Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-13C6]-d-glucose tracer in mice

Previous genetic and proteomic studies identified altered activity of various enzymes such as those of fatty acid metabolism and glycogen synthesis after a single toxic dose of valproic acid (VPA) in rats. In this study, we demonstrate the effect of VPA on metabolite synthesis flux rates and the possible use of abnormal 13C labeled glucose-derived metabolites in plasma or urine as early markers of toxicity. Female CD-1 mice were injected subcutaneously with saline or 600 mg/kg) VPA. Twelve hours later, the mice were injected with an intraperitoneal load of 1 g/kg [U-13C]-d-glucose. 13C isotopomers of glycogen glucose and RNA ribose in liver, kidney and brain tissue, as well as glucose disposal via cholesterol and glucose in the plasma and urine were determined. The levels of all of the positional 13C isotopomers of glucose were similar in plasma, suggesting that a single VPA dose does not disturb glucose absorption, uptake or hepatic glucose metabolism. Three-hour urine samples showed an increase in the injected tracer indicating a decreased glucose re-absorption via kidney tubules. 13C labeled glucose deposited as liver glycogen or as ribose of RNA were decreased by VPA treatment; incorporation of 13C via acetyl-CoA into plasma cholesterol was significantly lower at 60 min. The severe decreases in glucose-derived carbon flux into plasma and kidney-bound cholesterol, liver glycogen and RNA ribose synthesis, as well as decreased glucose re-absorption and an increased disposal via urine all serve as early flux markers of VPA-induced adverse metabolic effects in the host

SPCA2 Regulates Orai1 Trafficking and Store Independent Ca²⁺ Entry in a Model of Lactation

<div><p>An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca²⁺-ATPase, and the Ca²⁺ influx channel Orai1, has previously been shown to contribute to elevated Ca²⁺ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca²⁺ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca²⁺ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca²⁺ entry (SICE), which mediates the massive basolateral Ca²⁺ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.</p></div

Ectopic expression of hSPCA2 constructs restores Ca²⁺ influx and Orai1 trafficking.

<p>A) Immunofluorescence staining of mOrai1 shows trafficking defect in shSPCA2 cells (top panel). When the C-terminal end of hSPCA2, hSPCA2-C is ectopically expressed mOrai1 staining appears more vesicular (middle panel). Full-length hSPCA2 fully rescues mOrai1 trafficking, showing both plasma membrane and vesicular localization seen (bottom panel). B) SOCE is restored for shSPCA2 treated cells ectopically expressing either full-length hSPCA2 or C-terminal domain, hSPCA2C (results averaged from n = 111 cells for EV/GST; n = 63 for shSPCA2/CMV-EV; n = 67 for shSPCA2/CMV-hSPCA2C and n = 75 for shSPCA2/CMV-shSPCA2). Fold change is the normalized change in fluorescence ratio (340/380 nm) of Fura-2. Error bars reflect standard deviation from the mean for each measurement and time point using a Student’s <i>t</i> test.</p

Expression profiles of calcium transporters in lactation.

<p>Mouse mammary tissue, starting from day 10 prior to parturition, was evaluated for the expression of (A) SPCA1, and (B) SPCA2 by Western blotting. Loading was normalized relative to tissue DNA concentrations, and expressed relative to starting levels at Day -10. C) RT-PCR of mRNA for isoforms of SPCA, Orai, PMCA and STIM proteins at distinct time points of mammary development, including pre-pregnancy (virgin), parturition (Day 0), lactation (Day 5) and involution (Day 5 after removal of pups). Transcripts are grouped as early, mid and unchanged, according to their time of induction following lactation on Day 0. D) SPCA2 was immunoprecipitated from lactating mouse mammary tissue using polyclonal rabbit anti-hSPCA2 peptide antibody or rabbit IgG as control. Orai1 was detected as a co-immunoprecipitate by immunoblotting (IB).</p

Depletion of SPCA2 blocks cell surface trafficking of Orai1.

<p>A–B) Immunofluorescence labeling of mSPCA2 shows less vesicular distribution of SPCA2 in cells treated with shOrai1. C-D) Immunofluorescence labeling of mOrai1 in SPCA2 knockdown cells shows retention to the perinuclear region only. SPCA2 and Orai1 signals from conjugated anti-rabbit and anti-mouse (AlexaFluor 288 and 388, respectively) secondary antibody were pseudocolored for ease of comparison.</p

Store Independent Ca²⁺ Entry requires SPCA2 and Orai1.

<p>A) SICE is virtually abolished in monolayer cells expressing shRNA constructs for SPCA2 and Orai1 constructs (results averaged from n = 91cells for shSc; n = 82 for shSPCA2; n = 92 for shOrai1). Error bars reflect standard deviation from the mean for each measurement at each time point. B) SICE is restored and elevated in shSPCA2 treated cells expressing either hSPCA2 or hSPCA2C (results averaged from n = 94 cells for EV/GST; n = 74 for shSPCA2/CMV-EV; n = 91 for shSPCA2/CMV-hSPCA2C and n = 104 for shSPCA2/CMV-shSPCA2). C) Fluorescence image of Fura-2AM loaded in monolayer cells, and mammospheres. (D) Store independent Ca²⁺ influx in monolayer cells and differentiated mammospheres. After a brief (∼20 s) incubation in nominally Ca²⁺ free extracellular medium, readdition of Ca²⁺ (2 mM) elicits Ca²⁺ entry that is transient in monolayer cells but elevated and sustained in mammospheres (n = 129 and n = 84 for monolayer and mammosphere cells, also respectively). E) Basal Ca²⁺ levels are similar in mammospheres, relative to monolayer cells as seen by Fura-2 fluorescence ratio. F) Store-dependent Ca²⁺ influx in monolayer cells and differentiated mammospheres. Addition of thapsigargin (Tg) empties the internal (ER) stores in nominally Ca²⁺-free medium. Upon readdition of extracellular Ca²⁺ (2 mM), Ca²⁺ influx is slightly higher in mammospheres (n = 89 and n = 67 for monolayer and mammosphere cells, respectively). Fold change is the normalized change in fluorescence ratio (340/380 nm) of Fura-2.</p

Immunofluorescence microscopy of SPCA1, SPCA2, Orai1 and STIM1 in lactating mouse tissue.

<p>Confocal microscopy imaging of sections treated as described under Experimental procedures. A) SPCA1 co-localizes with the Golgi marker, GM130. B) SPCA2 has a diffuse distribution, with little co-localization with Golgi marker, GM130. C) Orai1 (left panel) and STIM1 (right panel) show basolateral and reticular localization, respectively. Scale bar: 100 µm.</p

Expression of SPCA2 and Orai1 in mammospheres.

<p>A) Schema for induction of SCp2 cells into mammospheres. B) Cells in monolayer migrate and assemble into mammospheres following induction. C) Semi-quantitative RT-PCR showing that SPCA2, Orai1–3 and STIM1–2 are induced along with known markers β-casein and PMCA2 in the transition of SCp2 cell into differentiated mammospheres. D–F) Confocal sections of mammosphere showing indirect immunofluorescence of the indicated proteins; asterisks mark some areas of co-localization. Orthogonal sections are shown with the merged image. The cartoons indicate approximate location of optical plane through the mammosphere. D) Confocal section of a single layered mammosphere shows vesicular distribution of SPCA2 with some colocalization in the vicinity of E-cadherin as seen in the orthogonal view. E) Differentiated mammosphere showing Orai1 limited to the basal membrane with some SPCA2 punctae just below the plasma membrane, seen in orthogonal view. F) Top view of mammosphere shown in (E). Colocalized SPCA2 (green) and Orai1 (red) appear yellow. G) Secondary anti-mouse or anti-rabbit antibody alone shows no staining of mammosphere.</p