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1

Adipocyte-specific Bola3 Knockout Mice Metabolic Profiling

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Adipocyte-specific Bola3 KO mice (Adipo-Cre; Bola3^flox/flox mice) and littermate control mice (Bola3^flox/flox) were fed a regular diet (RD) or a high-fat/high-sucrose diet (HF/HS) (D12451, Research Diet) under an ambient temperature (22°C) for 40 weeks. No difference in body weight was seen between control and Adipo-Bola3 KO mice at 22˚C. Whole-body energy expenditure, food intake and locomotor activity (beam break counts) were monitored by a comprehensive lab animal monitoring system (CLAMS) (Columbus Instruments). Metabolic data were collected at thermoneutrality (30° C). During the study, oxygen consumption was evaluated after administration with β3 adrenergic receptor agonist at 1 mg kg⁻¹ (CL-316, 243 (Sigma)). Body-weight was monitored once a week. Fat mass and lean mass were measured by Body Composition Analyzer EchoMRI (Echo Medical Systems). For glucose tolerance test experiments, after more than 6 hours of fasting, the mice were injected intraperitoneally with glucose (1.5g kg⁻¹). For insulin tolerance test experiments, after 3 hours of fasting, the mice were injected intraperitoneally with insulin (0.75U kg⁻¹). Serum level of insulin (Millipore) was measured using commercially available kits. Blood samples were collected at the indicated time points, and glucose levels were measured using blood glucose test strips (Abbott).

Tajima K., Ikeda K., Chang H.Y., Chang C.H., Yoneshiro T., Oguri Y., Jun H., Wu J., Ishihama Y, & Kajimura S. (2019). Mitochondrial lipoylation integrates age-associated decline in brown fat thermogenesis. Nature metabolism, 1(9), 886-898.

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Plasma Metabolite Analysis in Mice

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Blood was extracted from ad libitum fed mice 2–6 months of age via cardiac puncture under isofluorane following one hour acclimatization period, at minimum. Extractions were performed near end of the light cycle. Blood was immediately centrifuged at 3,000 g for 5 min at 4°C, to separate plasma from blood cells. Plasma fatty acids were measured using the HR Series NEFA-HR kit (Wako Diagnostics). Plasma triglycerides were measured using the Infinity Triglyceride Reagent (Thermoscientific). Blood glucose was measured using an AlphaTrak glucose meter and blood glucose test strips (Abbott Laboratories). Plasma ketones were measured using a Ketone Body Assay Kit (Abnova Corporation). Plasma corticosterone, TSH, and insulin were measured using radioimmunoassay and quantified using a Packard Gamma counter, by the Vanderbilt Hormone Assay and Analytical Services Core, Vanderbilt University, Nashville, TN 37232.

McGlashon J.M., Gorecki M.C., Kozlowski A.E., Thirnbeck C.K., Markan K.R., Leslie K.L., Kotas M.E., Potthoff M.J., Richerson G.B, & Gillum M.P. (2015). Central serotonergic neurons activate and recruit thermogenic brown and beige fat and regulate glucose and lipid homeostasis. Cell metabolism, 21(5), 692-705.

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Metabolic Effects of Cold Exposure and β-Blocker in Mice

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Twelve-week-old animals (Control, Pparg^flox/flox and Pparg^MyoD KO, Myod-Cre^ERT2;Pparg^flox/flox) were chronically pre-treated with β-blocker for 5 days, and then the animals were transferred from room temperature to mild cold at 15°C for additional 5 days. Whole-body energy expenditure (VO₂, VCO₂), food intake, and locomotor activity (beam break counts) were monitored using the Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments) at 15°C. For glucose tolerance test, mice were pre-treated with β-blocker and mild cold at 15°C, and then the mice were acutely treated β-blocker. After 6-hours fasting, the mice were injected intraperitoneally with glucose (2.0 g/kg). Blood samples were collected at several time points, and glucose levels were measured using blood glucose test strips (Abbott).

Chen Y., Ikeda K., Yoneshiro T., Scaramozza A., Tajima K., Wang Q., Kim K., Shinoda K., Sponton C.H., Brown Z., Brack A, & Kajimura S. (2018). Thermal stress induces glycolytic beige fat formation via a myogenic state. Nature, 565(7738), 180-185.

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Metabolic Regulation in Transgenic Mice

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Six-week-old animals (control, Prdm16 Tg, Ucp1−/−, and Prdm16 Tg x Ucp1−/− mice) were fed a HFD (D12492, Research Diet) or a regular diet (RD) under the ambient temperature at 22°C or thermoneutrality at 30°C for 12–24 weeks. HFD feedings were started at 6 weeks old. Body weight was measured every week. Whole-body energy expenditure (VO₂, VCO₂), food intake, and locomotor activity (beam break counts) were monitored using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments). CLAMS experiment in response to cold exposure was performed in mice at 10–12 weeks old under RD. Locomotor activity was measured by CLAMS for 5 days in mice at 12 weeks of HFD. Fat mass and lean mass were measured by the Body Composition Analyzer EcoMRI (Echo Medical Systems). For GTT experiments, mice were fed HFD or RD for 10 weeks. After an overnight fast, the mice were injected intraperitoneally with glucose (1.5 g/kg). For ITT experiments, the mice on HFD or for 11 weeks were injected intraperitoneally with insulin (0.75 U/kg for mice under HFD and 0.2U/kg for mice under RD) after 3 hours of fasting. Blood samples were collected at indicated time points, and glucose levels were measured using blood glucose test strips (Abbott).

Ikeda K., Kang Q., Yoneshiro T., Camporez J.P., Maki H., Homma M., Shinoda K., Chen Y., Lu X., Maretich P., Tajima K., Ajuwon K.M., Soga T, & Kajimura S. (2017). UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis. Nature medicine, 23(12), 1454-1465.

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Metabolic Effects of Cold Exposure and β-Blocker in Mice

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Twelve-week-old animals (Control, Pparg^flox/flox and Pparg^MyoD KO, Myod-Cre^ERT2;Pparg^flox/flox) were chronically pre-treated with β-blocker for 5 days, and then the animals were transferred from room temperature to mild cold at 15°C for additional 5 days. Whole-body energy expenditure (VO₂, VCO₂), food intake, and locomotor activity (beam break counts) were monitored using the Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments) at 15°C. For glucose tolerance test, mice were pre-treated with β-blocker and mild cold at 15°C, and then the mice were acutely treated β-blocker. After 6-hours fasting, the mice were injected intraperitoneally with glucose (2.0 g/kg). Blood samples were collected at several time points, and glucose levels were measured using blood glucose test strips (Abbott).

Chen Y., Ikeda K., Yoneshiro T., Scaramozza A., Tajima K., Wang Q., Kim K., Shinoda K., Sponton C.H., Brown Z., Brack A, & Kajimura S. (2018). Thermal stress induces glycolytic beige fat formation via a myogenic state. Nature, 565(7738), 180-185.

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6

Metabolic Regulation in Transgenic Mice

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Six-week-old animals (control, Prdm16 Tg, Ucp1−/−, and Prdm16 Tg x Ucp1−/− mice) were fed a HFD (D12492, Research Diet) or a regular diet (RD) under the ambient temperature at 22°C or thermoneutrality at 30°C for 12–24 weeks. HFD feedings were started at 6 weeks old. Body weight was measured every week. Whole-body energy expenditure (VO₂, VCO₂), food intake, and locomotor activity (beam break counts) were monitored using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments). CLAMS experiment in response to cold exposure was performed in mice at 10–12 weeks old under RD. Locomotor activity was measured by CLAMS for 5 days in mice at 12 weeks of HFD. Fat mass and lean mass were measured by the Body Composition Analyzer EcoMRI (Echo Medical Systems). For GTT experiments, mice were fed HFD or RD for 10 weeks. After an overnight fast, the mice were injected intraperitoneally with glucose (1.5 g/kg). For ITT experiments, the mice on HFD or for 11 weeks were injected intraperitoneally with insulin (0.75 U/kg for mice under HFD and 0.2U/kg for mice under RD) after 3 hours of fasting. Blood samples were collected at indicated time points, and glucose levels were measured using blood glucose test strips (Abbott).

Ikeda K., Kang Q., Yoneshiro T., Camporez J.P., Maki H., Homma M., Shinoda K., Chen Y., Lu X., Maretich P., Tajima K., Ajuwon K.M., Soga T, & Kajimura S. (2017). UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis. Nature medicine, 23(12), 1454-1465.

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7

Adipocyte-specific Bola3 Knockout Mice Metabolic Profiling

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Adipocyte-specific Bola3 KO mice (Adipo-Cre; Bola3^flox/flox mice) and littermate control mice (Bola3^flox/flox) were fed a regular diet (RD) or a high-fat/high-sucrose diet (HF/HS) (D12451, Research Diet) under an ambient temperature (22°C) for 40 weeks. No difference in body weight was seen between control and Adipo-Bola3 KO mice at 22˚C. Whole-body energy expenditure, food intake and locomotor activity (beam break counts) were monitored by a comprehensive lab animal monitoring system (CLAMS) (Columbus Instruments). Metabolic data were collected at thermoneutrality (30° C). During the study, oxygen consumption was evaluated after administration with β3 adrenergic receptor agonist at 1 mg kg⁻¹ (CL-316, 243 (Sigma)). Body-weight was monitored once a week. Fat mass and lean mass were measured by Body Composition Analyzer EchoMRI (Echo Medical Systems). For glucose tolerance test experiments, after more than 6 hours of fasting, the mice were injected intraperitoneally with glucose (1.5g kg⁻¹). For insulin tolerance test experiments, after 3 hours of fasting, the mice were injected intraperitoneally with insulin (0.75U kg⁻¹). Serum level of insulin (Millipore) was measured using commercially available kits. Blood samples were collected at the indicated time points, and glucose levels were measured using blood glucose test strips (Abbott).

Tajima K., Ikeda K., Chang H.Y., Chang C.H., Yoneshiro T., Oguri Y., Jun H., Wu J., Ishihama Y, & Kajimura S. (2019). Mitochondrial lipoylation integrates age-associated decline in brown fat thermogenesis. Nature metabolism, 1(9), 886-898.

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8

Streptozotocin-Induced Diabetes in Mice

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After one week of being housed in a new environment, the C57BL/6 mice were fasted but free for water for 16 h before the induction of diabetes. Streptozotocin (STZ) (Sigma, MN, USA), freshly prepared in buffer solution (0.1mol/L sodium citrate and 0.1 mol/L citric acid, pH 4.2–4.5), was intraperitoneally injected into the mice in a dose of 100 mg/kg for two successive days (day1 and day2). The diabetes induction method was mildly modified according to the reference [23 (link)]. After 72 h, the mice display polydipsia and polyuria. After one week (day 9), postprandial blood glucose levels were measured by blood glucose test strips (Abbott). Mice with blood glucose levels higher than 16.8mmol/L were defined diabetic and chosen for experiments. Ten sex- and age-matched mice were intraperitoneally injected with the buffer solution as the controls.

Pan L., Weng H., Li H., Liu Z., Xu Y., Zhou C., Lu X., Su X., Zhang Y, & Chen D. (2015). Therapeutic Effects of Bupleurum Polysaccharides in Streptozotocin Induced Diabetic Mice. PLoS ONE, 10(7), e0133212.

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9

Glucose and Hormone Metabolism Evaluation

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Blood glucose levels were determined using a FreeStyle Lite^® glucometer and blood glucose test strips (Abbott Diabetes Care) via tail tipping. Intraperitoneal glucose tolerance tests (IP-GTT) were conducted following an overnight fast (16 h) with access to water. The following day mice were weighed and fasting blood glucose measurements taken. Subsequently, mice were injected intraperitoneally with 2 g/kg 20% dextrose (Sigma Aldrich). Blood glucose levels were measured from the tail vein at 15, 30, 60 and 120 min post-dextrose administration. Intravenous (IV) GTT was conducted in a similar manner; however, glucose (1 g/kg) was administered intravenously into the tail vein and blood glucose measurements taken at 0, 5, 10, 15, 20, 30, and 60 min post-injection. During the IV-GTT blood samples were also taken for the determination of insulin content via ELISA, conducted as per the manufacture’s instructions (Cayman Chemical). Serum ELISA for 17β-estradiol (Cayman Chemical; 501890), progesterone (Cayman Chemical; 582601) and Luteinizing hormone (Elabscience; E-EL-M3053) were performed as per manufacturers’ instructions

Zammit N.W., McDowell J., Warren J., Muskovic W., Gamble J., Shi Y.C., Kaczorowski D., Chan C.L., Powell J., Ormandy C., Brown D., Oakes S.R, & Grey S.T. (2022). TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation. Frontiers in Immunology, 13, 811525.

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10

Islet Transplantation in Diabetic Mice

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In brief, and as described previously [13 (link)], 80 hand-picked isolated islets were transplanted under the kidney capsule of syngenic C57BL/6 mice with streptozotocin-induced hyperglycaemia. Diabetes was defined as a blood glucose level ≥16 mmol/l on 2 consecutive days following i.v. injection of alloxan (110 mg/kg). Blood glucose levels of non-fasted mice were determined using a FreeStyle Lite glucometer and blood glucose test strips (Abbott Diabetes Care) via tail tipping.

Zammit N.W., Wong Y.Y., Walters S.N., Warren J., Barry S.C, & Grey S.T. (2023). RELA governs a network of islet-specific metabolic genes necessary for beta cell function. Diabetologia, 66(8), 1516-1531.

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Blood glucose test strips

Lab products found in correlation

11 protocols using blood glucose test strips

Adipocyte-specific Bola3 Knockout Mice Metabolic Profiling

Plasma Metabolite Analysis in Mice

Metabolic Effects of Cold Exposure and β-Blocker in Mice

Metabolic Regulation in Transgenic Mice

Metabolic Effects of Cold Exposure and β-Blocker in Mice

Metabolic Regulation in Transgenic Mice

Adipocyte-specific Bola3 Knockout Mice Metabolic Profiling

Streptozotocin-Induced Diabetes in Mice

Glucose and Hormone Metabolism Evaluation

Islet Transplantation in Diabetic Mice

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