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. 2001 Mar;21(5):1573-80.
doi: 10.1128/MCB.21.5.1573-1580.2001.

VAMP3 null mice display normal constitutive, insulin- and exercise-regulated vesicle trafficking

C Yang  1 , S Mora, J W Ryder, K J Coker, P Hansen, L A Allen, J E Pessin
Affiliations

VAMP3 null mice display normal constitutive, insulin- and exercise-regulated vesicle trafficking

C Yang et al. Mol Cell Biol. 2001 Mar.

Abstract

To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.

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Figures

FIG. 1
FIG. 1
Generation of VAMP3-deficient mice by homologous recombination in ES cells. (A) Schematic representation of the wild-type murine VAMP3 locus, the targeting construct, and the targeted locus. A 7.8-kb region including exon 2 (E2) and exon 3 (E3) (black boxes) was replaced by a neo cassette inserted in the reverse orientation. The 5′ and 3′ homologous regions of the targeting construct were 2.3 and 3.3 kb, respectively. TK, thymidine kinase. Southern blots show genomic DNA extracted from ES cells (B) and mouse tail (C), using a diagnostic probe (as indicated in panel A). The wild-type locus generates a 3.5-kb XbaI fragment, whereas the targeted allele produces a 7-kb XbaI fragment.
FIG. 2
FIG. 2
VAMP3-deficient mice do not express VAMP3 but maintain normal expression levels of GLUT4 and other related proteins. Tissue extracts from heart (Hrt), skeletal muscle (Skm), white adipose tissue (Wat), brain (Br), liver (Liv), testis (Tes), kidney (Kid), and lung (Lu) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted for VAMP3, GLUT4, insulin-responsive aminopeptidase (IRAP), transferrin receptor (TfR), syntaxin 4 (Syn4), VAMP2, and insulin receptor (IR).
FIG. 3
FIG. 3
Deletion of the VAMP3 gene does not affect growth of VAMP3-deficient mice. Female (F) and male (M) offspring from heterozygous breedings were weighed every day from days 1 to 21 (A) and thereafter every 4 weeks from weeks 5 to 25 (B).
FIG. 4
FIG. 4
VAMP3-deficient mice display normal glucose and insulin tolerance. (A) GTT of 16-week-old male mice. d-Glucose (20% solution, 2 g/kg of body weight) was injected intraperitoneally into mice fasted for 12 h. Blood glucose was measured at 0, 30, 60, 90, and 120 min postinjection using a glucometer as described in Materials and Methods. (B) ITT of 16 week-old male mice. Insulin (0.75 U/kg of body weight) was injected intraperitoneally to mice that were fasted for 6 h. Blood glucose was measured immediately before and at 15, 30, and 60 min after injection as described in Materials and Methods. The experimental groups consisted of 10 and 6 mice for GTT and ITT, respectively, and were performed a minimum of three times with similar results.
FIG. 5
FIG. 5
Insulin-stimulated glucose uptake in isolated primary adipocytes does not differ significantly between wild-type and VAMP3 null mice. Epididymal adipocytes were isolated from 10-week-old males and incubated in the presence or absence of 100 nM insulin for 30 min. 2-Deoxyglucose transport activity was determined as described in Materials and Methods. Results are the average ± standard error from three independent experiments each performed in duplicate.
FIG. 6
FIG. 6
Insulin-, hypoxia-, and exercise-contraction-stimulated glucose uptake in skeletal muscle is unaffected by the absence of VAMP3. (A) Soleus muscles were isolated from male wild-type or VAMP3-deficient mice and incubated for 45 min under oxygenated or hypoxic (Hyp) conditions with 5 mM glucose at 30°C. The muscles were transferred to oxygenated glucose-free medium and incubated for an additional 15 min prior to the initiation of 2-deoxyglucose uptake as described in Materials and Methods. In parallel, sets of soleus muscle were incubated in the absence or presence of 120 nM insulin (Ins) for 30 min and assayed for 2-deoxyglucose uptake. (B) Mice either remained sedentary or were strenuously exercised by swimming as described in Materials and Methods. EDL muscles were isolated and assayed for 2-deoxyglucose uptake. In parallel, sets of isolated EDL muscles from sedentary mice were incubated with 120 nM insulin for 30 min and assayed for 2-deoxyglucose uptake.
FIG. 7
FIG. 7
VAMP3 null BMMs phagocytose inert particles and bacteria. (A) Detection of VAMP2 and VAMP3 in wild-type (WT) and VAMP3 knockout (KO) BMM lysates. (B) H. pylori (Hp), zymosan particles (Zymo), IgG-opsonized zymosan (IgGZ), or complement-opsonized zymosan (COZ) was added to cultures of adherent BMMs, and phagocytosis was synchronized by centrifugation. After 30 min at 37°C, ingestion of cell-associated particles was scored as described in Materials and Methods. Data shown are the average ± standard deviation of three independent experiments performed in triplicate. The absence of VAMP3 did not affect particle binding to BMMs, and attachment indices varied by less than 15% for each stimulus (data not shown).
FIG. 8
FIG. 8
VAMP3 is not essential for transferrin receptor recycling or pinocytosis in isolated primary mouse embryo fibroblasts. Embryonic fibroblasts from wild-type and VAMP3-deficient mice were depleted of bovine transferrin and labeled with [125I]transferrin (3 nM; 1 μCi/μg) at 4°C for 2 h. Unbound ligand was removed, and then cells were incubated at 37°C for the times indicated. Rates of transferrin internalization (endocytosis) (A), transferrin release (recycling) (B), and HRP uptake (pinocytosis) (C) were determined as described in Materials and Methods. Data shown are the average ± standard deviation of three independent experiments.
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