Localization of low density lipoprotein receptor-related protein 1 to caveolae in 3T3-L1 adipocytes in response to insulin treatment

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitope-tagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.     

Glut-4 is translocated to both caveolae and non-caveolar lipid rafts, but is partially internalized through caveolae in insulin-stimulated adipocytes

Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.     

Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specific proteins and functions

Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.     

A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization

Cavin (PTRF) has been shown to be a highly abundant protein component of caveolae, but its functional role there is unknown. Here, we confirm that cavin co-localizes with caveolin-1 in adipocytes by confocal microscopy and co-distributes with caveolin-1 in lipid raft fractions by sucrose gradient flotation. However, cavin does not directly associate with caveolin-1 as solubilization of caveolae disrupts their interaction. Cholesterol depletion with beta-cyclodextrin causes a significant down-regulation of cavin from plasma membrane lipid raft fractions. Overexpression of cavin in HEK293-Cav-1 cells and knockdown of cavin in 3T3-L1 adipocytes enhances and diminishes caveolin-1 levels, respectively, indicating an important role for cavin in maintaining the level of caveolin-1. A truncated form of cavin, eGFP-cavin-1-322, which lacks 74 amino acids from the C-terminal, reveals a microtubular network localization by confocal microscopy. Disruption of cytoskeletal elements with latrunculin B or nocodazole diminishes cavin expression without affecting the caveolin-1 amount. We propose that the presence of cavin on the inside surface of caveolae stabilizes these structures, probably through interaction with the cytoskeleton, and cavin therefore plays an important role in caveolae formation and organization.     

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.     

Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocytes

Phosphodiesterase 3B (PDE3B), is known to play an important role in acute insulin and cAMP-mediated regulation of lipid metabolism, and PDE4 are the main PDE types expressed in adipocytes. Here, we show that members of all PDE4 isoforms are expressed in 3T3-L1 and primary mouse adipocytes. Long-term treatment of 3T3-L1 adipocytes with insulin induced up-regulation of PDE3B and PDE4D in a phosphatidylinositol 3-kinase-dependent manner whereas long-term treatment with beta-adrenergic agonists induced down-regulation of PDE3B and up-regulation of PDE4D. Thus, PDE3B and PDE4D can be added to the list of genes regulated by insulin and cAMP-increasing hormones. Altered expression of PDE3B and PDE4D in response to long-term treatment with insulin and catecholamines may contribute to altered regulation of metabolism in diabetes.     

Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes

In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.     

Targeting of PKCzeta and PKB to caveolin-enriched microdomains represents a crucial step underpinning the disruption in PKB-directed signalling by ceramide

Elevated ceramide concentrations in adipocytes and skeletal muscle impair PKB (protein kinase B; also known as Akt)-directed insulin signalling to key hormonal end points. An important feature of this inhibition involves the ceramide-induced activation of atypical PKCzeta (protein kinase C-zeta), which associates with and negatively regulates PKB. In the present study, we demonstrate that this inhibition is critically dependent on the targeting and subsequent retention of PKCzeta-PKB within CEM (caveolin-enriched microdomains), which is facilitated by kinase interactions with caveolin. Ceramide also recruits PTEN (phosphatase and tensin homologue detected on chromosome 10), a 3'-phosphoinositide phosphatase, thereby creating a repressive membrane microenvironment from which PKB cannot signal. Disrupting the structural integrity of caveolae by cholesterol depletion prevented caveolar targeting of PKCzeta and PKB and suppressed kinase-caveolin association, but, importantly, also ameliorated ceramide-induced inhibition of PKB. Consistent with this, adipocytes from caveolin-1-/- mice, which lack functional caveolae, exhibit greater resistance to ceramide compared with caveolin-1+/+ adipocytes. We conclude that the recruitment and retention of PKB within CEM contribute significantly to ceramide-induced inhibition of PKB-directed signalling.     

Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B

Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.     

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.     

Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor

Phosphatidylinositol 3-kinase mediates several actions of insulin including its antilipolytic effect. This effect is elicited by the insulin-stimulated serine phosphorylation and activation of cGMP-inhibited phosphodiesterase (PDE3B). In human adipocytes, we found that insulin differentially stimulated phosphatidylinositol 3-kinase activity; the lipid kinase activity was associated with IRS-1, whereas the serine kinase activity was associated with the insulin receptor and phosphorylated a number of proteins including p85, p110, and a 135-kDa protein identified as PDE3B. PDE3B phosphorylation was associated with enzyme activation, thus initiating the antilipolytic effect of insulin. These results show a novel pathway for intracellular signaling through the insulin receptor leading to the serine phosphorylation of key proteins involved in insulin action.     

Tumour necrosis factor alpha-induced adipose-related protein (TIARP): co-localization with caveolin-1

We previously identified TIARP (TNF(alpha)-induced adipose-related protein, where TNF(alpha) stands for tumour necrosis factor alpha), a novel plasma-membrane protein that is induced during 3T3-L1 preadipocytes differentiation by TNF(alpha). Whereas the biological function of TIARP is currently unknown, its protein sequence is reminiscent of transporter protein and/or NAD(P)/NAD(P)H-dependent oxidoreductase activities. We hypothesized that TIARP could be associated with the 3T3-L1 adipocyte plasma-membrane caveolae domains that contain many proteins involved in cellular trafficking and signalling processes. Studies by confocal microscopy showed that TIARP and caveolin-1, a major protein of caveolae, co-localized as patches at the plasma membrane. Immunoblot analysis of cell extracts indicated that TIARP was completely detergent-extractible from membranes, whereas caveolin-1 was present as both detergent-extractible and -insoluble pools. Since TIARP is compartmentalized with caveolin-1 within caveolae domains, we suggest this protein to be part of a signalling complex in association with caveolin-1 and regulatory proteins.     

Cholesterol depletion inhibits fatty acid uptake without affecting CD36 or caveolin-1 distribution in adipocytes

Caveolin-1 and CD36 are plasma membrane fatty acid binding proteins that participate in adipocyte fatty acid uptake and metabolism. Both are associated with cholesterol-enriched caveolae/lipid rafts in the plasma membrane that are important for long chain fatty acid uptake. Depletion of plasma membrane cholesterol reversibly inhibited oleate uptake by adipocytes without altering the amount or the cell surface distribution of either caveolin-1 or CD36. Cholesterol levels thus regulate fatty acid uptake by adipocytes via a pathway that does not involve altered cell surface localization of caveolin-1 or CD36.     

Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background

The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.

Methodology/Principal Findings

We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t_1/2<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.

Conclusion

It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.     

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.     

Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocytes

In adipocytes, phosphorylation and activation of PDE3B is a key event in the antilipolytic action of insulin. The role of PDE4, another PDE present in adipocytes, is not yet known. In this work we investigate the role of PDE3B and PDE4 in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis. Inhibition of PDE3 (OPC3911, milrinone) but not PDE4 (RO 20-1724) lowered insulin-induced glucose uptake and lipogenesis, especially in the presence of isoproterenol (a general beta-adrenergic agonist), CL316243, a selective beta3-adrenergic agonist, and pituitary adenylate cyclase-activating peptide. The inhibitory effect of OPC3911 was associated with reduced translocation of GLUT-4 from the cytosol to the plasma membrane. Both OPC3911 and RO 20-1724 increased protein kinase A (PKA) activity and lipolysis. H89, a PKA inhibitor, did not affect OPC3911-mediated inhibition of insulin-induced glucose uptake and lipogenesis, whereas 8-pCPT-2'-O-Me-cAMP, an Epac agonist which mediates PKA independent cAMP signaling events, mimicked all the effects of OPC3911. Insulin-mediated activation of protein kinase B, a kinase involved in insulin-induced glucose uptake, was apparently not altered by OPC3911. In summary, our data suggest that PDE3B, but not PDE4, contributes to the regulation of insulin-induced glucose uptake, GLUT-4 translocation, and lipogenesis presumably by regulation of a cAMP/Epac signalling mechanisms.     

Regulated interaction of endothelin B receptor with caveolin-1.

The peptide hormone endothelin transmits various signals through G protein-coupled receptors, the endothelin type A (ETAR) and B (ETBR) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ETBR with caveolin-1, expressed in Sf9, COS-1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ETBR. ETBR formed a complex with caveolin-1 in cells in which these two proteins were coexpressed and in the mixture after purification and reconstitution (as examined by immunoprecipitation) suggesting the direct binding of ETBR with caveolin-1. The complex formed efficiently only when the ETBR was ligand-free or bound to an antagonist, RES-701-1, whereas the addition of ET-1 or another antagonist, BQ788, dissociated the complex, suggesting the structural recognition of ETBR by caveolin-1. In contrast, the ETAR bound to caveolin-1 regardless of ligand binding. Caveolin-1 utilized its scaffolding domain (residues 82-101) and the C-terminal domain (residues 136-178) to bind to ETBR, as for other signalling molecules. Furthermore, the amount of ETBR localized in caveolae increased significantly with the expression of caveolin-1 and decreased with the addition of ET-1. The disruption of caveolae by filipin reduced the ET-1-derived phosphorylation of ERK1/2. These results suggest the possibility that the binding to caveolin-1 retains the ligand-free ETBR in caveolae and regulates the ET signal.     

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.