Hepatic FoxO1 Acetylation Is Involved in Oleanolic Acid-Induced Memory of Glycemic Control: Novel Findings from Study 2

Our recent study (referred as Study 1) showed that the triterpenoid oleanolic acid (OA) was able to produce a sustained correction of hyperglycemia beyond treatment period in type 2 diabetes (T2D) mice with liver as a responsible site. To follow up the previous observations, the present study (referred as Study 2) investigated the possible role of acetylation of FoxO1 and associated events in this therapeutic memory by characterizing the pathways regulating the acetylation status during and post-OA treatments. OA treatment (100 mg/kg/day for 4 weeks, during OA treatment) reduced hyperglycemia in T2D mice by ∼87% and this effect was largely (∼70%) maintained even 4 weeks after the cessation of OA administration (post-OA treatment). During OA treatment, the acetylation and phosphorylation of FoxO1 were markedly increased (1.5 to 2.5-fold) while G6Pase expression was suppressed by ∼80%. Consistent with this, OA treatment reversed pyruvate intolerance in high-fat fed mice. Histone acetyltransferase 1 (HAT1) content was increased (>50%) and histone deacetylases (HDACs) 4 and 5 (not HDAC1) were reduced by 30–50%. The OA-induced changes in FoxO1, G6Pase, HAT1 and HDACs persisted during the post-OA treatment period when the increased phosphorylation of AMPK, SIRT1 content and reduced liver triglyceride had subsided. These results confirmed the ability of OA to control hyperglycemia far beyond treatment period in T2D mice. Most importantly, in the present study we demonstrated acetylation of FoxO1 in the liver is involved in OA-induced memory for the control of hyperglycemia. Our novel findings suggest that acetylation of the key regulatory proteins of hepatic gluconeogenesis is a plausible mechanism by the triterpenoid to achieve a sustained glycemic control for T2D.     

Physiological and pathological changes in glucose regulate brain Akt and glycogen synthase kinase-3

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.     

Forkhead Box O6 (FoxO6) Depletion Attenuates Hepatic Gluconeogenesis and Protects against Fat-induced Glucose Disorder in Mice

Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.     

Combined effect of nicotinamide and streptozotocin on diabetic status in partially pancreatectomized adult BALB/c mice.

Pancreatic regeneration after pancreatectomy has been well documented in the animal models. We have recently reported that STZ diabetic animals operated for partial pancreatectomy showed normoglycemic status after the operation as compared to uncontrolled hyperglycemia and even death in the diabetic sham operated animals. In drug and virus-induced experimental diabetic models there is a high mortality of animals due to uncontrolled destruction of the beta-cells. In order to destroy sufficient beta-cell mass so as to induce diabetes but prevent mortality, we designed present studies to investigate the combined effect of pancreatectomy, nicotinamide, and streptozotocin (STZ) on diabetic status of BALB/c mice. BALB/c mice of either sex were subjected to 50% pancreatectomy. These were then treated with nicotinamide (350 mg/kg body weight) before and after streptozotocin (200 mg/kg body weight) administration. The changes in body weight, blood glucose levels, serum and pancreatic insulin contents of these animals were monitored in experimental and control group for 12 weeks, and follow up studies were made of these animals for further 12 weeks. It was found that there was a drastic loss of body weight, decreased serum and pancreatic insulin levels coupled with sustained and low levels of hyperglycemia in the experimental group as opposed to the control group. The results indicate that partial pancreatectomy followed by nicotinamide and streptozotocin treatment leads to a long-lasting hyperglycemic state, depicting the clinical symptom of NIDDM without mortality. The study probably reveals a new model for experimental diabetes.     

The soybean peptide aglycin regulates glucose homeostasis in type 2 diabetic mice via IR/IRS1 pathway

It has been previously reported that aglycin, a natural bioactive peptide isolated from soybean, is stable in digestive enzymes and has an antidiabetic potential. With a view to explore the pharmacological activity of aglycin in vivo, studies have been conducted to examine its therapeutic effect in diabetic mice, in which it was administered intragastrically as an oral agent. Diabetes was induced in BALB/c mice fed with a high-fat diet and a single intraperitoneal injection of streptozotocin. With onset of diabetes, the mice were administered daily with aglycin (50 mg/kg/d) for 4 weeks. Blood glucose was monitored once a week. Subsequently, skeletal muscle was isolated for assessment in terms of levels of gene and protein IR, IRS1, Akt and glucose transporter 4 (GLUT4). In addition, C2C12 muscle cells as an in vitro diabetic model were used to investigate the effect of aglycin on glucose uptake. Treatment with aglycin was found to be significantly effective in controlling hyperglycemia and improving oral glucose tolerance. Furthermore, aglycin enhanced glucose uptake and glucose transporter recruitment to the C2C12 cell surface in 10 min in vitro. Consistent with these effects, aglycin restored insulin signaling transduction by maintaining IR and IRS1 expression at both the mRNA and protein levels, as well as elevating the expression of p-IR, p-IRS1, p-Akt and membrane GLUT4 protein. The results hence demonstrate that oral administration of aglycin can potentially attenuate or prevent hyperglycemia by increasing insulin receptor signaling pathway in the skeletal muscle of streptozotocin/high-fat-diet-induced diabetic mice.     

Altered REDD1, myostatin, and Akt/mTOR/FoxO/MAPK signaling in streptozotocin-induced diabetic muscle atrophy

Type 1 diabetes, if poorly controlled, leads to skeletal muscle atrophy, decreasing the quality of life. We aimed to search highly responsive genes in diabetic muscle atrophy in a common diabetes model and to further characterize associated signaling pathways. Mice were killed 1, 3, or 5 wk after streptozotocin or control. Gene expression of calf muscles was analyzed using microarray and protein signaling with Western blotting. We identified translational repressor protein REDD1 (regulated in development and DNA damage responses) that increased seven- to eightfold and was associated with muscle atrophy in diabetes. The diabetes-induced increase in REDD1 was confirmed at the protein level. This result was accompanied by the increased gene expression of DNA damage/repair pathways and decreased expression in ATP production pathways. Concomitantly, increased phosphorylation of AMPK and dephosphorylation of the Akt/mTOR/S6K1/FoxO pathway of proteins were observed together with increased protein ubiquitination. These changes were especially evident during the first 3 wk, along with the strong decrease in muscle mass. Diabetes also induced an increase in myostatin protein and decreased MAPK signaling. These, together with decreased serum insulin and increased serum glucose, remained altered throughout the 5-wk period. In conclusion, diabetic myopathy induced by streptozotocin led to alteration of multiple signaling pathways. Of those, increased REDD1 and myostatin together with decreased Akt/mTOR/FoxO signaling are associated with diabetic muscle atrophy. The increased REDD1 and decreased Akt/mTOR/FoxO signaling followed a similar time course and thus may be explained, in part, by increased expression of genes in DNA damage/repair and possibly also decrease in ATP-production pathways.     

A model for diabetic nephropathy: advantages of the inducible cAMP early repressor transgenic mouse over the streptozotocin-induced diabetic mouse

We have previously found progressive diabetic nephropathy in inducible cAMP early repressor (ICER Igamma) transgenic (Tg) mice. The ICER Igamma Tg mouse is an interesting model of sustained hyperglycemia due to its low production of insulin and insulin-producing beta cells. Here in a longitudinal study we further analyzed diabetic nephropathy and structural and functional alterations in other organs, comparing our model with streptozotocin (STZ)-diabetic model mice. The high-dose STZ-diabetic model showed marked variation in blood glucose levels and severe toxicity of STZ in the liver and kidney. The low-dose STZ-diabetic model showed less toxicity, but the survival rate was very low. STZ-diabetic mice had much more variation of glomerular hypertrophy and sclerosis. Furthermore, non-specific toxicity of STZ or insulin injections to maintain optimal blood glucose levels might have another effect upon the diabetic renal changes. In contrast, ICER Igamma Tg mice exhibited a stable and progressive phenotype of diabetic kidney disease solely due to chronic hyperglycemia without other modulating factors. Thus, ICER Igamma Tg mouse has advantages for examining diabetic renal disease, and offers unique and very different perspectives compared to STZ model.     

Hepatocyte growth factor preserves beta cell mass and mitigates hyperglycemia in streptozotocin-induced diabetic mice

Type I diabetes is an autoimmune disease that results in destructive depletion of the insulin-producing beta cells in the islets of Langerhans in pancreas. With the knowledge that hepatocyte growth factor (HGF) is a potent survival factor for a wide variety of cells, we hypothesized that supplementation of HGF may provide a novel strategy for protecting pancreatic beta cells from destructive death and for preserving insulin production. In this study, we demonstrate that expression of the exogenous HGF gene preserved insulin excretion and mitigated hyperglycemia of diabetic mice induced by streptozotocin. Blood glucose levels were significantly reduced in mice receiving a single intravenous injection of naked HGF gene at various time points after streptozotocin administration. Consistently, HGF concomitantly increased serum insulin levels in diabetic mice. Immunohistochemical staining revealed a marked preservation of insulin-producing beta cells by HGF in the pancreatic islets of the diabetic mice. This beneficial effect of HGF was apparently mediated by both protection of beta cells from death and promotion of their proliferation. Delivery of HGF gene in vivo induced pro-survival Akt kinase activation and Bcl-xL expression in the pancreatic islets of diabetic mice. These findings suggest that supplementation of HGF to prevent beta cells from destructive depletion and to promote their proliferation might be an effective strategy for ameliorating type I diabetes.     

Adiponectin gene therapy of streptozotocin-induced diabetic mice using hydrodynamic injection

BACKGROUND: Adiponectin (Adipo), an adipocyte hormone involved in the regulation of glucose and lipid metabolism, has already been identified as a potential therapeutic target for the treatment of diabetes. However, successful delivery of Adipo to the receptors is difficult due to their peptide characteristics. Receptors for Adipo are abundantly expressed in the liver and skeletal muscle. METHODS: Uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) in hepatoblastoma HepG2 cells expressing Adipo was examined. Adipo-expressing plasmid DNA (10-50 microg) in saline solution (0.1 ml/g body weight) was rapidly injected into the tail vein of 4-week-old diabetic mice after 4-6 weeks of treatment with streptozotocin (STZ). Uptake of glucose in diabetic mice also was measured using a planar positron imaging system featuring 18-fluorodeoxyglucose. RESULTS: HepG2 cells expressing Adipo exhibited significantly increased 2-NBDG uptake compared with cells transfected with control plasmid even in the absence of insulin. STZ-induced diabetic mice showed decreased serum Adipo levels compared with non-diabetic mice. A single hydrodynamic injection of 10-50 microg Adipo-expressing plasmid DNA into diabetic mice led to approximately 10-15-fold elevation in serum Adipo levels, and resulted in decreased serum levels of glucose and triglyceride. As well as exhibiting higher levels of Adipo expression, diabetic mice also had higher hepatic glucose uptake than similar mice injected with control plasmid. CONCLUSIONS: We report that STZ-induced diabetic mice exhibited decreased Adipo levels and hyperglycemia which may be alleviated by hydrodynamic injection of the Adipo gene. This type of gene delivery system to the liver offers a different approach in developing novel treatments for type 1 and 2 diabetes.     

Antidiabetic potential of oleanolic acid from Ligustrum lucidum Ait

Ligustrum lucidum Ait. has been used in traditional Chinese medicine for over 1000 years because of its anti-tumor, antimutagenic, antidiabetic, and hepatoprotective properties. The aim of this study was to determine whether oleanolic acid (OA) is the principal active compound of L. lucidum responsible for its antidiabetic properties, and to examine its effect on the expression of thyroid hormones and insulin secretion, thus revealing the mechanism by which L. lucidum modulates insulin levels in diabetes. When rats with streptozotocin-induced diabetes were treated with OA (100 and 200 mg/kg body mass per day, for 40 days), the changes in blood glucose levels and in oral glucose tolerance tests showed that hypoglycemia was more pronounced in OA-treated groups than in the diabetic control rats, and that the levels of triglyceride, total cholesterol, and low-density lipoportein cholesterol in OA-treated rats were lower than those in the diabetic control rats, whose high-density lipoprotein cholesterol increased. OA-treated rats also gained weight, and exhibited increased serum insulin levels. In contrast, OA treatment did not effect the levels of thyroid hormone or TSH in rats with streptozotocin-induced diabetes. These results indicate that OA has hypoglycemic and hypolipidemic effects. OA treatment might stimulate insulin release, and consequently, results in the modulation of glucose levels and regulation of lipid metabolism.     

Tocotrienol-rich fraction supplementation reduces hyperglycemia-induced skeletal muscle damage through regulation of insulin signaling and oxidative stress in type 2 diabetic mice

Chronic hyperglycemia induces impairment of muscle growth and development of diabetes mellitus (DM). Since skeletal muscle is the major site for disposal of ingested glucose, impaired glucose metabolism causes imbalance between protein synthesis and degradation which adversely affects physical mobility.In this study, we investigated the effect of tocotrienol-rich fraction (TRF) supplementation on skeletal muscle damage in diabetic mice. Diabetes was induced by a high-fat diet with streptozotocin (STZ) injection (100 mg/kg) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose levels≥250 mg/dl), normal control (CON) and diabetic control (DMC) groups were administrated with olive oil, while TRF treatment groups were administrated with TRF (dissolved in olive oil) at low dose (100 mg/kg BW, LT) or high dose (300 mg/kg BW, HT) by oral gavage for 12 weeks.TRF supplementation ameliorated muscle atrophy, plasma insulin concentration and homeostatic model assessment estimated insulin resistance in diabetic mice. Moreover, TRF treatment up-regulated IRS-1 and Akt levels accompanied by increased translocation of GLUT4. Furthermore, TRF increased mitochondrial biogenesis by activating SIRT1, SIRT3 and AMPK in diabetic skeletal muscle. These changes were in part mechanistically explained by reduced levels of skeletal muscle proteins related to oxidative stress (4-hydroxynonenal, protein carbonyls, Nrf2 and HO-1), inflammation (NFkB, MCP-1, IL-6 and TNF-α), and apoptosis (Bax, Bcl₂ and caspase-3) in diabetic mice. Taken together, these results suggest that TRF might be useful as a beneficial nutraceutical to prevent skeletal muscle atrophy associated with diabetes by regulating insulin signaling via AMPK/SIRT1/PGC1α pathways in type 2 diabetic mice.     

Characterization of an insulinotropic peptide from skin secretions of Odorrana andersonii

Insulinotropic peptide agents are regarded as potential candidates for anti‐diabetic treatment. In the present study, a novel insulinotropic peptide, termed OA‐A1, was purified from frog skin secretions of Odorrana andersonii. Mature OA‐A1 was determined to be a 1965.049 Da peptide with an amino acid sequence of LVGKLLKGAVGDVCGLLPIC, in which an intramolecular disulfide bridge was formed by two cysteine residues. At the cellular level, OA‐A1 exhibited potent proliferation promoting effects on mouse‐derived pancreatic β‐TC‐6 cells and significantly stimulated insulin release in β‐TC‐6 cells at a minimum concentration of 1 nM. In the animal model, OA‐A1 also showed a dose‐dependent insulin‐releasing role in mice. At concentrations ranging from 1 nmol/kg to 1 μmol/kg, OA‐A1 had a significant acute hypoglycemic effect on streptozotocin (STZ)‐induced diabetic mice. The pancreatic islet areas of diabetic mice increased dose‐dependently after 21 days of OA‐A1 treatment (1–100 nmol/kg) compared with those of the saline control group. Moreover, OA‐A1 significantly improved the oral glucose tolerance of STZ‐induced diabetic mice. Taken together, these results suggest that OA‐A1 provides an excellent template for the development of novel anti‐diabetic therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

熊果酸对糖尿病小鼠肝损伤的作用及其可能机制

目的：研究熊果酸对高脂饲料联合链脲佐菌素（STZ）诱导的糖尿病小鼠肝损伤的影响,探讨其可能的作用机制。方法：随机选取20只小鼠高脂饲料喂养6周后,STZ （30 mg/kg）腹腔注射连续5 d,9 d后测空腹血糖,大于11.1 mmol/L视为糖尿病模型,将其随机分为模型组和熊果酸组（100 mg/kg）（n=10）;另取10只小鼠设为对照组。连续给药8周。小鼠称重,测定空腹血糖（FBG）,血清总胆固醇（TC）、甘油三酯（TG）含量,谷丙转氨酶（ALT）、谷草转氨酶（AST）、超氧化物歧化酶（SOD）活性,丙二醛（MDA）含量,HE染色观察肝组织病理变化。结果：模型组与对照组比较,FBG、血清TC、TG含量,ALT、AST活性、MDA含量明显升高（P<0.05,P<0.01）;SOD活性明显降低（P<0.01）;HE染色显示部分肝细胞水肿,轻度脂肪变性,门管区可见淋巴细胞浸润。熊果酸组与模型组比较,FBG,血清TC、TG含量,ALT、AST活性、MDA含量明显降低（P<0.05,P<0.01）;SOD活性明显升高（P<0.01）;HE染色显示熊果酸组肝细胞排列较为整齐,水肿不明显,淋巴细胞少量存在。结论：熊果酸对高脂饲料联合STZ诱导的糖尿病小鼠肝损伤具有保护作用,其机制可能与降血糖、调节血脂、降低肝组织氧化应激水平,提高肝脏抗氧化能力有关。     

Systemic administration of HVJ viral coat-liposome complex containing human insulin vector decreases glucose level in diabetic mouse: A model of gene therapy

In this study, we examined the feasibility of a systemic administration of HVJ-liposome complex containing human insulin construct into the blood in mice via the tail vein. Transfection of human insulin vector resulted in a transient decrease in serum glucose in streptozotocin (SZT)-induced diabetic mice, accompanied by the detection of human insulin in the liver and spleen. In accordance with the decreased glucose, plasma immunoreactive insulin could be detected up to 14 days after a single transfection in mice transfected with insulin vector. Repeated intravenous injection of human insulin vector every week resulted in a sustained decrease in serum glucose over a 4-week period, accompanied by the detection of C-peptide fragments and a significant decrease in BUN and creatinine. Here, we demonstrated the feasibility of intravenous systemic administration of an insulin vector that results in a sustained improvement of diabetic glucose metabolism.     

Melatonin and taurine reduce early glomerulopathy in diabetic rats

Oxidative stress occurs in diabetic patients and experimental models of diabetes. We examined whether two antioxidants, melatonin and taurine, can ameliorate diabetic nephropathy. Enhanced expression of glomerular TGF-beta1 and fibronectin mRNAs and proteinuria were employed as indices of diabetic nephropathy. Experimental diabetes was induced by intravenous injection of streptozotocin 50 mg/kg. Two days after streptozotocin, diabetic rats were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% in drinking water; or iii) taurine supplement by 1% in drinking water. Four weeks after streptozotocin, diabetic rats (n = 6: plasma glucose 516+/-12 mg/dl) exhibited 6.1 fold increase in urinary protein excretion, 1.4 fold increase in glomerular TGF-beta1 mRNA, 1.7 fold increase in glomerular fibronectin mRNA, 2.2 fold increase in plasma lipid peroxides (LPO), and 44 fold increase in urinary LPO excretion above the values in control rats (n = 6: plasma glucose 188+/-14 mg/dl). Chronic administration of melatonin (n = 6) and taurine (n = 6) prevented increases in glomerular TGF-beta1 and fibronectin mRNAs and proteinuria without having effect on blood glucose. Both treatments reduced lipid peroxidation by nearly 50%. The present data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress.     

Modulation of Glucose Metabolism by Balanced Deep-Sea Water Ameliorates Hyperglycemia and Pancreatic Function in Streptozotocin-Induced Diabetic Mice

The aim of this study was to determine the effects of balanced deep-sea water (BDSW) on hyperglycemia and glucose intolerance in streptozotocin (STZ)-induced diabetic mice. BDSW was prepared by mixing DSW mineral extracts and desalinated water to yield a final hardness of 1000–4000 ppm. Male ICR mice were assigned to 6 groups; mice in each group were given tap water (normal and STZ diabetic groups) or STZ with BDSW of varying hardness (0, 1000, 2000, and 4000 ppm) for 4 weeks. The STZ with BDSW group exhibited lowered fasting plasma glucose levels than the STZ-induced diabetic group. Oral glucose tolerance tests showed that BDSW improves impaired glucose tolerance in STZ-induced diabetic mice. Histopathological evaluation of the pancreas showed that BDSW restores the morphology of the pancreatic islets of Langerhans and increases the secretion of insulin in STZ-induced diabetic mice. Quantitative real-time PCR assay revealed that the expression of hepatic genes involved in gluconeogenesis, glucose oxidation, and glycogenolysis was suppressed, while the expression of the genes involved in glucose uptake, β-oxidation, and glucose oxidation in muscle were increased in the STZ with BDSW group. BDSW stimulated PI3-K, AMPK, and mTOR pathway-mediated glucose uptake in C₂C₁₂ myotubes. BDSW increased AMPK phosphorylation in C₂C₁₂ myotubes and improved impaired AMPK phosphorylation in the muscles of STZ-induced diabetic mice. Taken together, these results suggest that BDSW is a potential anti-diabetic agent, owing to its ability to suppress hyperglycemia and improve glucose intolerance by modulating glucose metabolism, recovering pancreatic islets of Langerhans and increasing glucose uptake.