Brown adipose tissue–specific insulin receptor knockout shows diabetic phenotype without insulin resistance (original) (raw)
Creation of the UCP 1-Cre transgenic mice. To produce a mouse in which IR was selectively inactivated in BAT, we have used the UCP-1 gene promoter, a gene that is expressed exclusively in BAT, to direct the expression of the Cre cDNA. The UCP-1-Cre transgene vector was constructed as described in Methods, and its sequence was verified by complete sequencing (Figure 1a). Two transgenic lines (lines 13 and 14) of UCP-1-Cre mice with a similar copy number of the transgene were identified by Southern blot and PCR analysis. Analysis of the mRNA for the Cre recombinase by Northern blotting revealed that both lines had a restricted expression of Cre in BAT as compared with brain, white adipose tissue, liver, or skeletal muscle (Figure 1b), although the levels of the Cre mRNA were much lower in line 13 than in line 14. For efficient production of the knockout mice, we therefore used line 14 of the UCP-1-Cre mice and always as hemizygous (T/+).
Structure and expression of the UCP-1-Cre transgene. (a) The Cre cDNA sequence was included at the BglII site of the first exon of UCP-1 gene. The length of 5′ UTR of the UCP-1 gene was 8.4 kb. Exons 3, 4, 5, and 6 of the UCP-1 gene were also incorporated downstream of the Cre cDNA sequence. (b) Representative Northern blot analysis of total RNA isolated from several tissues of the two transgenic lines (lines 13 and 14) of mice carrying a UCP-1-Cre DNA construct described in a and C57BL/6J (B6) control mouse. Blots were hybridized with a full-length Cre cDNA probe.
Creation of the BAT-specific IR knockout mice. To create specific BAT inactivation of the IR, we bred IRLox/+ (17–20) and UCP-1-Cre (T/+) obtaining the F1 out of four individual crosses at the expected ratio [nine IRLox/+;UCP-1-Cre (T/+), ten IRLox/+, eight UCP-1-Cre (T/+), and nine wild-type]. Interbreeding of the F1 generation IRLox/+;UCP-1-Cre (T/+) and IRLox/+ was performed to create a BATIRKO mouse. These mice [F2 generation IRLox/Lox;UCP-1-Cre (T/+)] were obtained at the expected ratio of 12.5% for a trait determined by two independent loci. This strategy of breeding [IRLox/+;UCP-1-Cre (T/+) × IRLox/+] was required to obtain all the control littermates necessary for the study [UCP-1-Cre (T/+), wild-type (WT), and IRLox/Lox]. To obtain enough numbers of mice with the same age and gender to carry out all these studies with BATIRKO and their three control mice, it was necessary to work with 12 individual crosses [IRLox/Lox;UCP-1-Cre (T/+) × IRLox/+ mice] during the 3 years of the study. All the mice used were in a mixture background of FVB/NJ, C57BL/6J, and 129 sv.
Although newborn BATIRKO mice were indistinguishable from the IRLox/Lox (IRloxP), UCP-1-Cre (Cre), and WT littermates, grew normally, and no differences were seen in young mice after weaning (data not shown), in adult BATIRKO mice the BAT appeared reduced in an age-dependent manner. Thus, the amount of interscapular BAT normalized by body weight (BAT/body weight mass ratio) was reduced by 50% in 3-month-old BATIRKO mice and by 75% in 6- and 12-month-old BATIRKO mice as compared with controls (Figure 2a). This phenotype was shown by 100% of BATIRKO mice regardless of gender. By contrast, no significant differences were observed in the amount of white adipose tissue (WAT) as normalized by body weight (WAT/body weight-mass ratio) between BATIRKO mice and controls at any age studied (results not shown). Histological hematoxylin and eosin staining of interscapular BAT slides revealed a marked reduction of cell and fat droplet size and an increased eosinophilia in BATIRKO mice as compared with controls (Figure 2b, lower panel). These data are fully consistent with the marked interscapular BAT lipoatrophy induced in 6-month-old BATIRKO mice as compared with controls (Figure 2b, upper panel). However, no apoptosis was visualized by TUNEL assay in BAT sections as compared with controls (results not shown).
BAT content and IR expression. Interscapular brown fat was obtained from WT, Cre, IR_lox_P, and BATIRKO mice at 3, 6, and 12 months of age. (a) BAT weight vs. body weight is represented. Results are expressed as a mean ± SEM (n = 10–20). #P < 0.00001, BATIRKO vs. IR_lox_P; **P < 0.0001, BATIRKO vs. IR_lox_P or Cre; *P < 0.01, BATIRKO vs. IR_lox_P or Cre or WT. Representative interscapular BAT from IR_lox_P and BATIRKO mice at 6 months of age (b) (upper panel). Hematoxylin and eosin stain of BAT from IR_lox_P and BATIRKO mice at 6 months of age (b) (lower panel). RT-PCR analysis of RNA prepared from BAT from BATIRKO mice at different ages (1 day and 1, 3, 6, and 12 months) and IR_lox_P at 6 months of age, to study IR expression. The RT reaction was carried out as described in Methods. A larger band (585 bp) was observed in the IR_lox_P lane, while a smaller band of 435 bp was observed in BATIRKO mice lanes, suggesting a recombination event (c) (upper panel). Protein extracts of BAT from IR_lox_P and BATIRKO mice at different ages (1, 3, and 6 months) were subjected to immunoprecipitation with an mAb against IR β-chain and analyzed by Western blot. This is representative of three experiments (c) (lower panel). Protein extracts from different tissues of 6-month-old WT, IR_loxP_, Cre, and BATIRKO mice were immunoprecipitated with the same Ab and analyzed by Western blot. This is representative of three experiments (d). φx, DNA marker.
To assess the degree of recombination of the IR due to the excision by Cre recombinase of exon 4 (149 bp) floxed by loxP sequences, an age-dependent study by RT-PCR was performed. These data revealed that in control mice (IR_loxP_) only one band of larger size was observed. Conversely, in BATIRKO mice throughout development (from 1 day to 12 months) only one band of smaller size was visualized, a faint band of larger size due to the presence on non–brown adipocytes in the tissue also being present (Figure 2c, upper panel). Our data strongly suggest that the _UCP1_-Cre transgene is regulated in a very similar developmental fashion to the endogenous gene.
To validate the effect of the IR gene knockout on IR protein expression, protein extracts from BAT were subjected to immunoprecipitation with a monoclonal IR-specific antiserum, followed by Western blot analysis using a polyclonal IR-specific antiserum. IR expression was unaltered in BAT from the control IR_loxP_ mice as compared with that from the WT animal, indicating that the introduction of the loxP sites in the IR allele or Cre transgene under the UCP-1 promoter did not interfere with the endogenous IR expression (Figure 2d). By contrast, IR expression was reduced by 95% in BAT from BATIRKO mice (Figure 2d). The levels of IR protein in other tissues (liver, skeletal muscle, WAT, or pancreas) in 6-month-old BATIRKO mice as determined by Western blot analysis ranged from 83% to 120% of levels observed in WT, IR_lox_P, and Cre controls (Figure 2d). In addition, a time-course study showed the lack of expression of IR in BATIRKO mice at 1, 3, and 6 months, respectively, as compared with the corresponding control mice. (Figure 2c, lower panel). Thus, expression of the Cre transgene under control of the UCP-1 promoter was sufficient to direct recombination between the loxP sites flanking exon 4 (149 bp) of the IR with high efficiency in BAT and was highly effective in abolishing IR expression specifically in this tissue.
To assess the effect of IR on the expression of genes related to thermogenesis and lipogenesis, we performed Northern blot analysis on RNA from BAT, WAT, or skeletal muscle of BATIRKO mice and from IRloxP and Cre controls. A densitometric analysis was performed in mRNA expression of BAT from several genotypes throughout development (Figure 3, lower panels). Interestingly, at 3 months of age, expression of the major thermogenic protein, UCP-1, was normal in BAT of BATIRKO mice, despite the reduced BAT mass. Furthermore, as the BATIRKO mice aged, the expression of UCP-1 remained high as compared with IR_lox_P and Cre controls in which UCP-1 expression per microgram of RNA tended to decrease (Figure 3, a–c, lower panels). The expression of UCP-2 was also higher in brown fat of the BATIRKO mice as compared with controls throughout development and was even more evident in 12-month-old than in 3- to 6-month-old mice (Figure 3c, lower panel). We have shown previously that BAT undergoes a complete adipogenic program of gene expression in which insulin is the main signal involved in induction of lipogenic genes, such as FAS, glycerol-3-phosphate dehydrogenase (G3PD), ME, and the Glut-4 (21). Densitometric analysis also revealed a marked decrease in FAS expression in remnant BAT from 3- to 12-month old BATIRKO mice as compared to controls. A decrease was also observed in ME and to a lesser extent in G3PD and Glut-4 expression in BATIRKO mice as compared with controls (Figure 3, lower panels). These changes in gene expression were age dependent, with the largest effect being observed in 6- to 12-month-old mice (Figure 3, b and c, lower panels). There was also a slight decrease in the expression of PPARγ and a marked decrease in the CCAAT/enhancer-binding protein-α (C/EBP-α) in 6- and 12-month old BATIRKO mice as compared with controls (Figure 3, b and c, lower panels).
Gene expression throughout development. Skeletal muscle (Muscle), interscapular BAT, and WAT were obtained at 3 (a) (upper panel), 6 (b) (upper panel), and 12 months (c) (upper panel) of age from IR_lox_P, Cre, and BATIRKO mice. Total RNA was submitted to Northern blot analysis and hybridized with labeled UCP-1, UCP-2, ME, FAS, G3PD, PPARγ, C/EBP-α, or Glut-4. A final hybridization with 18S ribosomal RNA (rRNA) cDNA was performed for normalization. Densitometric analysis of the autoradiograms corresponding to BAT from controls (filled bars) and BATIRKO mice (open bars) after normalization of arbitrary units with the amount of 18S rRNA detected is shown in lower panels. Results are expressed as mean ± SEM (n = 5–6). *P < 0.05; **P < 0.005; ***P < 0.001 BATIRKO vs control. Autoradiograms are representative of five experiments.
Insulin-stimulated signaling events in liver, muscle, and BAT from WT, IRloxP, Cre, and BATIRKO mice. To assess the molecular consequences of BAT-specific knockout of IR on insulin signaling, we compared the early steps of insulin signaling in liver, skeletal muscle, and BAT of WT, IR_loxP_, Cre, and BATIRKO mice. To investigate IR phosphorylation, mice were starved overnight and injected with either saline or insulin (5 U) via the inferior vena cava. Protein extracts were prepared from liver, muscle, and BAT and subjected to immunoprecipitation with a monoclonal anti-IR Ab followed by Western blot analysis with an anti-Tyr (P) Ab. Insulin rapidly stimulated tyrosine phosphorylation of the IR in liver and muscle from the WT, IR_loxP_, Cre, and BATIRKO mice (Figure 4a). In contrast, no insulin-induced tyrosine phosphorylation of the IR was observed in the BAT of BATIRKO mice. Reblotting of these membranes with an anti-IR Ab revealed that, as expected, IR expression was absent in the BAT of BATIRKO mice, but remained normally expressed in liver and muscle of these animals (Figure 4a).
Insulin-stimulated tyrosine phosphorylation of the IR β-chain and PI 3-kinase activity. Six-month-old mice were anesthetized by intraperitoneal injection of pentobarbital and injected with either saline (–) or insulin (+) via the inferior vena cava. Protein extracts from liver, muscle, and BAT were subjected to immunoprecipitation with the monoclonal anti-IR Ab (a) or with a monoclonal anti-Tyr (P) Ab (b). The resulting immune complexes were separated by SDS-PAGE and analyzed by Western blot with the anti-Tyr (P) Ab or with the polyclonal anti-IR Ab (a), or washed and immediately used for an in vitro PI 3-kinase assay (b). This is representative of three experiments. WB, Western blot; pY, phospho-tyrosine; PIP, phosphatidylinositol phosphate.
To determine the effect of IR deletion on the activation of phosphatidylinositol (PI) 3-kinase, WT, IR_loxP_, Cre, and BATIRKO mice were injected with insulin. Protein extracts from various tissues were immunoprecipitated with an anti-Tyr (P) Ab and immediately used for an in vitro PI 3-kinase assay as described in Methods. Insulin-stimulated PI 3-kinase activation was normal in liver, muscle, and BAT from WT, IR_loxP_, and Cre mice (Figure 4b). Again, as expected, BAT from BATIRKO mice did not show PI 3-kinase activity in response to insulin, while insulin-induced PI 3-kinase activity in liver and muscle of these animals remained normal as compared with WT, IR_loxP_, and Cre controls (Figure 4b). These data reinforce the fact that the knockout of the IR was both tissue specific and quite complete.
BATIRKO mice exhibited an age-dependent impaired glucose tolerance, without insulin resistance. To determine the physiological consequence of tissue-specific inactivation of the IR in brown fat, glucose disposal was assessed by intraperitoneal injection of glucose (2 g/kg body weight) following an overnight fast. Upon glucose challenge, at 3 months of age both male and female BATIRKO mice showed a normal fasting blood glucose concentration and normal glucose tolerance as compared with their controls (Figure 5, a and b). However, beyond 6 months of age BATIRKO males showed fasting hyperglycemia. In addition, glucose tolerance was severely impaired in BATIRKO males at 6 months of age as compared with their controls, and by 12 months of age glucose levels following intraperitoneal glucose challenge were from to 1.5-fold to twofold higher in BATIRKO mice than in controls (Figure 5, c and e). BATIRKO females beyond 6 months of age also showed mild hyperglycemia and moderately impaired glucose tolerance as compared with controls. By 12 months of age, glucose levels 30 minutes after intraperitoneal glucose were 1.5-fold higher in BATIRKO mice than in their controls (Figure 5, d and f). Overall, this diabetic phenotype was shown by 60% of BATIRKO mice. Intravenous insulin tolerance (ITT) testing (1 U/kg body weight), on the other hand, indicated that both 6-month-old male and female BATIRKO mice showed the same glucose disposal rate in response to insulin than controls (results not shown).
BATIRKO mice demonstrate a progressive glucose intolerance. The ability to handle a glucose load was assessed by carrying out a glucose tolerance test at 3, 6, and 12 months of age in WT (×), Cre (triangles), IR_lox_P (diamonds), and BATIRKO (squares) male (a, c, and e) and female (b, d, and f) mice. An age-dependent glucose intolerance was observed in both male and female mice. Results are expressed as mean ± SEM (n = 10–20). ***P < 0.0005; #P < 0.001; **P < 0.005; *P < 0.05; BATIRKO vs. WT or IR_lox_P or Cre.
BATIRKO mice exhibited an insulin-secretion defect. The normal ITT, but abnormal glucose tolerance test, suggested that the defect in glucose homeostasis might be secondary to altered insulin secretion, rather than altered insulin action. Indeed, acute glucose-stimulated insulin secretion (3 g of glucose/kg body weight) was dramatically impaired in fasted 6-month-old male BATIRKO mice as compared with controls (Figure 6a). At this stage, fasted male BATIRKO mice were hyperglycemic (Figure 6b, upper panel) and showed a marked hypoinsulinemia (Figure 6b, lower panel), without significantly altered levels of free fatty acids (0.52 ± 0.07 mM in BATIRKO vs. 0.43 ± 0.07 mM in controls) and triglycerides (87.8 ± 4.4 mg/dl in BATIRKO vs. 96.5 ± 5.8 mg/dl in controls). However, fed male BATIRKO mice showed similar blood glucose concentration to controls, plasma insulinemia being slightly lower (Figure 6b).
BATIRKO male mice show an insulin-secretion defect. A representative insulin-secretion test is shown. Results are expressed as mean ± SEM (n = 7–12). *P < 0.001; #P < 0.005 (a). Blood glucose level (b)(upper panel) and blood insulin level (b) (lower panel) were determined in fasted and fed control and BATIRKO mice. Results are expressed as mean ± SEM (n = 25–30), *P < 0.000001, #P < 0.01 BATIRKO vs. control. Islet morphology was studied by immunohistochemical analysis in BATIRKO and control mice, as shown in Methods. Representative pancreatic sections from control and diabetic and nondiabetic 6-month-old BATIRKO mice stained for insulin are shown (c)(upper panels). β-cell area was measured by immunohistochemical analysis. Results are expressed as the percentage of the total surveyed area containing cells positive for insulin and are mean ± SEM (n = 10). *P < 0.01. Both diabetic and nondiabetic animals were studied (c)(lower panel). Insulin secretion in control and BATIRKO isolated islets is shown. Results are expressed as mean ± SEM (n = 4). *P < 0.05 (d). RT-PCR analysis of total RNA from BAT of BATIRKO mice (BB) and islets from control (CI) and BATIRKO mice (BI) was performed, and IR expression was studied (e)(left panel). Total islet RNA from control (C) and BATIRKO (B) mice was submitted to Northern blot analysis and hybridized with labeled insulin and UCP-2 cDNAs. A representative experiment out of three is shown. Densitometric analysis was performed using 18S ribosomal rRNA for normalization (e)(right panels).
Histologic examination of islet morphology revealed that BATIRKO diabetic mice showed a decrease in the islet size. However, islet size was quite similar in BATIRKO nondiabetic mice as compared with controls (Figure 6c, upper panels). Overall, quantitative analysis of pancreatic sections revealed that male BATIRKO mice showed around 35% decrease in beta islet mass (area) as compared with controls (Figure 6c, lower panel). In addition, TUNEL assay revealed the absence of apoptotic β cells in BATIRKO pancreatic sections as compared with controls (results not shown).
A 1-hour time course of insulin secretion in isolated islets upon 16.7 mM glucose stimulation was also studied (Figure 6d). Thus, insulin secretion was blunted in isolated islets from BATIRKO male mice as compared with controls. This effect is entirely consistent with data from the acute insulin-secretion test seen above. However, the Lox-P-IR recombination assay performed by RT-PCR showed no recombination in islet RNA from BATIRKO mice as compared with controls (Figure 6e, left panel), suggesting that the defect in the insulin secretion in BATIRKO mice seems to be an indirect effect. Northern blot analysis of islet RNA, normalized by 18S rRNA expression, pointed out that the insulin mRNA expression was quite similar in BATIRKO mice as compared with control. More importantly, the UCP-2 mRNA expression did not show significant differences between BATIRKO and control mice (Figure 6e, right panels).