Berberine-induced activation of 5'-adenosine monophosphate-activated protein kinase and glucose transport in rat skeletal muscles
Journal Title (Medline/Pubmed accepted abbreviation): Metabol Clin Exp
Year: 2010
Volume: 59
Page numbers: 1619-1627
doi: 10.1016/j.metabol.2010.03.009

Summary of Background and Research Design

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.

Summary of research findings:
  • BBR treatment increased
    • Thr172 phosphorylation of the catalytic α-subunit of AMPK in a dose- and time-dependent manner .
      • This phosphorylation is an essential step for full kinase activation.
    • Ser79 phosphorylation of ACC.
      • ACC is an intracellular substrate of AMPK activity.
  • BBR treatment activated both the α1 and α2 isoforms of AMPK.
  • Increased enzyme activity was associated with
    • Increased rate of 3MG uptake (glucose transport) in the absence of insulin.
    • Phosphorylation of AS160, a signaling precursor to GLUT4 translocation to the cell surface.
  • BBR decreased the total intracellular energy status (as estimated from phosphocreatine concentration).
    • Changes in ATP, however, were not significant.

Interpretation of findings/Key practice applications:

Three novel findings on the effect of BBR on skeletal muscle were observed:
  • BBR treatment increased phosphorylation of residues required for full activation of AMPK, suggesting an increase in AMPK activity
  • BBR elicited these effects in both fast-twitch (epitrochlearis) and slow-twitch (soleus) muscles
  • Activation of AMPK in response to BBR reduced fuel status of skeletal muscle
These results suggest that BBR increases glucose transport in skeletal muscle and reduces intracellular energy in a manner similar to insulin, caffeine administration, or muscle contraction (although not necessarily of the same magnitude). These results also suggest that BBR exerts its actions nonspecifically in both faster glycolytic and slower oxidative muscles. The authors speculate that these data, when viewed in light of data from other, longer-term studies (up to 8-wk), suggest potential long-term metabolic benefits of BBR in reducing residual energy (ie, hyperglycemia) and potentially protecting against the development of type 2 DM and obesity. A key limitation of this study is that it was an ex vivo study on rat tissues. It is not known if the doses used could be representative of human exposure.
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