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. 2023 Jan 3;120(1):e2214874120.
doi: 10.1073/pnas.2214874120. Epub 2022 Dec 27.

Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases

Affiliations

Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases

Wenqiang Song et al. Proc Natl Acad Sci U S A. .

Abstract

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

Keywords: PIK3C3/VPS34; adipocyte; autophagy; lipodystrophy; metabolic disease.

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Conflict of interest statement

L.V.K. is a member of the scientific advisory board of Isu Abxis Co., Ltd. (South Korea). The other authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.
Adipocyte-specific deletion of Pik3c3 disturbs autophagy and causes generalized lipodystrophy. Mice at W8 and W24 were used except for F. A and B. PIK3C3 mRNA and protein (A), P62, LC3, and LAMP1 protein (B) were examined (n = 4 to 8 in each group). C. Autophagic flux (LC3-II with inhibitor/LC3-II without inhibitor, Left and LC3-II with inhibitor—LC3-II without inhibitor, Right) was evaluated (n = 6 to 13 in each group). D and E. RFP representing LC3 (D) and autophagy progression (E) were examined (n = 3 to 4 in each group). F. Mice at the specified age were examined (n = 3 to 26 in each group at each time point). G. Collagen proteins (Left) and mRNAs (Right) were examined (n = 3 in each group). (Scale bar, 100 µm.) AU: arbitrary unit; NL: NH4Cl + leupeptin.
Fig. 2.
Fig. 2.
Adipocyte-specific deletion of Pik3c3 causes metabolic disorders. Mice at W24 were used except for A. A. IPITT was performed on mice at the indicated age (n = 7 to 19 in each group at each age). B. Fasting blood glucose and plasma insulin were examined (n = 7 to 9 in each group). C. IPGTT was performed (n = 15 to 21 in each group). D. Serum lipids were examined (n = 7 to 12 in each group). E. Liver lipids (Left, n = 8 to 9 in each group), H&E-stained (Middle, scale bar, 100 µm), and Oil red O-stained (Right, scale bar, 50 µm) liver sections (n = 4 in each group) were examined.
Fig. 3.
Fig. 3.
Adipocyte-specific deletion of Pik3c3 impairs adipocyte differentiation, survival, and function. A. SVFs were analyzed by flow cytometry for preadipocytes fraction F 1 to 3 (n = 6 in each group). B. Preadipocytes from iBAT were in vitro differentiated and stained with Oil red O (n = 3 in each group). C. CASP3 was examined by western blotting. Band intensity was quantified to reflect protein level. DI. The indicated mRNAs or proteins (quantification of band intensity) were examined in mice at W8 (n = 3 to 7 in each group for mRNAs and n = 7 to 8 in each group for proteins).
Fig. 4.
Fig. 4.
Adipocyte-specific deletion of Pik3c3 alters AT lipidome and circulating sRNA. Mice at W24 were used. A–C. Global untargeted lipidomics was performed. Shown are PCA and heatmap (A), DALs (B), and affected pathways (C, GPI: glycosylphosphatidylinositol) with n = 4 in each group. D and E. sRNAs in plasma were analyzed by sRNA sequencing. Shown are the length distribution (D) and heatmap using TPM of DESs (E) with n = 4 in each group.
Fig. 5.
Fig. 5.
Adipocyte-specific deletion of Pik3c3 compromises thermogenic response. Mice at W8 were used. A. Mice were housed at 6 °C for 1 wk with free access to food and water. Body temperature was measured (n = 5 to 13 in each group). B. iBAT was analyzed for indicated mRNAs and proteins (n = 2 to 4 in each group). C. WT mice were housed as indicated for 6 h and analyzed for LC3 II/LC3 I in iBAT by western blotting (n = 6 to 9 in each group). D. Mice were housed at 6 °C with free access to only water. Body temperature was recorded (n = 6 to 9 in each group). iBAT was analyzed for indicated mRNAs and proteins (n = 2 to 5 in each group). E and F. Mice were treated as indicated. iWAT was analyzed for the indicated mRNAs and proteins (n = 2 to 5 in each group).
Fig. 6.
Fig. 6.
Pik3c3-deleted preadipocyte cell lines mirror ATs of cKO mice. A. Cell line models are listed. B–F. IAC-BAT cells were induced to delete Pik3c3 during differentiation and analyzed on day 10 for Pik3c3 mRNA (B), the indicated proteins (C), apoptotic cells (D), lipid content (E), and mitochondrial content (F, MFI: mean fluorescence intensity) with n = 3 in each group. G. IRC-BAT cells were induced to delete Pik3c3, differentiated, and analyzed for autophagic flux and endocytosis/vesicle trafficking (n = 3 in each group). H–K. Control and Pik3c3-deleted 3T3-L1 cells were differentiated and analyzed on day 6 for indicated mRNAs and proteins (H), Oil red O staining (I, scale bar, 100 μm), lipid content (J), autophagic flux and endocytosis/vesicle trafficking (K) with n = 2 to 5 in each group. CQ: chloroquine.
Fig. 7.
Fig. 7.
Dominant phenotype of PIK3C3 deficiency over ATG7 deficiency in murine adipocytes. Mice (WT, wild-type; cKO, Adipoq-Cre;Pik3c3f/f; cKOAtg7, Adipoq-Cre;Atg7f/f; DKO, Adipoq-Cre;Pik3c3f/f;Atg7f/f) at W8 were used. AD. iBATs were examined for indicated proteins (A), tissue weight (B), indicated mRNAs (C), and UCP1 protein (D). E–G, pWATs were examined for tissue weight (E), UCP1 protein (F), and indicated mRNAs (G) with n = 4 to 7 in each group, except for tissue weight in which n = 9 to 18. H. Body temperature in response to cold and food deprivation was recorded (n = 4 in each group).

Comment in

  • PIK3C3/VPS34 keeps body fats healthy.
    Song W, Postoak JL, Wu L, Van Kaer L. Song W, et al. Autophagy. 2023 Aug;19(8):2398-2400. doi: 10.1080/15548627.2023.2166275. Epub 2023 Jan 18. Autophagy. 2023. PMID: 36629752 Free PMC article.

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