Evaluation of β-cell mass and function in the Göttingen minipig

Increased knowledge about β-cell mass and function is important for our understanding of the pathophysiology of type 2 diabetes (T2DM). The relationship between the two is difficult to study in humans, whereas animal models allow studies of consequences of, for example, reduction of β-cell mass and induction of obesity and procurement of the pancreas for histological examination. An overview of results obtained in the Göttingen minipig in relation to β-cell function, and mass is provided here. Effects of a primary reduction of β-cell mass have indicated that not all of the defects of pulsatile insulin secretion in human T2DM can be explained by reduced β-cell mass. Furthermore, induction of obesity has shown deterioration of β-cell function and morphological changes in the pancreas. As in humans, obesity leads to an increased β-cell volume in the minipig, and based on the increased number of islets, neogenesis of islets is an important factor in expansion of β-cell mass in this species. Measurement of β-cell function as an estimate of β-cell mass is, at present, the only method possible in humans, and this approach has been validated using lean and obese minipigs with a range of β-cell mass. The effects on β-cell function and mass of obesity of longer duration and/or more pronounced hyperglycaemia remains to be determined, but the models developed so far represent a valuable tool for such investigations.     

Balancing needs and means: the dilemma of the β-cell in the modern world

The insulin resistance of type 2 diabetes mellitus (T2DM), although important for its pathophysiology, is not sufficient to establish the disease unless major deficiency of β-cell function coexists. This is demonstrated by the fact that near-physiological administration of insulin (CSII) achieved excellent blood glucose control with doses similar to those used in insulin-deficient type 1 diabetics. The normal β-cell adapts well to the demands of insulin resistance. Also in hyperglycaemic states some degree of adaptation does exist and helps limit the severity of disease. We demonstrate here that the mammalian target of rapamycin (mTOR) system might play an important role in this adaptation, because blocking mTORC1 (complex 1) by rapamycin in the nutritional diabetes model Psammomys obesus caused severe impairment of β-cell function, increased β-cell apoptosis and progression of diabetes. On the other hand, under exposure to high glucose and FFA (gluco-lipotoxicity), blocking mTORC1 in vitro reduced endoplasmic reticulum (ER) stress and β-cell death. Thus, according to the conditions of stress, mTOR may have beneficial or deleterious effects on the β-cell. β-Cell function in man can be reduced without T2DM/impaired glucose tolerance (IGT). Prospective studies have shown subjects with reduced insulin response to present, several decades later, an increased incidence of IGT/T2DM. From these and other studies we conclude that T2DM develops on the grounds of β-cells whose adaptation capacity to increased nutrient intake and/or insulin resistance is in the lower end of the normal variation. Inborn and acquired factors that limit β-cell function are diabetogenic only in a nutritional/metabolic environment that requires high functional capabilities from the β-cell.     

Regulation of β-cell mass and function by the Akt/protein kinase B signalling pathway

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of β-cell mass and function, demonstrated both in vitro and in vivo . The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of β cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of β-cell mass and function by Akt.     

Type 2 diabetes – a matter of failing β-cell neogenesis? Clues from the GK rat model

Now that reduction in β-cell mass has been clearly established in humans with type 2 diabetes mellitus (T2D), the debate focuses on the possible mechanisms responsible for decreased β-cell number. Appropriate inbred rodent models are essential tools for this purpose. The information available from the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2D, is reviewed in such a perspective. We propose that the defective β-cell mass in the GK model reflects mostly a persistently decreased β-cell neogenesis. The data discussed in this review are consistent with the notion that poor proliferation and/or survival of the endocrine precursor cells during GK foetal life will result in a decreased pool of endocrine precursors in the pancreas, and hence an impaired capacity of β-cell neogenesis (either primary in the foetus or compensatory in the newborn and the adult). As we also demonstrated that β-cell neogenesis can be pharmacologically reactivated in the GK model, our work supports, on a more prospective basis, the concept that facilitation of T2D treatment may be obtained through β-cell mass expansion after stimulation of β-cell regeneration/neogenesis in diabetic patients.     

Towards better understanding of the contributions of overwork and glucotoxicity to the β-cell inadequacy of type 2 diabetes

Type 2 diabetes (T2D) is characterized by reduction of β-cell mass and dysfunctional insulin secretion. Understanding β-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when β-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, β-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. β-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains β-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human β-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how β-cell phenotype evolves as T2D develops and progresses.     

Islet gene expression and function in type 2 diabetes; studies in the Goto-Kakizaki rat and humans

Defective β-cell function with resulting impairment of glucose-stimulated insulin release is a critical factor in the pathogenesis of type 2 diabetes. Accumulated studies in pancreatic islets of the spontaneously diabetic Goto-Kakizaki (GK) rat suggest that this is a useful animal model of type 2 diabetes. The GK rat is non-obese, and abnormal glucose regulation develops early in life in association with impaired insulin secretion. There are some differences in islet morphology and function reported between different GK rat colonies. In addition to reduction of β-cell mass, a number of β-cell defects have been described with possible relevance for the reduced insulin secretion. Interestingly, some of these defects have also been shown in isolated islets from type 2 diabetic humans. The polygenic nature of diabetes heredity in the GK rat may well resemble the genetic basis in the majority of patients with type 2 diabetes. Here, we review studies concerning β-cell function and islet gene expression in the GK rat and compare it with the limited number of investigations on similar topics in isolated islets from patients with type 2 diabetes.     

No deterioration in insulin sensitivity,but impairment of both pancreatic β-cell function and glucose sensitivity,in Japanese women with former gestational diabetes mellitus

To identify the primary pathogenic factors involved in the development of Type 2 diabetes mellitus (DM), we studied Japanese women with former gestational diabetes mellitus (GDM) who are at risk for the later development of Type 2 DM. We used the minimal model analysis derived from frequently sampled intravenous glucose tolerance test (FSIGT). The subjects consisted of eight non-obese women with a history of GDM and eight non-obese normal women as control subjects. The 75 g oral glucose tolerance test (75 g OGTT) performed within 6 months of delivery confirmed that all the subjects with former GDM had a normal glucose tolerance. Insulin sensitivity (SI) derived from the minimal model analysis was not different between the two groups. Glucose effectiveness at zero insulin (GEZI), reflecting tissue glucose sensitivity, was significantly lower in former GDM patients than in control subjects (1.18 ± 0.34 vs 2.26 ± 0.29 × 10⁻² min⁻¹, p < 0.05). The early phase insulin secretion found in FSIGT was markedly reduced to 56 % of that observed in control subjects (1250 ± 187.4 vs 2223 ± 304.3 pmol l⁻¹ min, p < 0.01). Our results indicate that in former GDM patients, who are Japanese and non-obese, impairment of the acute insulin response to glucose and a decrease in tissue glucose sensitivity rather than insulin sensitivity are the primary pathogenic factors involved. Copyright © 1998 John Wiley & Sons, Ltd.     

Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α-granule membrane

The vitronectin receptor (VnR or α_vβ₃) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β₃ subunit in common: GP IIb–IIIa complexes (or α_IIbβ₃) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the α_v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, α_vβ₃ was mostly detected in internal membrane systems including those of α-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of α_vβ₃ was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb–IIIa, although intracellular staining was present in EM and α-granules were again labelled.     

Thiazolidinedione therapy in the prevention/delay of type 2 diabetes in patients with impaired glucose tolerance and insulin resistance

AIM: The second-generation thiazolidinediones (TZDs), rosiglitazone and pioglitazone, significantly decrease fasting plasma glucose and glycosylated haemoglobin (HbA(1c)) levels in patients with diabetes. Recent studies suggest that early treatment with TZDs may prevent the progression from insulin resistance (IR) to type 2 diabetes mellitus (T2DM). This prospective analysis examined the effect of early TZD treatment in the prevention or delay of T2DM in a multiethnic population with impaired glucose tolerance (IGT) and IR. METHODS: The analysis included 172 patients (aged 29-86 years) with IGT and IR (normal or borderline HbA(1c), C-peptide levels > 2 mg/ml, fasting blood sugar 100-125 mg/dl, and 2-h postprandial blood glucose levels 140-200 mg/dl). Patients in the active treatment group (n = 101) had received troglitazone for an average of 10 months before being randomly switched to rosiglitazone (4 mg/day) or pioglitazone (30 mg/day). Patients were switched when troglitazone was withdrawn from the US market because of liver toxicity concerns. Patients with IGT and IR who received no antidiabetic medication served as a control group (n = 71). HbA(1c) and C-peptide levels were measured at baseline (2 years) and study end point (3 years). Kaplan-Meier testing, using time to outcome as the main outcome variable, determined risk reduction in the TZD group relative to the control group. RESULTS: Mean HbA(1c) and C-peptide levels decreased for patients receiving either TZD at the 2-year assessment, and reductions were maintained at study end point. After 2 years, none of the patients receiving TZD therapy progressed to T2DM; three patients progressed to T2DM by study end point. In the control group, 11 patients became diabetic after 2 years and 19 patients became diabetic by the end of the study. The incidence (risk reduction) of diabetes after 3 years was 88.9% lower in the TZD group compared with the control group (p < 0.001). CONCLUSIONS: The TZDs, rosiglitazone and pioglitazone, were effective in reducing HbA(1c) and C-peptide levels in patients with IGT/IR. Progression of IR/IGT to T2DM appears to be significantly delayed or prevented with early TZD treatment.     

Glucose regulation of islet stress responses and β-cell failure in type 2 diabetes

Pancreatic β-cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional β-cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine-induced apoptosis and hypoxia. The glucose regulation of oxidative stress-response and integrated stress-response genes in cultured rat islets follows an asymmetric V-shaped profile parallel to that of β-cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional β-cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, β-cell demise under glucotoxic conditions may also result from β-cell hypoxia and, in vivo , from their exposure to inflammatory cytokines released locally by non-endocrine islet cells. A better understanding of the relative contribution of each type of stress to β-cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional β-cell mass in glucose intolerant and type 2 diabetic patients.     

Prevalence and significance of anticardiolipin, anti-β2 glycoprotein I and anti-prothrombin antibodies in chronic hepatitis C

Antiphospholipid antibodies have been demonstrated in chronic hepatitis C, but their clinical and pathogenetic significance remains elusive. We prospectively studied 115 patients (85 men, mean age 36.9 years) with chronic hepatitis C without cirrhosis and treated by α-interferon (α-IFN). Antiphospholipid determinations comprised anticardiolipin (ACA), anti-β₂-glycoprotein I and anti-prothrombin antibodies of the IgG and IgM classes. At entry, 24 patients (21%) were found to possess low to moderate ACA levels (18 IgG, two IgM and four both isotypes) compared with only 4/115 age- and sex-matched control subjects (3.5% P  = 0.001). ACA positivity rate increased to 31% ( P  = 0.01) after a 6-month course of α-IFN treatment. In contrast, the prevalence of anti-β₂-glycoprotein I and anti-prothrombin antibodies was not significantly different from controls at either time point. The presence of ACA correlated with that of antinuclear antibodies ( P  = 0.0002), but was not associated with parameters such as histological activity, viral burden and response to α-IFN, nor with a history of thrombosis or pregnancy loss. However, a non-significant trend of higher incidence of mild thrombocytopenia among ACA-positive patients was observed. We conclude that low-titre ACA positivity is a common finding in patients with chronic hepatitis C, especially following α-IFN treatment, but does not select a category with different clinical features. These data are in keeping with the absence of associated anti-β₂GPI and anti-prothrombin antibodies, and do not support a role for HCV infection in the pathogenesis of the antiphospholipid syndrome.     

Activating point mutations in the βC chain of the GM-CSF, IL-3 and IL-5 receptors are not a major contributory factor in the pathogenesis of acute myeloid leukaemia

A number of mutant growth factor receptors have been described which are constitutively activated and confer factor independence on growth factor dependent cells, possibly through constitutive dimerization in the absence of ligand or induction of a conformational change. Mutations in receptor chains may therefore contribute to the pathogenesis of haemopoietic malignancies, for example by causing constitutive receptor activation or uncontrolled downstream signalling. Since most of the activated mutants reported for the β_C chain of the GM-CSF/IL-3/IL-5 receptor involve point mutations or truncations of the extracellular domain, we have analysed the coding sequence of this region using RT-PCR-SSCP of RNA from blast cells of 31 patients with acute myeloid leukaemia (AML). Two point mutations detected were silent, C³⁰¹ → T (Cys91) and C¹³⁰⁶ → T (Ser426). The latter had previously been identified with an allele frequency of 0.13 in the general population. Two further point mutations detected led to amino acid substitutions, G⁷⁷³ → C (Glu249Gln), which is equivalent to the mouse sequences, and G⁹⁶² → A (Asp312Asn), both of which were found at similar frequencies in normal controls. Activating mutations of the β_C chain which might contribute to the pathogenesis of the disease are therefore rare in AML.     

Resistance to activated protein C activity of an anti-/2 -glycoprotein I antibody in the presence of β-glycoprotein I

Some researchers claim that lupus anticoagulant-positive plasma may cause a false-positive reaction in the test for activated protein C (APC) resistance, a hereditary thrombophilic state characterized by abnormal factor V, which frequently causes venous thrombosis, We investigated whether anti-/3₂ -glycoprotein I antibody (aGPI), which has recently come to be regarded as an anti-cardiolipin antibody (aCL) itself, might have an effect on the APC resistance test.     

Effects of resveratrol therapy on glucose metabolism,insulin resistance,inflammation, and renal function in the elderly patients with type 2 diabetes mellitus: A randomized controlled clinical trial protocol

Background:Diabetes mellitus is a spectrum of metabolic disorders characterized by hyperglycemia and shows a growing global public health problem in the elderly. Resveratrol presents antiaging, anti-inflammatory, antitumor antioxidant, and cardioprotective activities. The purpose of this study was to investigate the ameliorative effects of resveratrol on blood glucose, insulin metabolism, lipid profile, renal function, inflammation, and nutrient sensing systems in the elderly patients with type 2 diabetes mellitus.Methods:The study is a single-blind, parallel-group, randomized controlled clinical trial consisting of a 6-month treatment period. A total of 472 elderly patients with type 2 diabetes mellitus were enrolled, and included participants will be randomized into 2 groups: resveratrol (n = 242) and placebo (n = 230). The clinical efficacy and changes in clinical parameters in each group will be measured at the indicated time. Clinical parameters included blood glucose, insulin resistance index, blood lipid index, proinflammatory cytokines, renal function, and nutrient sensing systems.Results:Resveratrol treatment greatly improved glucose metabolism, insulin tolerance, and insulin metabolism compared to placebo. Resveratrol relieved symptoms through enhancing nutrient sensing systems, which in turn reduced production and activity of glucose-6-phosphatase. Compared with placebo, resveratrol treatment significantly decreased proinflammatory cytokines glycated hemoglobin/hemoglobin A1c, interleukin-6, tumor necrosis factor-alpha, and interleukin-1beta in the elderly diabetes. Resveratrol treatment decreased blood glucose parameters, improved the lipid profile (total cholesterol, low-density lipoprotein, high-density lipoprotein, and triglycerides), and renal function compared to placebo.Conclusion:In conclusion, resveratrol treatment improves inflammation, renal function, blood glucose parameters, inflammation, insulin resistance, and nutrient sensing systems in the elderly patients with type 2 diabetes mellitus, indicating resveratrol may be a potential therapeutic drug for the treatment of the elderly patients with type 2 diabetes mellitus.     

Efficacy of the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide (P32/98) in fatty Zucker rats with incipient and manifest impaired glucose tolerance

Aim: Incretin enhancers are a new class of antidiabetic drugs with promising therapeutic potential for type 2 diabetes. Therapeutic intervention in prediabetes is an attractive strategy for preventing or delaying diabetes onset. The aim of the present study was to investigate the therapeutic effects of incretin enhancement on incipient impaired glucose tolerance (iIGT) and manifest IGT (mIGT) using the dipeptidyl peptidase IV (DPP‐4) inhibitor P32/98‐ and fatty Zucker rat (ZR, fa/fa) as a model. Methods: ZRs were classified into groups with iIGT and mIGT (n = 10 per group). P32/98 (21.61 mg/kg body weight) was administered orally to ZR with iIGT and mIGT once daily for 6 and 3 weeks respectively. Assessments included body weight, morning blood glucose and insulin, oral glucose tolerance test (oGTT; 2 g glucose/kg), plasma parameters and blood glucose day–night profile (DNP). In addition, glucose responsiveness of isolated islets and islet morphology were analysed. Results: P32/98 decreased non‐fasting morning blood glucose more effectively in ZR with iIGT than in ZR with mIGT. Compared with study entry, P32/98 improved DNP of blood glucose in ZR with mIGT and nearly normalized DNP in ZR with iIGT. An acute bolus of inhibitor reduced glucose load during oGTT in rats chronically treated with placebo or P32/98. In contrast to placebo‐treated rats, rats receiving long‐term treatment with P32/98 required less insulin during oGTT. This effect was larger in rats with iIGT vs. rats with mIGT. In isolated pancreatic islets of ZR with mIGT, treatment with P32/98 decreased pancreatic insulin content and increased glucose responsiveness, while the β‐cell volume density was unaffected. P32/98 significantly reduced triglycerides and non‐esterified fatty acids. Intestinal growth was comparable between inhibitor‐ and placebo‐treated fatty rats. Conclusions: Enhancement of incretin with the DPP‐4 inhibitor P32/98 has therapeutic effects in hyperinsulinaemia, hyperglycaemia and IGT in ZR with iIGT and mIGT. Apparently, administration of P32/98 in ZR with iIGT results in more efficient β‐cell function, which is associated with less need for insulin to cope with deterioration of glucose tolerance. Importantly, P32/98 has a strong effect on dyslipidaemia in mIGT. P32/98 has no side effect on intestinal growth. Daily intake of P32/98 is a promising strategy for treatment of glucose intolerance and has the potential to prevent type 2 diabetes.