Long-Term Cold Adaptation Does Not Require FGF21 or UCP1.

Brown adipose tissue (BAT)-dependent thermogenesis and its suggested augmenting hormone, FGF21, are potential therapeutic targets in current obesity and diabetes research. Here, we studied the role of UCP1 and FGF21 for metabolic homeostasis in the cold and dissected underlying molecular mechanisms using UCP1-FGF21 double-knockout mice. We report that neither UCP1 nor FGF21, nor even compensatory increases of FGF21 serum levels in UCP1 knockout mice, are required for defense of body temperature or for maintenance of energy metabolism and body weight. Remarkably, cold-induced browning of inguinal white adipose tissue (iWAT) is FGF21 independent. Global RNA sequencing reveals major changes in response to UCP1- but not FGF21-ablation in BAT, iWAT, and muscle. Markers of mitochondrial failure and inflammation are observed in BAT, but in particular the enhanced metabolic reprogramming in iWAT supports the thermogenic role of UCP1 and excludes an important thermogenic role of endogenous FGF21 in normal cold acclimation.

Aspalathin, a dihydrochalcone C-glucoside, protects H9c2 cardiomyocytes against high glucose induced shifts in substrate preference and apoptosis.

SCOPE: Energy deprivation in the myocardium is associated with impaired heart function. This study aims to investigate if aspalathin (ASP) can ameliorate hyperglycemic-induced shift in substrate preference and protect the myocardium against cell apoptosis. METHODS AND RESULTS: H9c2 cells were exposed to, either normal (5.5 mM) or high (33 mM) glucose concentrations for 48 h. Thereafter, cells exposed to 33 mM glucose were treated with metformin (1 µM) or ASP (1 µM), as well as a combination of metformin and ASP for 6 h. In vitro studies revealed that ASP improved glucose metabolism by decreasing fatty acid uptake and subsequent ß-oxidation through the decreased expression of adenosine monophosphate-activated protein kinase threonine 172 (pAMPK (Thr172)) and carnitine palmitoyltransferase 1 (CPT1), while increasing acetyl-CoA carboxylase (ACC) and glucose transporter 4 (GLUT4) expression. ASP inhibited high glucose induced loss of membrane potential in H9c2 cells as observed by an increase in 5' ,6,6'-tetrachloro-1,1',3,3' -tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) ratio (orange\red fluorescence) and decreased apoptosis by reducing intracellular reactive oxygen species and DNA nick formation, while increasing glutathione, superoxide dismutase, uncoupling protein 2 (UCP2), and Bcl-2\Bax ratio. CONCLUSION: Our study provides evidence that ASP increases glucose oxidation and modulates fatty acid utilization producing a favorable substrate shift in H9c2 cardiomyocytes exposed to high glucose. Such a favorable shift will be of importance in the protection of cardiomyocytes in the diabetic heart.

Assessing similarity analysis of chromatographic fingerprints of Cyclopia subternata extracts as potential screening tool for in vitro glucose utilisation.

Similarity analysis of the phenolic fingerprints of a large number of aqueous extracts of Cyclopia subternata, obtained by high-performance liquid chromatography (HPLC), was evaluated as a potential tool to screen extracts for relative bioactivity. The assessment was based on the (dis)similarity of their fingerprints to that of a reference active extract of C. subternata, proven to enhance glucose uptake in vitro and in vivo. In vitro testing of extracts, selected as being most similar (n = 5; r ≥ 0.962) and most dissimilar (n = 5; r ≤ 0.688) to the reference active extract, showed that no clear pattern in terms of relative glucose uptake efficacy in C2C12 myocytes emerged, irrespective of the dose. Some of the most dissimilar extracts had higher glucose-lowering activity than the reference active extract. Principal component analysis revealed the major compounds responsible for the most variation within the chromatographic fingerprints, as mangiferin, isomangiferin, iriflophenone-3-C-ß-D-glucoside-4-O-ß-D-glucoside, iriflophenone-3-C-ß-D-glucoside, scolymoside, and phloretin-3',5'-di-C-ß-D-glucoside. Quantitative analysis of the selected extracts showed that the most dissimilar extracts contained the highest mangiferin and isomangiferin levels, whilst the most similar extracts had the highest scolymoside content. These compounds demonstrated similar glucose uptake efficacy in C2C12 myocytes. It can be concluded that (dis)similarity of chromatographic fingerprints of extracts of unknown activity to that of a proven bioactive extract does not necessarily translate to lower or higher bioactivity.

Aspalathin improves glucose and lipid metabolism in 3T3-L1 adipocytes exposed to palmitate.

SCOPE: Saturated-free fatty acids, such as palmitate, are associated with insulin resistance. This study aimed to establish if an aspalathin-enriched green rooibos extract (GRE) and, its major flavanoid, aspalathin (ASP) could contribute significantly to the amelioration of experimentally induced insulin resistance in 3T3-L1 adipocytes. METHODS AND RESULTS: 3T3-L1 adipocytes were cultured in DMEM containing 0.75 mM palmitate for 16 h to induce insulin resistance before treatment for 3 h with GRE (10 µg/mL) or ASP (10 µM). GRE and ASP reversed the palmitate-induced insulin resistance. At a protein level GRE and ASP suppressed nuclear factor kappa beta (NF-κB), insulin receptor substrate one (serine 307) (IRS1 (Ser (307) )) and AMP-activated protein kinase phosphorylation and increased serine/threonine kinase AKT (AKT) activation, while only GRE increased glucose transporter four (Glut4) protein expression. Peroxisome proliferator-activated receptor alpha and gamma (PPARα and γ), and carnitine palmitoyltransferase one (CPT1) expression were increased by ASP alone. CONCLUSION: Together these effects offer a plausible explanation for the ameliorative effect of GRE and ASP on insulin-resistance, an underlying cause for obesity and type 2 diabetes.

In vitro antihyperlipidemic potential of triterpenes from stem bark of Protorhus longifolia.

Two lanostane triterpenes, 3ß-hydroxylanosta-9,24-dien-21-oic acid (1) and methyl-3ß-hydroxylanosta-9,24-dien-21-oate (2), were isolated from the stem bark of Protorhus longifolia. Their structures were deduced on the basis of spectroscopic analysis (NMR, HRMS, IR). This study investigated the in vitro anti-adipogenic activity of the two triterpenes. Their inhibitory activity was evaluated on selected lipid digestive enzymes (pancreatic lipase and cholesterol esterase). The inhibitory activity of the compounds on hormone-sensitive lipase and their ability to bind bile acids were also evaluated. The effect of the compounds on glucose uptake in C2C12 muscle cells and 3T3-L1 adipocytes, and on triglyceride accumulation in 3T3-L1 adipocytes was investigated. The triterpenes effectively inhibited the activities of the enzymes with IC50 values ranging from 0.04 to 0.31 mg/mL. The compounds showed a high affinity for secondary bile acids. Both compounds stimulated glucose uptake in C2C12 muscle cells and 3T3-L1 adipocytes. Compound 1 significantly reduced triglyceride accumulation in mature differentiated 3T3-L1 adipocytes. It is apparent that these lanostane triterpenes enhance glucose uptake and suppress adipogenesis, which together with their inhibitory effects on lipid digestive enzymes suggests that they have antihyperlipidemic potential.

Benzophenone C- and O-glucosides from Cyclopia genistoides (Honeybush) inhibit mammalian α-glucosidase.

An enriched fraction of an aqueous extract prepared from the aerial parts of Cyclopia genistoides Vent. yielded a new benzophenone di-C,O-glucoside, 3-C-ß-d-glucopyranosyl-4-O-ß-d-glucopyranosyliriflophenone (1), together with small quantities of a known benzophenone C-glucoside, 3-C-ß-d-glucopyranosylmaclurin (2). The isolated compounds showed α-glucosidase inhibitory activity against an enzyme mixture extracted from rat intestinal acetone powder. Compound 2 exhibited significantly (p < 0.05) higher inhibitory activity (54%) than 1 (43%) at 200 µM. In vitro tests in several cell models showed that 1 and its 3-C-monoglucosylated derivative (3-C-ß-d-glucopyranosyliriflophenone) were marginally effective (p ≥ 0.05) in increasing glucose uptake.

Effects of fermented rooibos (Aspalathus linearis) on adipocyte differentiation.

Rooibos (Aspalathus linearis) contains a rich complement of polyphenols, including flavonoids, considered to be largely responsible for its health promoting effects, including combatting obesity. The purpose of this study was to examine the effect of fermented rooibos hot water soluble solids on in vitro adipocyte differentiation by using differentiating 3T3-L1 adipocytes. Hot water soluble solids were obtained when preparing an infusion of fermented rooibos at "cup-of-tea" strength. The major phenolic compounds (>5 mg/g) were isoorientin, orientin, quercetin-3-O-robinobioside and enolic phenylpyruvic acid-2-O-ß-D-glucoside. Treatment of 3T3-L1 adipocytes with 10 µg/ml and 100 µg/ml of the rooibos soluble solids inhibited intracellular lipid accumulation by 22% (p<0.01) and 15% (p<0.05), respectively. Inhibition of adipogenesis was accompanied by decreased messenger RNA (mRNA) expression of PPARγ, PPARα, SREBF1 and FASN. Western blot analysis exhibited decreased PPARα, SREBF1 and AMPK protein expression. Impeded glycerol release into the culture medium was observed after rooibos treatment. None of the concentrations of rooibos hot water soluble solids was cytotoxic, in terms of ATP content. Interestingly, the higher concentration of hot water soluble solids increased ATP concentrations which were associated with increased basal glucose uptake. Decreased leptin secretion was observed after rooibos treatment. Our data show that hot water soluble solids from fermented rooibos inhibit adipogenesis and affect adipocyte metabolism, suggesting its potential in preventing obesity.