Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae

Abstract

Caveolin-2 is a member of the caveolin gene family with no known function. Although caveolin-2 is coexpressed and heterooligomerizes with caveolin-1 in many cell types (most notably adipocytes and endothelial cells), caveolin-2 has traditionally been considered the dispensable structural partner of the widely studied caveolin-1. We now directly address the functional significance of caveolin-2 by genetically targeting the caveolin-2 locus (Cav-2) in mice. In the absence of caveolin-2 protein expression, caveolae still form and caveolin-1 maintains its localization in plasma membrane caveolae, although in certain tissues caveolin-1 is partially destabilized and shows modestly diminished protein levels. Despite an intact caveolar membrane system, the Cav-2-null lung parenchyma shows hypercellularity, with thickened alveolar septa and an increase in the number of endothelial cells. As a result of these pathological changes, these Cav-2-null mice are markedly exercise intolerant. Interestingly, these Cav-2-null phenotypes are identical to the ones we and others have recently reported for Cav-1-null mice. As caveolin-2 expression is also severely reduced in Cav-1-null mice, we conclude that caveolin-2 deficiency is the clear culprit in this lung disorder. Our analysis of several different phenotypes observed in caveolin-1-deficient mice (i.e., abnormal vascular responses and altered lipid homeostasis) reveals that Cav-2-null mice do not show any of these other phenotypes, indicating a selective role for caveolin-2 in lung function. Taken together, our data show for the first time a specific role for caveolin-2 in mammalian physiology independent of caveolin-1.

FIG. 1.

Targeted disruption of the caveolin-2 gene produces a null mutation. (A) The caveolin-2 locus (containing the first two exons) and the targeting construct (containing the neomycin resistance cassette [NEO] with flanking segments homologous to the locus) are shown in schematic format in this ≈13-kb map. The transcriptional orientations of the neomycin resistance cassette and the caveolin-2 locus are delineated by arrows. Note that homologous recombination would eliminate a 2.5-kb genomic segment containing caveolin-2 exons 1 and 2 and introduce a new restriction site (SpeI), which was used to screen for positive ES cell clones. The 600-bp XbaI-PstI probe used for Southern blot analysis is located 5′ of the targeting vector, as shown. (B) Southern blot analysis of SpeI-digested tail DNA from offspring of caveolin-2 heterozygote (Het) intercrosses. The ≈8-kb band signifiesthe appropriate targeted disruption of the caveolin-2 locus. The absence of a wild-type (WT) ≈10.0-kb band signifies the generation of the Cav-2-knockout (KO) animal. An alternative PCR-based strategy used to determine the genotype of animals is also shown. The absence of a ≈500-bp wild-type band signifies the generation of a Cav-2-knockout animal. (C) Lysates from three tissue types with various levels of caveolin-2 expression (fat, lung, and heart) were prepared for mice of all three genotypes. Protein (30 μg) was loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-2 MAb (clone 26). A longer exposure of the same blot is also shown. Equal protein loading was assessed by using the anti-β-tubulin MAb (clone 2.1).

FIG. 2.

Caveolae are not disrupted in caveolin-2-deficient mice. Lung and perigonadal adipose tissue were processed from wild-type and Cav-2-null mice for transmission electron microscopy (EM), as detailed in the text. Analyses were performed at 30,000× and 16,000× magnifications for lungs and adipose tissue, respectively (for ease of view, the images shown were magnified further). The plasma membranes of numerous cells were scanned for caveolae, defined as uniform 50- to 100-nm flask-shaped membrane invaginations. The scale bar is shown at the lower left corner. RBC, red blood cell.

FIG. 2.

Caveolae are not disrupted in caveolin-2-deficient mice. Lung and perigonadal adipose tissue were processed from wild-type and Cav-2-null mice for transmission electron microscopy (EM), as detailed in the text. Analyses were performed at 30,000× and 16,000× magnifications for lungs and adipose tissue, respectively (for ease of view, the images shown were magnified further). The plasma membranes of numerous cells were scanned for caveolae, defined as uniform 50- to 100-nm flask-shaped membrane invaginations. The scale bar is shown at the lower left corner. RBC, red blood cell.

FIG. 3.

Caveolin-1 expression is mildly reduced but caveolin-1 remains localized to plasma membrane caveolae in Cav-2-null mice. (A) Lysates (30 μg) from the tissues shown in Fig. 1C were loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-3 MAb (clone 65). A longer exposure of the same blot is also shown. Both caveolin-1 and caveolin-3 are expressed in Cav-2-knockout (KO) mice; however, caveolin-1 expression shows a mild reduction in some tissues (≈50% of wild-type levels in lung and heart). Equal protein loading was assessed using the anti-β-tubulin MAb (clone 2.1). (B) Lung tissue from wild-type (WT) and Cav-2-knockout mice was homogenized thoroughly in lysis buffer containing 1% Triton X-100 and subjected to sucrose gradient centrifugation, a procedure that separates caveolar microdomains from other cellular constituents (23). Twelve fractions, of which fractions 4 to 5 and 8 to 12 are considered of caveolar and noncaveolar origin, respectively, were collected and subjected to SDS-PAGE. Immunoblotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297) was used to determine the localization of caveolin-2 and caveolin-1 in these gradient fractions. Arrows point at the caveola-enriched gradient fractions (4 and 5). (C) Formaldehyde-fixed wild-type and Cav-2-knockout MEFs were doubly immunostained with anti-caveolin-1 PAb (N20) and anti-caveolin-2 MAb (clone 26). Bound primary antibodies were visualized with distinctly tagged secondary antibodies (see text). (D) Lung tissue samples from wild-type and Cav-2-knockout mice were homogenized thoroughly in lysis buffer containing 60 mM octylglucoside and subjected to velocity gradient centrifugation, an established procedure that allows the assessment of the size of caveolin oligomers (40, 42, 44, 49). Briefly, solubilized material was loaded atop a 5 to 40% sucrose density gradient and subjected to centrifugation for 10 h. Twelve fractions were recovered, and a 20-μl aliquot from each fraction was analyzed by SDS-PAGE and Western blotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297). Arrows mark the positions of molecular mass standards. Note that caveolin-1 migrates as a high-molecular-mass oligomer of ≈200 to 400 kDa (see fractions 6 and 7) in both wild-type and Cav-2-knockout lung tissue samples. Thus, caveolin-2 expression is not required for the formation of high-molecular-mass caveolin-1 oligomers. Upper panels, caveolin-2 immunoblots; lower panels, caveolin-1 immunoblots.

FIG. 3.

Caveolin-1 expression is mildly reduced but caveolin-1 remains localized to plasma membrane caveolae in Cav-2-null mice. (A) Lysates (30 μg) from the tissues shown in Fig. 1C were loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-3 MAb (clone 65). A longer exposure of the same blot is also shown. Both caveolin-1 and caveolin-3 are expressed in Cav-2-knockout (KO) mice; however, caveolin-1 expression shows a mild reduction in some tissues (≈50% of wild-type levels in lung and heart). Equal protein loading was assessed using the anti-β-tubulin MAb (clone 2.1). (B) Lung tissue from wild-type (WT) and Cav-2-knockout mice was homogenized thoroughly in lysis buffer containing 1% Triton X-100 and subjected to sucrose gradient centrifugation, a procedure that separates caveolar microdomains from other cellular constituents (23). Twelve fractions, of which fractions 4 to 5 and 8 to 12 are considered of caveolar and noncaveolar origin, respectively, were collected and subjected to SDS-PAGE. Immunoblotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297) was used to determine the localization of caveolin-2 and caveolin-1 in these gradient fractions. Arrows point at the caveola-enriched gradient fractions (4 and 5). (C) Formaldehyde-fixed wild-type and Cav-2-knockout MEFs were doubly immunostained with anti-caveolin-1 PAb (N20) and anti-caveolin-2 MAb (clone 26). Bound primary antibodies were visualized with distinctly tagged secondary antibodies (see text). (D) Lung tissue samples from wild-type and Cav-2-knockout mice were homogenized thoroughly in lysis buffer containing 60 mM octylglucoside and subjected to velocity gradient centrifugation, an established procedure that allows the assessment of the size of caveolin oligomers (40, 42, 44, 49). Briefly, solubilized material was loaded atop a 5 to 40% sucrose density gradient and subjected to centrifugation for 10 h. Twelve fractions were recovered, and a 20-μl aliquot from each fraction was analyzed by SDS-PAGE and Western blotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297). Arrows mark the positions of molecular mass standards. Note that caveolin-1 migrates as a high-molecular-mass oligomer of ≈200 to 400 kDa (see fractions 6 and 7) in both wild-type and Cav-2-knockout lung tissue samples. Thus, caveolin-2 expression is not required for the formation of high-molecular-mass caveolin-1 oligomers. Upper panels, caveolin-2 immunoblots; lower panels, caveolin-1 immunoblots.

FIG. 3.

Caveolin-1 expression is mildly reduced but caveolin-1 remains localized to plasma membrane caveolae in Cav-2-null mice. (A) Lysates (30 μg) from the tissues shown in Fig. 1C were loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-3 MAb (clone 65). A longer exposure of the same blot is also shown. Both caveolin-1 and caveolin-3 are expressed in Cav-2-knockout (KO) mice; however, caveolin-1 expression shows a mild reduction in some tissues (≈50% of wild-type levels in lung and heart). Equal protein loading was assessed using the anti-β-tubulin MAb (clone 2.1). (B) Lung tissue from wild-type (WT) and Cav-2-knockout mice was homogenized thoroughly in lysis buffer containing 1% Triton X-100 and subjected to sucrose gradient centrifugation, a procedure that separates caveolar microdomains from other cellular constituents (23). Twelve fractions, of which fractions 4 to 5 and 8 to 12 are considered of caveolar and noncaveolar origin, respectively, were collected and subjected to SDS-PAGE. Immunoblotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297) was used to determine the localization of caveolin-2 and caveolin-1 in these gradient fractions. Arrows point at the caveola-enriched gradient fractions (4 and 5). (C) Formaldehyde-fixed wild-type and Cav-2-knockout MEFs were doubly immunostained with anti-caveolin-1 PAb (N20) and anti-caveolin-2 MAb (clone 26). Bound primary antibodies were visualized with distinctly tagged secondary antibodies (see text). (D) Lung tissue samples from wild-type and Cav-2-knockout mice were homogenized thoroughly in lysis buffer containing 60 mM octylglucoside and subjected to velocity gradient centrifugation, an established procedure that allows the assessment of the size of caveolin oligomers (40, 42, 44, 49). Briefly, solubilized material was loaded atop a 5 to 40% sucrose density gradient and subjected to centrifugation for 10 h. Twelve fractions were recovered, and a 20-μl aliquot from each fraction was analyzed by SDS-PAGE and Western blotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297). Arrows mark the positions of molecular mass standards. Note that caveolin-1 migrates as a high-molecular-mass oligomer of ≈200 to 400 kDa (see fractions 6 and 7) in both wild-type and Cav-2-knockout lung tissue samples. Thus, caveolin-2 expression is not required for the formation of high-molecular-mass caveolin-1 oligomers. Upper panels, caveolin-2 immunoblots; lower panels, caveolin-1 immunoblots.

FIG. 3.

Caveolin-1 expression is mildly reduced but caveolin-1 remains localized to plasma membrane caveolae in Cav-2-null mice. (A) Lysates (30 μg) from the tissues shown in Fig. 1C were loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-3 MAb (clone 65). A longer exposure of the same blot is also shown. Both caveolin-1 and caveolin-3 are expressed in Cav-2-knockout (KO) mice; however, caveolin-1 expression shows a mild reduction in some tissues (≈50% of wild-type levels in lung and heart). Equal protein loading was assessed using the anti-β-tubulin MAb (clone 2.1). (B) Lung tissue from wild-type (WT) and Cav-2-knockout mice was homogenized thoroughly in lysis buffer containing 1% Triton X-100 and subjected to sucrose gradient centrifugation, a procedure that separates caveolar microdomains from other cellular constituents (23). Twelve fractions, of which fractions 4 to 5 and 8 to 12 are considered of caveolar and noncaveolar origin, respectively, were collected and subjected to SDS-PAGE. Immunoblotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297) was used to determine the localization of caveolin-2 and caveolin-1 in these gradient fractions. Arrows point at the caveola-enriched gradient fractions (4 and 5). (C) Formaldehyde-fixed wild-type and Cav-2-knockout MEFs were doubly immunostained with anti-caveolin-1 PAb (N20) and anti-caveolin-2 MAb (clone 26). Bound primary antibodies were visualized with distinctly tagged secondary antibodies (see text). (D) Lung tissue samples from wild-type and Cav-2-knockout mice were homogenized thoroughly in lysis buffer containing 60 mM octylglucoside and subjected to velocity gradient centrifugation, an established procedure that allows the assessment of the size of caveolin oligomers (40, 42, 44, 49). Briefly, solubilized material was loaded atop a 5 to 40% sucrose density gradient and subjected to centrifugation for 10 h. Twelve fractions were recovered, and a 20-μl aliquot from each fraction was analyzed by SDS-PAGE and Western blotting with anti-caveolin-2 MAb (clone 26) and anti-caveolin-1 MAb (clone 2297). Arrows mark the positions of molecular mass standards. Note that caveolin-1 migrates as a high-molecular-mass oligomer of ≈200 to 400 kDa (see fractions 6 and 7) in both wild-type and Cav-2-knockout lung tissue samples. Thus, caveolin-2 expression is not required for the formation of high-molecular-mass caveolin-1 oligomers. Upper panels, caveolin-2 immunoblots; lower panels, caveolin-1 immunoblots.

FIG. 4.

Caveolin-2-deficient mice show lung abnormalities identical to those of caveolin-1-deficient mice. (A) Routine histology (H&E) was performed on lung tissue from mice of four different genotypes, wild type (WT), Cav-2-knockout (KO), Cav-1-knockout, and caveolin-1 heterozygote (Het), and examined using a Zeiss Axiophot with a 40× objective. (B) Lysates from lung tissue were prepared from two mice of each of the four different genotypes (wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote). A total of 30 μg of protein was loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-2 MAb (clone 26). Note that caveolin-1 heterozygote mice and Cav-2-null mice show the same levels of caveolin-1 protein expression (asterisks). (C) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections stained with H&E were examined using a Zeiss Axiophot. Via the 40× objective, five random fields were photographed for each genotype, and all the nuclei within those regions were manually tabulated with a hand-held counter. (D) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections were subjected to reticulin staining (a major component of basement membranes) and examined using a Zeiss Axiophot with a 40× objective. (E) Lung paraffin sections from wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mice were immunostained with an endothelial marker, anti-VEGF-R (Flk-1) IgG. Bound primary antibodies were detected with a fluorescently labeled secondary antibody.

FIG. 4.

Caveolin-2-deficient mice show lung abnormalities identical to those of caveolin-1-deficient mice. (A) Routine histology (H&E) was performed on lung tissue from mice of four different genotypes, wild type (WT), Cav-2-knockout (KO), Cav-1-knockout, and caveolin-1 heterozygote (Het), and examined using a Zeiss Axiophot with a 40× objective. (B) Lysates from lung tissue were prepared from two mice of each of the four different genotypes (wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote). A total of 30 μg of protein was loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-2 MAb (clone 26). Note that caveolin-1 heterozygote mice and Cav-2-null mice show the same levels of caveolin-1 protein expression (asterisks). (C) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections stained with H&E were examined using a Zeiss Axiophot. Via the 40× objective, five random fields were photographed for each genotype, and all the nuclei within those regions were manually tabulated with a hand-held counter. (D) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections were subjected to reticulin staining (a major component of basement membranes) and examined using a Zeiss Axiophot with a 40× objective. (E) Lung paraffin sections from wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mice were immunostained with an endothelial marker, anti-VEGF-R (Flk-1) IgG. Bound primary antibodies were detected with a fluorescently labeled secondary antibody.

FIG. 4.

Caveolin-2-deficient mice show lung abnormalities identical to those of caveolin-1-deficient mice. (A) Routine histology (H&E) was performed on lung tissue from mice of four different genotypes, wild type (WT), Cav-2-knockout (KO), Cav-1-knockout, and caveolin-1 heterozygote (Het), and examined using a Zeiss Axiophot with a 40× objective. (B) Lysates from lung tissue were prepared from two mice of each of the four different genotypes (wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote). A total of 30 μg of protein was loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-2 MAb (clone 26). Note that caveolin-1 heterozygote mice and Cav-2-null mice show the same levels of caveolin-1 protein expression (asterisks). (C) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections stained with H&E were examined using a Zeiss Axiophot. Via the 40× objective, five random fields were photographed for each genotype, and all the nuclei within those regions were manually tabulated with a hand-held counter. (D) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections were subjected to reticulin staining (a major component of basement membranes) and examined using a Zeiss Axiophot with a 40× objective. (E) Lung paraffin sections from wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mice were immunostained with an endothelial marker, anti-VEGF-R (Flk-1) IgG. Bound primary antibodies were detected with a fluorescently labeled secondary antibody.

FIG. 4.

Caveolin-2-deficient mice show lung abnormalities identical to those of caveolin-1-deficient mice. (A) Routine histology (H&E) was performed on lung tissue from mice of four different genotypes, wild type (WT), Cav-2-knockout (KO), Cav-1-knockout, and caveolin-1 heterozygote (Het), and examined using a Zeiss Axiophot with a 40× objective. (B) Lysates from lung tissue were prepared from two mice of each of the four different genotypes (wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote). A total of 30 μg of protein was loaded in each lane, subjected to SDS-PAGE, and immunoblotted with anti-caveolin-1 MAb (clone 2297) and anti-caveolin-2 MAb (clone 26). Note that caveolin-1 heterozygote mice and Cav-2-null mice show the same levels of caveolin-1 protein expression (asterisks). (C) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections stained with H&E were examined using a Zeiss Axiophot. Via the 40× objective, five random fields were photographed for each genotype, and all the nuclei within those regions were manually tabulated with a hand-held counter. (D) Wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mouse lung tissue sections were subjected to reticulin staining (a major component of basement membranes) and examined using a Zeiss Axiophot with a 40× objective. (E) Lung paraffin sections from wild-type, Cav-2-null, Cav-1-null, and caveolin-1 heterozygote mice were immunostained with an endothelial marker, anti-VEGF-R (Flk-1) IgG. Bound primary antibodies were detected with a fluorescently labeled secondary antibody.

FIG. 5.

Caveolin-2-deficient mice do not show abnormal nitric oxide-dependent modulation of aortic contraction. (A) Representative traces of wild-type, Cav-2-knockout (KO), and Cav-1-knockout concentration-dependent acetylcholine (Ach, 10⁻⁸ to 10⁻⁴)-induced relaxation, followed by addition of l-NAME (100 μM), which induced further contraction of the mouse aorta. Note the appearance of spontaneous oscillatory contractions in the acetylcholine dose-response curve in aortic rings from the wild-type and Cav-2-knockout mice, and their absence from tracings obtained on aortic rings from the Cav-1-null mouse. As such, for comparative purposes, the percent relaxation (B) was calculated from the steady-state trough of relaxation observed for each acetylcholine concentration. The arrows indicate the times of addition of increasing amounts of acetylcholine (10⁻⁸, 3 × 10⁻⁸, 10⁻⁷, 3 × 10⁻⁷, 10⁻⁶, 3 × 10⁻⁶, 10⁻⁵, 3 × 10⁻⁵, and 10⁻⁴ M). PE, phenylephrine. (B) Concentration-dependent relaxation induced by acetylcholine (Ach) in aortas constricted with 10 μM phenylephrine from wild-type (⧫), Cav-1-knockout (KO) (▪), and Cav-2-knockout (□) mice. Points represent mean ± standard error of the mean for 11 (wild-type), 6 (Cav-1-knockout), and 7 (Cav-2-knockout) rings from three mice each. ∗∗∗, P < 0.0001, Cav-1 knockout versus wild type. +++, P < 0.0001, Cav-1 knockout versus Cav-2 knockout. (C) l-NAME (100 μM) modulation of phenylephrine (PE)-induced contraction in mouse aorta from wild-type, Cav-1-null, and Cav-2-null mice. Points represent the mean ± standard error of the mean for 11 (wild-type), 6 (Cav-1-knockout), and 7 (Cav-2-knockout) rings from three mice each. ∗, P < 0.05, and ∗∗∗, P < 0.0001 versus wild-type control with no l-NAME treatment, two-way analysis of variance for repeated measures.

FIG. 6.

Caveolin-2-deficient mice exhibit inadequate exercise capacity. Weight-matched and age-matched (4.5 months) male wild-type (n = 8), Cav-2-knockout (KO) (n = 5), and Cav-1-knockout (n = 5) mice were subjected to a swimming test (see Materials and Methods). Note that wild-type animals were able to swim for up to 40 min (gauged as the point of exhaustion), while both caveolin-1- and caveolin-2-deficient animals swam for only approximately 8 to 12 min (a ≈3- to 4-fold change). Thus, both caveolin-1- and caveolin-2-deficient mice clearly show exercise intolerance, as would be predicted from their similar morphological lung abnormalities.

FIG. 7.

Cav-2-null mice have normal body weights, adipose tissue morphology, and serum metabolites. (A) Routine histology (H&E) was performed on several different adipose tissue regions (perigonadal fat pads are shown) of 1-year-old wild-type and Cav-2-null mice, an age at which the adipose tissue of Cav-1-null mice has been shown to display heterogeneously sized adipocytes, marked interstitial fibrosis, and hypercellularity. No differences between wild type and Cav-2 knockouts (KO) are apparent. (B) The entire left-side mammary gland 4 of wild-type, Cav-2-knockout, and Cav-1-knockout females was dissected and subjected to whole-mount preparation. Carmine dye staining of the mammary tissue allowed visualization of the ductal architecture and density. (C) Fasting blood samples were collected at 7:00 a.m. (12 h after removal of food) and postprandial blood was collected at 12:00 a.m. (after 3 h of feeding in the dark) from 3-month-old wild-type, Cav-2-knockout (KO), and Cav-1-knockout cohorts. Glucose, cholesterol, triglyceride, and free fatty acid levels in plasma were measured colorimetrically.

FIG. 7.

Cav-2-null mice have normal body weights, adipose tissue morphology, and serum metabolites. (A) Routine histology (H&E) was performed on several different adipose tissue regions (perigonadal fat pads are shown) of 1-year-old wild-type and Cav-2-null mice, an age at which the adipose tissue of Cav-1-null mice has been shown to display heterogeneously sized adipocytes, marked interstitial fibrosis, and hypercellularity. No differences between wild type and Cav-2 knockouts (KO) are apparent. (B) The entire left-side mammary gland 4 of wild-type, Cav-2-knockout, and Cav-1-knockout females was dissected and subjected to whole-mount preparation. Carmine dye staining of the mammary tissue allowed visualization of the ductal architecture and density. (C) Fasting blood samples were collected at 7:00 a.m. (12 h after removal of food) and postprandial blood was collected at 12:00 a.m. (after 3 h of feeding in the dark) from 3-month-old wild-type, Cav-2-knockout (KO), and Cav-1-knockout cohorts. Glucose, cholesterol, triglyceride, and free fatty acid levels in plasma were measured colorimetrically.

FIG. 7.

Cav-2-null mice have normal body weights, adipose tissue morphology, and serum metabolites. (A) Routine histology (H&E) was performed on several different adipose tissue regions (perigonadal fat pads are shown) of 1-year-old wild-type and Cav-2-null mice, an age at which the adipose tissue of Cav-1-null mice has been shown to display heterogeneously sized adipocytes, marked interstitial fibrosis, and hypercellularity. No differences between wild type and Cav-2 knockouts (KO) are apparent. (B) The entire left-side mammary gland 4 of wild-type, Cav-2-knockout, and Cav-1-knockout females was dissected and subjected to whole-mount preparation. Carmine dye staining of the mammary tissue allowed visualization of the ductal architecture and density. (C) Fasting blood samples were collected at 7:00 a.m. (12 h after removal of food) and postprandial blood was collected at 12:00 a.m. (after 3 h of feeding in the dark) from 3-month-old wild-type, Cav-2-knockout (KO), and Cav-1-knockout cohorts. Glucose, cholesterol, triglyceride, and free fatty acid levels in plasma were measured colorimetrically.