Hyperfunction of the entero-PP axis in non-insulin dependent diabetes mellitus

In a previous study we have found that the plasma pancreatic polypeptide (PP) response to oral glucose loading is exaggerated in diabetic patients compared with normal subjects. We have investigated, therefore, the effects of a protein-rich meal or meat extract ingestion on plasma PP secretion and examined also the effects of intravenous arginine administration on PP levels in normal subjects and in patients with noninsulin-dependent diabetes mellitus (NIDDM). Following a 600 Kcal meal ingestion, plasma PP levels increased immediately and showed biphasic secretion in normal subjects and in NIDDM, but the response was exaggerated in NIDDM. A 50 g meat extract administration also produced an exaggerated PP response in NIDDM compared with normal subjects. In NIDDM and normal subjects, plasma PP levels did not change significantly during an arginine infusion (30 g for 45 min) but after the end of the infusion PP levels increased significantly compared with basal levels. In normal subjects, plasma PP rose abruptly after a bolus arginine injection (4 g for 2 min) and then remained at significantly high levels even 30 min after the injection. In NIDDM, however, plasma PP levels tended to increase, but not significantly, after the bolus arginine injection. Since in NIDDM the protein-rich meal and meat extract ingestion produced an exaggerated rise in plasma PP while the PP responses to the intravenous arginine administration were rather impaired compared with normal subjects, we suggest that the entero-PP axis is overactive in NIDDM.     

Early reduction of circulating homocysteine levels in Goto-Kakizaki rat, a spontaneous nonobese model of type 2 diabetes

Diabetes mellitus is associated with increased risk for cardiovascular disorders, which are major causes of mortality in this disease. Hyperhomocysteinemia, defined by high plasma homocysteine levels, is an independent risk factor for the development of cardiovascular diseases. Type 2 diabetic patients have higher circulating homocysteine levels than healthy subjects and these levels are even higher in plasma of obese than nonobese diabetic patients. Homocysteine metabolism that has been studied in 2 animal models of type 2 diabetes with obesity led to conflicting data. The aim of the present study was to analyze homocysteine metabolism in a spontaneous nonobese model of type 2 diabetes, the Goto-Kakizaki rats at various successive and well characterized stages of the disease: during early postnatal normoglycemia, at the onset of hyperglycemia (around weaning), and during chronic mild hyperglycemia with progressive insulin resistance. Compared to age-matched Wistar controls, Goto-Kakizaki rats showed lower plasma levels of homocysteine and a falling trend in its major byproduct antioxidant, glutathione, from the prediabetic stage onwards. Concomitantly, Goto-Kakizaki rats exhibited increased liver activity of cystathionine beta synthase, which catalyzes the condensation of homocysteine with serine in the first step of the transsulfuration pathway. These results emphasize a strong association between homocysteine metabolism and insulin via the first step of the hepatic transsulfuration pathway in Goto-Kakizaki rats.     

Diabetic cataract of the musk shrew (Suncus murinus, Insectivora) exhibiting spontaneous non-insulin dependent diabetes mellitus (NIDDM).

The EDS colony, developed as a new laboratory colony of the musk shrew, is characterized by a high incidence of early-onset spontaneous non-insulin dependent diabetes mellitus (NIDDM). In this colony, a few diabetic shrews exhibited a cataract at 1 month after the onset of diabetes, and all diabetic shrews had bilateral cataracts at 5 months after the onset of diabetes. In contrast, cataractous animals were never observed among non-diabetic shrews. These results suggest that the cataract in the EDS colony is a diabetic complication.     

Differential glucose-regulation of microRNAs in pancreatic islets of non-obese type 2 diabetes model Goto-Kakizaki rat

Background

The Goto-Kakizaki (GK) rat is a well-studied non-obese spontaneous type 2 diabetes (T2D) animal model characterized by impaired glucose-stimulated insulin secretion (GSIS) in the pancreatic beta cells. MicroRNAs (miRNAs) are short regulatory RNAs involved in many fundamental biological processes. We aim to identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats and investigate both their short- and long term glucose-dependence during glucose-stimulatory conditions.

Methodology/Principal Findings

Global profiling of 348 miRNAs in the islets of GK rats and Wistar controls (females, 60 days, N = 6 for both sets) using locked nucleic acid (LNA)-based microarrays allowed for the clear separation of the two groups. Significant analysis of microarrays (SAM) identified 30 differentially-expressed miRNAs, 24 of which are predominantly upregulated in the GK rat islets. Monitoring of qPCR-validated miRNAs during GSIS experiments on isolated islets showed disparate expression trajectories between GK and controls indicating distinct short- and long-term glucose dependence. We specifically found expression of rno-miR-130a, rno-miR-132, rno-miR-212 and rno-miR-335 to be regulated by hyperglycaemia. The putative targets of upregulated miRNAs in the GK, filtered with glucose-regulated mRNAs, were found to be enriched for insulin-secretion genes known to be downregulated in T2D patients. Finally, the binding of rno-miR-335 to a fragment of the 3′UTR of one of known down-regulated exocytotic genes in GK islets, Stxbp1 was shown by luciferase assay.

Conclusions/Significance

The perturbed miRNA network found in the GK rat islets is indicative of a system-wide impairment in the regulation of genes important for the normal functions of pancreatic islets, particularly in processes involving insulin secretion during glucose stimulatory conditions. Our findings suggest that the reduced insulin secretion observed in the GK rat may be partly due to upregulated miRNA expression leading to decreased production of key proteins of the insulin exocytotic machinery.     

Characterization of the insulin resistance in liver cirrhosis: a comparison with non-insulin dependent diabetes mellitus.

To characterize the mechanisms of insulin resistance in liver cirrhosis (LC), we estimated the peripheral tissue sensitivity and responsiveness to insulin using the euglycemic clamp technique and determined the insulin binding to erythrocytes in patients with compensated LC as well as in patients with non-insulin dependent diabetes mellitus (NIDDM). The insulin dose-response curves of the glucose metabolic clearance rates (MCR) were shifted to the right and downward both in patients with LC and NIDDM, indicating a reduced sensitivity and responsiveness to insulin. In the cirrhotics, MCR at the maximally effective insulin level, an index of insulin responsiveness, was correlated with fasting insulin levels (r = -0.57, P < 0.01) and sigma BG in 75 gOGTT (r = -0.43, P < 0.05), but no correlations were found between them and the diabetics. Although specific insulin bindings to erythrocytes were significantly lower in patients both with LC and NIDDM, Scatchard analysis revealed a significant decrease in the number of insulin receptors in the cirrhotics, and a decrease in the empty-site affinity in the diabetics. These findings suggest that insulin resistance in LC consists of a combination of binding and postbinding defects. The latter defect may be caused by basal hyperinsulinemia and contribute to the development of glucose intolerance. Although binding and postbinding abnormalities are also found in NIDDM, the mechanisms of insulin resistance in LC and NIDDM may be different.     

Postprandial glycaemic effects of a long-acting somatostatin analogue (octreotide) in non-insulin dependent diabetes mellitus

Postprandial changes in blood glucose, insulin and glucagon were examined in 7 non-insulin dependent diabetic patients, before and after 3 days' treatment with the somatostatin analogue, octreotide (50 ug injected subcutaneously thricedaily). After octreotide injection, postprandial rises in plasma insulin and glucagon were significantly flattened. The postprandial glycaemic rise was delayed but the area under the glycaemic curve was not increased. Animal studies have suggested that octreotide inhibits growth hormone and glucagon secretion much more powerfully than native somatostatin, while relatively sparing insulin secretion. However, the present findings suggest that this analogue is not sufficiently selective to be therapeutically useful in non-insulin dependent diabetes.     

Developing a new model for non-insulin dependent diabetes mellitus (NIDDM) by using the Philippine wild mouse, Mus musculus castaneus.

The Philippine wild-caught castaneus mouse (Mus musculus castaneus) and laboratory mouse (C57BL/6J: B6) were used to develop a new non-insulin dependent diabetes mellitus (NIDDM) model. Offspring from the cross between a wild male and B6 female were backcrossed to the sire. One male which exhibited highest fasting hyperglycemia (190 mg/dl) among eighty-seven backcross offspring was selected at 10 weeks of age, and crossed with a B6 female to comprise the fundamental stock (F0). Thereafter, full-sib mating was performed to develop a new inbred strain named CBD (Castaneus-B6 diabetic) mouse. Mice with relatively higher fasting hyperglycemia among F0 and F1 generations were selected for breeding. From the F2 generation, mice were defined as diabetic when blood glucose levels exceeded 200 mg/dl at 120 min in intraperitoneal glucose tolerance test (IPGTT) at 10 weeks of age, and have been selectively bred. The incidence of diabetic males from the F3-F6 generation fluctuated 45-75% at 10 weeks of age and 59-72% at 20 weeks of age. Diabetic males had about two-fold higher fasting glucose and insulin levels than B6 males. Glucose-stimulated insulin secretion was impaired in diabetic CBD mice compared to B6 males at 20 weeks. Moreover, diabetic mice had slight obesity compared to B6 mice. These facts indicated that diabetic features of CBD mice resemble NIDDM in humans. The CBD strain, characterized by high incidence and early onset of diabetes with mild obesity would be of value as a new NIDDM model. The method, utilizing wild castaneus mouse of different origin from laboratory mice, maybe useful in the development of other animal models.     

The combined use of insulin and sulfonylurea therapy in patients with non-insulin dependent diabetes mellitus

This study was initiated in order to evaluate the clinical efficacy of glipizide treatment in 18 patients with non-insulin dependent diabetes mellitus in poor glycemic control with insulin. Insulin dose was kept constant, and various facets of carbohydrate and lipid metabolism were evaluated before and from 4-6 months after the addition of glipizide. The results indicated that fasting and post-prandial glucose concentration were significantly (P less than 0.001) reduced following glipizide treatment, associated with a commensurate fall in glycosylated hemoglobin concentration. The average fall in fasting plasma glucose concentration in the total patient group approximated 60 mg/dl, and the mean decrement in 8 of the 18 patients who had a fall of more than 70 mg/dl in fasting glucose was 93 mg/dl. These results demonstrate that the addition of glipizide to the treatment program of patients with non-insulin dependent diabetes mellitus poorly controlled on insulin can lead to substantial clinical benefit.     

Effect of pravastatin on serum lipids, apolipoproteins and lipoprotein (a) in patients with non-insulin dependent diabetes mellitus.

In 43 patients with non-insulin dependent diabetes mellitus (NIDDM) associated with hypercholesterolemia, the effect of pravastatin, a potent HMG CoA-reductase inhibitor, on serum lipids, apolipoproteins and lipoprotein (a) was examined. After 1 to 3 months administration of 10 mg per day of pravastatin, the serum levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol (LDL-C) were significantly decreased, while the serum level of high density lipoprotein cholesterol (HDL-C) was significantly increased in patients with NIDDM. The levels of apolipoproteins B (apo B) and E were significantly decreased, while apolipoprotein AI (apo A-I) was not changed by the administration of pravastatin. The atherogenic indices (LDL-C/HDL-C and apo B/apo A-I) were significantly decreased by the administration of this drug. The serum lipoprotein (a), which was increased in the diabetic patients, was not affected by the pravastatin treatment. Plasma glucose and hemoglobin A1c levels were not affected by the treatment. We concluded that pravastatin is a potentially useful agent in the treatment of hypercholesterolemia in patients with NIDDM.     

DNA polymorphism of Pvu II site in the lipoprotein lipase gene in patients with non-insulin dependent diabetes mellitus

We studied the effect of variation at the lipoprotein lipase (LPL) gene locus on the susceptibility of individuals with non-insulin dependent diabetes mellitus (NIDDM) in a population of 110 NIDDM patients and 91 controls. Our objective was to study the relationship between the LPL-Pvu II polymorphism and NIDDM and lipid metabolism. PCR-RFLP was used to determine the DNA polymorphism of the sixth intron of the LPL gene. The frequencies of the genotypes in case and control groups were 29.1 and 30.8% for P+/P+; 45.5 and 36.3% for P+/P-; 25.5 and 33% for P-/P- respectively. There was no significant difference in frequencies of genotypes between the two groups. Logistic regression analysis revealed that triacylglycerol (TAG) and apolipoprotein E levels were associated with NIDDM, whereas Pvu II genotypes were not found as independent risk factors for the disease. Overall this study demonstrates the role of the Pvu II polymorphism in the LPL gene in modulating plasma lipid/lipoprotein levels in patients with NIDDM.     

Mucosal inflammation in a genetic model of spontaneous type I diabetes mellitus

The BioBreeding (BB) rat provides a model of spontaneous type I diabetes mellitus that closely resembles the human disease. Diabetes-prone BB rats demonstrate increased intestinal permeability prior to the development of insulinitis. Studies suggest that alterations in intestinal permeability can lead to increased intestinal inflammatory activity. Diabetes-prone (BBdp) and diabetes-resistant (BBdr) BB rats were examined at 45 days and at >70 days of age following the development of clinical disease (BBd). In separate experiments, tissue was assayed for myeloperoxidase (MPO) or fixed for histological assessment and immunohistochemistry. Blood was obtained for leukocyte MPO measurements and morphological assessment of circulating leukocytes. MPO activity was significantly elevated in the distal small intestine of 45-day-old BBdp rats. In contrast, at >70 days of age, MPO activity was significantly increased throughout the small intestine of BBd and non-diabetic BBdp rats. Subsequently, all measurements were performed in >70-day-old rats. An increase in inflammatory infiltrate was noted in the distal small intestine of BBd rats by light microscopy. Infiltrating cells were identified as bands (a maturing cell type of the neutrophil lineage) and mature neutrophils. The findings suggest diabetes susceptibility is associated with an increase in intestinal inflammatory activity.     

Alterations in the growth hormone-insulin-like growth factor axis in insulin dependent diabetes mellitus.

The growth hormone (GH)-insulin-like growth factor (IGF) axis and insulin are major anabolic effectors in promoting weight gain and linear growth. These two anabolic systems are interlinked at many levels, thus abnormalities in one of these systems effect the other causing disordered metabolic homeostasis. Insufficient portal insulinization in insulin dependent diabetes mellitus (IDDM) results in hepatic GH resistance and increased production of IGF-binding proteins-1 (IGFBP-1) and IGFBP-2. GH resistance is reflected by decreased hepatic IGF-I production. In addition, changes in other GH-dependent proteins are also observed in IDDM. Increased proteolysis of IGFBP-3 results in reduction of intact IGFBP-3. Serum ALS levels are also slightly diminished in untreated diabetic patients. Hepatic resistance to GH is, at least in part, caused by diminished GH receptors as reflected by diminished circulating GHBP levels. In addition, there is also evidence from experimental and human studies suggesting post-receptor defect(s) in GH action. As a result of these changes, circulating total and free IGF-I levels are decreased during insulinopenia. Lack of negative feed-back effect of IGF-I on GH secretion causes GH hypersecretion which increases hyperglycemia by decreasing sensitivity to insulin. GH hypersecretion in poorly controlled diabetic patients may play a role in the pathogenesis of diabetic vascular complications. Most of these abnormalities in the GH-IGF axis in diabetes are reversed by effective insulinization of the patient. Addition of IGF-I treatment to insulin in adolescents with IDDM allows correction of GH hypersecretion, improves insulin sensitivity and glycemic control, and decreases insulin requirements. The effect of IGF-I treatment on diabetic complications has yet to be seen.     

Low dose streptozotocin diabetes after partial pancreatectomy in dogs. Histological findings in a new type of experimental diabetes

Permanent diabetes was produced in 16 out of 55 dogs by partial pancreatectomy (77% of the calculated organ weight) and simultaneous infusion of 2 mg/kg streptozotocin into the superior pancreaticoduodenal artery. The animals exhibited hyperglycemia, absolute lack of endogenous B-cell function, and ketosis, but no exocrine pancreatic insufficiency. 21 animals needed up to 7 additional subsequent intravenous streptozotocin injections (15 mg/kg each at intervals of 3 days). In 18 animals the procedure failed to render them diabetic; they died mainly from toxic effects of the drug. There were severe pathohistological changes in all streptozotocin-treated animals. Besides the well known alterations of the islets of Langerhans, lymphocytic inflammations were found in numerous organs including the exocrine pancreas. In most cases they were combined with degenerative changes of the organ parenchyma, particularly in kidney and liver. These findings were not correlated to the sex of the animals, to the occurrence and severity if diabetes, to the time of survival, or to the streptozotocin dose applied. But they were obviously related to the clinical picture existing besides diabetes. It is concluded that the model of experimental diabetes presented might be useful in a carnivorous big animal species but that toxic streptozotocin effects are to be expected when the dose administered exceeds 2 mg/kg.     

Genetic dissection of ``OLETF', a rat model for non-insulin-dependent diabetes mellitus

To elucidate the genetic factors underlying non-insulin-dependent diabetes mellitus (NIDDM), we performed genome-wide quantitative trait locus (QTL) analysis, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF rat is an excellent animal model of NIDDM because the features of the disease closely resemble human NIDDM. Genetic dissection with two kinds of F2 intercross progeny, from matings between the OLETF rat and non-diabetic control rats F344 or BN, allowed us to identify on Chromosome (Chr) 1 a major QTL associated with features of NIDDM that was common to both crosses. We also mapped two additional significant loci, on Chrs 7 and 14, in the (OLETF × F344)F₂ cross alone, and designated these three loci as Diabetes mellitus, OLETF type Dmo 1, Dmo2 and Dmo3 respectively. With regard to suggestive QTLs, we found loci on Chrs 10, 11, and 16 that were common to both crosses, as well as loci on Chrs 5 and 12 in the (OLETF × F344)F₂ cross and on Chrs 4 and 13 in the (OLETF × BN)F₂ cross. Our results showed that NIDDM in the OLETF rat is polygenic and demonstrated that different genetic backgrounds could affect ``fitness' for QTLs and produce different phenotypic effects from the same locus. Received: 9 October 1997 / Accepted: 29 January 1998