Plasma measurements

Whole blood was collected from the tail tips of iC-GHRKO (n = 8) and littermate controls (n = 24) after a 12-hour overnight fast by clipping approximately 1 mm of the tail tip and collecting approximately 250 μL of blood using heparinized capillary tubes. Plasma was collected after centrifugation for 10 minutes at 7000 × g at 4°C. Circulating levels of insulin were determined using the mouse insulin ELISA (number 80-INSMS-E01; ALPCO) following the manufacturer's protocol. Levels of circulating IGF-1 were measured using the mouse IGF-1 ELISA (number 22-IG1MS-E01; ALPCO) following the manufacturer's directions. C-peptide, leptin, resistin, monocyte chemoattractant protein 1 (MCP-1), and IL-6 were measured in samples from 12.5-month-old mice using a Milliplex mouse metabolic panel (number MMHMAG-44K; Millipore) according to the manufacturer's instructions.