Transient knock-down of kcna2 reduces sleep in larval zebrafish.

In the current study we set out to determine the effects of morpholino oligonucleotide (MO) knock-down of kcna2 on sleep-wake cycles in zebrafish. The results were compared to a non-overlapping MO injection, Dec2, who's mutant is also linked with a short sleep phenotype. Four groups of fish were used in the experiment: naïve fish, and fish injected with either control, kcna2, or Dec2 MO. All groups underwent 24-h behavioral monitoring of sleep-wake cycles at four and seven days-post-fertilization (dpf). First, we established an immobility dependent, sleep related, increase in arousal thresholds at both 4 and 7 dpf. Secondly, we show that kcna2 MO injected fish exhibit significantly less sleep behavior than controls and naïve fish, whereas Dec2 MO injections had similar but less severe effects. Finally, using kcna2 MO injected fish only, we turn to local field recordings at the level of the telencephalon and tectum opticum and rule out that the knock-down resulted in a non-specific increase in neural excitability that would mask sleep behavior.

v- and t-SNARE protein expression in models of insulin resistance: normalization of glycemia by rosiglitazone treatment corrects overexpression of cellubrevin, vesicle-associated membrane protein-2, and syntaxin 4 in skeletal muscle of Zucker diabetic fatty rats.

Insulin stimulation of adipose and muscle cells results in the translocation of GLUT4 from an intracellular location to the plasma membrane; this translocation is defective in insulin resistance. Studies have suggested an important role for synaptobrevin and syntaxin homologues in this event, particularly the v-soluble N-ethylmaleimide attachment protein receptors (SNAREs) cellubrevin and vesicle-associated membrane protein-2 (VAMP-2) and the t-SNARE syntaxin 4, but the expression of these proteins has not been studied in insulin-resistant tissues. Therefore, we examined SNARE protein content in skeletal muscle from Zucker diabetic fatty (ZDF) rats compared with lean controls and determined the effect of the thiazolidinedione insulin sensitizer rosiglitazone on these proteins. GLUT4 levels in skeletal muscle from ZDF rats were similar to those in lean control animals. In contrast, cellubrevin, VAMP-2, and syntaxin 4 protein levels were elevated (2.8-fold, P = 0.02; 3.7-fold, P = 0.01; and 2.2-fold, P < 0.05, respectively) in skeletal muscle from ZDF rats compared with lean controls. Restoration of normoglycemia and normoinsulinemia in ZDF rats with rosiglitazone (30 micromol/kg) normalized cellubrevin, VAMP-2, and syntaxin 4 protein to levels approaching those observed in lean control animals. These data show that elevated v- and t-SNARE protein levels are associated with insulin resistance in skeletal muscle and that these increases may be reversed by rosiglitazone treatment concomitant with a restoration of glycemic control. Such increases in SNARE protein levels were not observed in streptozotocin-induced diabetic rats, which suggests that hyperinsulinemia rather than hyperglycemia may be more important in modulating SNARE protein expression in rodent models of insulin resistance. Consistent with this hypothesis, elevated levels of SNARE proteins were also observed in 3T3-L1 adipocytes chronically treated with insulin (500 nmol/l for 24 h). These data argue that SNARE protein levels may be altered in insulin-resistant states and that the levels of these proteins are modulated by agents that increase insulin sensitivity. Moreover, these data demonstrate for the first time altered expression of proteins known to regulate GLUT4 translocation in a model of diabetes.

Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport.

AIMS/HYPOTHESIS: Insulin stimulates glucose transport in adipose and muscle tissue by the translocation of a specialised pool of intracellular GLUT4-containing vesicles to the cell surface. It is well established that defective insulin-stimulated GLUT4 translocation is associated with insulin resistance. Long-term insulin treatment (500 nmol/l for 24 h) of 3T3-L1 adipocytes has previously been shown to decrease cellular GLUT4 content and reduce insulin-stimulated GLUT4 translocation. Here, we test the hypothesis that the insulin resistance observed after long-term insulin treatment arises by the selective loss of GLUT4 from a specific intracellular compartment. METHODS: Using iodixanol gradient centrifugation we have separated intracellular GLUT4 containing membranes into two distinct populations corresponding to recycling endosomes and a distinct intracellular compartment which probably represents GLUT4 storage vesicles (GSVs). RESULTS: A short-term insulin stimulation reduced the content of GLUT4 in the GSV fraction (51 +/- 3.5%) with only a modest decrease from the endosomal fraction (23 +/- 2.6%). Long-term insulin treatment decreased cellular GLUT4 content by about 40% and diminished the ability of a short-term insulin challenge to promote GLUT4 translocation. We further show that this depletion of cellular GLUT4 is selectively from the GSV fraction (68 +/- 7% decrease compared to untreated cells). CONCLUSIONS/INTERPRETATION: Such data argue that long-term insulin treatment results in the mis-targeting of GLUT4 such that it no longer accesses the GSV compartment. These data imply that defective targeting of GLUT4 away from the GSV compartment plays an important role in the aetiology of insulin resistance.

Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport.

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.

The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4.

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.