Mito-Ob: a transgenic mouse model for obesity
09781909 · 2017-10-10
Assignee
Inventors
Cpc classification
A01K67/0275
HUMAN NECESSITIES
C12N2830/008
CHEMISTRY; METALLURGY
A01K2267/0362
HUMAN NECESSITIES
International classification
Abstract
An obese mouse model was developed by overexpressing the mitochondrial protein prohibitin (PHB) in white adipose tissue (WAT) specific manner driven by adipocyte protein 2 (aP2) promoter. These mice begin to develop obesity as a result of mitochondrial remodeling (upregulation of mitochondrial biogenesis and function) in WAT.
Claims
1. A transgenic mouse whose genome comprises a nucleic acid sequence encoding prohibitin operably linked to an adipocyte protein 2 (aP2) promoter, wherein the mouse has increased adipose tissue mass as compared to a wild type mouse.
2. The transgenic mouse according to claim 1 wherein the prohibitin gene comprises the nucleotide sequence as set forth in SEQ ID No:2.
3. The transgenic mouse according to claim 1 wherein the aP2 promoter comprises the nucleotide sequence as set forth in SEQ ID No:1.
4. The transgenic mouse according to claim 1 wherein the exogenous nucleic acid construct is integrated into the mouse genome.
5. A transgenic mouse, comprising a transgene, said transgene comprising a polynucleotide encoding a mouse prohibitin protein operably linked to a mouse adipocyte protein 2 (aP2) promoter, wherein said transgenic mouse develops increased adipose tissue mass and obesity compared to a wild type mouse of the same strain.
6. The transgenic mouse according to claim 5 wherein the prohibitin protein comprises an amino acid sequence deduced from the nucleotide sequence as set forth in SEQ ID No:2.
7. The transgenic mouse according to claim 5 wherein the aP2 promoter comprises the nucleotide sequence as set forth in SEQ ID No:1.
8. The transgenic mouse according to claim 5 wherein the transgene is integrated into the mouse genome.
9. A mouse transgenic fertilized egg comprising an expression construct comprising (a) a nucleotide sequence encoding prohibitin and (b) a mouse adipocyte protein 2 (aP2) promoter said mouse transgenic fertilized egg capable of becoming a transgenic mouse developing increased adipose tissue mass and obesity as compared to a wild type mouse of the same strain.
10. The mouse transgenic fertilized egg according to claim 9 wherein the nucleotide sequence encoding prohibitin comprises the nucleotide sequence as set forth in SEQ ID No:2.
11. The mouse transgenic fertilized egg according to claim 10 wherein the aP2 promoter comprises the nucleotide sequence as set forth in SEQ ID No:1.
12. The mouse transgenic fertilized egg according to claim 9 wherein the expression construct is integrated into the genome of the mouse transgenic fertilized egg.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3) ). Scale bars, 20 μm. Lower panel: Representative photomicrographs showing hematoxylin-eosin staining of brown adipose tissue (BAT) from 6 months old mice (n=6 mice in each group). Large unilocular lipid droplets are shown with asterisk (*). Scale bars, 20 μm. Data are presented as mean±SEM. Asterisks indicate comparison between sex matched Mito-Ob vs Wt. * P<0.05, ** P<0.01, *** P<0.001 by Student's t test. NS, not significant.
(4) ) in adipocytes from Mito-Ob and Wt mice (n=5 mice in each group). * Indicates unilocular lipid droplets, n indicates nucleus. Further magnified view is shown in the inset in each case. Scale bars, 1 μm. (B) Representative transmission electron micrographs (magnifications 19,000×) of BAT showing changes in mitochondrial cristae structure (white arrowhead) and larger fat droplet size (white asterisk) in Mito-Ob mice in comparison with Wt mice (n=5 mice in each group). Scale bars 500 nm. (C) Histograms showing mitochondrial numbers, and mtDNA copy numbers in the WAT of Mito-Ob and Wt mice as determined by real-time PCR (n=5). (D) Representative immunoblots and histograms showing the expression levels of SDH-A and COX-I in the adipose tissue of Mito-Ob mice (the ratio of SDH-A or COX-I in relation to beta-actin; n=4 in each group). (E) Representative immunoblots showing expression level of marker proteins of mitochondrial biogenesis, fission and fusion (n=4). * P<0.05, ** P<0.01, *** P<0.001 by Student's t test. Data are presented as mean±SEM.
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DESCRIPTION OF THE PREFERRED EMBODIMENTS
(6) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
(7) As discussed above, most of obese animal models currently used have been either selected through inbreeding or characterized following spontaneously arising mutations and are often associated with increased food intake and reduced energy expenditure (Shafrir and Ziv, 2009). Irrespective of the origin, obesity is characterized by increase in adipose tissue mass and involves corresponding changes in adipose tissue to synthesize and store excess fat. However, an obese animal model based on primary changes in adipose tissue or other defects is not available.
(8) As discussed herein, the inventors have developed an obese mouse model by overexpressing the mitochondrial protein prohibitin (PHB) in white adipose tissue (WAT) specific manner driven by adipocyte protein 2 (aP2) promoter. The inventors have named these mice “Mito-Ob” because they begin to develop obesity as a result of mitochondrial remodeling (upregulation of mitochondrial biogenesis and function) in WAT, thus are comparable to polygenic obese rodent models.
(9) According to an aspect of the invention, there is provided a transgenic mouse comprising increased adipose tissue mass as compared to a wild-type mouse of the same strain and an exogenous nucleic acid construct that comprises a promoter operably linked to a gene encoding prohibitin.
(10) As discussed below, preferably the prohibitin gene comprises the nucleotide sequence as set forth in SEQ ID No:2.
(11) The promoter may be adipocyte protein 2 (aP2) promoter and the aP2 promoter may comprise the nucleotide sequence as set forth in SEQ ID No:1.
(12) The exogenous nucleic acid construct may be integrated into the mouse genome.
(13) According to another aspect of the invention, there is provided a transgenic mouse, comprising a transgene, said transgene comprising a polynucleotide encoding a mouse prohibitin protein operably linked to at least a portion of a regulatory region of a mouse aP2 promoter, wherein said transgenic mouse develops obesity compared to a wild type mouse of the same strain. As will be appreciated by one of skill in the art, obesity in this context refers to at least an increase in adipose tissue. As discussed herein, the male transgenic mice described are also insulin resistant.
(14) Preferably, the prohibitin protein comprises an amino acid sequence deduced from the nucleotide sequence as set forth in SEQ ID No:2.
(15) Preferably, the aP2 promoter comprises the nucleotide sequence as set forth in SEQ ID No:1.
(16) The transgene may be integrated into the mouse genome.
(17) According to another aspect of the invention, there is provided a transgenic mouse whose genome comprises: a DNA transgene encoding prohibitin.
(18) The prohibitin may be encoded by the nucleotide sequence as set forth in SEQ ID No:2.
(19) The transgene may be operably linked to aP2 promoter and the aP2 promoter may have the nucleotide sequence as set forth in SEQ ID No:1.
(20) According to another aspect of the invention, there is provided a mouse transgenic fertilized egg comprising an expression construct comprising (a) a nucleotide sequence encoding prohibitin and (b) a transcription-regulating sequence operatively linked to the nucleotide sequence.
(21) As will be appreciated by one of skill in the art, such a fertilized egg comprising the expression construct may be generated using a variety of means known in the art. The fertilized egg may then be implanted into a suitable host for generation of a transgenic mouse as described above.
(22) Preferably, the nucleotide sequence encoding prohibitin comprises the nucleotide sequence as set forth in SEQ ID No:2.
(23) The transcription-regulating sequence may be aP2 promoter.
(24) Preferably, the aP2 promoter comprises the nucleotide sequence as set forth in SEQ ID No:1.
(25) Preferably, the expression construct is integrated into the genome of the mouse transgenic fertilized egg.
(26) According to another aspect of the invention, there is provided an isolated totipotent mouse cell comprising an exogenous nucleic acid construct that comprises prohibitin operably linked to a suitable promoter.
(27) Preferably, the exogenous nucleic acid construct encoding prohibitin comprises the nucleotide sequence as set forth in SEQ ID No:2.
(28) Preferably, the suitable promoter comprises aP2 promoter.
(29) Preferably, the aP2 promoter comprises the nucleotide sequence as set forth in SEQ ID No:1.
(30) Preferably, the exogenous nucleic acid construct is integrated into the genome of the mouse cell.
(31) As discussed herein, Mito-Ob male mice develop insulin resistance in addition to obesity and they do not develop overt diabetes. In this aspect, the Mito-Ob mice share similarities with a large portion of human obese population, the group who are both obese and insulin resistance but are not diabetic, as discussed below. The Mito-Ob mice therefore are a valuable animal model for obesity and metabolic syndrome. Further, as discussed herein, Mito-Ob male mice frequently show hyperglycemia thus in this aspect are similar to ZDF, UCD-T2DM rats and NZO mice, which are widely used for studies of anti-obesity and anti-diabetic drugs (Nilsson et al 2011). Thus, Mito-Ob mice are unique in their initiation of obesity that shares features of both polygenic DIO models and a number of monogenic obese rodent models.
(32) Consequently, the Mito-Ob mice are very useful not only in the discovery and development of anti-obesity drugs but also in revealing the role of WAT mitochondria in systemic energy homeostasis and in the identification of novel target genes for the treatment of obesity.
(33) During the last three decades, the prevalence of obesity has grown rapidly including children and youth (Pulgaron 2013). Although factors that led to such a rapid increase in obesity are not understood, transgenerational inherited factors appear to play an important role. For example, epidemiological and animal studies suggest that pre- or peri-conceptional obesity of the mother or environmental exposures early in life may affect metabolic programming in the offspring (Tanumihardjo et al. 2007; Levin 2006; Boerschmann et al 2010). In addition, new evidence suggests that paternal metabolic state may have similar influence in offspring. Irrespective of its origin from maternal or paternal health status, these transgenerational effects are now being explained by disruptions at the level of epigenetic machinery (Soubry et al. 2013). The Mito-Ob mice provide an opportunity to determine the effect of parental obesity, alone or combined together, with and without insulin resistance on the development of obesity in adult offspring in both F1 and F2 generations.
(34) For example, Mito-Ob male mice may be crossed with wild-type female mice of similar genetic background or vice versa. In this way the offspring will be exposed to either paternal or maternal obese environment; making it possible to then observe and compare health problems in Mito-Ob and non-Mito-Ob adult offspring. Another scenario would be to cross heterozygous Mito-Ob male and female mice and then observe health problems in adult offspring as a consequence of exposure to the obese environment of both parents. It is of note that suitable partners for mating will be readily apparent to one of skill in the art and/or may be readily determined, depending of course on the specific environment desired.
(35) The novel Mito-Ob mouse models provide unique opportunities for the discovery and testing new drugs and identification of novel targets for the treatment of obesity and potentially type 2 diabetes.
(36) As described herein, the Mito-Ob male mice develop obesity, insulin resistance and hyperglycemia in a progressive manner thus provide a larger and distinct windows for the discovery and testing of anti-obesity and anti-diabetic drugs.
(37) As will be readily apparent to one of skill in the art, a wide variety of specific, well-defined diets for animal research are available commercially. For example, high fat, high protein or high carbohydrate diets may be used. Consequently, as discussed herein, improved new models of progressive development of obesity and type 2 diabetes can be developed by manipulating diet or by crossing Mito-Ob mice with other mice models of obesity and type 2 diabetes.
(38) In addition, the Mito-Ob mice are useful to define if the impact is mediated through epigenetic machinery (beyond F2 generations) or simply a manifestation of exposures to parental obesity during pre-conceptional and prenatal life.
(39) For example, transgenerational obesity may be mediated through an epigenetic mechanism or simply by toxic effects of environmental exposures including the obese environment of the parents. To distinguish the precise mechanism involved and to confirm the involvement of epigenetics, it is important to study the effect in offspring beyond second (F2) generation. For example, the germ cells develop during fetal life. So an obese pregnant mother can potentially affect/expose not only her baby (F1 generation) but also the germ cells of the baby that are going to produce the second generation (F2).
(40) Furthermore, the transgenic mice can also be used to determine whether therapeutic intervention in obese parents during or before pregnancy would reduce obesity development in adult offspring.
(41) The transgenic mice can also be used to investigate how the offspring born to obese parents would respond to postnatal environmental determinants (e.g. diet) on the development of obesity and type 2 diabetes.
(42) The transgenic mice can be used to study the underlying mechanisms involved in these processes and in the identification of obesity biomarkers with special emphasis on WAT mitochondria.
(43) The transgenic mice can be used to investigate the underlying mechanisms involved in the crosstalk between the nucleus and mitochondria that is critical for mitochondrial biogenesis.
(44) On the basis of observed phenotype of Mito-Ob mice, it is anticipated that these mice will also develop obesity and type 2 diabetes associated health complications thus provide additional use of this novel mouse model.
(45) In general, excess calorie intake along with reduced energy expenditure has been considered as the cause of obesity and this is supported by a number of rodent obese models (Chua et al. 2007; Hummel et al. 1966; Trayhurn et al. 1982). However, the initiation of obesity development independent of excess calorie intake has not been well explored. For example, changes in the structural and functional components of WAT mitochondria, which have been suggested to have a role in the cellular homeostasis of the adipose tissue and whole body energy homeostasis, may create such a condition. However, there remain critical gaps in our understanding of the relevance of WAT mitochondria in adipose tissue energy homeostasis and their impact upon systemic energy homeostasis. This gap in knowledge may be attributed in part to the lack of obese animal model based on mitochondria remodeling in WAT specific manner, because the majority of the obese animal models used for research studies develop obesity as a result of excess calorie intake. To reduce this critical gap in our knowledge and to explore the possibility of the initiation of obesity independent of high calorie intake, we developed “Mito-Ob” mouse models by overexpressing the mitochondrial protein PHB in WAT specific manner. PHB is an important protein in mitochondrial biogenesis and function and the consequences of its expression in adipocytes are hitherto unknown. Our preliminary observations suggest that the Mito-Ob mice begin to develop obesity as a result of mitochondrial remodeling that is independent of increased food intake. Thus, our novel obese mouse models provide a unique opportunity to study the development of obesity from WAT perspective especially WAT mitochondria.
(46) The inventors hypothesized that upregulation of mitochondrial biogenesis and function in WAT specific manner will lead to increased adipose tissue mass. The obese phenotype of Mito-Ob mice overexpressing mitochondrial protein prohibitin (PHB), known to be involved in mitochondrial biogenesis and function, in WAT specific manner confirms this hypothesis (
(47) It is of note that obese rodent modes based on primary changes in white adipose tissue were not previously available. Furthermore, it was not previously possible to investigate the role of WAT mitochondria in adipose tissue and systemic metabolic regulation. However, this model opens the possibility of initiation of obesity development due to primary changes in adipose tissue including adipose tissue mitochondria. Increased food intake may be a manifestation of such changes in the body.
(48) To further establish the role of PHB in adipose tissue homeostasis at the systems level and the role of WAT mitochondria in whole body metabolism, we developed Mito-Ob mouse models by overexpressing PHB in WAT specific manner. As expected, both male and female transgenic mice start to gain weight from 3-4 weeks onward, become obese by 3-4 months, however only male transgenic mice become insulin resistant between 6-9 months of age in comparison to wild-type (Wt) control littermates (
(49) As discussed herein, the obese phenotype of Mito-Ob mice affirms the emerging notion that prohibitin and adipocyte mitochondria have a role in the regulation of adipose tissue homeostasis and metabolic regulation (Wilson-Fritch et al., 2004; Lu et al., 2010; De pauw et al., 2009; Ande et al., 2012; Artal-Sanz et al., 2009; Liu et al., 2012; Kang et al., 2013). However, the metabolic consequences of obesity in the Mito-Ob mice are sex specific. This would suggest that intrinsic differences exist in the way adipose tissues are regulated and/or respond to obesity development in male and female mice despite similar underlying cause. The aP2 promoter used in this study is primarily expressed in adipocytes and it has been used recently in a similar work (Kusminski et al., 2012). In addition, it is expressed in immune cells such as macrophages (Fu et al., 2006). However, a similar effect of PHB manipulation in adipocytes in vitro and in vivo (Ande et al., 2012; Artal-Sanz et al., 2009; Liu et al., 2012; Kang et al., 2013) would suggest that the phenotype observed is most likely due to the role of PHB in adipocytes.
(50) PHB translocates between mitochondria to the nucleus in response to estrogen (Dong et al, 2013). In addition, PHB has been associated with the function of mitochondrial transcription factor A (Tfam) and nuclear factor-like 2 (Nrf-2) (Kasashima et al., 2008; Theiss et al., 2009). Tfam has important roles in mitochondrial biology and Nrf-2 regulates transcription of nuclear-encoded mitochondrial proteins (Kasashima et al., 2008; Athale et al., 2012). These evidences along with the finding of upregulation of mitochondrial biogenesis in the WAT of the Mito-Ob mice would suggest a potential role of PHB in mito-nuclear crosstalk that is required for mitochondrial biogenesis. Furthermore, the upregulation mitochondrial biogenesis markers in the WAT of Mito-Ob mice suggest that PHB indeed has a role in this process. An enhanced mito-nuclear crosstalk may be the underlying mechanism behind increased mitochondrial biogenesis in the WAT of Mito-Ob mice.
(51) Men and women have distinct distributions of their body fat, where subcutaneous fat tends to be predominant in females, whereas visceral fat is predominant in males (Bjorntorp, 1996). It is the visceral fat that is related to obesity and its complications (Bjorntorp, 1996). However, the paradox of normal insulin sensitivity along with increased visceral obesity in the Mito-Ob female mice would suggest that it is not the visceral obesity per se, but rather the functional status of the adipose tissue such as the dynamics of lipid handling and adipokines secretion that leads to obesity associated disorders. For example, a differential regulation of adiponectin in Mito-Ob male and female mice could be related to their different metabolic status, as adiponectin promotes adipocyte differentiation, insulin sensitivity, and lipid accumulation (Fu et al., 2005); and adiponectin secretion has been reported to correlate with mitochondrial function (Wang et al., 2013). In this context it should be noted that estrogens, which have protective effects against obesity also have a role in mitochondrial biogenesis (Bjorntorp, 1996; Chen et al., 2009; Yao et al., 2013). However, it is not known whether the role of estrogen in mitochondrial biology is linked to its protective effect against obesity. The Mito-Ob female mice provide a unique opportunity to dissect the relationship between estrogen, prohibitin, and mitochondria in adipose tissue biology and in metabolic regulation.
(52) In summary, the obese phenotype and sex specific metabolic dysregulation in Mito-Ob mice established for the first time an important role of PHB in adipose tissue biology in mammals. Mito-Ob mice provide a valuable tool for obesity research. Mito-Ob mice can be used to better define the sex differences in obesity and associated health problems, and the potential role of adipose tissue mitochondria.
(53) The invention will now be further described by way of examples. However, the invention is not necessary limited to or by the examples.
(54) Cloning of PHB in aP2 Promoter Containing Vector:
(55) The aP2 promoter-containing vector (pBS-aP2 promoter (5.4 kb) polyA) was obtained from Addgene (Cambridge, Mass.). Full length PHB clone was obtained from Origene, USA (Cat # SC110973). This clone was digested with Not1 to release the full-length cDNA of PHB. Subsequently PHB cDNA was sub-cloned into the Not1 site of pBS-aP2 promoter vector. The authenticity of the construct was confirmed by DNA sequencing at the Cancer Care Manitoba, Canada.
(56) TABLE-US-00001 pBS-aP2 promoter (SEQ ID NO: 1) AAAGGGAACA AAAGCTGGAG CTCCACCGCG GTGGAGCTCG AGTCAGTGAG CGAGGAAGCG GAAGAGTCTA GAGTCGACCA GACATGATAA GATACATTGA TGAGTTTGGA CAAACCACAA CTAGAATGCA GTGAAAAAAA TGCTTTATTT GTGAAATTTG TGATGCTATT GCTTTATTTG TAACCATTAT AAGCTGCAAT AAACAAGTTC TGCTTTAATA AGATCTGATT CGAATTCCAA GCTTGGATCC GAATTCGCCC TATAGTGAGT CGTATTACGC GGCCGCTCTA GAACTAGTGG ATCCCCCGGG CTGCAGCACA GGAGGGTGCT ATGAGCCTCT GAAGTCCAGA TAGCTCACTT TTAAAGATGC CCTGACCATG TGACTGTAGG AGTGACCAAT GGGGGCCAGA TCATTTCCTT CATGACCAGA CCCTGTATGT TTTCCTCTGA GTCATGTTTT TAATAGAAAT TTCTCAACTT TGGTTCTCCC TGGCAATGAT CACTGGACTT AGAGTACAAA TTATTTTTAA CCATGAACAG AGTATTTTAA AGGTTCCTGT TTTGACTGTC AAAAGCTAAT GCATTGAACT TCCCCCCATT ATTCCTTATG GATTTGCCTC ATTGTGGAGG AGACAATTAT CTTGGACACA TTTGACCTTC TTATCTTGAG TTTTTATTTT ATTAATACTG CAATAATGTG TTTAGTTCTT CTGAATTTGA GAACATAAAA ACTATCTTAG AGATTCTTAG TCTTAATGGC TCTTTTGTTA GAATAGTGTT TATCTCACGA ATTTTAACAA AATAAATAAT GACATTTTAA AGTAGC 826
>OriGene sequence for PHB (NM_002634) (SEQ ID NO:2)
PHB sequence begins here at “G” (shown with arrow); translation initiation site (ATG) shown below in “underline”
(57) TABLE-US-00002 ↓ GAATTCGGCA CGAGGGGAAT TCATGTGGAG GTCAGAGTGG AAGCAGGTGT GAGAGGGTCC AGCAGAAGGA AACATGGCTG CCAAAGTGTT TGAGTCCATT GGCAAGTTTG GCCTGGCCTT AGCTGTTGCA GGAGGCGTGG TGAACTCTGC CTTATATAAT GTGGATGCTG GGCACAGAGC TGTCATCTTT GACCGATTCC GTGGAGTGCA GGACATTGTG GTAGGGGAAG GGACTCATTT TCTCATCCCG TGGGTACAGA AACCAATTAT CTTTGACTGC CGTTCTCGAC CACGTAATGT GCCAGTCATC ACTGGTAGCA AAGATTTACA GAATGTCAAC ATCACACTGC GCATCCTCTT CCGGCCTGTC GCCAGCCAGC TTCCTCGCAT CTTCACCAGC ATCGGAGAGG ACTATGATGA GCGTGTGCTG CCGTCCATCA CAACTGAGAT CCTCAAGTCA GTGGTGGCTC GCTTTGATGC TGGAGAACTA ATCACCCAGA GAGAGCTGGT CTCCAGGCAG GTGAGCGACG ACCTTACAGA GCGAGCCGCC ACCTTTGGGC TCATCCTGGA TGACGTGTCC TTGACACATC TGACCTTCGG GAAGGAGTTC ACAGAAGCGG TGGAAGCCAA ACAGGTGGCT CAGCAGGAAG CAGAGAGGGC CAGATTTGTG GTGGAAAAGG CTGAGCAACA GAAAAAGGCG GCCATCATCT CTGCTGAGGG CGACTCCAAG GCAGCTGAGC TGATTGCCAA CTCACTGGCC ACTGCAGGGG ATGGCCTGAT CGAGCTGCGC AAGCTGGAAG CTGCAGAGGA CATCGCGTAC CAGCTCTCAC GCTCTCGGAA CATCACCTAC CTGCCAGCGG GGCAGTCCGT GCTCCTCCAG CTGCCCCAGT GAGGGCCCAC CCTGCCTGCA CCTCCGCGGG CTGACTGGGC CACAGCCCCG ATGATTCTTA ACACAGCCTT CCTTCTGCTC CCACCCCAGA AATCACTGTG AA 992
(58) It is of note that this promoter was selected because it has been used before to develop WAT specific animal models.
(59) The inventors have shown that prohibitin has an important role in adipogenesis/overexpression of prohibitin in 3T3-L1 cell culture system increases adipogenesis. Furthermore, mitochondrial biogenesis is involved in adipogenesis and prohibitin is known to have a role in mitochondrial biogenesis. Thus, the inventors' hypothesis was that overexpression of prohibitin in WAT will upregulate mitochondrial biogenesis and will leads to obesity development.
(60) Generation of Mito-Ob Transgenic Mice
(61) The aP2 promoter containing PHB clone was digested with Sac1 and Kpn1 to release the fragment containing aP2 promoter with full length PHB. The aP2-PHB DNA was purified and used to generate Mito-Ob mice by pro-nuclear injection of aP2-PHB DNA in CD1 zygotes. This procedure was carried out at the transgenic and knockout mice facility at University of Manitoba, Winnipeg, Canada. Founder animals were identified by PCR amplification of tail genomic DNA using the following primers. Forward Primer: 5′ GCAGCCCGGGGGATCCACTA 3′ (SEQ ID NO:3) and Reverse Primer: 5′ GCACACGCTCATCAAAGTCCTCTCCGATGCTG 3′ (SEQ ID NO:4).
(62) Founder male mice were mated with CD1 female mice to obtain the Mito-Ob transgenic progeny as per standard protocol. The Mito-Ob transgenic mice were identified by genotyping the tail DNA by PCR using above-mentioned primers. All the procedures involving Mice were done according to the animal use protocol of the University of Manitoba, Winnipeg, Canada.
(63) As discussed above,
(64)
(65)
(66) As can be seen in
(67) As can be seen in
(68) Growth Curve and Adipose Tissue Weight
(69) Both male and female Mito-Ob mice initially weighed similar to their wild-type littermates; however, they started to gain weight around one month onwards and became significantly obese (P<0.001) by 2-3 months of age (
(70) Female Mito-Ob mice accumulated more visceral fat, but less BAT, than male Mito-Ob mice (
(71) Histology of Adipose Tissue
(72) Consistent with an increase in the adipose tissue mass in Mito-Ob mice, adipocyte hypertrophy was apparent in the WAT from Mito-Ob mice (
(73) Ultrastructural Analysis of Adipose Tissue
(74) TEM showed an increase in mitochondrial number and their distribution throughout the cytoplasmic rim of white adipocytes in Mito-Ob mice vs. wild-type controls (
(75) To confirm mitochondrial biogenesis in the WAT of Mito-Ob mice, mitochondrial DNA copy numbers and mitochondrial protein (SDH-A, Cox-I, (PGC-1α, DNA poly-γ A, Tfam etc.) levels were determined. Both were significantly upregulated (P<0.05-0.001) in Mito-Ob mice (
(76) Systemic Metabolism in Mito-Ob Mice
(77) Only Mito-Ob males were found to have significantly impaired glucose (P<0.05-0.001) and insulin (P<0.05-0.01) tolerance (
(78) Serum insulin levels were significantly higher (P<0.01) in Mito-Ob male mice vs. wild-type littermates (Table 1). However, Mito-Ob female mice had insulin levels as wild-type littermates (Table 1). Among adipokines, adiponectin levels were increased (P<0.05) only in Mito-Ob female mice (Table 1), whereas leptin levels were higher in Mito-Ob male mice than in females (Table 1). Resistin levels were unchanged in the Mio-Ob male mice (Table 1).
(79) WAT Lipase Levels in the Mito-Ob Mice
(80) Next, the expression levels of adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) in WAT were analyzed. ATGL and HSL were upregulated in both male and female Mito-Ob mice vs. wild-type mice; however, their levels were higher in Mito-Ob male mice than Mio-Ob female mice (
(81) Materials and Methods
(82) Body Weight and Food Intake.
(83) The animals were given normal chow (LabDiet, St. Louis, Mo.). Body weight of Mito-Ob and wild-type mice was recorded on a weekly basis after weaning and food intake during 3 to 6 months of age was determined (Chen and Nyomba, 2003).
(84) Physical Activity.
(85) Horizontal activity levels were measured using a metabolic cage system (AccuScan, Columbus, Ohio), and data were collected for every 5 minutes for 24 hours (Kim et al., 2008).
(86) Histology.
(87) Adipose tissues from six months old Mito-Ob and Wt littermates were fixed in buffered formaldehyde and subsequently dehydrated, embedded in paraffin, and 5 μm sections were stained with hematoxylin-eosin (Nguyen at al., 2011).
(88) Western Immunoblotting.
(89) Adipose tissue lysates from Mito-Ob and wild-type mice containing equal amount of proteins (˜15 μg/lane) were separated by SDS-PAGE and subsequently analyzed by immunoblotting (Ande et al., 2012; Nguyen et al., 2011). MitoBiogenesis™ Western blot cocktail (Abcam Inc.) was used to determine succinate dehydrogenase-A (SDH-A) and cytochrome c oxidase-I (COX-I) protein levels in adipose tissue.
(90) Transmission Electron Microscopy (TEM).
(91) Adipose tissues were excised into small pieces (<1 mm.sup.3) and fixed with 2.5% glutaraldehyde in 0.1 M PBS buffer (pH 7.4) for 3 hours. Each specimen was post-fixed in 1% osmium tetroxide for 1 hour before embedding in Epon resin. TEM was performed with a Philips CM10, at 80 kV, on ultra-thin sections (100 nm) and stained with uranyl acetate and counterstained with lead citrate.
(92) Mitochondrial DNA (mtDNA).
(93) mtDNA copy number in adipose tissue was determined by real-time PCR (Kelly et al., 2012).
(94) Measurement of Adipokines and Hormones.
(95) Serum adipokines and hormones were measured using mouse Bio-Plex Pro™ Assays Diabetes panel and Bio-Plex 200™ multiplex suspension array systems (Bio-Rad, Hercules, Calif.) as per manufacturer's protocols.
(96) Glucose and Insulin Tolerance Tests (GTT and ITT).
(97) GTT and ITT in 6 months old mice were performed as s described (Nguyen et al., 2011).
(98) Measurement of Cholesterol, Triglycerides and Free Fatty Acids (FFA).
(99) Serum cholesterol (Molecular Probes, Eugene, Oreg.), triglycerides and free fatty acid levels were measured using kits from BioAssay Systems (Hayward, Calif.). FFA uptake and oxidation were measured using kits from (Abcam Inc.).
(100) Statistical Analysis.
(101) All statistical analyses were performed using GraphPad Prism 6. Experimental results are shown as mean±SEM. Two-tailed Student's unpaired t-tests were performed to compare sex-matched Mito-Ob and wild-type littermates, unless indicated otherwise. P<0.05 was considered significant.
(102) The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
(103) TABLE-US-00003 TABLE 1 Serum hormones, adipokines and lipid profile in Mito-Ob and Wt mice at 6 months of age. Data are presented as mean ± SEM (n = 5 to 9 in each group). Female Male Wt Mito-Ob Wt Mito-Ob Fasting Fed Fasting Fed Fasting Fed Fasting Fed Insulin 1.1 ± 0.1 3.1 ± 0.7 1.3 ± 0.2 3.8 ± 0.9 1.6 ± 0.3 7.5 ± 0.9 6.58 ± 0.9** 14.3 ± 1.5** (ng/ml) Glucagon 0.7 ± 0.1 0.6 ± 0.1 0.5 ± 0.04 0.6 ± 0.1 0.3 ± 0.04 0.4 ± 0.03 0.4 ± 0.03 0.3 ± 0.03 (ng/ml) Ghrelin 5.3 ± 0.5 2.6 ± 0.5* 11.6 ± 1.6* 2.8 ± 0.8 4.8 ± 0.4 2.3 ± 0.3 2.7 ± 0.3*** 0.7 ± 0.1*** (ng/ml) GIP 0.4 ± 0.08 0.3 ± 0.04 0.3 ± 0.03 0.3 ± 0.06 0.4 ± 0.09 0.3 ± 0.03 0.3 ± 0.04 0.6 ± 0.08* (ng/ml) GLP-1 0.3 ± 0.05 0.2 ± 0.02 0.2 ± 0.02 0.2 ± 0.05 0.1 ± 0.01 0.2 ± 0.02 0.2 ± 0.03 0.2 ± 0.03 (ng/ml) Adiponectin 12.3 ± 0.5 6.0 ± 0.6 14.3 ± 1.9 8.3 ± 0.6* 6.3 ± 0.2 4.8 ± 0.2 7.2 ± 0.4 5.6 ± 0.6 (μg/ml) Leptin 7.5 ± 1.5 4.9 ± 1.0 6.1 ± 1.7 8.6 ± 1.3 8.6 ± 2.1 11.0 ± 2.8 14.4 ± 1.0* 14.2 ± 1.6 (ng/ml) Resistin 2.4 ± 0.3 1.1 ± 0.1 1.8 ± 0.2 1.9 ± 0.1 1.1 ± 0.1 1.2 ± 0.1 0.8 ± 0.1 1.1 ± 0.1 (ng/ml) Cholesterol 648 ± 19.0 716 ± 18.8 558 ± 69.1 594 ± 35.3* 508 ± 59.4 748 ± 58.8 600 ± 59.2.sub. 643 ± 20.6.sub. (μg/ml) Free fatty acids 0.8 ± 0.1 0.7 ± 0.1 0.9 ± 0.2 0.4 ± 0.1 0.9 ± 0.2 0.9 ± 0.1 0.8 ± 0.1 1.6 ± 0.1** (mM) Glycerol 1.4 ± 0.08 1.1 ± 0.12 0.6 ± 0.04*** 0.5 ± 0.04*** 0.6 ± 0.14 1.0 ± 0.04 0.8 ± 0.02 0.7 ± 0.09 (mM) Triglycerides 1.4 ± 0.23 1.4 ± 0.23 0.4 ± 0.04*** 0.3 ± 0.05*** 1.1 ± 0.14 1.5 ± 0.15 1.5 ± 0.08 1.0 ± 0.16 (mM) Asterisk signs applicable to both sexes (Mito-Ob) in comparison with respective wild-type mice. Comparison shown are sex matched fasting vs. fasting or fed vs fed between Wt and Mito-Ob. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test.
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