Free Fatty Acids - Clinical Trial

Effect of Wang's Ketsumeisei on diabetes, including changes of serum free fatty acids and TNF-a (‘a’ represents alpha)

Xianen WANGI,2, Nobuhiro SATOI, Guangying HUANG2, Fuer LU2,
Jiangong HE3, Gangsan LIU3, Hua ZHANG3, and Sumio WATANABEI

I Juntendo University School of Medicine, Department of Gastroenterology

2 Huazhong University of Science and Technology, Department of Sino-Western Medical Synergy

3 North China Pharmaceutical Company, Ltd.

Summary

It is known that the development of type II diabetes mellitus is related to insulin resistance that is promoted by free fatty acids (FFA) and tumor necrosis factor alpha (TNF-a). A new Chinese herbal medicine, Wang's Ketsumeisei (WK), has been reported to be effective in an animal model of diabetes and in patients with type II diabetes. We studied the changes of FFA and TNF-a due to WK treatment in 55 patients who had type II diabetes without complications. The patients were divided into a WK group (n=30) and a control group (n=25). The control group do not receive WK while the WK group took WK at a dose of 6 g/day for 60 days together with their previous anti-diabetic therapy.

Treatment with WK lowered the fasting and 2-hour postprandial blood glucose levels, and also reduced the serum insulin level and improved the symptoms of patients with type II diabetes. In the WK group, the TNF-a level decreased slightly, while the FFA level decreased significantly.  In the control group, both TNF-a and FFA levels showed no obvious changes. These results suggest that WK is effective for type II diabetes mellitus, and that its activity may be related to the amelioration of FFA-induced insulin resistance.

Keywords:

FFA, TNF-a, diabetes mellitus, Wang's Ketsumeisei, Chinese herbal medicine.

I. Introduction

Hyperglycemia is one of the cardinal features of type II diabetes mellitus, but the goals of treating diabetes should include improvement of the constitution as well as reduction of the blood glucose level. Patients with obesity, type II diabetes mellitus, fatty liver, hypertension, dyslipidemia, and other relevant conditions are considered to have metabolic syndrome, and the common factor among these disease is insulin resistance. This is close to the concept of `Kikyoketsuohsho,' which means a predisposition to metabolic-circulatory disorders, in traditional Chinese medicine. It is known that the pathogenesis of type II diabetes involves the induction of insulin resistance by free fatty acids (FFA) and tumor necrosis factor a (TNF-a).  Chinese herbal medicines are frequently used for the treatment of diabetes, and we previously reported the efficacy of a new Chinese medicine known as Wang's Ketsumeisei (WK) for diabetes mellitus. In the present study, we investigated the change of FFA and TNF-a after administration of WK.

II. Materials and Methods

1. Test drug

Commercially available Wang's Ketsumeisei (WK; also known as Wang's Juemingjing) is a preparation that is extracted from pure Chinese herbal medicines, including Cassia obtusifolia L., Discoreae Rhizoma, Carthami Flos, Astragali Radix, Gardeniae Fructus, Puerariae Radix, Panax notoginseng, and Glycyrrhizae Radix.

2. Methods

(I) The diagnostic criteria for diabetes mellitus were a fasting blood glucose level7.0 mmol/L (126 mg/dL) or a 2-hour postprandial blood glucose level11.1 mmol/L(200 mg/dL).

(2) Observation methods: Fifty-five patients who had type II diabetes without complications such as cardiac, hepatic, or renal disorders (aged 35~65 years) were divided into a WK group (n=30) and a control group (n=25). The control group did not receive WK, while the WK group received WK at a dose of 6 g/day. In both groups, the previous anti-diabetic therapy was continued (no lipid-lowering agents were allowed). The fasting and 2-hour postprandial blood glucose levels and fasting serum insulin level
(ELISA) were measured before and 60 days after the start of the study. Fasting blood samples were obtained and serum was stored frozen at -80 until measurement of FFA (enzymatic method) and TNF-a (human TNF-a ELISA kit; Roche Diagnostics). The improvement of symptoms (including malaise, dry mouth,
polydipsia, polyuria, hyperphagia, nocturia, and erectile dysfunction) was assessed by using a questionnaire.

3.  Statistical analysis

Results are expressed as the mean+/-S.D., and statistical analysis was performed using analysis of variance and Student's t-test.

III. Results

1. Patient profile

The clinical characteristics of the subjects are show in Table 1.

2. Changes of blood glucose and symptoms

As shown in Figure 1, the fasting blood glucose levels of the control group before and after the study were 9.05 +/- 0.31 mmol/L and 9.16 +/- 0.34mmol/L, respectively, while the 2-hour postprandial blood glucose levels were 11.79+/-0.50 mmol/L and 11.73+/-0.46 mmol/L, respectively. Thus, there were no marked changes. In the WK group, the fasting blood glucose level after 60 days of administration (7.93+/-0.27 mmol/L) was significantly lower than the pretreatment level (9.03+/-0.29 mmol/L) (p<0.01), and was also lower than that of the control group (p<0.05). The 2-hour postprandial blood glucose level of the WK group (10.37+/-0.42 mmol/L) was significantly lower than the pro-treatment level (11.57+/-0.52 mmol/L) (p<0.05), and was also lower than that of the control group (but not significantly). In the WK group, symptoms were markedly improved, including malaise, dry mouth, and polyuria (Table 2).

Table 2   Improvment of major symptoms

3. Changes of insulin

In the control group, the fasting serum insulin levels measured before and after the study were 16.69 +/-1.93 µU/mL and 17.52+/-3.87 µU/mL, respectively. In the WK group, the fasting serum insulin level was 14.64+/-5.44 µU/mL after treatment, which was significantly lower than the pretreatment level (16.72+/-1.46 µU/mL, p<0.05).

4. Change of free fatty acids and TNF-a

In the control group, the TNF-a level measured after the study was 61.09+/-5.35 pg/mL, which did not differ much from the baseline level (60.45+/-5.05 pg/mL). The FFA level was 1,565.68+/-100.51 µmol/L, which also did not differ much from the baseline level (1,564.99+/-88.59 µmol/L). In theWK group, the final TNF-a level (58.61+/-4.65 pg/mL) was slightly lower than the pretreatment level (59.224.62 pg/mL). However, the FFA level measured after treatment (983.55+/-77.55 µmol/L) was about 37% lower than the pretreatment level Cl,570.44+/-79.02 µmol/L, p<0.01), and was restored to the reference range (300~1,100 µmol/L). The FFA level of the WK group was also significantly lower than that of the control group (p<0.01).

IV. Discussion

The pathogenesis of type II diabetes mellitus involves insulin resistance, which is assumed to cause a metabolic imbalance and compensatory hyperinsulinemia that is followed by exhaustion of Pancreatic
b-cells and decreased insulin secretion, eventually resulting in the onset of type II diabetes.

Insulin binds to the insulin receptor on target cells, and activates receptor tyrosine kinase that phosphorylates various proteins including IRS protein. These phosphorylated proteins are activated and bind to signaling molecules with a P13 kinase SH2 domain and signals are transduced further downstream, so that insulin has various effects on cellular metabolism and proliferation.

Insulin resistance is a condition in which more insulin than normal is required to obtain a given response. Metabolic syndrome includes multiple diseases resulting from insulin resistance (obesity type II diabetes, hypertension, fatty liver, and dyslipidemia), which together cause the progression of arteriosclerosisl(~4). TNF-a and free fatty acids (FFA) are known to promote insulin resistance. TNF-a is primarily produced by monocytes and macrophages, and also to a lesser extent by hypertrophic fat cells and muscle cells.

TNF-a suppresses tyrosine phosphorylation by IRS-I and reduces the tyrosine kinase activity of the insulin receptor, thus causing insulin resistance in skeletal muscles. FFAs are produced by fat cells through lipolysis. FFAs suppress the activity of P13 kinase bound to IRS-I in the liver and muscles, and also inhibit tyrosine phosphorylation by IRS- 1 that induces insulin resistance. This is followed by the promotion of hepatic lipid synthesis that causes hyperlipidemia and leads to enhancement of gluconeogenesis. Both TNF-a and FFAs act on the target cells of insulin to evoke insulin resistance(5~7).

In studies on diabetes mellitus and metabolic syndrome, fat cells have recently attracted attention, and it has been suggested that these diseases are attributable to insufficient secretion of leptin (anorexigen) and adiponectin by fat cellslo,(1), as well as excessive excretion of FFAs and TNF- a. The essential function of fat tissue is the storage of energy. These molecular changes are probably a secondary effect of the primary cause, which are considered to be a poor lifestyle (stress, overeating, and inadequate of exercise) and a genetic predisposition.

From the viewpoint of traditional Chinese medicine, the underlying predisposition to metabolic syndrome is Kikyoketsuoh. Kikyo indicates hypofunction and abnormal energy metabolism, while Ketsuoh indicates circulatory dysfunction.  Kikyo causes Ketsuoh, and Ketsuoh aggravates Kikyo(l2). Metabolic and microcirculatory disorders exist simultaneously(l3)' Kikyo is present in the spleen, but the spleen in traditional Chinese medicine means the systems involved in digestion, absorption, and

metabolism (including the pancreas).

According to Chinese medicine, type II diabetes is mainly caused by dysfunction of the spleen, liver, and kidneys, and is recognized as a condition in which Kanjininkyo is added to Kikyoketsuoh. As in the saying
that the liver regulates the muscles,' the metabolism and functioning of the tendon and muscles are controlled by the liver. Among the raw materials of WK, Cassia obtusifolia L. and Gardeniae Fructus are originally drugs for the liver.

Inkyo means a shortage of materials due to insufficient supplementation of Hikikyo and consumption of Shinkanetsudoku, and it is prevented by suppressing ‘Kyonetsu' in mild inflammation. Shinkanetsudoku is non-infectious inflammation due to stress, alcohol, and metabolic disorders. WK was compounded on the basis of the therapeutic principles of Hokikakketsu and Yoinseinetsu.

The results of this study show that WK alone or in combination with hypoglycemic agents could reduce fasting and postprandial blood glucose levels and improve symptoms in patients with type II diabetes. The serum insulin level was also decreased in the WK group. Although TNF-a only showed a slight decrease, FFAs were lowered by about 37%. TNF-a is an inflammatory factor, and it is known that increased systemic inflammatory activity is a basic feature of metabolic syndrome. Thus, TNF-a is Probably involved in the progression of diabetes and the onset of complications as an inflammatory factor. Free fatty acids are produced through degradation of neutral fat by hormone-sensitive lipase, released from fat cells, and primarily taken up by the liver. FFAs provide an energy source for the liver and muscles, and the FFA level closely reflects the state of glucose and metabolism. FFAs are considered to be more deeply involved in insulin resistance and metabolic abnormalities than TNF-a. WK probably reduced the FFA level by Hokikakketsu, that is, improvement of metabolism and strengthening of hepatic function, but further studies are needed to elucidate the exact mechanism.

Wang's Ketsumeisei (WK) is designed for improvement of Kikyoketsuoh, which is a predisposition to metabolic and circulatory disorders. The present study suggested that it can correct endogenous insulin resistance by reducing free fatty acid levels.

References

1)       Defronzo RA, Ferrannini E: Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 14: 173, 1991

2)      L6pez-Candales A: Metabolic syndrome X: A comprehensive review of the pathophysiology and recommended therapy. J Med. 32:283-300, 2001

3)       Kotronen A, Yki-Jrvinen H: Fatty Liver. A Novel component of the metabolic syndrome. Arterioscler Thromb Vase Bio 2007

4)      Qureshi K, Abrams GA: Metabolic liver disease of obesity and role of
adipose tissue in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol. 13(26): 3540-3553, 2007

5)      Hotamisligil GS, et al: IRS-I-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha and obesity-induced insulin resistance' Science 271: 665-668, 1996

6)       Alan D, et al: Effects of free fatty acids on glucose transport and IRS-l-
associated phosphatidylinositol 3-kinase activity. J Clin Invest 103: 253-259, 1999

7)      Delaru, Magnan C: Free fatty acids and insulin resistance. Curr Opin Clin Nutr Metab Care. 1O(2): 142-8, 2007

8)      Jia W,  Gao W, Tang L: Antidiabetic herbal drugs officially approved in China. Phytother Res. 17(10): 1127-1134, 2003

9)      Wang X, Huang G, Sato N: Therapeutic efficacy of a Chinese medicine, Wang's Ketsumeisei, in diabetes mellitus. Oriental Medicine 18(3); 43-49, 2002

10)   Minokosh Alquier T, Furukawa N, et al: AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature, 428: 569-574, 2004

11)    Lindsay RS, Funahash Hanson RL, et al: Adiponectin and
development of type 2 diabetes in the Pima Indian population. Lancet, 360: 57-58, 2002

12)   Wang X: Pathological change of blood. p115-121, Pictorial Traditional Chinese Medicine Concept. Sekibun-do, Tokyo, 2004

13)   Wiernsperger N, Nivoit P, De Aguiar LG: Microcirculation and the metabolic syndrome. Microcirculation. 14: 403-38, 2007