Antidiabetic effect of Lactobacillus casei CCFM0412 on mice with type 2 diabetes induced by a high-fat diet and streptozotocin

Antidiabetic effect of Lactobacillus casei CCFM0412 on mice with type 2 diabetes induced by a high-fat diet and streptozotocinPei Chen Ph.D, Qiuxiang Zhang Ph.D , Hui Dang Ph.D , Xiaoming Liu Ph.D, Fengwei Tian Ph.D , Jianxin Zhao Ph.D. , Yongquan Chen Ph.D , Hao Zhang M.A. Wei Chen Ph.D

AbstractObjective:

The aim of this study was to evaluate the antidiabetic effects of Lactobacillus casei CCFM0412 on mice with type 2 diabetes induced by a high-fat diet and streptozotocin.

AbstractMethods:

Thirty-two male C57 BL/6 J mice were assigned to four groups in this study. Type 2 diabetes was induced by feeding of a high-fat diet and injection of streptozotocin. L. casei CCFM0412 was administered to mice at a dose of 10 *9 cfu/d per mouse for 12 wk. Body weight, fasting and postprandial 2-h blood glucose, oral glucose tolerance, glycosylated hemoglobin, insulin, and glycogen in liver were measured. Endotoxin, tumor necrosis factora, and interleukin-10 levels were determined. Lipid metabolic parameters including triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were also measured. The activities of glutathione peroxides, reactive oxygen species, and superoxide dismutase, and the levels of glutathione and malondialdehyde in the liver also were determined. Pancreas injury was evaluated by histologic analysis.

Abstract

Results:

At 13 wk, L. casei CCFM0412 signicantly decreased fasting and postprandial 2-h blood glucose, glycosylated hemoglobin, endotoxin, tumor necrosis factora, triglycerides, total cholesterol, low-density lipoprotein cholesterol, reactive oxygen species, and malondialdehyde levels compared with the control group (P < 0.05). The values for insulin, interleukin-10, high-density lipoprotein cholesterol, glutathione peroxides, superoxide dismutase, glutathione, and glycogen were signicantly increased at 13 wk (P < 0.05). Islets of Langerhans in the L. casei CCFM0412 group were substantially protected from destruction compared with those in the control group.

AbstractConclusion: L. casei CCFM0412 signicantly improved glucose intolerance, dyslipidemia, immune- regulatory properties, and oxidative stress in mice with type 2 diabetes induced by a high-fat diet and streptozotocin. The results provide a sound rationale for future clinical trials of oral administration of L. Casei CCFM0412 for the primary prevention of type 2 diabetes.

IntroductionThe incidence of type 2 diabetes mellitus (T2DM), which is characterized by abnormally high blood glucose due to a relative deciency of insulin [1], has rapidly increased in the world during the past few decades. It has been suggested that insulin resistance (IR) precedes the development of overt hyperglycemia, although the molecular basis for this has not been identied.

IntroductionFive classes of oral antidiabetic agents are available: aglucosidase inhibitors, sulfonylurea, meglitinides, biguanides, and thiazolidinediones. However, the use of these medicines has serious side effects and causes secondary failure, including bloating, atulence, and diarrhea. Lactic acid bacteria (LAB) have none of these side effects, thus can be considered. In recent years, many studies have demonstrated that LAB are useful in preventing or decreasing the progression of diabetes.

IntroductionOne study found that the blood glucose levels in type 2 diabetic mice administered Lactobacillus gasseri BNR17 were lower than those in a control group. Another study demonstrated that L. rhamnosus GG cells signicantly lowered blood glucose levels and improved hyperglycemia in neonatal streptozotocin-induced diabetic rats compared with a control group. Previous studies have shown that LAB can signicantly decrease the oxidative stress of high fructose-induced diabetic rats and enhance pancreatic glutathione biosynthesis, thus reducing oxidative stress in the pancreas. LABprevented or delayed the onset of diabetes in various experimental models in those studies. We hypothesized that LAB have an antidiabetic ability by their a-glucosidase inhibitory activity in vivo, improving plasma lipids, and increasing antioxidant potential in mice.

IntroductionWe isolated L. casei CCFM0412 from yogurt in our lab. Previous experiments showed that this strain exhibited good probiotic properties, including acid and bile salt tolerance, and antioxidant ability. Moreover, its cell-free supernatant inhibition rate of rat intestinal a-glucosidase was 30%. Several studies have demonstrated that the association of oxidative stress markers with diabetes and reactive oxygen species (ROS) plays an important role in the regulation of IR. Meanwhile, dyslipidemia is a common and important character in T2DM. It has been demonstrated that probiotics can improve dyslipidemia and delay the progression of high fructose-induced glucose intolerance in rats. Ourinvitro study has demonstrated that L. casei has an excellent ability to contribute an antioxidant effect. So in this study, we investigated whether L. casei CCFM0412 affected blood glucose levels and ameliorated the oral glucose tolerance in mice with type 2 diabetes induced by a high-fat diet and streptozotocin by improving dyslipidemia, immunoregulatory properties, and oxidative stress.

Materials and methodsPreparation of bacteria suspensionsL. casei CCFM0412 samples for administration to animals were prepared by suspending lyophilized bacteria powered with skim milk as its protectant [11]. Colony counting was conducted before the animal experiments to ensure the numbers of surviving bacteria were adjusted to

Materials and methods (cont)Experimental animalsThirty-two, 3-wk-old male C57 BL/6 J mice were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). The mice were housed in an animal room at constant temperature (22C 2C) and humidity (55% 5%) under a 12-h light12-h dark cycle in the animal facility at the Jiangnan University. The mice were randomly divided into four groups (n 8 per group) as follows: normal (N) group : mice without diabetes; control (C) group: mice with diabetes but no treatment; pioglitazone (P) group: mice with diabetes and treated with pioglitazone (a drug for treating diabetes); and the L. casei CCFM0412 (L) group: mice with diabetes and treated with L. casei CCFM0412.

Experimental animalsA diet of normal chow and water were freely accessible to the mice for 1 wk .Then, the C, P, and L groups were fed a high-fat diet, and the N group continued to consume normal chow for 12 wk.L group mice were administered 4 10 9 cfu/mL L. casei CCFM0412 250 mL/d for 12 wk. At 3 wk, C, P, and L groups were intraperitoneally injected with streptozotocin(Sigma, St. Louis, MO, USA) freshly dissolved in 50 mmol/L citrate buffer (pH 4.5) at a dose of 100 mg/kg, whereas the N group mice received 0.9% saline alone. Materials and methods (cont)

Experimental animalsAt 3 wk, C, P, and L groups were intraperitoneally injected with streptozotocin(Sigma, St. Louis, MO, USA) freshly dissolved in 50 mmol/L citrate buffer (pH 4.5) at a dose of 100 mg/kg, whereas the N group mice received 0.9% saline alone. After 1 wk (week 4), pioglitazone was administered to the P group at a dose of 10 mg/ kg. Body weights were measured weekly. Fasting and postprandial 2-h blood glucose levels were analyzed with a glucometer (HMD Biomedical, Taiwan, China) weekly from blood collected from the tail vein of the mice. This study was approved by the animal Ethics Committee of Jiangnan University, China (JN No. 26 2012).Materials and methods (cont)

Oral glucose tolerance testThe oral glucose tolerance tests (OGTT) is a primary outcome that often has been used to derive estimates of the relative roles of insulin secretion and IR in population studies of glucose tolerance. OGTT were performed in weeks 4 and 13. Before each test, mice were fasted for 12 h and blood glucose was measured (time 0). The glucose solution was prepared at a concentration of 2 g/kg and administered to the mice. Then, blood glucose values were analyzed at 15, 30, 60, 90, and 120 min.Materials and methods (cont)

Blood and tissue sample collectionOn the nal day of the experiment (week 13), blood samples were collected from the orbital venous plexus of 12-h fasted and anesthetized animals. The blood samples were centrifuged at 4000g for 10 min at 4C, and serum was obtained to analyze levels of glycosylated hemoglobin (HbA ), insulin, endotoxin, TNFa, IL-10, triglycerides (TGs), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The mice were sacriced by cervical dislocation. Liver and pancreatic tissues were immediately removed and rinsed with cold 0.9% saline. The liver was thenchopped into pieces before being stored at 80 1c C until use, and the pancreas was xed in 10% formalin saline.Materials and methods (cont)

Determination of HbA The values of HbA1c , insulin, endotoxin, TNF-a, and IL-10 reect overall glycemic exposure over the past 2 to 3 mo and are determined by both fasting and postprandial 2-h blood glucose levels . Insulin is produced by the pancreas, which regulates the level of glucose in the blood. TNFa and endotoxin are two contributing factors to IR and dyslipidemia\ . IL-10 plays an important role in the regulation of the innate immune 1c system to prevent the development of diabetes in non-obese diabetic mice [24]. Levels of HbA , insulin, TNF-a, and IL-10 were measured according to the recommendations of the manufacturer using enzymatic kits purchased from AbcamMaterials and methods (cont)

Determination of plasma lipidsAbnormal plasma lipids in patients with diabetes include elevated LDL-C andTGs, and reduced levels of HDL-C. These are associated with the onset of diabetes . Serum levels of TG, TC, LDL-C, and HDL-C were determined by enzymatic kits produced by Boster Bio-engineering Company Ltd. (Wuhan, China).Determination of GSH-px, ROS, SOD, GSH, MDA, and glycogen Accumulating evidence indicates that the generation of oxidative stress may play an important role in the etiology of diabetic complications [. Glycogen is mainly stored in the liver and the muscles, which is useful for providing a readily available source of glucose for the body. The activities of glutathione peroxides (GSH-px), ROS, superoxide dismutase (SOD), and the levels of glutathione (GSH), malondialdehyde (MDA), and glycogen in the mice livers were measured accordingto the recommendations of the manufacturer, using enzymatic kits produced by Boster Bio engineering Company Ltd.Histopathologic examination The pancreases were xed for 48 h in 10% formalin saline, embedded in parafn, serially cut at 5mm thickness with a rotary microtome, and routinelystained with hematoxylin-eosin (H-E) for light microscopy examination (DM2000; Leica, Bensheim, Germany).Materials and methods (cont)

Oral glucose tolerance testThe oral glucose tolerance tests (OGTT) is a primary outcome that often hasbeen used to derive estimates of the relative roles of insulin secretion and IR inpopulation studies of glucose tolerance [20,21]. OGTT were performed in weeks 4and 13. Before each test, mice were fasted for 12 h and blood glucose wasmeasured (time 0). The glucose solution was prepared at a concentration of2 g/kg and administered to the mice. Then, blood glucose values were analyzed at15, 30, 60, 90, and 120 min [13].