* Department of Cell Biology, Medical School, Utrecht University, 3584 CX Utrecht, The Netherlands; Centre for Molecular
and Cellular Biology, University of Queensland, St. Lucia, QLD, 4072, Australia; § Garvan Institute of Medical Research, St.
Vincents Hospital, Darlinghurst, NSW, 2010, Australia;
Cell Biology of Hypertension, Clinical Research Institute of Montreal,
Montreal, Quebec, H2W 1R7, Canada; and ¶ Endocrinology and Metabolic Diseases, Institute of Medicine, University of Bari,
70124 Bari, Italy
The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4-containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.
Glucose entry into mammalian cells occurs in most
cases by facilitative transport, a process that is mediated by a family of glucose transporter proteins.
The individual members of this family, GLUTs 1-7, are
variably expressed in different tissues (Bell et al., 1993 Despite considerable progress in our understanding of
insulin-regulated GLUT-4 movement, the nature of the intracellular GLUT-4 storage compartment, and the intracellular trafficking pathway(s) undertaken by GLUT-4,
remain to be fully defined. Morphological studies have
provided important clues concerning GLUT-4 trafficking in insulin-sensitive cells (Slot et al., 1991a In an effort to further explore this question we have
studied a bona fide regulated secretory cell type, atrial myocytes, in which GLUT-4 is endogenously expressed. The
main secretory product of the atrial cardiomyocyte is the
precursor of atrial natriuretic factor (ANF), an antihypertensive hormone (De Bold, 1985; Ruskoaho, 1992 Materials
Polyclonal rabbit antiserum against the cytoplasmic carboxy terminus of
GLUT-4 (James et al., 1989 Immunocytochemistry
Male Wistar rats were fasted overnight, unless indicated otherwise. Stimulated animals were injected intraperitoneally with a mixture of insulin (8 U/kg) and d-glucose (1 g/kg) 30 min before fixation. Whole body fixation
was performed on animals that were anesthetized with pentobarbitone as
described (Slot et al., 1991a Immunoblotting
Different regions of the heart were dissected from rats and frozen at
Equal amounts of protein (25 µg) were separated by SDS-PAGE using
a 10% acrylamide resolving gel. Proteins resolved by SDS-PAGE were
electrophoretically transferred to nitrocellulose. Nitrocellulose sheets
were incubated with the polyclonal antibodies diluted 1/1,000 (vol/vol) in
1% dried milk in PBS, pH 7.4. Detection of immunoreactive bands was
achieved using ECL and quantitation was performed using an imaging
densitometer (GS-670; Bio-Rad Laboratories, Richmond, CA) and the
molecular analyst program.
[3H]-2-Deoxyglucose Uptake
Male Wistar rats were fitted with cannulae introduced into the carotid artery and jugular vein as previously described (Kraegen et al., 1985 At steady-state euglycemia (~75 min after commencement of the
clamp or 2 min after intravenous saline injection), an intravenous bolus of
[3H]-2-deoxyglucose (80 µCi) (3H-2DG) (Amersham) was administered.
Blood samples (200 µl) were obtained after administration of the bolus
for estimation of plasma tracer and glucose concentration. Plasma samples
for determination of tracer concentration were deproteinized immediately
in 5.5% ZnSO4 and saturated Ba(OH)2. At completion of the study (45 min after 3H-2DG administration), rats were anesthetized (pentobarbitone; 60 mg/kg, intravenously) and the right and left atria and ventricles were rapidly removed and frozen. An estimate of tissue glucose uptake
(the glucose metabolic index, Rg') was calculated from the tissue accumulation of phosphorylated 3H-2DG (Kraegen et al., 1985 Plasma ANF Determinations
To assess the effect of insulin on ANF secretion, the plasma level of the
NH2-terminal fragment (1-98) of pro-ANF was determined 10 min before
and 10, 40, and 70 min after commencement of the euglycemic hyperinsulinemic clamp in 24-h fasted rats by radioimmunoassay as described previously (Thibault et al., 1988 In the present study our primary goal was to characterize
the intracellular GLUT-4 compartment. Therefore, most
of the localization studies were performed in atrial myocytes of nonstimulated, overnight-fasted animals.
Immunofluorescence Microscopy
The alignment of myocytes in atrial tissue (Fig. 1, A and
B) is ordered in a much more complicated fashion than in
the ventricle (Fig. 1 C), and so the atrial fibers are sectioned much more randomly. Ventricular cells were viewed
in longitudinally cut sections. We observed no significant
difference in ANF or GLUT-4 labeling between the right
and left atrium. As reported previously (Cantin et al., 1990
Expression of GLUT-4 in Heart
The immunofluorescence signal for GLUT-4 was consistently stronger in atrium than in ventricle. This was confirmed by immunoblotting (Fig. 1 D), which indicated a
higher expression of GLUT-4 in atrial tissue. When expressed per unit of protein, the immunoreactivity for
GLUT-4 was approximately two times higher in the left
and right atrium than in either ventricle.
Immuno-EM of ANF
The ANF localization observed in the present study was
similar to that described previously (Thibault et al., 1989
Immuno-EM of GLUT-4
A significant proportion of the total GLUT-4 labeling (50-
60%) was found in ANF secretory granules (Table I) in
regular sections. In overstretched sections, where GLUT-4
labeling was enhanced (Fig. 2 B), virtually all secretory
granules were labeled for GLUT-4, whether they were in
the Golgi area or toward the cell periphery. The secretory
granules of the atrial myocyte are unusual in that their
membrane is often coated (Jamieson and Palade, 1964 Table I.
Relative Distribution of GLUT-4 Gold Particles in
Myocytes of the Right Atrium of Overnight-fasted Rats
The structure of the Golgi complex in the rat atrial cell
has been studied in detail by Rambourg and colleagues
(1984). They described the entire structure as a continuum
of plate-shaped stacks of cisternae that are interconnected
by tubular regions in such a way that they form a beltlike
structure around the nucleus. This belt is complicated by
the formation of loops at both nuclear poles. A characteristic stack comprises five cisternae. Each cisterna has specific morphological and cytochemical features. In the perinuclear stacks the first or cis-most cisternae always faces
the nucleus, which facilitates the recognition of the cis-
trans orientation, making the use of specific markers for
either side unnecessary. The fifth cisterna, which is often
partly detached from the stack, probably represents part
of the TGR as defined by Griffiths and Simons (1986) Effects of Cyclohexamide Treatment
To determine if GLUT-4 within the trans-Golgi originated
from the biosynthetic pathway or the recycling pathway,
we performed experiments using the protein synthesis inhibitor cyclohexamide. To test the efficacy of the cyclohexamide treatment we first examined its effects on albumin labeling in liver. After 1 h of cyclohexamide treatment
there was a substantial reduction in liver albumin labeling (compare Fig. 5, A and B). Longer periods of cyclohexamide
treatment (2 h) did not further change this situation. Similarly, in atrial cardiomyocytes from the same animals
cyclohexamide treatment completely abolished the presence of newly forming granules in the Golgi cisternae.
Also the disperse labeling for ANF in the cisternae that we
observed in control cells was virtually absent (Fig. 5 C).
These data indicate that the cyclohexamide treatment effectively blocked protein synthesis in the atrial myocytes.
Despite this, GLUT-4 labeling at the trans side of the
Golgi was not affected by cyclohexamide treatment, indicating that GLUT-4 at this location is not derived from the
biosynthetic pathway.
Insulin Stimulation
We have previously shown that insulin stimulates the
movement of GLUT-4 from the T-V elements and TGR to
the cell surface in other insulin-sensitive cells (Slot at al.,
1991a; James et al., 1994 To further confirm the response to insulin in atrium we
next examined the effect of insulin on 2-deoxyglucose
(2DG) uptake in different heart regions using the hyperinsulinemic euglycemic clamp technique in whole animals
(Table II). This technique enables measurement of 2DG
uptake, an estimate of glucose transport, in individual tissues of the rat in vivo after the infusion of insulin and variable amounts of glucose to prevent the onset of hypoglycemia (Kraegen et al., 1985 Table II.
Influence of Insulin on [3H]-2-Deoxyglucose Uptake
in Heart Regions of 24-h-fasted Rats
).
GLUT-4 is expressed in cell types, such as skeletal muscle,
cardiac muscle, white and brown adipose tissue, that exhibit acute changes in glucose transport (Birnbaum, 1989
; James et al., 1989
). Most glucose transporter isoforms constitutively reside at the cell surface to optimize exposure of
the cell to the extracellular glucose (James and Piper,
1994
). In contrast, GLUT-4 is found in an intracellular
compartment from where it can be rapidly translocated to
the cell surface in response to insulin, thus allowing the
cell to transiently increase its access to extracellular glucose (Cushman and Wardzala, 1980
; Suzuki and Kono, 1980
).
This process plays an important role in the postabsorptive removal of glucose from the bloodstream in mammals and
also during enhanced energy consumption, such as exercise in the case of muscle.
,b; James et al., 1994
). GLUT-4, like a number of other cell surface recycling proteins, is internalized from the cell surface via
clathrin-coated pits (Robinson et al., 1992
). Thereafter,
the transporter is sorted from the lysosomal pathway into
a compartment comprising tubules and vesicles that we
have referred to as tubulo-vesicular (T-V)1 elements. The
relationship of this compartment to other secretory compartments is currently not known. It has been suggested
that it has specialized properties that distinguish it from
the endosomal/trans-Glogi reticulum (TGR) system (Martin at al., 1994; Herman et al., 1994
). However, it is not
clear if it arises like small synaptic vesicles from the endosomal system, or like secretory granules from the biosynthetic pathway (Rindler, 1992
). One way to address this issue is to study the trafficking of GLUT-4 in a cell type that possesses one or more of these secretory systems. GLUT-4
has been expressed in PC12 cells, a neuroendocrine cell
line that contains two regulated secretory systems: small
synaptic vesicles, which evolve from endosomes, and
dense core granules, which are formed in the biosynthetic
pathway. In one study (Hudson et al., 1993
), a small proportion (~14%) of GLUT-4 was targeted to dense core granules with the remainder being found in T-V elements
distinct from synaptic vesicles. In contrast, Herman et al.
(1994)
did not detect GLUT-4 in dense core granules, the
majority being found in a small, vesicular compartment
distinct from synaptic vesicles or recycling endosomes.
). In the
present study we have shown that GLUT-4 is expressed at
high levels in atrial cardiomyocytes. This presented the opportunity to study the distribution of GLUT-4 in a cell
type that contains both an insulin-regulatable transport system and a regulated secretory pathway. Our data show
that a large proportion of GLUT-4 (50-60%) is targeted
to the ANF-containing secretory granules. GLUT-4 appears to enter this compartment as it recycles through the
TGR and so we suggest that this may represent an important and unique aspect of the function of this molecule.
Materials and Methods
) has been described previously. Rabbit antisera
against the NH2 and COOH termini of the pro-ANF polypeptide were
used. Since only the prohormone of ANF could be detected in the myocytes the immunolabeling pattern for both antisera was similar (Cantin et
al., 1990
). This is referred to as ANF labeling. Rabbit anti-gamma adaptin
antibodies were kindly provided by Margaret Robinson, Cambridge University, UK. Goat anti-rabbit IgG conjugated to CY3 was obtained from
Jackson ImmunoResearch Laboratories, West Grove, PA. HRP-conjugated goat anti-rabbit IgG and enhanced chemiluminescence (ECL) detection kits were from Amersham (Aylesbury, UK). Colloidal gold was
prepared by tannic acid-citrate reduction (Slot and Geuze, 1985
) and coupled to protein A (Roth et al., 1978
; Slot et al., 1988
).
), using a mixture of 2% paraformaldehyde
and 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. In
some experiments glutaraldehyde was omitted or fresh atrial tissue was
dissected and fixed by immersion. For cryosectioning (Slot et al., 1991a
) small fragments of the fixed tissue were prepared from the rim of the left
or right atrium and immersed in 10% gelatin for ~15 min at 37°C. The gelatin was then solidified on ice and small gelatin-embedded tissue blocks
were immersed in 2.3 M sucrose at 4°C overnight. Tissue blocks from the
left ventricle were processed similarly to the atrial tissue. Liver tissue
blocks were prepared without gelatin. Omission of the gelatin-embedding
step resulted in serious damage to the myocyte ultrastructure, probably
due to overstretching of the sections when they were thawed on sucrose (Liou et al., 1996
). On the other hand, this overstretching resulted in increased accessibility of the antigens as the immunolabeling efficiency was
generally higher in these sections. Thus, in some experiments the gelatin
embedding of heart tissue was omitted to take advantage of this increased
labeling efficiency. For light microscopy (LM), ~300-nm-thick cryosections were cut at
90°C, and for EM, 50-70-nm sections were cut at
120°C using an Ultracut S/FCS (Leica Inc., Vienna, Austria) equipped
with an antistatic device (Diatome, Biel, Switzerland) and a diamond
knife (Drukker, Cuyck, The Netherlands). The EM sections were immunolabeled using protein A-gold as the marker for single and double labeling (Slot et al., 1991a
). LM sections were placed on poly-l-lysine-coated glass slides and similarly immunolabeled, using CY3-conjugated goat
anti-rabbit IgG as a fluorescent marker.
80°C. Frozen tissue was thawed into ice-cold PBS containing protease
inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 250 µM phenylmethane sulphonylfluoride), washed free of blood and homogenized thoroughly in the same buffer (1 ml) first using a polytron homogenizer and
then by passage through a 22-gauge needle (Polytron; Brinkmann Instruments, Westbury, NY). Aliquots of the homogenate were removed and
solubilized for 1 h in 1% Triton X-100 (final concentration) at 4°C. The insoluble material was pelleted for 10 min in a microfuge (Sorvall, Wilmington, DE) and the supernatant retained for immunoblotting. The protein
concentration of the homogenates and the solubilized extracts was determined using the BCA reagent (Pierce Chemical Co., Rockford, IL).
). Cardiac glucose uptake was assessed 7 d after surgery. Basal studies or euglycemic hyperinsulinemic clamps were performed after a 24-h fast as
described previously (Kraegen et al., 1985
). Briefly, insulin (Actrapid-HM
Neutral; Novo Nordisk, Bagsvaerd, Denmark) was infused via the carotid
cannula, at 0.25 U/kg/h for 2 h while the blood glucose concentration was
maintained at basal fasting levels by a variable rate glucose infusion (dextrose 30 g/100 ml; Astra Pharmaceuticals, North Ryde, NSW, Australia).
).
). The NH2-terminal fragment, being cosecreted with ANF in the blood, reflects the secretory function of the atria
(Itoh et al., 1988
). The major advantage of measuring the NH2-terminal
fragment rather than ANF is that the former requires less plasma (0.1 ml vs
2.0 ml), allowing multiple samples to be obtained from the same animal.
Results
;
Thibault et al., 1989
), the localization of ANF was restricted to secretory granules which appear by LM as dots scattered throughout the cells, but primarily concentrated
near the nucleus (Fig. 1 A). The labeling pattern observed
for GLUT-4 in the atrium was more complex (Fig. 1 B).
GLUT-4 labeling, like that of ANF, occurred in large
punctate structures both in the perinuclear region and
elsewhere. However, beyond the perinuclear region very
fine granular structures were predominantly labeled for
GLUT-4. In longitudinal-sectioned myocytes these appeared arranged parallel to the cross-striation of the myofibers. In the ventricle a similar grainy labeling pattern is
evident for GLUT-4, again with clear cross arrangement
with a periodicity similar to the sarcomeres (Fig. 1 C). In
addition, some arrangement in the longitudinal direction
was apparent, the dots either following the cell surface or
the spaces between the myofibrils (Fig. 1 C). Previously,
we observed that this fine granular labeling is due to small
T-V elements that occur predominantly at the Z-line level
in cardiomyocytes (Slot et al., 1991b
). Typically, the
GLUT-4 labeling pattern in the atrium, at the level of LM,
resembled a composite image of atrial ANF labeling and
ventricular GLUT-4 labeling.
Fig. 1.
Distribution of ANF and GLUT-4 in cardiac tissues. (A) Fluorescence distribution of ANF in the right atrium. Most of the labeling was observed in small punctate dots that seemed to accumulate in a juxta-nuclear (n) position (arrowheads). (B) Distribution of
GLUT-4 in right atrium. As well as positive dots that resemble the ANF labeling pattern, GLUT-4 reactivity was also found in a fine
granular pattern that displays a periodicity in places where the cells are cut longitudinally. In the GLUT-4 staining pattern of the left ventricle (C), the fine granular staining is more predominant and clearly follows the sarcomeric periodicity. At some places it seemed to follow the plasma membrane (arrrowheads) or other longitudinal lines (arrows). Concentrations of GLUT-4 were occasionally observed
near the nucleus (n) but these were less significant than in the atrium. Bars, 10 µm. (D) Immunoblots of GLUT-4 from tissue samples of
right atrium (RA), left atrium (LA), right ventricle (RV), and left ventricle (LV). The results are from two experiments (a and b) in each
of which equivalent samples from three rats were pooled. The optical density of the GLUT-4 reaction, underneath the bands of each tissue, is given as the average of the two experiments in percentages of the value measured for the right atrium.
[View Larger Version of this Image (113K GIF file)]
;
Cantin et al., 1990
). Most of the labeling occurred in secretory granules (Fig. 2 A). Forming granules in the Golgi
complex were smaller than the secretory granules and
were labeled with similar intensity. Besides the forming
granules there was also some disperse labeling in the Golgi
cisternae. In general, ANF labeling in both of these locations was more exaggerated in tissues from animals fed ad lib. However, labeling of the Golgi complex was rather
variable between individual animals and cells. Diffuse labeling was often more obvious in the cisternae at the cis
side of the Golgi stack (Fig. 4 E).
Fig. 2.
Immuno-EM images of GLUT-4 and ANF labeling in atrial myocytes. (A) Immunogold labeling of a cryosection of the right
atrium for ANF (10 nm gold). Most gold is in the spherical secretory granules (s). Similar labeling is also associated with smaller structures (see center part of image), which are presumably forming granules. The profile of one of these (arrow) suggests its connection to
the Golgi cisternae (g). Some disperse labeling of the Golgi cisternae is present as well. (B) GLUT-4 labeling of a section taken from
right atrial tissue that was not embedded in gelatin (see Materials and Methods). Such sections became overstretched during thawing
(Liou and Slot, 1996), which resulted in considerable structural damage, but also a more efficient labeling due to better penetration conditions for the immunoreagents. GLUT-4 labeling is clear around all of the secretory granules. m, mitochondria; n, nucleus. C, as B, but
this section is from gelatin-embedded tissue. GLUT-4 labeling (10 nm gold) is clearly associated with the secretory granule membranes.
Forming granules (arrows) attached to Golgi cisternae (g) occasionally show GLUT-4 labeling (left), but this was rare (right; see also
Fig. 4). The Golgi stacks at the right are negative for GLUT-4, but GLUT-4 is clearly present at the trans side of the stacks at the left.
Bars, 200 nm.
[View Larger Version of this Image (166K GIF file)]
Fig. 4.
Immuno-EM of ANF and GLUT-4 labeling in the Golgi region of atrial myocytes. Cryosections are double labeled for GLUT-4
(10 nm) and ANF (5 nm). A and B are from overnight-fasted rats, whereas C-E are from rats fed ad lib. Disperse labeling of the Golgi
cisternae (g) for ANF is sometimes almost absent (A and B), sometimes low (C and D), and occasionally quite abundant (E). In the latter case, disperse ANF labeling seemed to be present mainly in the cis cisternae of the Golgi. The cis side of the Golgi faces the nucleus
(n). GLUT-4 labeling is present in trans-Golgi elements (arrowheads in A, C, and D). In B and more clearly in E, one or two cisternae at
the trans side of the Golgi stack are labeled for GLUT-4. Secretory granules show solid ANF and peripheral GLUT-4 labeling, but
ANF-positive forming granules (arrows), which are attached to the first (D), second (E), and third (A and C) cisternae from the cis side,
are GLUT-4 negative. f, myofibrils; z, Z-line. Bars, 200 nm.
[View Larger Version of this Image (136K GIF file)]
;
Newman and Severs, 1992
). In the cryosections these coats appeared as fuzzy layers, which were most clear in the
overstretched sections of tissue fixed without glutaraldehyde (Fig. 3). The coats were present on granules in the
perinuclear (Fig. 3 A) as well as in the peripheral (Fig. 3 B)
regions of the cell. These coats were labeled with antibodies specific for both clathrin (data not shown) and for AP1,
the gamma adaptor protein (Fig. 3 C). The density of
GLUT-4 labeling was similar within coated and noncoated
regions of the granules (Fig. 3, A and B). Apart from the
granule labeling, the distribution of GLUT-4 within the atrial myocyte was similar to that reported previously in
ventricular myocytes (Slot et al., 1991b
). Small GLUT-4-
positive T-V elements were scattered throughout the cells,
often associated with the Z-line zones. These probably
cause the cross-striated pattern in the fluorescence observations (Fig. 1, B and C). These structures, with which
~30% of the GLUT-4 labeling was associated (Table I), were morphologically indistinguishable from the GLUT4-containing T-V elements in the ventricle myocytes (Slot
et al., 1991b). Labeling of similar vesicles and tubules was
often observed at the trans side of the Golgi complex in
the atrial cell (Fig. 4, A, C, and D). These we considered as
part of the TGR.
Fig. 3.
Clathrin coats on ANF granules. Sections of atrial tissue
fixed without glutaraldehyde and not embedded in gelatin. (A and B)
Sections treated as in Fig. 2 B. Coated parts can be seen on granules
(arrows), in A, in the cell center (g, Golgi complex) and, in B, near the
cell surface (ex, extracellular space). GLUT-4 labeling (10 nm gold) is
similar in coated segments and other parts of the granule surface. (C)
Section labeled for the TGR-type adaptor protein, AP1. Coats on
ANF granules are labeled (10 nm gold) for AP1 (arrows). n, nucleus.
Bars, 200 nm.
[View Larger Version of this Image (132K GIF file)]
.
Rambourg and colleagues also focused on the biogenesis of ANF-secretory granules in the atrial myocyte. They
found forming granules attached to all, except the cis-most,
Golgi cisternae. The attachment is preferentially via tubular extensions, which explains why such connections are
rarely seen in thin sections (Fig. 2 A). We found GLUT-4
labeling in the TGR (Fig. 4, A, C, and D) and sometimes
in the trans-most cisternae of the Golgi stack (Fig. 4, B and
E) but not in the medial and cis cisternae. Forming secretory granules in the trans-Golgi were often labeled for
GLUT-4 (Fig. 2 C), but this was not the case for ANF-positive granules that were connected to the medial or cis cisternae of the Golgi stack (Fig. 4, A, C, D, and E). In nonstimulated cells there was no detectable (<1%) GLUT-4
labeling at the cell surface (Table I). The internal plasma
membrane of the transverse tubules was excluded from
the counting since these structures could not always be
recognized in the sections. However, distinct transverse tubule profiles were usually not labeled for GLUT-4.
Fig. 5.
Effects of cyclohexamide on ANF and GLUT-4 labeling. Cryosections are from rats fed ad lib. (A and B) Liver sections immunolabeled for rat serum albumin (10 nm gold). (C) Right atrium, double labeled for GLUT-4 (10 nm gold) and ANF (5 nm gold). A
is from a control rat; B and C are from a rat treated for 1 h with cyclohexamide. In normal rat liver, albumin marks the biosynthetic
secretory route, with diffuse labeling in the RER (r) and dense labeling in Golgi cisternae and secretory vesicles (v). After cyclohexamide treatment albumin is much less present in these compartments and secretory vesicles are not seen. In atrial myocytes (C) of the same rat
as in B, Golgi cisternae (g) are devoid of diffuse ANF labeling and ANF-positive forming granules are rarely seen. GLUT-4 labeling is
still associated with elements at the trans side of the Golgi stack (arrowheads) and with the trans-most cisterna (arrow) like in control animals (Fig. 4). n, nucleus. Bars, 200 nm.
[View Larger Version of this Image (154K GIF file)]
). In the present study it was of
considerable interest to examine the effects of insulin on
exocytosis of the GLUT-4/ANF-containing granules in
atrial myocytes. Insulin caused a significant increase in
GLUT-4 labeling all along the plasma membrane in atrial
cardiomyocytes. However, this effect was less than that
observed in ventricular cells. In five measurements from
longitudinally cut cells from left ventricle and right atrium
of the same animals, we observed 0.96 ± 0.03 and 0.41 ± 0.06 gold particles per micrometer of plasma membrane,
respectively. In basal myocytes of ventricle as well as
atrium, GLUT-4 labeling of the plasma membrane was at
undetectable low level (Table I).
). The basal 2DG uptake rate was
very low, consistent with the predominant intracellular
distribution of GLUT-4 observed by EM under these conditions (Table I; Slot et al., 1991b
). In addition, there was
no significant difference in 2DG uptake among the various
heart regions in basal animals. Insulin caused a substantial
increase in 2DG uptake in both atrium and ventricle.
However, quantitatively the effect was significantly lower
in the atrium than in the ventricle, once again consistent with the EM observations. Together with the observation
that GLUT-4 expression is twofold higher in atrial tissue
(Fig. 1 D), this suggests that insulin-induced translocation
of the transporter is less efficient there (we estimate five to
eight times) than in the ventricular myocyte. Indeed, initial immunocytochemical observations indicated that the
GLUT-4 labeling that could be measured at the cell surface after insulin stimulation represented no more than
2-4% of the total cellular labeling (data not shown). This
seems rather low when compared with the ~25% surface
labeling that we found previously in stimulated ventricular
myocytes, but that was in response to a maximal stimulation involving both insulin and exercise. Taking that into
account together with the five to eight times lower response of the atrial cell to insulin, one cannot expect more
than a few percent GLUT-4 labeling at the cell surface after insulin treatment. Such minor changes were too inconspicuous for establishing immunocytochemically to what
extent each of the intracellular pools (T-V elements; TGR;
ANF granules) contributed to the translocation of GLUT-4.
For that reason we did not pursue such studies in insulinstimulated atrium.
We also explored if insulin treatment had an effect on
ANF secretion which could imply a certain participation
of the secretory granule-associated GLUT-4 pool in insulin-dependent GLUT-4 translocation. However, we could
not detect a significant effect of insulin on ANF secretion
in the rat. There was no substantial difference in the
amount of the prohormone detected by immunoblotting in homogenates of atrial tissue, obtained from nonstimulated
and insulin-treated (30 min) animals (data not shown).
Furthermore, the serum concentration of NH2-terminal
ANF, which reflects the atrial secretory fraction (Itoh et
al., 1988), was measured. Severalfold raises of serum concentration of ANF have been reported after appropriate
stimuli of ANF release (Horky et al., 1985
; Manning et al.,
1985
; Lang et al., 1985
), which are mostly related to atrial
stretch the major stimulator of ANF secretion (Ruskoaho, 1992
). No detectable changes in serum levels of ANF
could be detected after insulin stimulation (Table III).
Table III. Effect of Insulin on ANF Secretion |
In muscle and adipose tissue, GLUT-4 is localized to an intracellular compartment comprising small tubules and vesicles clustered in the vicinity of the endosomal/TGR system (Slot et al., 1991a,b; Rodnick et al., 1992
; James et al.,
1994
). Despite partial overlap with recycling endosomes
and the TGR (Hanpeter and James, 1995
; Livingstone et
al., 1996
; Martin et al., 1996
) a large proportion of intracellular GLUT-4 appears to be segregated into a population of vesicles that contain the neuronal v-SNARE, VAMP-2,
and an amino peptidase, vp165 (Cain et al., 1992
; Kandror
and Pilch, 1994
; Martin et al., 1996
; Mastick et al., 1994
).
This compartment may represent a specialized intracellular storage depot possibly analogous to small synaptic vesicles (Herman et al., 1994
; Martin et al., 1996
; Verhey et al.,
1995
). How is this storage compartment formed and what
is its relationship to other regulated secretory compartments? By examining the location of GLUT-4 in an insulinregulated cell type that also contains a bona fide regulated
secretory system we hoped to address these questions.
Using immunoelectron microscopy to localize GLUT-4 in
atrial cardiac myocytes we have found that a substantial
proportion (50-60%) of the total GLUT-4 found in these
cells is targeted to ANF-containing secretory granules with the remainder localized to the TGR and T-V elements,
similar to those identified in other insulin-sensitive cells
(Slot et al., 1991a
,b).
Regulated secretory granules of the type found in atrial
myocytes are synthesized along the biosynthetic route of
the secretory pathway. In the atrial cell secretory granules
at various stages of maturation are readily detected throughout the Golgi apparatus, in the TGR and scattered throughout the cytoplasm. GLUT-4 may enter this organelle either
during the course of its own biosynthesis or via recycling,
presumably through the trans-Golgi elements. The earliest
point along the secretory pathway where we could detect
ANF labeling was in the cis and medial Golgi cisternae. At
this point ANF labeling is usually dispersed or just starting to aggregate (Fig. 4), presumably representing the early
stages of granule formation, as suggested previously
(Jamieson and Palade, 1964). The membranes around
these forming granules were not labeled for GLUT-4, but
they became GLUT-4 positive at the trans side of the
Golgi stacks and in the TGR. To determine if this was
newly synthesized GLUT-4 accumulating in the TGR we
examined the effects of protein synthesis inhibitors on
GLUT-4 targeting. Cyclohexamide caused a pronounced
inhibition of protein synthesis in atrial myocytes as determined by the complete disappearance of ANF labeling throughout the Golgi apparatus. Despite this, GLUT-4
levels in the TGR remained unaffected by this treatment.
It is possible that newly synthesized GLUT-4 is actively retained in the TGR for long periods. However, several observations indicate that TGR-derived GLUT-4 stems from
the recycling pathway rather than the biosynthetic pathway: (a) In response to acute insulin treatment in adipocytes and cardiac myocytes (Slot et al., 1991a
,b) there is a
significant decline in GLUT-4 levels in the TGR, suggesting that GLUT-4 in the TGR readily exchanges with the
cell surface and presumably endosomes. (b) After expression of GLUT-4 in fibroblasts using a viral expression system we have detected GLUT-4 labeling throughout the
biosynthetic pathway. In response to cyclohexamide treatment for 45 min, GLUT-4 labeling in the ER and Golgi
cisternae of these cells was completely depleted whereas
the level of GLUT-4 in the TGR remained constant (Piper
et al., 1992
). (c) Previous studies have shown that cyclohexamide does not alter the localization of other recycling
proteins, such as the asialoglycoprotein receptor and the
mannose 6-phosphate receptor (MPR), in the TGR (Geuze et al., 1984
) consistent with the data reported here for
GLUT-4. Collectively, these findings indicate that GLUT-4
traffics through the TGR and this explains its presence in
this organelle in all of these cell types. Thus, it is most
likely that GLUT-4 enters the ANF granules via this recycling pathway rather than the biosynthetic route.
The recycling of membrane proteins through the TGR is
quite specific as most endosomal proteins appear to avoid
this route. Using resialylation as an index of sorting through
the TGR, Duncan and Kornfeld (1988) showed that both
MPRs are resialylated with a t1/2 of ~3 h whereas other cell
surface glycoproteins acquire sialic acid at a 10-fold slower
rate. The most likely explanation for this is that a small
subset of surface glycoproteins cycle through the TGR at a
similar rate to the MPR while the remainder do not cycle
through this compartment at all. This being the case, the
fact that we observe such a large proportion of the total GLUT-4 complement within secretory granules in myocytes implies that the majority of GLUT-4 probably recycles via the TGR in these cells. Further studies using approaches similar to that used for the MPRs will be
required to test this hypothesis and to determine if a similar trafficking pathway is used by GLUT-4 in other insulin-sensitive cell types.
The recycling of GLUT-4 via the TGR may be central to
the unique character of this protein. GLUT-4 has been
shown to have a much slower exocytic rate than other recycling proteins such as the transferrin receptor (for review see James et al., 1994) and this may be in part because it follows a completely distinct trafficking pathway
through the TGR. Immuno-EM studies taught us that
GLUT-4 is concentrated in the TGR in all insulin-sensitive cells (Slot et al., 1991 a,b; James et al., 1994
) consistent
with it possibly being retained in this organelle. Also it is
conceivable that the intracellular GLUT-4 storage depot
(Herman et al., 1994
; Martin et al., 1996
; Verhey et al.,
1995
) may form at the level of the TGR rather than endosomes. This may explain the significant colocalization between GLUT-4 and MPR in insulin-sensitive cell types
(Hanpeter and James, 1995
; Martin et al., 1996
). In addition, we have recently shown that there is significant colocalization between AP1, the Golgi-specific adaptor protein, and GLUT-4 in vesicles isolated from adipocytes
(Martin, S., and D. James, unpublished data). Furthermore, insulin stimulates the efflux of a number of TGR recycling proteins as well as constitutive secretory proteins such as adipsin (for review see Lienhard, 1989
). It has
been reported that the TGR-specific protein TGN38 does
not colocalize with GLUT-4 in adipocytes (Martin et al.,
1994
). This may reflect the heterogeneous composition of
the TGR as shown recently (Glickman et al., 1996
). In our
present study we could not confirm this, being unsuccessful in detecting TGN38 in the sections of the atrial cells.
Despite the targeting of GLUT-4 to the ANF-containing secretory granules there was also a significant amount
in T-V elements that were morphologically indistinguishable from those found in other cell types. Hence, it is likely
that if both organelles bud from the TGR they do so via
different sorting mechanisms. The presence of GLUT-4 in
ANF granules does not appear to reflect a default pathway
in these cells because vp165, which colocalizes with GLUT-4
in adipocytes, is not targeted to the ANF granules in the
atrial cells, but colocalizes there with GLUT-4 in the T-V
elements (Martin et al., 1997). Furthermore, it has been
reported that GLUT-4 but not GLUT-1 is targeted to
secretory granules in PC12 cells (Hudson et al., 1993
).
Thus, this implies the presence of a specific sorting signal
in GLUT-4 that targets it for entry into this organelle.
The targeting of GLUT-4 into regulated secretory granules appears to be cell specific. In PC12 cells or insulinoma
cells transfected with the GLUT-4 cDNA, relatively low
levels (Hudson et al., 1993), or no GLUT-4 (Herman et al.,
1994
; Thorens and Roth, 1996
), were detected in the secretory compartment. In renal arteriolar cells of the juxta
glomerular apparatus, where GLUT-4 is also expressed endogenously, there is very little GLUT-4 labeling of the
renin-containing secretory granules, most being found in
T-V elements (Anderson, T.J., S. Martin, D.E. James, J.W.
Slot, J.L. Berka, and J. Stow, manuscript submitted for
publication). However, similar differences have been reported for other secretory proteins. For instance, in the case
of P-selectin, which is very efficiently targeted to
-granules
in platelets (Stenberg et al., 1985
), only a small proportion
is found in secretory granules in AtT-20 cells (Koedam et al., 1992
). Such differences likely reflect differences between individual cell types in GLUT-4 expression, the rate
of granule formation and their average lifetime before secretion, or the rate of GLUT-4 TGR recycling. The atrial
cell is in some respects an atypical secretory cell because
granule formation begins at a very early stage of the biosynthetic pathway (Fig. 4, and Rambourg et al., 1984
) and
there is not much indication of further maturation of the
granule after its exit from the TGR. For example, many ANF granules appear to retain the AP1-specific coat in
the atrial myocyte (Fig. 3 C) until secretion (Newman and
Severs, 1992
). This is quite different from other endocrine
cells where granules begin to form in the TGR and continue to mature after budding from the TGR (Arvan and
Castle, 1992
; Rindler, 1992
; Tooze, 1991
), during which
process they tend to lose clathrin coats (Dittié et al., 1996
).
Thus, it is possible that GLUT-4 is efficiently targeted to
the regulated secretory pathway in all cases but is transported out of the secretory granules during their postTGR maturation to different extent in individual cell
types. Therefore, the proportion of GLUT-4 that is found
in the secretory granule compartment in a particular cell
will reflect a balance of each of these parameters which in
all likelihood vary considerably from one cell to another.
Does insulin stimulate the movement of GLUT-4 from
ANF-containing secretory granules to the cell surface in
atrial myocytes? Answers to this question may provide insight into the physiological basis for the targeting of GLUT-4
to this organelle, which at present remains unknown. In
theory GLUT-4 could move from the granules to the cell
surface in two ways. First, as the granules fuse with the cell
surface during secretion the granule membrane protein
cargo presumably inserts into the surface membrane.
However, as described above we have been unable to detect an effect of insulin on ANF secretion and therefore
regulation of this route by insulin seems unlikely. The second possibility is that small GLUT-4-containing vesicles
may bud from the ANF granules. This would probably result in GLUT-4 translocation without substantial amounts
of ANF secretion. Such budding via coated vesicles commonly occurs from immature secretory granules in other
endocrine cell types. The presence of AP1-positive clathrin coats on the ANF granules, with some decreasing frequency during their lifetime (Newman and Severs, 1992),
indicates that there may be a route for GLUT-4 out of the
granules by budding. It remains to be seen if this is regulated by insulin. The insulin effects on GLUT-4 distribution were too small to measure shifts of GLUT-4 labeling either from the ANF granules or from other cellular pools
(T-V elements; TGR). Therefore we cannot yet determine
the precise intracellular origin of the GLUT-4 that is
translocated to the cell surface by insulin in this cell. However, the involvement of the ANF granules via these budding vesicles would place them functionally in line with the
TGR, as is suggested for the regulated secretory compartment at an immature stage in other cell types (Arvan and
Castle, 1992
).
Received for publication 16 August 1996 and in revised form 27 March 1997.
1. Abbreviations used in this paper: ANF, atrial natriuretic factor; LM, light microscopy; MPR, mannose 6-phosphate receptor; 2DG, 2-deoxyglucose; TGR, trans-Golgi reticulum; T-V, tubulo-vesicular.We thank Jenny Stow, Rob Parton, Hans Geuze, and Peter Peters for helpful discussions and for reading the manuscript, and Russel Wilson for his contribution to the practical part of this study.
This work was supported by grants from the National Health and Medical Research Council of Australia and the Juvenile Diabetes Foundation International. D.E. James is a Wellcome Trust Professorial Research Fellow.
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