Effects of insulin therapy on weight gain and fat distribution in the HF/HS-STZ rat model of type 2 diabetes.

BACKGROUND/OBJECTIVES: Insulin therapy is required for many patients with the obesity-related disorder type 2 diabetes, but is also associated with weight gain. The specific location of adipose tissue location matters to cardiovascular disease (CVD) risk. We investigated effects of exogenous insulin on fat distribution in the high-fat/high-sucrose fed rat treated with streptozotocin (HF/HS-STZ) rat model of type 2 diabetes. We also examined effects of insulin therapy on circulating CVD markers, including adiponectin, triglycerides (TGs), total cholesterol and high-density lipoprotein. SUBJECTS/METHODS: Male SD rats were HF/HS fed for 5 weeks followed by STZ treatment to mimic the hallmarks of human obesity-associated insulin resistance followed by hyperglycemia. Magnetic resonance imaging and computed tomography were used to determine total fat, abdominal fat distribution and liver fat before and after insulin therapy in HF/HS-STZ rats. HbA1c%, TGs, cholesterol, high-density lipoprotein and adiponectin were analyzed by conventional methods adapted for rats. RESULTS: Insulin therapy lowered HbA1c (P<0.001), increased body weight (P<0.001), increased lean mass (P<0.001) and led to a near doubling of total fat mass (P<0.001), with the highest increase in subcutaneous adipose tissue as compared with visceral adipose tissue (P<0.001). No changes in liver fat were observed after insulin therapy, whereas plasma TG and cholesterol levels were decreased (P<0.001, P<0.01), while high-density lipoprotein (HDL) and adiponectin levels were elevated (P<0.01, P<0.001). CONCLUSIONS: Using the HF/HS-STZ rat as an animal model for type 2 diabetes, we find that insulin therapy modulates fat distribution. Specifically, our data show that insulin has a relatively positive effect on CVD-associated parameters, including abdominal fat distribution, lean body mass, adiponectin, TGs and HDL in HF/HS-STZ rats, despite a modest gain in weight.

Cellular mechanism of nutritionally induced insulin resistance in Psammomys obesus: overexpression of protein kinase Cepsilon in skeletal muscle precedes the onset of hyperinsulinemia and hyperglycemia.

The sand rat (Psammomys obesus) is an animal model of nutritionally induced diabetes. We report here that several protein kinase C (PKC) isoforms (alpha, epsilon, and zeta, representing all three subclasses of PKC) are overexpressed in the skeletal muscle of diabetic animals of this species. This is most prominent for the epsilon isotype of PKC. Interestingly, increased expression of PKCepsilon could already be detected in normoinsulinemic, normoglycemic (prediabetic) animals of the diabetes-prone (DP) line when compared with a diabetes-resistant (DR) line. In addition, plasma membrane (PM)-associated fractions of PKCalpha and PKCepsilon were significantly increased in skeletal muscle of diabetic animals, suggesting chronic activation of these PKC isotypes in the diabetic state. The increased PM association of these PKC isotypes revealed a significant correlation with the diacylglycerol content in the muscle samples. Altered expression/activity of PKCepsilon, in particular, may thus contribute to the development of diabetes in these animals; along with other PKC isotypes, it may be involved in the progression of the disease. This may possibly occur through inhibition of insulin receptor (IR) tyrosine kinase activity mediated by serine/threonine phosphorylation of the IR or insulin receptor substrate 1 (IRS-1). However, overexpression of PKCepsilon also mediated down-regulation of IR numbers in a cell culture model (HEK293), resulting in attenuation of insulin downstream signaling (reduced protein kinase B [PKB]/Akt activity). In accordance with this, we detected decreased 125I-labeled insulin binding, probably reflecting a downregulation of IR numbers, in skeletal muscle of Psammomys animals from the DP line. The number of IRs was inversely correlated to both the expression and PM-associated levels of PKCepsilon. These data suggest that overexpression of PKCepsilon may be causally related to the development of insulin resistance in these animals, possibly by increasing the degradation of IRs.

Early growth restriction leads to down regulation of protein kinase C zeta and insulin resistance in skeletal muscle.

Epidemiological studies have revealed a relationship between early growth restriction and the subsequent development of type 2 diabetes. A rat model of maternal protein restriction has been used to investigate the mechanistic basis of this relationship. This model causes insulin resistance and diabetes in adult male offspring. The aim of the present study was to determine the effect of early growth restriction on muscle insulin action in late adult life. Rats were fed either a 20% or an isocaloric 8% protein diet during pregnancy and lactation. Offspring were weaned onto a 20% protein diet and studied at 15 Months of age. Soleus muscle from growth restricted offspring (LP) (of dams fed 8% protein diet) had similar basal glucose uptakes compared with the control group (mothers fed 20% protein diet). Insulin stimulated glucose uptake into control muscle but had no effect on LP muscle. This impaired insulin action was not related to changes in expression of either the insulin receptor or glucose transporter 4 (GLUT 4). However, LP muscle expressed significantly less (P<0.001) of the zeta isoform of protein kinase C (PKC zeta) compared with controls. This PKC isoform has been shown to be positively involved in GLUT 4-mediated glucose transport. Expression levels of other isoforms (betaI, betaII, epsilon, theta) of PKC were similar in both groups. These results suggest that maternal protein restriction leads to muscle insulin resistance. Reduced expression of PKC zeta may contribute to the mechanistic basis of this resistance.

Secretory unit of islet transplant objects (SUITO) index can predict outcome of intravenous glucose tolerance test.

BACKGROUND: Simple monitoring of engrafted islet function is important for follow-up of recipients after islet transplantation. We previously developed a simple assessment tool for islet graft function; the secretory unit of islet transplant objects (SUITO) index. The aim of this study was to clarify the relationship between the SUITO index and the outcomes of intravenous glucose tolerance tests (IVGTT). METHODS: Fifteen series of blood samples from 6 islet recipients were collected before 3, 5, 10, 20, and 30 minutes after injection of 0.5 g/kg 50% dextrose. The SUITO index was calculated using plasma C-peptide and glucose level at fasting baseline. Samples were divided into the following 3 groups; low-SUITO (SUITO index <10; n = 3); middle-SUITO (SUITO index > or =10 to <26; n = 4); and high-SUITO (SUITO index > or =26; n = 8). RESULTS: A threshold SUITO index of 26 showed good sensitivity (85.7%) and specificity (75.0%) to predict a blood glucose level of >10 mmol/L at 30 minutes. Blood glucose levels in the low-SUITO group were significantly higher than among the other 2 groups at baseline and 10, 20 and 30 minutes (P < .05). Glucose-level areas under the receiver-operating characteristic curve during IVGTT in the low-SUITO group were also significantly larger than among the other 2 groups (P < .05). CONCLUSION: The SUITO index, using only a fasting blood sample, predicted IVGTT outcomes.

Assessment of human islet isolation with four different collagenases.

BACKGROUND: The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. METHODS: The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). RESULTS: There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003). CONCLUSION: Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

Improved method of human islet isolation for young donors.

BACKGROUND: Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. METHODS: We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. RESULTS: Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. CONCLUSION: Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Induction of insulin-producing cells from human pancreatic progenitor cells.

INTRODUCTION: We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. MATERIALS AND METHOD: Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. RESULTS: The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. CONCLUSION: Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.

Comparison of fresh and cultured islets from human and porcine pancreata.

INTRODUCTION: For clinical islet transplantation, many centers have recently introduced of human islet cultures prior to transplantation. They provide flexibility to evaluate isolated islets and pretreat patients. However, isolated islets deteriorate rapidly in culture. In the present study, we compared fresh human and porcine islets with cultured islets for c-Jun NH(2)-terminal kinase (JNK) activity. MATERIALS AND METHODS: Islet isolations from human and porcine pancreata were performed using the standard Ricordi technique with a modified Edmonton protocol. Isolated islets cultured for 24 hours at 37 degrees C with 5% CO(2) in culture medium were evaluated for counts and JNK activity. RESULTS: After 24 hours of culture, the percentages of surviving islets were 86.9% for human and 47.3% for porcine sources. JNK activity in isolated islets declined to a low baseline level after 24-hour culture. CONCLUSION: Both human and porcine islets deteriorated rapidly in 24-hour cultures, although the in vitro conditions did not induce JNK activation.

Insulin signaling is inhibited by micromolar concentrations of H(2)O(2). Evidence for a role of H(2)O(2) in tumor necrosis factor alpha-mediated insulin resistance.

Both hyperglycemia and tumor necrosis factor alpha (TNFalpha) were found to induce insulin resistance at the level of the insulin receptor (IR). How this effect is mediated is, however, not understood. We investigated whether oxidative stress and production of hydrogen peroxide could be a common mediator of the inhibitory effect. We report here that micromolar concentrations of H(2)O(2) dramatically inhibit insulin-induced IR tyrosine phosphorylation (pretreatment with 500 microM H(2)O(2) for 5 min inhibits insulin-induced IR tyrosine phosphorylation to 8%), insulin receptor substrate 1 phosphorylation, as well as insulin downstream signaling such as activation of phosphatidylinositol 3-kinase (inhibited to 57%), glucose transport (inhibited to 36%), and mitogen-activated protein kinase activation (inhibited to 7.2%). Both sodium orthovanadate, a selective inhibitor of tyrosine-specific phosphatases, as well as the protein kinase C inhibitor Gö6976 reduced the inhibitory effect of hydrogen peroxide on IR tyrosine phosphorylation. To investigate whether H(2)O(2) is involved in hyperglycemia- and/or TNFalpha-induced insulin resistance, we preincubated the cells with the H(2)O(2) scavenger catalase prior to incubation with 25 mM glucose, 25 mM 2-deoxyglucose, 5.7 nM TNFalpha, or 500 microM H(2)O(2), respectively, and subsequent insulin stimulation. Whereas catalase treatment completely abolished the inhibitory effect of H(2)O(2) and TNFalpha on insulin receptor autophosphorylation, it did not reverse the inhibitory effect of hyperglycemia. In conclusion, these results demonstrate that hydrogen peroxide at low concentrations is a potent inhibitor of insulin signaling and may be involved in the development of insulin resistance in response to TNFalpha.