Labassay glucose kit

Glycogen levels in the muscles collected from the mice were measured using a method based on that of Chan et al. (24 (link)). Briefly, the tissues were weighed using a microanalytical balance, dissolved in 30% KOH at 95°C, 2% Na₂SO₄, and 66% ethanol were then added, and the final solution was centrifuged (13,000 × g, 5 min at 4°C) to obtain a pellet. The pellet was washed twice with 66% ethanol, 0.2 M acetate buffer (pH 4.5) and amyloglucosidase (Sigma-Aldrich, Tokyo, Japan) were added, and the solution was incubated at 37°C for 30 min. The glucose content was measured using the LabAssay™ Glucose kit (Wako, Tokyo, Japan) and the amount of glycogen per gram of tissue (wet weight) was calculated.

For the unilateral ureteral obstruction (UUO) model, C57BL/6 J male mice (n = 4–5, age, 8 weeks) were anesthetized under isoflurane and the left ureter was accessed dorsally and ligated (or not, for sham-operated controls) [11 (link)]; blood was collected from the abdominal aorta 7 days after ligation. For streptozocin (STZ)-induced diabetic mice, C57BL/6 J male mice (n = 5–6, age, 8 weeks) received STZ (50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) or citrate buffer (pH 4.5) intraperitoneally once daily for 5 consecutive days; 11 weeks after the last dose of STZ, blood was collected from the abdominal femoral artery. The urinary protein level in each mouse was measured by using Micro TP-Test photometric (FujiFilm Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer’s protocol. Blood glucose concentration was measured by using a LabAssay Glucose kit (FujiFilm Wako Pure Chemical Corporation) according to manufacturer’s protocol.

Blood was collected from a tail vein before and after exercise to measure the blood lactate concentration, as an indicator of exercise intensity (Lactate Pro2 LT‐1730, Arklay). Serum lipopolysaccharide (LPS) was measured using a Glucoshield Buffer kit (Associates of Cape Cod Inc.). The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using standard protocols (Oriental Yeast Co., Ltd.). Serum glucose was measured using a LabAssay Glucose kit (Wako) and serum insulin was measured using a Mouse Insulin ELISA kit (Morinaga). Serum DHEA was measured using a DHEA ELISA kit (Enzo Life Sciences Inc.) and serum corticosteroids were measured using a Corticosterone ELISA kit (Cayman Chemical Company). Serum myostatin was measured using a GDF‐8/Myostatin Quantikine ELISA kit (R&D Systems) and serum follistatin was measured using a Mouse Follistatin ELISA kit (Raybiotech Life Inc.). Absorbances were measured using a Varioskan microplate reader (Thermo Fisher Scientific).

Cells grown under continuous light conditions were harvested, washed twice with distilled water, and resuspended in 500 µl of 3.5% (v/v) sulfuric acid, followed by incubation at 100 °C for 120 min. Glycogen was quantified using a LabAssay Glucose Kit (Wako, Japan) as described previously^{27 (link)}.

For diabetic nephropathy therapeutic tests, urine and blood were sampled on each of the following days: 0, 20, 60, and 120 after the final administration of STZ on day 5. Each serum sample was prepared from blood by centrifugation at 1200× g for 20 min at 4 °C. Urine albumin, urine creatinine, serum creatinine, and serum albumin were measured using an Albuwell M Test kit (#1011; Ethos Biosciences, Logan Township, NJ, USA), the creatinine Companion (#1012; Ethos Biosciences), and the Creatinine Assay kit QuantiChrom (#DICT-500, BioAssay Systems, Hayward, CA, USA), respectively. Blood glucose levels in the serum samples were measured using a LabAssay Glucose kit (#298-65701, FUJIFILM Wako, Osaka, Japan).

Twenty-four-hour urine was collected in a metabolic cage at day 42. Urinary glucose levels were measured using an enzymatic colorimetric assay (LabAssay Glucose Kit; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Non-esterified fatty acid (NEFA), triglycerides (TG), 3-hydroxybutyrate (3-OHBA) levels were measured at day 39 by enzymatic colorimetric assays (LabAssay NEFA Kit and LabAssay Triglyceride Kit; Wako Pure Chemical Industries, Ltd., Osaka, Japan; Ketorex Kit; Sanwa Kagaku Kenkyusho Co.Ltd., Nagoya, Japan) after overnight fasting. Plasma insulin was measured using a commercial ELISA kit (Ultra Sensitive Mouse Insulin ELISA Kit; Morinaga Institute of Biological Science Inc. Yokohama, Japan). Glycated hemoglobin (HbA1c) levels were measured using DCA Vantage Analyzer (Siemens, Munich, Germany) at day 53.