5-aminolevulinic acid combined with ferrous ion reduces adiposity and improves glucose tolerance in diet-induced obese mice via enhancing mitochondrial function

Abstract

Background: Mitochondrial dysfunction is associated with obesity and various obesity-associated pathological conditions including glucose intolerance. 5-Aminolevulinic acid (ALA), a precursor of heme metabolites, is a natural amino acid synthesized in the mitochondria, and various types of cytochromes containing heme contribute to aerobic energy metabolism. Thus, ALA might have beneficial effects on the reduction of adiposity and improvement of glucose tolerance through its promotion of heme synthesis. In the present study, we investigated the effects of ALA combined with sodium ferrous citrate (SFC) on obesity and glucose intolerance in diet-induced obese mice.

Methods: We used 20-weeks-old male C57BL/6J diet-induced obesity (DIO) mice that had been fed high-fat diet from 4th week or wild-type C57BL/6J mice. The DIO mice were orally administered ALA combined with SFC (ALA/SFC) for 6 weeks. At the 4th and 5th week during ALA/SFC administration, mice were fasted for 5 h and overnight, respectively and used for oral glucose tolerance test. After the ALA/SFC administration, the plasma glucose levels, weight of white adipose tissue, and expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes were examined. Furthermore, the effects of ALA/SFC on lipid content and glucose uptake were examined in vitro.

Results: Oral administration of ALA/SFC for 6 weeks reduced the body weight by about 10% and the weight of white adipose tissues in these animals. In vitro, ALA/SFC reduced lipid content in mouse 3T3-L1 adipocytes in a dose dependent manner, and enhanced glucose uptake in 3T3-L1 adipocytes by 70-90% and rat L6 myoblasts by 30% at 6 h. Additionally, oral administration of ALA/SFC reduced plasma glucose levels and improved glucose tolerance in DIO mice. Furthermore, ALA/SFC enhanced the expression of OXPHOS complexes III, IV, and V by 40-70% in white adipose tissues of DIO mice, improving mitochondrial function.

Conclusions: Our findings indicate that ALA/SFC is effective in the reduction of adiposity and improvement of glucose tolerance, and that the induction of mitochondrial OXPHOS complex III, IV, and V by ALA/SFC might be an essential component of the molecular mechanisms underlying these effects. ALA/SFC might be a useful supplement for obesity and obesity-related metabolic disease such as type 2 diabetes mellitus.

Keywords: 5-aminolevulinic acid; Diet-induced obese mice; Glucose tolerance; Glucose uptake; Mitochondrial oxidative phosphorylation complex; Obesity; White adipose tissue.

Fig. 1

Heme synthesis pathway in animal cells. ALA is synthesized by the condensation of glycine and succinyl CoA via mitochondrial ALA synthase. The polymerization of 8 molecules of ALA in several subsequent steps produces protoporphyrin IX (PpIX). Heme is synthesized by the insertion of a ferrous ion into PpIX, and incorporated into proteins to produce heme-proteins such as hemoglobin, cytochrome, and P450

Fig. 2

ALA/SFC reduces adiposity in DIO mice. DIO mice were orally administered ALA/SFC for 6 weeks. The numbers of mice in HFD + vehicle, HFD + ALA/SFC, and ND + vehicle groups were 5, 5, and 8, respectively. a Body weight. *p < 0.05 vs. vehicle-administered DIO mice (Student’s t-test). b Food intake (Student’s t-test). c Weights of epididymal, retroperitoneal, and mesenteric fat tissues normalized by body weight. **p < 0.01 vs. vehicle-administered DIO mice (Student’s t-test)

Fig. 3

ALA/SFC reduces lipid content in 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in adipocyte maintenance medium with or without ALA/SFC for the indicated times, stained with Oil Red O, and the extracted Oil red O was quantified by its absorbance at 540 nm. Results are shown as means ± SD relative to the control (set to 1.0). a Cell staining was observed after ALA (1 mM)/SFC (0.5 mM) treatment for 24 h. Representative figures are shown (original magnification, × 400). b Time-dependent effects of ALA/SFC. The cells were treated with ALA (1 mM)/SFC (0.5 mM) for the indicated times and then stained with Oil Red O. **p < 0.01, ***p < 0.001 vs. control (Dunnett’s test, n = 3). c Effects of 24-h treatment of various ALA/SFC concentrations on lipid content. **p < 0.01, ***p < 0.001 vs. control (Dunnett’s test, n = 4). d Effects of 24-h treatments with ALA (1 mM) alone, SFC (0.5 mM) alone, or ALA (1 mM)/SFC (0.5 mM) in combination on lipid content. *p < 0.05, ***p < 0.001 vs. control (Bonferroni’s test, n = 4)

Fig. 4

ALA/SFC improves glucose tolerance. DIO mice were orally administered ALA/SFC for 6 weeks. The numbers of mice in HFD + vehicle, HFD + ALA/SFC, and ND + vehicle groups were 5, 5, and 8, respectively. a Plasma glucose levels after 6-weeks administration of ALA/SFC. *p < 0.05, **p < 0.01 vs. vehicle-administered DIO mice (Dunnett’s test). b, c At 4th week after ALA/SFC administration, mice were fasted for 5 h and then OGTT was performed. b Fasting plasma glucose levels. c Plasma glucose levels and AUC. *p < 0.05, **p < 0.01 vs. vehicle-administered DIO mice (Dunnett’s test). d, e At 5th week of ALA/SFC administration, mice were fasted overnight and then OGTT was performed. d Fasting plasma glucose levels. e Plasma glucose levels and AUC. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle-administered DIO mice (Dunnett’s test)

Fig. 5

ALA/SFC induces glucose uptake in cells. Differentiated 3T3-L1 or L6 cells were incubated in KRPH buffer for 18 min at 37 °C and then further incubated with KRPH buffer containing 1 mM 2DG with or without insulin for 20 min. Results are shown as means ± SD relative to the 2DG uptake of controls (set to 1.0). a The effect of treatment of ALA (0.5 mM)/SFC (0.25 mM) on glucose uptake for the indicated times in 3T3-L1 adipocytes. *p < 0.05, **p < 0.01, vs. control (Dunnett’s test, n = 3). b The effect of only ALA (0.5 mM), SFC (0.25 mM), or ALA (0.5 mM)/SFC (0.25 mM) treatment for 6 h on glucose uptake in 3T3-L1 adipocytes. **p < 0.01, ***p < 0.001 vs. control (Bonferroni’s test, n = 3). c The effect of treatment of ALA (0.5 mM)/SFC (0.25 mM) on glucose uptake for 6 h in L6 myotubes. Insulin (100 nM) was used as a positive control. ***p < 0.001 vs. control (Dunnett’s test, n = 3). d The effect of ALA (0.5 mM)/SFC (0.25 mM) treatment on insulin action to glucose uptake in 3T3-L1 adipocytes. The concentration of insulin was 10 nM. *p < 0.05, ***p < 0.001 vs. control (Dunnett’s test, n = 4)

Fig. 6

Expression levels of mitochondrial complexes I–V in epididymal WAT of DIO mice. a The expression levels of complex I–V proteins in WAT were analyzed by immunoblotting. b The relative expression levels of complexes I–V to β-actin. The expression levels of ALA/SFC-administered mice are shown as relative values compared to those of the vehicle-administered DIO mice. *p < 0.05, **p < 0.01 vs. vehicle-administered DIO mice (Student’s t-test, n = 5)