The role of adipocyte XBP1 in metabolic regulation during lactation

Abstract

The adipocyte is central to organismal metabolism and exhibits significant functional and morphological plasticity during its formation and lifespan. Remarkable transformations of this cell occur during obesity and lactation, and thus it is essential to gain a better understanding of adipocyte function in these two metabolic processes. Considering the critical importance of the cellular organelle endoplasmic reticulum (ER) in adapting to fluctuations in synthetic processes, we explored the role of XBP1, a central regulator of ER adaptive responses, in adipocyte formation and function. Unexpectedly, deletion of adipocyte-XBP1 in vivo in mice (XBP1ΔAd) had no effect on adipocyte formation or on systemic homeostatic metabolism in mice fed a a regular or high-fat diet. However, during lactation, XBP1ΔAd dams displayed increased adiposity, decreased milk production, and decreased litter growth as compared with control dams. Moreover, we demonstrate that XBP1 is regulated during lactation and responds to prolactin to alter lipogenic gene expression. These results demonstrate a role for adipocyte-XBP1 in the regulation of lactational metabolism.

Figure 1. Targeted deletion of XBP1 in the adipocyte does not affect tissue formation or function in homeostatic metabolism

(A-C) Lentiviral suppression of XBP1 mRNA in 3T3L1 preadipocytes. (A) Xbp1 mRNA levels were measured by real-time quantitative RT-PCR (QPCR). Preadipocytes carrying control or XBP1 shRNA (iXBP1) were induced to differentiate with or without rosiglitazone (10uM). (B) On day 8 of differentiation, mRNA levels of the Adiponectin gene were measured by QPCR and (C) bright phase microscopy images were taken. (D) XBP1 protein levels in fat explants from WT and XBP1^ΔAd mice after treatment with or without protease inhibitor MG132 (25μM) for 20 hours to stabilize XBP1 protein. Protein extracts were probed using XBP1 or Actin antibody (Santa Cruz). *denotes a non-specific band. (E-K) were performed with male mice (n=7-12) on high fat diet (HFD). (E) Body weight of WT and XBP1^ΔAd mice over time on HFD. (F) Percent fat, (G) Lean Mass, and Fat Mass of WT and XBP1^ΔAd mice (n=5-11) as measured by DEXA analysis. (H) Hematoxylin and Eosin staining (H&E) of adipose tissue sections from WT and XBP1^ΔAd mice (Magnification 100x). Inguinal or epididymal white adipose tissue (IWAT or EWAT). (I) serum insulin and (J) adiponectin levels in WT and XBP1^ΔAd mice (n=5,6). (K) Glucose tolerance test performed after 16 weeks on HFD with (1.0 g/kg glucose injection, n=6). All error bars indicate +/- SEM. See also Figure S1.

Figure 2. Adipocyte XBP1 deletion disturbs metabolic flux during lactation

(A) mRNA levels of Xbp1 in gonadal adipose tissue from WT mice that were non-pregnant (NonPG), in late pregnancy (d18PG), or in day 5 or 10 of lactation(L). (B) Fully differentiated adipocytes were treated with or without prolactin for 6 hours and mRNA levels of UPR genes were measured by QPCR. (C) Both spliced and unspliced forms of XBP1 are shown. (D) Gonadal white adipose tissue (GWAT) wet weights of WT and XBP1^ΔAd dams from non-pregnant (NonPG) and day 1, 10 and 20 lactation (L) timepoints (n=4-9). (E) Representative images from CT scans of WT and XBP1^ΔAd dams during peak lactation (day 12). Adipose tissue density is displayed. (F) Serum insulin and leptin levels of WT and XBP1^ΔAd dams at day 20 lactation (n=3). (G) Pup weights of litters nursed by WT or XBP1^ΔAd dams during lactation. All litters are mixtures of pup genotypes (WT and XBP1^ΔAd) (n=6-10 litters per genotype). (H) Cross-foster of XBP1^ΔAd and WT litters. Average pup weight during lactation from cross-fostered litters switched on day 1 lactation (n=8-10, litters were pared to 6 pups). All error bars indicate +/- SEM. *p<0.05, **p<0.01 using standard t-test. † denotes p<0.007 as measured by regular 2-way ANOVA. See also Figure S2.

Figure 3. XBP1 is deleted specifically in the adipocytes of the mammary gland

(A) Xbp1 mRNA levels in fractionated mammary tissue from day 1 and day 10 lactation. Tissues from WT and XBP1^ΔAd mice were separated into adipocyte and stromal-vascular (SV) fractions (n=4-5). (B) Laser Capture Microdissection (LCM) of mammary epithelial cells from mammary glands on day 1 lactation. Total RNA was extracted from the microdissected cells to determine levels of Xbp1 deletion. Pictures of histology illustrate the area of capture before and after dissection. (C) Average pup weights of litters nursed by WT and XBP1Δ^MAC(LysM Cre) dams throughout lactation (n=5-6). (D) Average pup weights of litters nursed by WT and Adiponectin Cre XBP1^ΔAd dams throughout lactation (n=5-6). (E) Mammary gland wet weights of WT and XBP1^ΔAd dams (n=4-9). (F) Whole mount stains of mammary glands from WT and XBP1^ΔAd mice at different stages of mammary development (non-pregnant (NonPG), day 1 or day 10 lactation). All error bars indicate +/- SEM. *denotes p<0.05 using standard t-test. See also Figure S3.

Figure 4. Adipocyte XBP1 affects milk quantity and lipogenic gene expression

Milk was collected from WT and XBP1^ΔAd dams at day 12 lactation. (A) non-esterified fatty acid (NEFA) and (B) triglyceride levels in milk. (C) Heatmap of lipidomics analysis in XBP1^ΔAd and WT milk. (D) Lactose and (E) total protein concentrations in WT and XBP1^ΔAd milk. (F) Milk quantitation assay. Amount of milk produced from each dam was measured as difference in pup weight before and after a controlled 1.5 hour feeding. Litters were pared to 6 pups. (G) Serum prolactin in NP and d10L WT and XBP1^ΔAd mice. *p<0.02 using standard t-test. (H) Heatmap of gene expression from F442A adipocytes overexpressing sXBP1 or GFP control. (I) QPCR demonstrating suppression of Xbp1 mRNA by adenoviral shRNA. (J) Graph of densitometry data for protein levels of SCD1 in cultured adipocytes during low or high (50-500ng/ml) prolactin treatments. SCD1 protein levels were normalized to adiponectin. Basal WT SCD1 protein level was set at 1. (K) Western blot analysis of FAS, SCD1, and Tubulin proteins in gonadal adipose tissue from d10 lactation. Densitometry measures are provided in the graph to the right. All error bars indicate +/- SEM. *p<0.05, **p<0.004 using standard t-test. † denotes p<0.004 as measured by regular 2-way ANOVA. See also Figure S4.