3T3-L1 differentiation-induced UPR accompanies increased secretion. A–C, 3T3-L1 cells were harvested at full confluence (day −2), just prior to the addition of hormonal mixture (day 0), and at the indicated days thereafter. A, cell extracts were analyzed by Western blotting with antibodies to the proteins indicated. B, culture medium was analyzed by Western blotting with anti-adiponectin (Acrp30). C, cell extracts were analyzed by Western blotting with antibodies to the proteins indicated. CytC, cytochrome c. D, Western blotting of mouse tissues with an antibody to mouse AK2. W, white adipose tissue; M, muscle; L, liver; B, brain; H, heart; P, pancreas; D, diaphragm; Lu, lung; K, kidney; Sp, spleen; St, stomach.

AK2 depletion leads to inhibition of UPR activation and adiponectin secretion. A, B, and E–F, cells were transfected with scrambled (Scr) or AK2-targeting (AK2) siRNA at day 2 of differentiation, and experiments were performed at day 5. A, the degree of knockdown was determined for the protein level by Western blot (top panel) and for mRNA expression by qRT-PCR (lower panel). B, -fold change of mRNAs for adipocyte proteins determined by qRT-PCR upon depletion of AK2 relative to scrambled. C and D, cells were transfected with scrambled or AK2-targeting siRNA at day 5 of differentiation and experiments were performed at day 7. C, -fold change of basal glucose transport assessed by 2-deoxyglucose (2-DOG) uptake. D, -fold change of oxygen consumption relative to untreated (Con) scrambled cells. Olig, oligomycin; Gluc, glucose; Palm, palmitate. E, -fold change of mRNAs for mitochondrial proteins determined by qRT-PCR upon depletion of AK2 relative to scrambled. F, -fold change of mRNAs for specific UPR proteins determined by qRT-PCR upon depletion of AK2 relative to scrambled. G, level of protein expression in cell lysates was determined by Western blot analysis with antibodies against the proteins indicated. Calretic., calreticulin; Acrp30, anti-adiponectin. H, culture medium was removed at days 4, 5, and 6 of differentiation as indicated, and secretion of adiponectin (Acrp30) over the previous 24 h was determined by Western blot. A representative blot is shown (top panel) as well as the average of all experiments (bottom panel). * indicates p < 0.05 by paired two-tailed Student's t test. All values shown are means of a minimum of three independent experiments. Error bars indicate S.E.

AK2 depletion leads to inhibition of UPR activation and IgM secretion. A, Western blot analysis of AK2 protein expression in BCL1 cells during LPS induced differentiation at days indicated. B, AK2 mRNA expression by qRT-PCR in BCL1 cells during LPS-induced differentiation at days indicated. D0, day 0. C–I, BCL1 cells were nucleofected with either scrambled (Scr) or AK2-directed (AK2) siRNA and then induced to differentiate 24 h later by incubation with LPS. C, Western blot analysis for AK2 protein expression in scrambled (S) or AK2-directed (A) siRNA-transfected cells. D, -fold change of mRNAs for AK2 and BCL6 determined by qRT-PCR upon depletion of AK2 relative to scrambled at day 4 of differentiation. E, -fold change of mRNAs for mitochondrial proteins determined by qRT-PCR upon depletion of AK2 relative to scrambled at day 4 of differentiation. F, -fold change in oxygen consumption relative to untreated (Con) scrambled cells. Olig, oligomycin; Gluc, glucose; Palm, palmitate. G, -fold change of mRNAs for UPR proteins and IgM determined by qRT-PCR upon depletion of AK2 relative to scrambled at day 4 of differentiation. H, Western blot analysis with antibodies against BiP and calreticulin (Calret) in scrambled or AK2-directed siRNA-transfected cells during BCL1 differentiation. I, Western blot analysis for IgM protein expression in cell lysate (top panel) or culture medium (middle panel) in scrambled or AK2-directed or siRNA-transfected cells during BCL1 differentiation. The bottom panel shows the quantification of IgM secretion through analysis by densitometry. % of Max, percentage of maximum; D1–D4, days 1–4. * indicates p < 0.05 by paired two-tailed Student's t test. All values shown are means of a minimum of three independent experiments. Error bars indicate S.E.

Model for the interactions between AK2 and IRE1. AK2, localized to the mitochondrial intermembrane space, catalyzes the reversible interconversion of adenine nucleotides. In this location, AK2 can draw AMP into the high energy nucleotide pool under conditions of high ATP production and ADP limitation. Conversely, under conditions of ADP accumulation, such as after FCCP-mediated uncoupling or oligomycin-mediated ATPase inhibition, AK2 can catalyze the synthesis of ATP from ADP to support IRE1 activity and other essential functions. In addition, AK2 may be necessary for channeling ATP from its site of synthesis to sites of contact with the ER, facilitating energy flux to IRE1. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane.

Tfam depletion does not inhibit induction of UPR. Cells were transfected with scrambled (Scr) or Tfam targeting (Tfam) siRNA at day 2 of differentiation. Experiments were performed on day 5 of differentiation. A, level of Tfam mRNA determined by qRT-PCR. B, level of mtDNA copy number. C, -fold change of mRNAs for specific UPR proteins determined by qRT-PCR upon depletion of Tfam relative to scrambled. D, time course of IRE1 activation by tunicamycin, measured by qRT-PCR of spliced XBP1 (sXBP1). E, time course of BiP accumulation upon tunicamycin treatment determined by qRT-PCR of BiP mRNA. * indicates p < 0.05 by paired two-tailed Student's t test. All values shown are means of a minimum of three independent experiments. Error bars indicate S.E.

AK2 depletion mitigates ER stress signaling. Cells were transfected at day 5 of differentiation with scrambled (Scr) or AK2-directed (AK2) siRNA and analyzed 48 h later. A, the degree of knockdown was determined for the mRNA expression by qRT-PCR (left panel), and the protein level was determined by Western blot (right panel). * indicates p < 0.05 by paired two-tailed Student's t test. B, time course of IRE1 activation by tunicamycin, measured by qRT-PCR of spliced XBP1 (sXBP1). Paired two-tailed Student's t tests yielded a p = 0.00058, with means of 35.5 and 23.1 times basal values (t = 0) for scrambled and AK2 groups, respectively. C, time course of BiP accumulation upon tunicamycin (Tuni) treatment measured by Western blotting of BiP with β-actin shown as a loading control (upper panel) and qRT-PCR of BiP mRNA (bottom panel); p = 0.0029, with means of 5.8 and 3.6 times basal values for scrambled and AK2 groups. S, scrambled siRNA-transfected cells; A, AK2-directed siRNA-transfected cells. D, cell lysates and culture medium from multiple experiments were analyzed by Western blot with antibodies to AK2 and adiponectin (Acrp30). Band intensities were expressed as the ratio between AK2 and scrambled and plotted against each other. Regression analysis yielded an adjusted R² of 0.326 and a significance of 0.012. E, time course of CHOP accumulation upon tunicamycin treatment measured by Western blotting (upper panel) and qRT-PCR of CHOP mRNA (bottom panel). p = 0.00028, with means of 3.4 and 2.2 times basal values for scrambled and AK2 groups. All values shown are means of a minimum of three independent experiments. Error bars indicate S.E.

IRE1 activation is highly sensitive to ATP levels. A–C, level of adenine nucleotides isolated from cell lysates by TCA extraction expressed as relative luminescent units (RLU). A, 3T3-L1 cells were transfected with scrambled (Scr) or AK2-targeting (AK2) siRNA at day 2 of differentiation, and nucleotides were extracted at day 5. B, 3T3-L1 cells were transfected with scrambled or AK2-targeting siRNA at day 5 of differentiation, and nucleotides were extracted at day 7. C, BCL1 cells were transfected with scrambled or AK2-targeting siRNA prior to differentiation, and nucleotides were extracted at day 4. D, 3T3-L1 adipocytes at day 7 of differentiation were exposed to oligomycin (Olig) or FCCP for 2–4 h, and ATP levels in whole cell extracts were measured. Values are expressed as a function of the value in untreated cells (Con). E, -fold change of spliced XBP1 (sXBP1) mRNA levels was measured by qRT-PCR for 3T3-L1 adipocytes at day 7 of differentiation treated with oligomycin or FCCP for 2–4 h as compared with untreated cells. F, cells were treated with tunicamycin (Tuni), with oligomycin present from the start (t = 0 h) or added after 5.5 h of incubation. Spliced XBP1 was then measured by qRT-PCR at the times indicated. Values are expressed as a function of untreated levels. * indicates p < 0.05 by paired two-tailed Student's t test. All values shown are means of a minimum of three independent experiments. Error bars indicate S.E.