title: Amelioration of palmitate-induced insulin resistance in C₂C₁₂ muscle cells by rooibos (Aspalathus linearis).
abstract: Increased levels of free fatty acids (FFAs), specifically saturated free fatty acids such as palmitate are associated with insulin resistance of muscle, fat and liver. Skeletal muscle, responsible for up to 80% of the glucose disposal from the peripheral circulation, is particularly vulnerable to increased levels of saturated FFAs. Rooibos (Aspalathus linearis) and its unique dihydrochalcone C-glucoside, aspalathin, shown to reduce hyperglycemia in diabetic rats, could play a role in preventing or ameliorating the development of insulin resistance. This study aims to establish whether rooibos can ameliorate experimentally-induced insulin-resistance in C₂C₁₂ skeletal muscle cells. Palmitate-induced insulin resistant C₂C₁₂ cells were treated with an aspalathin-enriched green (unfermented) rooibos extract (GRE), previously shown for its blood glucose lowering effect in vitro and in vivo or an aqueous extract of fermented rooibos (FRE). Glucose uptake and mitochondrial activity were measured using 2-deoxy-[³H]-D-glucose, MTT and ATP assays, respectively. Expression of proteins relevant to glucose metabolism was analysed by Western blot. GRE contained higher levels of all compounds, except the enolic phenylpyruvic acid-2-O-glucoside and luteolin-7-O-glucoside. Both rooibos extracts increased glucose uptake, mitochondrial activity and ATP production. Compared to FRE, GRE was more effective at increasing glucose uptake and ATP production. At a mechanistic level both extracts down-regulated PKC θ activation, which is associated with palmitate-induced insulin resistance. Furthermore, the extracts increased activation of key regulatory proteins (AKT and AMPK) involved in insulin-dependent and non-insulin regulated signalling pathways. Protein levels of the glucose transporter (GLUT4) involved in glucose transport via these two pathways were also increased. This in vitro study therefore confirms that rooibos can ameliorate palmitate-induced insulin resistance in C₂C₁₂ skeletal muscle cells. Inhibition of PKC θ activation and increased activation of AMPK and AKT offer a plausible mechanistic explanation for this ameliorative effect.
authors: Diabetes Discovery Platform, South African Medical Research Council, P.O. Box 19070, Tygerberg 7505, South Africa. Sithandiwe.mazibuko@mrc.ac.za
authors: S E
authors: SE
authors: Mazibuko
authors: C J F
authors: CJ
authors: Muller
authors: E
authors: E
authors: Joubert
authors: D
authors: D
authors: de Beer
authors: R
authors: R
authors: Johnson
authors: A R
authors: AR
authors: Opoku
authors: J
authors: J
authors: Louw
mesh_terms: D000818:Animals; D036903:Aspalathus / Q000737:chemistry; D002460:Cell Line; D006946:Hyperinsulinism / Q000139:chemically induced / Q000188:drug therapy; D007333:Insulin Resistance / Q000502:physiology*; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D019308:Palmitic Acid / Q000494:pharmacology / Q000627:therapeutic use*; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D010936:Plant Extracts; D019308:Palmitic Acid
keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5′ adenosine monophosphate-activated protein kinase; AKT; AMPK; ATP; Aspalathin; Aspalathus linearis; BSA; DAG; DMEM; DPBS; Dulbecco's Modified Eagle Medium; Dulbecco's phosphate buffered saline; FCS; FFAs; FRE; GLUT4; GRE; Glucose uptake; HPLC-DAD; HPR; IR; IRS1; IgG; Insulin resistance; MTT; PKC θ; PMSF; Palmitate; RNAse; Rooibos; T2D; TBST; Tris-buffered saline with Tween; adenosine triphosphate; aspalathin-enriched green rooibos extract; bovine serum albumin; diacylglycerol; fermented rooibos extract; fetal calf serum; free fatty acids; glucose transporter type 4; high-performance liquid chromatography with diode-array detection; horseradish peroxidise; immunoglobulin G; insulin receptor substrate 1; insulin resistance; pAKT; pAMPK; pPKC θ; phenylmethanesulfonylfluoride; phosphorylated 5′ adenosine monophosphate-activated protein kinase; phosphorylated protein kinase; phosphorylated protein kinase C theta; protein kinase B; protein kinase C theta; ribonuclease; tAKT; tAMPK; tPKC θ; total 5′ adenosine monophosphate-activated protein kinase; total protein kinase B; total protein kinase C theta; type 2 diabetes