Antidiabetic effect of Lactobacillus casei

CCFM0412 on mice with type 2 diabetes

induced by a high-fat diet and
streptozotocin
Pei 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

Abstract
Objective:
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.

Abstract
Methods:
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.

Abstract
Conclusion:
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.

Introduction
The 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.

Introduction
Five 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.

Introduction
One 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.

Introduction
We 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 methods

Preparation of bacteria suspensions
L. 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 animals
Thirty-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:
1. normal (N) group : mice without diabetes;
2. control (C) group: mice with diabetes but no treatment;
3. pioglitazone (P) group: mice with diabetes and treated with pioglitazone (a drug for
treating diabetes); and
4. the L. casei CCFM0412 (L) group: mice with diabetes and treated with L. casei
CCFM0412.

Materials and methods (cont)

Experimental animals
A 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 animals
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.
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 test
The 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 collection
On 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 then
chopped 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 HbA
1c , 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 Abcam

Materials and methods (cont)

Determination of plasma lipids
Abnormal plasma lipids in patients with diabetes include elevated LDL-C and
TGs, 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 Bioengineering 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 according
to 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 routinely
stained with hematoxylin-eosin (H-E) for light microscopy examination (DM2000; Leica,
Bensheim, Germany).

 Oral glucose tolerance test

The 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 [20,21]. 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 [13].