Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization (original) (raw)
Munc18c–/– mice die either in utero or as neonates. We generated mice in which the Munc18c gene was disrupted as a result of homologous recombination. A targeting vector was designed to replace the exon that includes the ATG initiation codon with the neomycin resistance gene and an internal ribosome entry site (IRES) sequence (Figure 1). Screening of neomycin-resistant ES cells by Southern blot analysis identified 4 cell clones that had undergone appropriate recombination. One line of chimeric mice was obtained from these recombinant ES cells. We generated Munc18c+/– mice by mating the chimeric animals with C57BL/6J mice. All Munc18c−/− mice died either in utero or within 6 hours after birth. At 13.5 days postcoitum (dpc), the Munc18c–/– embryos were 17% shorter than were wild-type embryos (mean body lengths, 8.9 mm and 10.8 mm, respectively) (Figure 2A). Live newborn Munc18c−/− mice were also 30% shorter than Munc18c+/+ animals (mean body lengths, 19.1 mm and 27.3 mm, respectively) (Figure 2B). The intermediate zone of the cerebral cortex of newborn Munc18c−/− mice contained poorly formed neural fibers and a greater number of cellular components compared with Munc18c+/+ animals (Figure 2C). Furthermore, the border between the subventricular and intermediate zones was indistinct in Munc18c−/− mice. These results thus suggest that Munc18c is essential for normal brain development. The precise mechanisms by which Munc18c deficiency gives rise to intrauterine growth retardation and embryonic or neonatal death remain to be defined.
Targeted deletion of Munc18c in mice. The wild-type Munc18c allele, the targeting vector, and the targeted allele after homologous recombination are shown. Probe A is a DNA fragment used for Southern blot analysis of BamHI-digested genomic DNA from ES cells; the wild-type and mutant alleles give rise to 9.0- and 6.4-kb hybridizing fragments, respectively.
Reduced size and disorganized brain structure of Munc18c–/– mice. (A) Gross morphology of Munc18c+/+, Munc18c+/–, and Munc18c–/– embryos at 13.5 dpc. (B) Gross morphology of Munc18c+/+, Munc18c+/–, and Munc18c–/– newborn mice. (C) Coronal sections of the brain of newborn Munc18c+/+ and Munc18c−/− mice. The sections were stained with Nissl solution. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone. Scale bar: 200 μm.
Generation of Munc18c–/– adipocytes. To determine the role of Munc18c in insulin-stimulated GLUT4 exocytosis, we generated adipocytes that lack this protein by inducing the differentiation of MEFs isolated from Munc18c−/− animals at 13.5 dpc. We first examined adipogenesis in the Munc18c–/– cells by staining with oil red O, which detects triglycerides. The proportion of MEFs that differentiated into adipocytes was similar in populations of cells isolated from Munc18c+/+, Munc18c+/–, and Munc18c–/– embryos (Figure 3A). Furthermore, the triglyceride content of the differentiated cells did not differ significantly among the 3 genotypes (Figure 3B), which suggests that Munc18c does not affect adipogenesis in vitro.
Lack of effect of Munc18c on adipocyte differentiation in MEFs. (A) Adipocyte differentiation was induced in MEFs isolated from Munc18c+/+, Munc18c+/−, or Munc18c−/− embryos at 13.5 dpc. Twelve days after the onset of induction, adipocytes were fixed, stained with oil red O, and examined both macroscopically (Macro) and microscopically (Micro). Scale bars: 10 mm (left panels) and 50 μm (right panels). (B) Intracellular triglyceride (TG) content was quantified as described in Methods. The amount of TG content in adipocytes of each genotype was normalized to protein concentration. Data are mean ± SE of values from 4 independent experiments and are expressed relative to the value for Munc18c+/+.
SNARE protein expression in Munc18c–/– adipocytes. We next examined the expression of SNARE proteins in Munc18c−/− adipocytes. Northern blot analysis revealed that the amounts of syntaxin4, SNAP23, and VAMP2 mRNAs did not differ among Munc18c+/+, Munc18c+/−, and Munc18c−/− adipocytes (Figure 4A). The abundance of another isoform of Munc18, Munc18b, also did not differ among these adipocytes (Figure 4A). Immunoblot analysis, however, showed that the amount of syntaxin4, which forms a complex with Munc18c in adipocytes (6), was markedly reduced in Munc18c−/− adipocytes and slightly reduced in Munc18c+/− cells compared with that in wild-type cells (Figure 4B). In contrast, the abundance of SNAP23 or VAMP2 did not differ among adipocytes of the 3 genotypes. Furthermore, the expression of syntaxin2, which does not bind to Munc18c in adipocytes (6), was not affected by Munc18c deficiency. These results suggest that Munc18c might stabilize syntaxin4 by forming a complex with it in adipocytes.
Reduced abundance of syntaxin4 in Munc18c−/− adipocytes. (A) Northern blot analysis of mRNAs for SNARE proteins. Total RNA extracted from Munc18c+/+, Munc18c+/–, or Munc18c–/– adipocytes was analyzed for hybridization with probes specific for Munc18c, syntaxin4, SNAP23, VAMP2, or Munc18b mRNAs. The region of the ethidium bromide–stained gel containing 28S ribosomal RNA (rRNA) is also shown. (B) Immunoblot analysis of SNARE proteins. Lysates prepared from adipocytes of each genotype were subjected to immunoblot analysis with antibodies specific for the indicated proteins.
The binding of Munc18c to syntaxin4 inhibits the association of the latter protein with SNAP23 or VAMP2 (4, 5, 7), which suggests that, in the absence of Munc18c, syntaxin4 might be free to form a SNARE complex with SNAP23 and VAMP2. Moreover, the ratio of the amounts of syntaxin4 and Munc18c, rather than the absolute abundance of these proteins, has been shown to affect insulin-stimulated glucose uptake in skeletal muscle and adipose tissue (16). We therefore next examined the amount of Munc18c-free syntaxin4 in Munc18c+/+, Munc18c+/−, and Munc18c−/− adipocytes with the use of an mAb (A6C) that immunoprecipitates only syntaxin4 molecules that are not associated with Munc18c (Figure 5A). Whereas the total amount of syntaxin4 was significantly reduced in Munc18c−/− adipocytes (Figure 5B), the amount of syntaxin4 immunoprecipitated by the A6C antibody was increased by approximately 2.5-fold in these cells compared with that in Munc18c+/+ or Munc18c+/− adipocytes (Figure 5C). These results thus indicate that the amount of Munc18c-free syntaxin4 is increased in Munc18c−/− adipocytes even though the total amount of syntaxin4 is reduced.
Increased amount of Munc18c-free syntaxin4 in Munc18c–/– adipocytes. (A) Immunoprecipitation of Munc18c-free syntaxin4 by the mAb A6C. COS-7 cells were transfected with expression plasmids for full-length rat synatxin4 and mouse Munc18c, after which cell lysates were subjected to immunoprecipitation with the A6C antibody (lane 2) or with polyclonal antibodies (pAb) to syntaxin4 (lane 3). The resulting precipitates, as well as the original cell lysate (lane 1), were subjected to immunoblot analysis with polyclonal antibodies to syntaxin4 (lower panel) and with polyclonal antibodies to Munc18c (upper panel). Munc18c was not associated with syntaxin4 immunoprecipitated by A6C (lane 2) but was present in the immunoprecipitate prepared with the polyclonal antibodies to syntaxin4 (lane 3). (B and C) Quantitation of Munc18c-free syntaxin4 in adipocytes. Lysates of Munc18c+/+, Munc18c+/–, or Munc18c–/– adipocytes (B) as well as immunoprecipitates prepared from such lysates with mAb A6C (C) were subjected to immunoblot analysis with polyclonal antibodies to syntaxin4 or to Munc18c. The bands corresponding to total syntaxin4 (B) and to Munc18c-free syntaxin4 (C) were quantitated and expressed relative to the corresponding values for Munc18c+/+ cells. Data are mean ± SE of values from 4 independent experiments. *P < 0.05, **P < 0.001 versus Munc18c+/+ cells (Student’s t test).
Deficiency of Munc18c does not affect proximal insulin signaling. We examined whether proximal insulin signaling implicated in GLUT4 exocytosis in adipocytes was affected by the lack of Munc18c. No marked difference in the extent either of tyrosine phosphorylation of the insulin receptor or insulin receptor substrate (IRS) or of serine phosphorylation of the downstream kinase Akt induced by stimulation with insulin (1 or 100 nM) for 5 minutes was apparent among Munc18c+/+, Munc18c+/–, and Munc18c–/– adipocytes (Figure 6). The absence of Munc18c thus does not affect the activity of major molecules that mediate proximal insulin signaling.
Lack of effect of Munc18c deficiency on proximal insulin signaling in adipocytes. Munc18c+/+, Munc18c+/–, or Munc18c–/– adipocytes were deprived of serum for 2 hours and then stimulated with insulin (1 or 100 nM) for 5 minutes. For detection of tyrosine-phosphorylated forms of IRS (pY-IRS) and the β subunit of the insulin receptor (pY-IRβ), cell lysates were subjected to immunoprecipitation and subsequent immunoblot analysis with mAb PY20 to phosphotyrosine (upper panel). For detection of serine-phosphorylated Akt, cell lysates were subjected to immunoblot analysis with antibodies specific for Akt phosphorylated on serine 473 (lower panel).
Enhancement of insulin-stimulated GLUT4 externalization in Munc18c–/– adipocytes. We next determined the subcellular localization of endogenous GLUT4 in Munc18c–/– adipocytes. Subcellular fractionation and subsequent immunoblot analysis with antibodies to GLUT4 revealed that, upon stimulation with 100 nM insulin for 20 minutes, GLUT4 translocated from intracellular storage sites to the plasma membrane to a similar extent in Munc18c+/+, Munc18c+/–, and Munc18c–/– adipocytes (Figure 7, A and B), which suggests that Munc18c does not contribute to this process. To distinguish between the movement of GLUT4 vesicles to the cell periphery and the subsequent fusion of the vesicles with the plasma membrane and GLUT4 externalization, we studied adipocytes that had been infected with a retroviral vector encoding a form of GLUT4 (GLUT4-myc7-GFP) that contains 7 Myc epitope tags in its first extracellular loop and is fused with GFP at its intracellular COOH-terminus (17). The insulin-induced movement of GLUT4 to the plasma membrane was monitored by measurement of GFP fluorescence at the cell periphery, and GLUT4 externalization was monitored by measurement of cell surface labeling with antibodies to the Myc tag. To exclude the possibility that GFP protein is not always fluorescent in adipocytes, we confirmed that insulin-induced localization of GLUT4 in Munc18c+/+ adipocytes was the same whether determined by immunodetection with anti-GFP antibody or GFP fluorescence (data not shown). The extent of GLUT4 movement to the cell periphery was similar upon 20-minute stimulation of cells with either 1 or 100 nM insulin, and similar results were obtained with adipocytes of each of the 3 genotypes (Figure 8, A and B). Munc18c thus does not appear to affect the insulin-stimulated translocation of GLUT4 to the plasma membrane. In Munc18c+/+ or Munc18c+/− cells, 10 or 100 nM insulin was required for maximal externalization of GLUT4 (Figures 8, A and C), even though 1 nM insulin was sufficient for maximal induction of GLUT4 movement to the cell periphery (Figure 8B). However, in Munc18c−/− adipocytes, both GLUT4 translocation to the cell periphery and GLUT4 externalization were maximal at 1 nM insulin (Figure 8, B and C). Thus, sensitivity to the induction of GLUT4 externalization by insulin stimulation was enhanced in Munc18c−/− adipocytes compared with Munc18c+/+ or Munc18c+/− cells, although the maximal responses did not differ significantly among the 3 genotypes. Furthermore, we investigated the insulin-stimulated glucose uptake in adipocytes of each genotype. One hundred nanomolar insulin was required for the remarkable increase of glucose uptake in Munc18c+/+ or Munc18c+/− cells, whereas in Munc18c–/– adipocytes, 1 nM insulin induced almost the same increase in glucose uptake as did 100 nM insulin (Figure 9). These results indicate that not only externalization of GLUT4 but also glucose uptake are enhanced in Munc18c−/− adipocytes compared with Munc18c+/+ or Munc18c+/− cells.
Insulin-stimulated translocation of endogenous GLUT4 to the plasma membrane in MEF-derived adipocytes. (A) Munc18c+/+, Munc18c+/–, or Munc18c–/– adipocytes were deprived of serum for 2 hours and then stimulated with 100 nM insulin for 20 minutes. Cell homogenates were subjected to subcellular fractionation, and plasma membrane and intracellular membrane fractions were subjected to immunoblot analysis with antibodies to GLUT4. (B) Immunoblot band intensity of GLUT4 was quantitated using NIH Image software and was expressed relative to the abundance of GLUT4 without insulin in cells of each genotypes. Data are mean ± SE of values from 3 or 4 independent experiments.
Enhancement of insulin-stimulated GLUT4 externalization, but not of GLUT4 translocation to the cell periphery, in Munc18c−/− adipocytes. (A) Munc18c+/+, Munc18c+/–, or Munc18c–/– adipocytes were infected with a retroviral vector encoding a GFP fusion protein of Myc epitope–tagged GLUT4 (GLUT4-myc7-GFP). The cells were stimulated with the indicated concentrations of insulin for 20 minutes, fixed, and subjected to indirect immunofluorescence staining with antibodies to the Myc tag and PE-conjugated secondary antibodies in order to detect externalized GLUT4 (red). GLUT4 translocation to the cell periphery was detected by GFP fluorescence (green) on analysis of the cells by confocal microscopy. Scale bar: 10 μm. (B) Movement of GLUT4 to the cell periphery in experiments similar to that shown in A was quantitated by determination of the percentage of GFP-positive cells that manifested GFP fluorescence at the cell periphery. Data are mean ± SE of values from 3 separate experiments, with 1,000 GFP-positive cells being examined in each experiment. (C) GLUT4 externalization in experiments similar to that shown in A was quantitated by determination of the percentage of GFP-positive cells that exhibited PE fluorescence at the cell surface. Data are mean ± SE of values from 4–6 independent experiments, with 1,000 GFP-positive cells being examined in each experiment. *P < 0.05, **P < 0.001 versus the corresponding values for Munc18c+/+ cells (Student’s t test).
Enhancement of insulin-stimulated glucose transport in Munc18c−/− adipocytes. Uptake of 2-deoxy-D-[1,2-3H]glucose was assayed in Munc18c+/+, Munc18c+/–, or Munc18c–/– adipocytes stimulated with the indicated concentrations of insulin. Data are mean ± SE of 6 independent experiments and are expressed as fold stimulation of glucose uptake relative to that without insulin. *P < 0.01 versus cells stimulated with 1 nM insulin (Student’s t test).
To further confirm the observed effects of Munc18c deficiency in adipocytes, we restored Munc18c expression in Munc18c−/− cells by infection with the adenovirus containing Munc18c that we have used previously (6). Restoration of Munc18c expression in the Munc18c−/− adipocytes increased the abundance of syntaxin4 to the level apparent in wild-type cells (Figure 10A). At this time, it seemed that the amount of Munc18c in Munc18c−/− cells by infection with adenovirus containing Munc18c was more abundant than that of endogenous Munc18c in Munc18c+/+ cells as determined by Western blot analysis (Figure 10A, upper panel). However, we suspected that this may have been be due to the much higher level of expression of adenovirus-mediated Munc18c proteins in undifferentiated cells which are scattered among differentiated adipocytes because of high efficiency of adenovirus infection of undifferentiated cells. Therefore, we investigated the expression level of Munc18c in adipocytes or in undifferentiated cells with immunofluorescence staining with anti-Munc18c antibody and confirmed that the abundance of adenovirus-mediated Munc18c proteins in Munc18c−/− adipocytes was almost the same as that of endogenous Munc18c in Munc18c+/+ adipocytes (Figure 10B, arrows) and that Munc18c proteins are expressed in much greater quantities in undifferentiated cells (Figure 10B, arrowhead). Furthermore, restoration of Munc18c in Munc18c−/− adipocytes expressing GLUT4-myc7-GFP reduced the insulin sensitivity of GLUT4 externalization in Munc18c−/− cells to a level similar to that apparent in wild-type adipocytes, without affecting the extent of GLUT4 movement to the cell periphery (Figure 10, C and D). These results thus confirmed that the insulin-induced fusion of GLUT4-containing vesicles with the plasma membrane and the consequent externalization of GLUT4, but not the translocation of these vesicles to the cell periphery, were enhanced by Munc18c deficiency in mouse adipocytes.
Normalization both of syntaxin4 expression and of the insulin sensitivity of GLUT4 externalization in Munc18c–/– adipocytes by restoration of Munc18c expression. (A) Syntaxin4 expression. Munc18c+/+ or Munc18c–/– adipocytes were infected with an adenoviral vector for Munc18c (adex-Munc18c) or with the corresponding empty vector (control adex), as indicated. After 2 days, cell lysates were subjected to immunoblot analysis with antibodies to Munc18c (upper panel) or to syntaxin4 (lower panel). (B) Immunofluorescence microscopic analysis of Munc18c in adipocytes. Munc18c+/+ adipocytes infected with control adex, Munc18c–/– adipocytes infected with control adex, or Munc18c–/– adipocytes infected with adex-Munc18c at an MOI of 5 were fixed, permeabilized, subjected to immunofluorescence staining with antibodies to Munc18c, and analyzed by confocal microscopy. Arrows mark the localizations of differentiated adipocytes, and the arrowhead indicates undifferentiated cells. Scale bar: 10 μm. (C and D) GLUT4 translocation and externalization. Cells infected as in A were assayed for the translocation (C) and externalization (D) of GLUT4 in response to insulin as described in Figure 8. Data are mean ± SE of values from 3 separate experiments. *P < 0.05 versus the value for Munc18c–/– cells infected with control adex and stimulated with 1 nM insulin (Student’s t test).
Insulin-stimulated GLUT4 externalization is wortmannin resistant in Munc18c–/– cells. Activation of a PI3K-dependent pathway is required for insulin-stimulated exocytosis of GLUT4-containing vesicles (18–20). In Munc18c+/+ adipocytes expressing GLUT4-myc7-GFP, the PI3K inhibitor wortmannin (100 nM) inhibited by approximately 50% the GLUT4 externalization induced by 100 nM insulin (Figure 11, A and C). This observation is consistent with the previous finding that the half-maximal concentration of wortmannin for inhibition of insulin-stimulated GLUT4 insertion into the plasma membrane in 3T3-L1 mouse adipocytes is 80 nM (21). However, the insulin-stimulated movement of GLUT4 to the cell periphery in Munc18c+/+ cells was not inhibited by 100 nM wortmannin (Figure 11, A and B), which is consistent with the previous observation that microtubule-dependent movement of GLUT4 elicited by insulin is insensitive to 100 nM wortmannin (22). Whereas 100 nM wortmannin inhibited insulin-stimulated GLUT4 externalization in WT cells, it did not do so in Munc18c–/– adipocytes (Figure 11, A and C), which suggests that the wortmannin-sensitive step of the insulin signaling pathway that regulates the fusion of GLUT4-containing vesicles with the plasma membrane and GLUT4 externalization involves Munc18c.
Wortmannin insensitivity of insulin-induced GLUT4 externalization in Munc18c−/− adipocytes. (A) Munc18c+/+ or Munc18c–/– adipocytes expressing GLUT4-myc7-GFP were incubated for 20 minutes in the absence or presence of 100 nM insulin and then for 20 minutes in the additional absence or presence of 100 nM wortmannin. The cells were then analyzed as described in Figure 8A. Scale bar: 10 μm. (B) Quantitation of GLUT4 movement to the cell periphery in experiments similar to that shown in A. Data are mean ± SE of values from 6 separate experiments, with 100 GFP-positive cells being examined in each experiment. (C) Quantitation of GLUT4 externalization in experiments similar to that shown in A. Data are mean ± SE of values from 6 independent experiments, with 100 GFP-positive cells being examined in each experiment. *P < 0.001 versus the corresponding value for cells exposed to insulin alone (Student’s t test).
Phosphatidylinositol 3-phosphate induces GLUT4 externalization in Munc18c–/– adipocytes. Given that we could not exclude the possibility that the insulin signaling pathway that is operative at 1 nM hormone and is insensitive to 100 nM wortmannin might also be required for GLUT4 externalization, we reconstituted the exocytosis of GLUT4-containing vesicles without insulin. The insulin-induced formation of phosphatidylinositol 3-phosphate (PI3P), which is mediated by the activation of TC10, is resistant to 100 nM wortmannin (23); PI3P was recently shown to trigger the movement of GLUT4-containing vesicles to the plasma membrane, but not glucose transport, in insulin-responsive cells (23). Exogenous delivery of PI3P with a polyamine carrier induced GLUT4 movement to the cell periphery in approximately 40–50% of Munc18c+/+ or Munc18c−/− adipocytes (Figure 12, A and B). However, PI3P also induced GLUT4 externalization in Munc18c−/− adipocytes but not in wild-type cells (Figure 12, A and C). Thus, a large proportion (approximately 80%) of GLUT4 molecules that were translocated to the cell periphery in Munc18c–/– adipocytes in response to PI3P was externalized in the absence of insulin (Figure 12D). These results suggest that the insulin-induced formation of PI3P is sufficient to trigger both the translocation of GLUT4-containing vesicles to the cell periphery and their fusion with the plasma membrane in the absence of Munc18c.
PI3P-induced externalization of GLUT4 in Munc18c−/− adipocytes. (A) Munc18c+/+ or Munc18c−/− adipocytes expressing GLUT4-myc7-GFP were exposed for 7 minutes to a mixture of PI3P and polyamine carrier or to the carrier alone and were then analyzed as described in Figure 8A. Scale bar: 10 &––––m. (B–D) Quantitation of GLUT4 movement to the cell periphery (B), of GLUT4 externalization (C), and of the proportion of GLUT4 molecules translocated to the cell periphery that undergo externalization (the percentage of cells with peripheral GFP fluorescence that also show surface PE fluorescence) (D) in experiments similar to that shown in A. Data are mean ± SE of values from 3 independent experiments. *P < 0.01, **P < 0.001 versus the corresponding values for Munc18c+/+ cells.