Antidiabetic and vasoprotective activity of lithium: Role of glycogen synthase kinase-3.

OBJECTIVES: Lithium is a drug of choice in maniac disorder. Lithium inhibits the glycogen synthase kinase-3 (GSK-3), an enzyme involved in the insulin signalling pathway. Elevated levels of GSK-3 were found in diabetic rats and humans. We aimed to determine the effect of lithium chloride in diabetes and associated vascular complications in diabetic rats.
MATERIALS AND METHODS: Type 2 diabetes was induced by high fat diet and low dose of streptozotocin. Diabetic rats were divided into diabetic control and lithium chloride treatment groups. Lithium chloride was used as a GSK-3 inhibitor. The treatment was given for 4 weeks. Various biochemical parameters were measured before initiation and the end of treatment. Systolic blood pressure was measured by the non-invasive tail-cuff method, while various biochemical and tissue parameters were estimated for efficacy. Vasoreactivity was performed by taking the contractile response of H(2)O(2) (10(-6) M to 10(-3)M) and angiotensin II (10(-11) to 10(-7) M) in rat thoracic aortas of different groups. Statistical comparisons between all groups were performed by using two tailed one-way ANOVA followed by the Dunnett test. P-values <0.05 were considered statistically significant.
RESULTS: Treatment with lithium chloride significantly reduced the augmented systolic blood pressure, various biochemical parameters, and antioxidant parameters in diabetic-treated rats. Treatment also showed the decrease in augmented responses of H(2)O(2) and angiotensin II in rat thoracic aortas of treated rats.
CONCLUSIONS: We can conclude that lithium chloride treatment reduces the diabetic state as well as diabetes-induced vascular dysfunction.

Introduction

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase phosphorylates and thereby inactivates glycogen synthase, resulting in reduced glycogenesis[1] and involved in the insulin signaling pathway.[23] Elevated levels of GSK-3 were found in type 2 diabetic rats as well as humans.[4] Shin and co-workers reported that GSK-3b is rapidly activated in response to H2O2 treatment.[5] Angiotensin II (Ang-II) produces oxidative stress by activating NADPH oxidase.[6] The aim of this study was to study the effect of GSK-3 inhibitors in diabetes and associated vascular dysfunction in rats.

Materials and Methods

Animals

Male Sprague Dawley (SD) rats weighing 250-300 g were procured from central animal facility of institute. The animals were maintained in controlled temperature as well as humidity. Animals were free to access water and food. The experimental protocol was approved by Institutional Animal Ethics Committee (IAEC).

Chemicals

Streptozotocin and Ang-II were purchased from the Sigma Chemicals Company (St. Louis, MO, USA), lithium chloride from MP Biomedicals, Inc., France, and H2O2 from Merck Ltd, India.

Dilutions will be made with Kreb-Hansellet solution that is free of glucose. Adjust the pH 7.4 and it was done by addition of 0.1N NaOH if required. Drug concentrations were expressed as final molar concentration in bath solution.

Induction of Diabetes in Rats

Healthy SD rats showing normal plasma glucose level in the range of 80-120 mg/dl were used. Animals were fed with high fat diet for two weeks prior to Streptozotocin (STZ) injection and were continued till the end of study. A single dose of streptozotocin (35 mg/kg, i.p.) was administered for induction of diabetes. Plasma glucose level was measured after 72 hours of streptozotocin treatment. Those animals showing fasting blood glucose more than or equal to 250 mg/dl were considered as diabetic and was used for further studies. Diabetic animals were also fed with the high fat diet till the experiment termination. Plasma glucose was measured again at the end of every week to confirm consistent hyperglycemia.

Study Design

Animals were grouped into normal control, diabetic control, and lithium chloride (5, 10, and 25 mg/kg, respectively) treated diabetic rats. The total duration of the study was 12 weeks. After the STZ administration diabetic animals were kept as such for 6 weeks without any treatment for the development of vascular complications. Development of vascular complications was confirmed by measuring the blood pressure using tail-cuff BP measurement with NIBP controller on Powerlab. Rats were acclimatized in rodent restrainer for half an hour before recording. An average of three recordings was made for each rat. After confirmation of vascular complications diabetic rats were treated with lithium chloride for 4 weeks.

Biochemical Analysis

The blood samples (approximately 0.3 ml) were collected from rat tail vein under light anesthesia in heparinized centrifuge tubes. The plasma was separated by centrifugation (5000 rpm, 5 min at 4°C) and analyzed for glucose (GOD-POD), triglycerides (GPO-POD) and total cholesterol (CHOD-POD) using commercially available spectrophotometric kits. The remaining plasma samples were stored at -20 °C till the insulin determination was made by the ELISA kit using rat insulin as standard.

Glycogen Estimation from Liver

Glycogen estimation was done according to method described by Osterberg.[7] It has been definitely established that 60% potassium hydroxide at 100 °C does not destroy glycogen, that glycogen is quantitatively precipitated from a 70% solution of alcohol, and that the optimal condition for its conversion to glucose is in 2.2% hydrochloric acid at 100 °C. Following the conversion of glycogen to glucose, glucose can be estimated with the help of the photometric method. Glycogen estimation was done in terms of glucose equivalent to glycogen in mg/gm of tissue unit.

Assay for SOD activity

Isolated thoracic aorta was cleaned of surrounding fat and homogenized in 50 mM PBS buffer pH 7.0 using homogenizer. Homogenate was then centrifuged at 4 °C; 15,000 rpm for 10 min. Supernatant was used for the estimation of SOD activity by the adrenaline auto-oxidation method.[8]

Assay for catalase activity

Catalase activity was measured according to Grover and co-workers.[9] Thoracic aorta was homogenized (20 mg of tissue/ml of PBS, pH 7.0) and centrifuged at 4 °C (15,000 rpm for 10 min). The supernatant obtained was used for the assay. The degradation pattern of exogenously added H2O2 by catalase enzyme present in 200 μl of tissue supernatant was monitored at 240 nm in spectrophotometer at 15 S intervals for 5 min and its activity calculated. Catalase activity is expressed as U/mg of protein. Protein was estimated by Lowry's method.

Lipid peroxidation assay

The concentration of MDA [thiobarbituric acid reactive substance (TBARS)] was assayed using the method described by Beltowski and co-workers.[10] 1 ml of tissue supernatant of thoracic aorta was mixed with 1 ml of 10% trichloroacetic acid and allowed to stand for 30 min at 37 °C. Then 1 ml of 0.67% (w/v) thiobarbituric acid and 20 μl of 20% butylated hydroxytoluene (BHT) and the sample were heated at 95 °C for 30 min in boiling water bath. After cooling to room temperature, 2 ml of n-butanol was added and vortex immediately and centrifuged for 5 min at 5000 rpm. The organic layer was removed and its absorbance was measured at 532 nm. The concentration of MDA is expressed as nM of MDA/mg of tissue.

Vascular reactivity study

Ten weeks post-STZ administration, the rats were sacrificed and thoracic aorta was isolated from the heart to the diaphragm and cleaned of surrounding fat and connective tissues. Care was taken not to stretch the vessel. Helical strips of aorta of 2-3mm in width and 22 mm in length was cut with sharp iris scissors and placed in 10 ml organ bath containing Krebs–Henseleit buffer (NaCl 118 mM, KCl 4.7 mM, KH2PO4 1.2 mM, MgSO4·7H2O 1.2 mM, CaCl2·2H2O 2.5 mM, NaHCO3 25 mM and glucose 5.5 mM) of pH 7.4 and osmolality (280–308 mOsmol). The solution was continuously aerated with 5% carbogen at 37 °C. A resting tension of 2 g was applied to the strips and allowed to equilibrate for 2 h. After 2 h of equilibration, two wake up responses of KCl (80 mM) were recorded following which concentration response curves (CRC) of H2O2(10–6 to 10–3 M) and Ang-II (10–11 to 10–7 M) were recorded in age matched normal and diabetic rat thoracic aortas. Changes in the isotonic contraction were recorded. The maximum vasoconstrictor response to the H2O2 or Ang-II in normal control aorta was considered as 100%. In case of lithium-treated rats, contractile responses of H2O2 and Ang-II were taken without incubation of any drug. These responses were compared with responses obtained from untreated diabetic rats.

Data and statistical analysis

Data were expressed as mean ± standard error of mean (SEM). Significance between two groups were determined using the unpaired student's t-test. Statistical comparisons between all groups were performed by using two-tailed one-way ANOVA followed by the Dunnett test. P-values <0.05 were considered statistically significant.

Results

Effect of Lithium on Various Biochemical Parameters

Treatment with lithium chloride in varying doses for 4 weeks reduces the systolic blood pressure, fasting blood glucose, insulin, and lipid profile dose-dependent manner. While lithium chloride (10 and 25 mg/kg) treatment showed a significant increase in glycogen content in diabetic rats [Table 1].

Table 1

Effect of lithium chloride on body weight, blood pressure and biochemical parameters

Effect of Lithium on Various Antioxidant Parameters

Treatment with lithium chloride in varying dosage for 4 weeks significantly increases the level of antioxidant enzymes like SOD and catalase and reduces the level of lipid peroxidation in terms of MDA content in a dose-dependent manner. The values are shown in Table 2.

Table 2

Effect of lithium chloride on various antioxidant parameters

Effect of Lithium Treatment on H2O2 and Angiotensin II-Induced Contraction

The comparison of H2O2 and Ang-II-induced contractions between normal rat thoracic aorta, diabetic rat thoracic aorta, and thoracic aortas of lithium-treated groups is shown in Figure 1, while pD2 values of H2O2 and Ang-II-induced contraction in different treatment groups mentioned in Table 3.

Figure 1

CRC of H2O2 and angiotensin II on aortic spiral preparations obtained from normal (■), diabetic (▲), lithium chloride 5 (▼), 10 (♦) and 25 mg/kg (●) treated diabetic rats. n=6. *P < 0.05, **P < 0.01, ***P < 0.001 versus the respective diabetic control group.

Table 3

pD2 values and % Emax of H2O2 and Ang-II induced contraction in different treatment groups

Contractile responses of H2O2 and Ang-II were significantly increased in diabetic rats compared to normal rats. % Emax of H2O2 and Ang-II were significantly high in diabetic rat thoracic aorta than the normal rat thoracic aorta and significantly reduced by lithium chloride treatment. While pD2 values of H2O2 was only significantly increased in diabetic rat thoracic aorta than the normal rat thoracic aorta. Treatment with lithium chloride did not reduce the pD2 values of H2O2 and Ang-II in diabetic rats significantly.

Discussion

This study showed that in diabetic rats, treatment with different dosage of lithium results an improvement in fasting blood glucose, insulin sensitivity, lipid profile and vascular oxidative stress. In addition, there is an improvement in hydrogen peroxide and Ang-II-induced contractile responses in thoracic aortas of lithium-treated diabetic rats. However there is no significant decrease in pD2 values of Ang-II in thoracic aorta of lithium-treated diabetic rats compared to diabetic rats.

After the STZ administration rats were kept for 6 weeks on the same high fat diet without any treatment for development of insulin resistance and vascular complications. Our findings show that 6 weeks were sufficient to the significant increase in fasting blood glucose, insulin level, and systolic blood pressure. As we know insulin resistance plays a primary role in the development of type-2 diabetes[11] and is a characteristic feature of other health disorders including obesity, dyslipidemias, hypertension, and cardiovascular disease.[1213] GSK-3 activity is increased in skeletal muscle and adipose tissues of obese rodents and in skeletal muscle of obese humans with type 2 diabetes[14] and this elevated GSK-3 activity is associated with decreased insulin sensitivity.[15] Lithium treatment in a dose-dependent manner decreases hyperglycemia, plasma insulin level, lipid profile, and systolic blood pressure, while lithium also promoted the glycogen synthesis in treated diabetic rats. This increase in glycogen synthesis can be correlated with GSK-3 inhibition by lithium.

Oxidative stress implies an imbalance between the production of reactive oxygen species and the antioxidant defense system. Markers of oxidative stress are increased in individuals with diabetes and insulin resistance.[16] Lipid peroxidation products such as MDA are generated under high levels of un-scavenged free radicals.[17] These products may be important in the pathogenesis of vascular complication in diabetes mellitus.[18] Our findings demonstrate that lithium has antioxidant activity which reduces development of free radicals inside the vasculature and it may be important for prevention of vascular complications.

Our findings also demonstrate that H2O2 and Ang-II induced enhanced contractile responses can be reduced by lithium treatment. However, lithium treatment did not show a significant decrease in pD2 values compared to diabetic rat thoracic aorta. From the above findings it can be concluded that lithium can be used as hypoglycemic agent in diabetes and also in maniac depressive patients with diabetes and associated diabetic vascular complications.

On chronic use lithium may cause unwanted effects like slurred speech, convulsions, and renal failure. The intension of this study was not only to prove lithium as an antidiabetic drug but to reinforce the research on GSK-3, as a target for treating the diabetes and associated vascular complications.