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High fat diet (55% fat by calories) will be fed ad libitum for 3 or more weeks to generate diet-induced obesity. Quality control studies have shown that a 3-week feeding of high fat diet increases whole body fat mass by more than 2-fold and causes insulin resistance in skeletal muscle, liver, adipose tissue and heart of male C57Bl6 mice. Longer term high fat feeding further increases whole body adiposity and causes more severe insulin resistance in these organs associated with alteration in insulin signaling and adipokines.
Acute intravenous lipid infusion (5 ml/kg/hr, triglyceride emulsion) and heparin (6 U/hr) will be performed for 5 hrs to induce hyperlipidemia that will be followed by in vivo experiments. Quality control studies have shown that a 5-hr lipid infusion increases circulating fatty acids to 2~4 mM and causes insulin resistance associated with defects in IRS-associated insulin signaling without alteration in adipokines.
Intraperitoneal injection of streptozotocin (50 mg/kg daily for 5 days), which selectively destroys the pancreatic β-cells with rapid and irreversible necrosis, will be applied to generate a chronic model of hyperglycemia/hypoinsulinemia. Following streptozotocin injection, blood glucose levels will be monitored for the onset and maintenance of hyperglycemia. For selected mouse models, other doses of streptozotocin may be required to maintain chronic hyperglycemia.
Acute or chronic treatment of phloridzin (100 µg/kg/min for acute treatment, 0.4 mg/kg body weight twice daily for chronic treatment) will be applied to reduce plasma glucose levels in hyperglycemic mice prior to the metabolic experiments. Insulin clamp requires maintenance of constant basal glucose levels (6~7 mM) which is difficult to achieve in severely hyperglycemic and insulin resistant mice. Thus, phloridzin treatment will be used to lower blood glucose levels.