1α inhibiting the NF‐κB subunit p65, suggesting an M2 phenotype. This inhibits the recruitment of neutrophils which could promote an M1 phenotype and inhibit lipogenic processes. The M2 phenotype is further promoted by interleukin (IL)‐5, which can induce eosinophils to produce pro‐M2 IL‐4. IL‐10 likely produced by macrophages can then stimulate adipocytes to be more insulin sensitive and dampen inflammatory processes, reducing pro‐obesity processes. Inhibition of key thermogenic genes in BAT predisposes the mice to obesity development, with overcompensation by the induction of PGC1α during HFD feeding.

Mentions: To further explore the metabolic changes in miR‐34a−/− iBAT, we examined miR‐34a expression over in vitro differentiation of WT brown adipocyte precursors, but found no change in miR‐34a (Figure 6A). However, we observed increases in miR‐34b* at day 4 (P = 0.0305) and both miR‐34b* and 34c* at day 6 (P < 0.05) and day 8 (P < 0.01) of differentiation (Figure 6B).


miR ‐ 34a − / − mice are susceptible to diet ‐ induced obesity
In vitro miR‐34a−/− brown adipocytes show an increase in percentage lipid+ cells but no change in mitochondrial markers. (A,B) RT‐qPCR quantification of miR‐34a, 34b*, and 34c* over in vitro WT brown adipocyte differentiation (day 0–8) from intrascapular (i)BAT SVF precursors, normalized to RNU6B; three mice were pooled together for each replicate, n = 3–4. (C,D) RT‐qPCR gene expression at day 8 of in vitro differentiation of SVF brown preadipocytes from miR‐34a−/− (KO) and WT murine iBAT, after a 4‐h stimulation with 0.1 µM noradrenaline (NA) or PBS control, normalized to 18s rRNA; three mice were pooled together for each replicate, n = 4. (E,F) Quantification of mitochondria in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 4 h, by FACS and MitroTracker Deep Red, shown as geometric mean fluorescence intensity (MFI) values representing content, and percentage positive cells; n = 3. (G,H) Quantification of lipid in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 20 h, by FACS and BODIPY, shown as geometric MFI values representing content, and percentage positive cells; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as 1/ΔCt. Statistics were calculated using a one‐way ANOVA, with Bonferroni's multiple comparisons post‐test, or an unpaired Student's t‐test for FACS measurements. *P < 0.05, **P < 0.01, ***P < 0.001. (I) A diagrammatic representation of the theoretical mechanism in miR‐34a−/− (KO) mice, predisposing them to diet‐induced obesity. The imbalance in WAT metabolic genes caused by chronic overexpression of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and mitochondrial dysfunction promotes lipid uptake/storage and weight gain when stressed with a high‐fat diet (HFD). As the tissue increases in size there is an increase in inflammatory cytokines, such as tumor necrosis factor (TNF)‐α, which polarize macrophages to an M1 phenotype. However, the KO macrophages are unresponsive to TNF‐α induction of CXCL1 and possibly other pro‐M1 genes, through overexpression of PGC1α inhibiting the NF‐κB subunit p65, suggesting an M2 phenotype. This inhibits the recruitment of neutrophils which could promote an M1 phenotype and inhibit lipogenic processes. The M2 phenotype is further promoted by interleukin (IL)‐5, which can induce eosinophils to produce pro‐M2 IL‐4. IL‐10 likely produced by macrophages can then stimulate adipocytes to be more insulin sensitive and dampen inflammatory processes, reducing pro‐obesity processes. Inhibition of key thermogenic genes in BAT predisposes the mice to obesity development, with overcompensation by the induction of PGC1α during HFD feeding.
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oby21561-fig-0006: In vitro miR‐34a−/− brown adipocytes show an increase in percentage lipid+ cells but no change in mitochondrial markers. (A,B) RT‐qPCR quantification of miR‐34a, 34b*, and 34c* over in vitro WT brown adipocyte differentiation (day 0–8) from intrascapular (i)BAT SVF precursors, normalized to RNU6B; three mice were pooled together for each replicate, n = 3–4. (C,D) RT‐qPCR gene expression at day 8 of in vitro differentiation of SVF brown preadipocytes from miR‐34a−/− (KO) and WT murine iBAT, after a 4‐h stimulation with 0.1 µM noradrenaline (NA) or PBS control, normalized to 18s rRNA; three mice were pooled together for each replicate, n = 4. (E,F) Quantification of mitochondria in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 4 h, by FACS and MitroTracker Deep Red, shown as geometric mean fluorescence intensity (MFI) values representing content, and percentage positive cells; n = 3. (G,H) Quantification of lipid in day 8 in vitro brown adipocytes stimulated with 0.1 µM NA or PBS for 20 h, by FACS and BODIPY, shown as geometric MFI values representing content, and percentage positive cells; n = 3. All graphs represent mean values with SEM, except for RT‐qPCR, represented as 1/ΔCt. Statistics were calculated using a one‐way ANOVA, with Bonferroni's multiple comparisons post‐test, or an unpaired Student's t‐test for FACS measurements. *P < 0.05, **P < 0.01, ***P < 0.001. (I) A diagrammatic representation of the theoretical mechanism in miR‐34a−/− (KO) mice, predisposing them to diet‐induced obesity. The imbalance in WAT metabolic genes caused by chronic overexpression of peroxisome proliferator‐activated receptor‐y coactivator (PGC)‐1α and mitochondrial dysfunction promotes lipid uptake/storage and weight gain when stressed with a high‐fat diet (HFD). As the tissue increases in size there is an increase in inflammatory cytokines, such as tumor necrosis factor (TNF)‐α, which polarize macrophages to an M1 phenotype. However, the KO macrophages are unresponsive to TNF‐α induction of CXCL1 and possibly other pro‐M1 genes, through overexpression of PGC1α inhibiting the NF‐κB subunit p65, suggesting an M2 phenotype. This inhibits the recruitment of neutrophils which could promote an M1 phenotype and inhibit lipogenic processes. The M2 phenotype is further promoted by interleukin (IL)‐5, which can induce eosinophils to produce pro‐M2 IL‐4. IL‐10 likely produced by macrophages can then stimulate adipocytes to be more insulin sensitive and dampen inflammatory processes, reducing pro‐obesity processes. Inhibition of key thermogenic genes in BAT predisposes the mice to obesity development, with overcompensation by the induction of PGC1α during HFD feeding.
Mentions: To further explore the metabolic changes in miR‐34a−/− iBAT, we examined miR‐34a expression over in vitro differentiation of WT brown adipocyte precursors, but found no change in miR‐34a (Figure 6A). However, we observed increases in miR‐34b* at day 4 (P = 0.0305) and both miR‐34b* and 34c* at day 6 (P < 0.05) and day 8 (P < 0.01) of differentiation (Figure 6B).

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