Background: Exercise and insulin stimulate the uptake of glucose into skeletal muscle via glucose transporter 4 (GLUT4).  Patients with diabetes mellitus (DM) develop insulin resistance and experience reduced uptake and metabolism of plasma glucose by skeletal muscle, resulting in hyperglycemia. Glucose metabolism in response to exercise is intact in patients with DM. Increased glucose transport in contracting skeletal muscles is mediated, in part, by 5′-adenosine monophosphate–activated protein kinase (AMPK), an energy and metabolite-sensing molecule in muscle cells that increases surface expression of GLUT4 and affects glycogen regulation, fatty acid oxidation, mitochondrial biogenesis, and insulin sensitivity. Skeletal muscle AMPK also regulates leptin and adiponectin activities on glucose and lipid homeostasis. Two isoforms of AMPK are expressed in skeletal muscle: AMPKα1 (the predominant isoform) and AMPKα2; however, it is unknown which isoform plays a role in regulating glucose uptake into skeletal muscle. Berberine (BBR) is the main alkaloid of Coptis chinensis, a folk medicine used in Asian countries to treat DM. Recent preclinical studies suggest a role for BBR in stimulation of glucose transport and activation of AMPK in cultured myocytes. Moreover, BBR and dihydroberberine, a biologically available derivative of BBR, may alter intracellular energy status by inhibiting the mitochondrial electron transport chain.

Hypothesis/purpose of study: The study was conducted to determine whether BBR enhances muscle glucose utilization in skeletal muscles via AMPKα1 and/or AMPKα2.

Subjects: Isolated epitrochlearis and soleus muscles from 5-week-old male Wistar rats were used for this preclinical trial.

Experimental design:Ex vivo BBR dose-response and muscle physiology study in isolated rat muscle

Treatments and protocol:Male rats were sacrificed and the epitrochlearis and soleus muscles, representative of fast-twitch and slow-twitch muscles, respectively, were surgically removed, sutured, and mounted in an incubation apparatus at 0.5 g tension. The epitrochlearis is a superficial muscle of the rodent arm and is not found in humans, whereas the soleus muscle is located in the calf of both rodents and humans. Muscles were washed with buffer to remove residual AMPK generated during muscle isolation. To measure the time course of changes in AMPK phosphorylation (activation), the muscles were preincubated with buffer (maintained at 5% CO2 and 37°C) for 30 minutes followed by treatment with BBR for 0, 15, 30, 45, or 60 minutes. The muscle was used fresh for glucose uptake analysis or snap frozen in liquid nitrogen prior to measurement of isoform-specific AMPK activity, adenosine triphosphate (ATP), phosphocreatine (PCr) concentrations, and Western blot analysis (ie, to determine change in specific protein expression). Western blot analysis was conducted for AMPKα1, AMPKα2, phosphorylated acetyl-coA carboxylase (pACC), p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated p44/42 MAPK. Immunoprecipitation of Akt substrate of 160 kd (AS160) was also conducted. Isoform-specific AMPK activity assays were conducted to assess which of the 2 AMPK isoforms were activated in response to treatment. Total cellular ATP and PCr were isolated and assessed for changes in response to treatment in each muscle type. Additionally, a 3-O-methyl-d-glucose (3MG) transport assay was performed to assess changes in muscle cell glucose uptake in response to treatment.