Sirong He

Comparison of single high-dose streptozotocin with partial pancreatectomy combined with low-dose streptozotocin for diabetes induction in rhesus monkeys.

Monkeys with insulin-dependent diabetes are important experimental models for islet xenotransplantation. However, with regard to diabetes induction, total pancreatectomy is a difficult operation with a high complication rate, while streptozotocin (STZ) administration may cause serious toxic effects and individual difference in metabolism. We compared two strategies involving pancreatectomy and STZ to successfully and safely induce diabetes in rhesus monkeys. Thirteen rhesus monkeys were divided into two groups: single high-dose STZ administration (80, 100 and 120 mg/kg, n = 3 for each dose) (group 1) and partial pancreatectomy (70–75%) combined with low-dose STZ (15 mg/kg, n = 4) (group 2). Induction of diabetes was evaluated by blood glucose, insulin, C-peptide, intravenous glucose tolerance test (IVGTT) and arginine stimulation test (AST). Detection of hematological and serum biochemical parameters and biopsies of pancreas, liver and kidney were periodically performed. In our study, animals in both groups developed diabetes. Serum C-peptide levels in groups 1 and 2 decreased to 0.08 ± 0.07 and 0.35 ± 0.06 nmol/L, respectively. IVGTT and AST indicated severely impaired glucose tolerance. Immunohistochemistry demonstrated that rare insulin-positive cells remained in the pancreas. In terms of STZ toxicity, four monkeys died 8–14 days after STZ administration (3 with 120 mg/kg STZ and 1 with 100 mg/kg STZ). Group 1 animals developed liver and kidney injury evidenced by increased alanine aminotransferase, aspartate aminotransferase, total cholesterol, LDL, triglyceride and blood urea nitrogen for one month, and histological abnormality including hepatic steatosis, renal glomerulus and tubular injury. Nevertheless, moderate histological injuries were seen in animals with 80 mg/kg STZ, with subsequent recovery. In contrast, group 2 animals displayed normal biochemical parameters and histology, with generally less risk of postoperative complications. We conclude that injection of 80 mg/kg STZ could induce diabetes with moderate injuries. Partial pancreatectomy with low-dose STZ is a safer and more reproducible method for inducing diabetes in rhesus monkeys.

Serum metabolic variables associated with impaired glucose tolerance induced by high-fat-high-cholesterol diet in Macaca mulatta

Dyslipidemia caused by ‘Western-diet pattern’ is a strong risk factor for the onset of diabetes. This study aimed to disclose the relationship between the serum metabolite changes induced by habitual intake of high-fat and high-cholesterol (HFHC) diet and the development of impaired glucose tolerance (IGT) and insulin resistance through animal models of Macaca mulatta. Sixteen M. mulatta (six months old) were fed a control diet or a HFHC diet for 18 months. The diet effect on serum metabolic profiles was investigated by longitudinal research. Islet function was assessed by intravenous glucose tolerance and hyperinsulinemic-euglycemic clamp test. Metabonomics were determined by 1 H proton nuclear magnetic resonance spectroscopy. Prolonged diet-dependent hyperlipidemia facilitated visceral fat accumulation in liver and skeletal muscle and disorder of glucose homeostasis in juvenile monkeys. Glucose disappearance rate (KGlu) and insulin response to the glucose challenge effects in HFHC monkeys were significantly lower than in control monkeys. Otherwise, serum trimethylamine-N-oxide (TMAO), lactate and leucine/isoleucine were significantly higher in HFHC monkeys. Sphingomyelin and choline were the most positively correlated with KGlu (R2 = 0.778), as well as negative correlation (R2 = 0.64) with total cholesterol. The HFHC diet induced visceral fat, abnormal lipid metabolism and IGT prior to weight gain and body fat content increase in juvenile monkeys. We suggest that increased serum metabolites, such as TMAO, lactate, branched-chain amino acids and decreased sphingomyelin and choline, may serve as possible predictors for the evaluation of IGT and insulin resistance risks in the prediabetic state.

Induction of diabetes with signs of autoimmunity in primates by the injection of multiple-low-dose streptozotocin

AIM To develop a preclinical large animal model of autoimmune diabetes to facilitate the translational research of autoimmune diabetes in human. MATERIALS AND METHODS Nine young rhesus monkeys received multiple-low-dose (MLD) intravenous injections of streptozotocin for five consecutive days, followed by two additional boosting injections of STZ given 1 week apart. The induction of autoimmune diabetes was evaluated by regular metabolic testing, serological assessment of islet-reactive autoantibodies and histological examination of pancreatic tissues. RESULTS Seven of nine treated animals became diabetic with moderate hyperglycemia initially and more severe hyperglycemia thereafter. All diabetic animals exhibited severely impaired glucose tolerance, limited islet function, and required insulin therapy to maintain relatively normal glucose metabolism and healthy status. Serological tests showed that all diabetic monkeys developed autoantibodies specifically against insulin and islet antigens. Furthermore, histological examination of the pancreata from diabetic animals revealed evidence of specific destruction of islet β cells and islets infiltrated with T lymphocytes. Overt and persistent diabetes can be induced in young rhesus monkeys by the injection of MLD-STZ, and autoimmune responses to pancreatic islet cells seem to be involved in the development of glucose intolerance and diabetes. CONCLUSION These data indicate for the first time that autoimmune diabetes can be induced in primates; this may serve as a valuable preclinical model for studying the pathogenesis of and potential therapies for autoimmune diabetes in humans.

1H NMR-based metabonomic analysis of serum and urine in a nonhuman primate model of diabetic nephropathy

Diabetic nephropathy (DN) is a serious metabolic disease, and comprehensive understanding of its complex mechanism will help in preventing the onset and progression of DN. To reveal the systemic metabolic changes associated with renal injury, we performed 1H NMR-based metabonomic and multivariate analyses to analyze serum and urine obtained from a nonhuman primate model of DN. Our results indicated that DN monkeys exhibited a distinct metabolic profile, including higher levels of VLDL/LDL, lipids, unsaturated lipids, uric acid, allantoin, fumarate and hippurate, as well as lower levels of HDL, alanine, glutamate, pyruvate, formate, tyrosine, histidine and NAD+. The disturbed metabolic pathways were further identified, including NAD+ metabolism, purine metabolism, oxidative stress, lipid metabolism, and renal tubular reabsorption. This study highlights that NMR-based metabonomics provides insight into the underlying pathways in the pathogenesis and progression of DN at the metabolic level.

Treatment and risk factor analysis of hypoglycemia in diabetic rhesus monkeys

In order to anticipate and promptly treat hypoglycemia in diabetic monkeys treated with insulin or other glucose-lowering drugs, the relationships between the incidence and symptoms of hypoglycemia in these animals, and many factors involved in model development and sustainment were analyzed. Different procedures were performed on 22 monkeys for the induction of diabetes. The monkey models were evaluated by blood glucose, insulin, C-peptide levels and intravenous glucose tolerance tests. A glucose treatment program for the diabetic monkeys was administered and laboratory tests were regularly performed. A standard procedure of hypoglycemia treatment was established and the risk factors of hypoglycemia were analyzed by a logistic regression model. Furthermore, the relationships between the four methods of diabetes induction, renal function, glycemic control and hypoglycemia were studied using one-way analysis of variance and t-test. We found that the hypoglycemic conditions of diabetic monkeys were improved rapidly by our treatment. The statistical analysis suggested that the modeling methods, renal function and glycemic control were related to the incidence of hypoglycemia. In detail, the progress of diabetes, effects of glycemic control and, particularly, the severity of the hypoglycemia differed according to the induction strategy used. The models induced by partial pancreatectomy with low-dose streptozotocin were not prone to hypoglycemia and their glycemic controls were stable. However, the models induced by total pancreatectomy were more vulnerable to severe hypoglycemia and their glycemic controls were the most unstable. Moreover, the levels of blood creatinine and triglyceride increased after the development of diabetes, which was related to the occurrence of hypoglycemia. In conclusion, we suggested that total pancreatectomy and renal impairment are two important risk factors for hypoglycemia in diabetic monkeys. More attention should be paid to daily care of diabetic monkeys, particularly monitoring and protecting their renal function.

Increasing glucagon secretion could antagonize the action of exogenous insulin for glycemic control in streptozocin-induced diabetic rhesus monkeys

Although intraislet insulin signaling is known to play a critical role in regulating glucagon secretion, it is unknown whether abnormal glucagon secretion influences the hypoglycemic effect of exogenous insulin with intraislet insulin deletion. We performed a longitudinal study using 16 streptozocin (STZ)-induced diabetic rhesus monkeys to explore α-cell function under the absence β-cells and to assess whether increasing glucagon secretion antagonizes the action of exogenous insulin for glycemic control. We found that although the α-cells were impaired and the basal secretion levels of glucagon decreased rapidly after STZ (80–90 mg/kg) administration, as based on long-term observation post-STZ injection, glucagon secretion and the number of α-cells were increased. Glycemic control was increasingly difficult, the insulin resistance (HOMA-IR) index was significantly higher, and the triglycerides (TG) levels were gradually decreased. Moreover, a significant correlation between the levels of glucagon and HOMA-IR was found. Under the long-term absence of β-cells, the inhibitory effect on α-cell activity is profoundly attenuated, leading to an increase in glucagon secretion and the amount of α-cells and even α-cell dysfunction. Increased glucagon levels have a serious impact on the insulin sensitivity in vivo and result in an antagonization of the hypoglycemic effect of exogenous insulin.

Human insulin versus porcine insulin in rhesus monkeys with diabetes mellitus

Monkeys with insulin‐dependent diabetes are important preclinical animal models for islet transplantation. Exogenous insulin should be administered to achieve good glycemic control and minimize the long‐term vascular complications associated with diabetes until the graft function recovered completely. However, the effect of multiple daily injections of porcine or human insulin and the long‐term effects of porcine insulin have not been studied in diabetic rhesus monkeys.

A preclinical evaluation of alternative site for islet allotransplantation

The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs).

MSCs promote the development and improve the function of neonatal porcine islet grafts

Deficient insulin secretion caused by immaturity is the predominant disadvantage of neonatal porcine islets (NPIs) when they serve as a source for islet xenotransplantation. We hypothesize that the transplantation of NPIs with a combination of mesenchymal stem cells (MSCs) can accelerate NPI maturation and improve the engraftment and function of NPIs. After indirect coculturing with monkey MSCs over 21 d, insulin secretion and the expression of regulatory genes relevant to development were assessed in NPIs. NPIs alone or in combination with allogeneic MSCs were intraportally transplanted into diabetic monkeys. Glycemic control was monitored, and graft function was evaluated. Our results suggest that MSCs benefit both the development and proliferation of NPIs in the coexisting systems in vitro and in vivo. These effects are dependent on platelet‐derived growth factor receptor‐a and are relevant to the inhibition of downstream target Notch1 signaling and the activation of PI3K/protein kinase B signaling.—He, S., Wang, C., Du, X., Chen, Y., Zhao, J., Tian, B., Lu, H., Zhang, Y., Liu, J., Yang, G., Li, L., Li, H., Cheng, J., Lu, Y. MSCs promote the development and improve the function of neonatal porcine islet grafts. FASEB J. 32, 3242–3253 (2018). www.fasebj.org