Generation of mice with overexpression of MIOX (MIOX-transgenic [MIOX-TG]) and MIOX knockout (MIOX-KO) has previously been described (30 (link)). Also, heterozygous Ins2Akita mice (The Jackson Laboratory) were mated with MIOX-KO to generate double mutant mice, MIOX-KO/Ins2Akita. The reagents were purchased from the vendors listed in Supplementary Table 1 .
Ins2akita
Manufactured by Jackson ImmunoResearch
Sourced in United States
Ins2Akita is a laboratory mouse strain that is used for research purposes. It is a model for type 2 diabetes and is characterized by hyperglycemia, obesity, and insulin resistance. The Ins2Akita mouse carries a mutation in the insulin 2 gene, which leads to the development of these metabolic abnormalities.
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10 protocols using ins2akita
1
Generation and Characterization of Transgenic Mice
2
Chronic MitoGamide Treatment in Akita Mice
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Wild-type C57BL/6 J (Charles River Laboratories, UK) and heterozygous Ins2Akita (The Jackson Laboratory, USA) male mice were used for experiments. Ins2Akita, hereafter called Akita, carry a single nucleotide substitution in the insulin 2 gene (Ins2). This mutation results in misfolding of proinsulin 2 and eventually leads to ER stress and pancreatic β cell failure and ultimately to severe hyperglycaemia. At 5 weeks of age, Akita mice developed hyperglycaemia with non-fasting blood glucose levels higher than 25 mM. For chronic MitoGamide treatment, 6-week-old mice were subjected to daily oral administration via gavage of MitoGamide (10 mg/kg) or vehicle (sterile water) for 12 weeks. Blood glucose and HbA1c levels (service provided by Core Biochemical Assay Laboratory, Cambridge University Hospital) were monitored during the treatment period. Cardiac function and morphology were evaluated using echocardiography (Vevo 3100, VisualSonics). The parasternal long-axis view (B mode), the short-axis view (M mode) and blood flow velocity (PW mode) at mitral valve were obtained, and measurements of cardiac structure and function were accessed using Vevo Lab (VisualSonics) in a completely blinded manner.
3
Characterizing Diabetic Cardiomyopathy in Mice
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Homozygotes for the hypermorphic (H) alleles for the ELMO1 gene (Elmo1H/H)1 (link),2 (link) with heterozygous diabetogenic Akita mutation in the Insulin 2 gene (Ins2Akita/+; Jackson Laboratory; Stock No: 003548) were generated by crossbreeding. All experiments used male mice on a C57BL/6J genetic background, because male Ins2Akita/+ mice develop type 1 diabetes, while female Ins2Akita/+ mice do not develop diabetes3 (link). Unless otherwise stated, vitamin B12 was provided through their drinking water at a dose to achieve an estimated daily B12 intake of 10 mg/kg bw/mouse, starting at 8 weeks of age for 16 weeks, echocardiograms were taken at 8, 16, and 24 weeks, and other biochemical analyses were performed on tissues collected at 24 weeks of age. All mice were kept under husbandry conditions conforming to the National Institutes of Health Guideline for Use and Care of Experimental Animals, and the protocols used were approved by the University of North Carolina Institutional Animal Care and Use Committee. All measurements were taken from distinct samples.
4
Diabetic Mouse Model Generation
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Lean (LepRdb/wt) male mice were obtained from Harlan Laboratories and crossed with Fpnwt/C326S female mice to obtain the founders (Fpnwt/C326S;LepRdb/wt and Fpnwt/wt;LepRdb/wt) used to generate the four experimental models herein described. Mice were housed at the SPF barrier of the Interfakultäre Biomedizinische Forschungseinrichtung (IBF) animal facility of the University of Heidelberg under a constant light–dark cycle and maintained on a standard mouse diet containing 200 ppm iron (LASQCdiet® Rod18) with ad libitum access to food and water. Diabetic heterozygous Ins2Akita+/− (Ins2Akita) mice, purchased from Jackson Laboratory (Charles River Laboratories, Germany), were bred at the animal facility of the University Hospital Mannheim, Heidelberg University. Age-matched non-diabetic homozygous Ins2Akita+/+ littermates served as control [32 (link)].
All animals were maintained following approval from the Animal Ethics Committee, in accordance with German Animal Welfare Act for the care and use of laboratory animals and the rules of the regulatory authorities in Baden-Württemberg (T-81/14, T-94/15).
All animals were maintained following approval from the Animal Ethics Committee, in accordance with German Animal Welfare Act for the care and use of laboratory animals and the rules of the regulatory authorities in Baden-Württemberg (T-81/14, T-94/15).
5
Characterization of Akita/miR-133a Transgenic Mice
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We procured WT (C57BL/6J, stock # 000664) and Ins2+/−Akita (stock # 003548) mice from The Jackson Laboratory. The cardiac-specific miR-133aTg mouse strain was a gift from Dr. Scot Matkovich from the Washington University, St. Louis, USA. These mice were maintained in the animal care facility of the University of Nebraska Medical Center. They were provided with standard rodent chow and water ad libitum. We created Akita/miR-133aTg by crossbreeding Akita with miR-133aTg mice in the same animal care facility. We used 13–15 week old male mice for these studies.
6
Diabetes Progression in Akita Mice
7
Diabetic Phenotype in Male Akita Mice
8
Akita Mouse Model for Cardiac Studies
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We procured WT (C57BL/6J, stock # 000664) and Ins2+/−Akita (stock # 003548) mice from The Jackson Laboratory (Bar Harbor, ME, USA). These mice were maintained and bred in the animal care facility of the University of Nebraska Medical Center (Omaha, NE, USA). They were provided with standard rodent chow and water ad libitum. We used the left ventricle (LV) heart tissue from 14–16-week-old male Akita and the age and gender matched littermates/siblings, normoglycemic Ins2+/+ WT mice. These studies were completed following the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use (IACUC) committee of the University of Nebraska Medical Center (protocol 19-054-06-FC, 5 June 2019).
9
Evaluating Bone Phenotypes in Mice
10
Evaluating Bone Phenotypes in Mice
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Five groups of male mice (N = 3 mice in each group) of various ages and genotypes were used. Group 1 included adult, 15-week-old wild-type (WT) C57BL/6J mice (Jackson Laboratory), which served as younger controls to Group 2. Group 2 included 32-week-old C57BL/6J mice (older adults), which also served as WT controls to the perlecan-deficient mice in Group 3. Group 3 consisted of 32-week-old C57BL/6J mice with homozygous C1532Yneo mutations in the PLN/HSPG2 gene. Initially developed to model human Schwartz-Jampel Syndrome, these mice (termed “Hypo” herein) exhibited a significant deficiency in perlecan expression25 (link) and were bred back to C57BL/6J mice background in-house. Bones from Hypo mice were found to have altered canalicular structure26 (link) and attenuated bone formation in response to tibial uniaxial loading11 (link) in our previous studies. Group 4 consisted of 20-week-old Akita mice with heterozygous Ins2Akita mutation, a spontaneous type 1 diabetes model (Jackson Laboratory). Akita mice developed severe hyperglycemia from 5 weeks of age. Our previous work has shown that they failed to respond to anabolic ulnar loading when compared to age-matched WT mice.27 Group 5 included age-matched WT controls to Group 4 (20-week-old C57BL/6J). The University of Delaware Institutional Animal Care and Use Committee approved the handling and use of all animals in this study.
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