Combination Effects of Metformin and a Mixture of Lemon Balm and Dandelion on High-Fat Diet-Induced Metabolic Alterations in Mice

Abstract

Metformin, the first-line drug for type 2 diabetes mellitus (T2DM), has additional effects on improvements of nonalcoholic fatty liver disease (NAFLD); however, there are no treatments for both T2DM and NAFLD. Previous studies have shown hepatoprotective effects of a mixture of lemon balm and dandelion (LD) through its antioxidant and anti-steatosis properties. Thus, combination effects of metformin and LD were examined in a high-fat diet (HFD)-induced metabolic disease mouse model. The model received an oral administration of distilled water, monotherapies of metformin and LD, or a metformin combination with LD for 12 weeks. The HFD-induced weight gain and body fat deposition were reduced more by the combination than either monotherapy. Blood parameters for NAFLD (i.e., alanine aminotransferase and triglyceride), T2DM (i.e., glucose and insulin), and renal functions (i.e., blood urea nitrogen and creatinine) were reduced in the combination. The combination further enhanced hepatic antioxidant activities, and improved insulin resistance via the AMP-activated protein kinase and lipid metabolism pathways. Histopathological analyses revealed that the metformin combination ameliorated the hepatic hypertrophy/steatosis, pancreatic endocrine/exocrine alteration, fat tissue hypertrophy, and renal steatosis, more than either monotherapy. These results suggest that metformin combined with LD can be promising for preventing and treating metabolic diseases involving insulin resistance.

Keywords: HFD; NAFLD; T2DM; combination; dandelion; dyslipidemia; kidney; lemon balm; metformin; obesity.

Figure 1

Combination effects on weight loss and energy metabolism. (a) Kinetic changes in body weights. (b) Total weight gains for 12 weeks. (c) Food consumption. (d,e) Fecal excretion of triglyceride (TG) and total cholesterol (Chol). Values are represented as the means ± standard deviations (SDs) (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 2

Combination effects on inhibition of body fat deposition. (a) Representative images in necropsy (upper) and live dual-energy X-ray absorptiometry (DEXA; lower). In live DEXA images, red, yellow, and blue indicate high-, intermediate-, and low-density fats, respectively. Scale bars = 2 cm. (b,c) Fat densities of total body and the abdominal region. Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 3

Combination effects on HFD-induced organ weights. Absolute organ weights of the liver, pancreas, kidney, and abdominal/periovarian fat mass, and the relative weights to the body weights, are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 4

Combination effects on parameters in blood biochemistry. (a–n) Blood levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), glucose, insulin, blood glycated hemoglobin (HbA1c), blood urea nitrogen (BUN), creatinine, triglyceride, total cholesterol (Chol), and low-/high-density lipoprotein cholesterol (LDL/HDL). Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 5

Combination effects on hepatic antioxidant defense system and glucose regulation. (a–d) Levels of malondialdehyde (MDA) and glutathione (GSH), activities of catalase and superoxide dismutase (SOD). (e–g) Glucose-regulating enzyme activities of glucokinase (GK), glucose-6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK). Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 6

Combination effects on gene expression involved in metabolic alteration. (a–c) mRNA expressions of acetyl-CoA carboxylase 1 (ACC1), AMP-activated protein kinase (AMPK)α1, and AMPKα2 in the liver. (d–l) mRNA expressions of leptin, adiponectin, uncoupling protein (UCP)2, CCAAT-enhancer-binding protein (C/EBP)α, C/EBPβ, sterol-regulatory-element-binding protein 1c (SREBP1c), peroxisome proliferator-activated receptor (PPAR)α, PPARγ, and fatty acid synthase (FAS) in the periovarian fat tissues. Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 7

Histopathological improvements in lesions of the liver. (a) Representative images in stains with hematoxylin and eosin and oil red O. CV = central vein. Scale bars = 50 μm. (b,c) Sizes of the hepatocytes and steatosis areas stained with oil red O. Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 8

Histopathological improvements in pancreatic lesions. (a) Representative images in stains with hematoxylin and eosin and immuno-stains for insulin and glucagon. In hematoxylin and eosin stains, pancreatic islet and exocrine duct (upper) are indicated, and the islets and acinar regions were highly magnified (middle and lower, respectively). Scale bars = 50 μm. (b,c) Numbers and sizes of the islets. (d) Acinar areas containing zymogen granules. (e–g) Numbers of insulin- (IR) and glucagon-immunoreactive (GR) cells, and ratio of IR to GR cells. Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.

Figure 9

Histopathological improvements in lesions in the kidney and fat tissue. (a) Representative images of the kidney and abdominal (A.)/periovarian (O.) fat tissue in stains with hematoxylin and eosin. Each image was highly magnified (lower rows). Scale bars = 50 μm. (b) Areas with renal tubular (Tubul.) vacuolation. (c–f) Thickness of the A./O. fat tissues and sizes of the adipocytes. Values are represented as the means ± SDs (n = 8). †, *, #, and $: p < 0.05 vs. the Intact, HFD control (HFD), Met, and LD(H) groups, respectively.