Sweating the Small Stuff: A Closer Look at the Endocrine Pancreas Throughout Stages of Type 1 Diabetes Progression.

In diabetes, pancreatic beta-cells play a key role. These cells are organized within structures called islets of Langerhans inside the pancreas and produce insulin. Insulin is one of the main hormones contributing to glucose homeostasis, which is the hallmark of diabetes. In vivo imaging of beta-cells and their function are required for diagnosis purposes and assessment of new treatments. However, this task is challenging due to the localization of the pancreas deep inside the abdominal cavity and the small size and heterogeneous distribution of islets of Langerhans throughout the organ. Indeed, islets of Langerhans represent only 1-2% of the pancreas' total volume. All state-of-the-art medical imaging techniques such as MRI, SPECT or PET cannot resolve individual islets. Furthermore, they require the use of tracers to detect the beta-cells. Hence, the research for imaging islets of Langerhans, including the identification of specific beta-cell tracers is a highly active field of research. Indeed, no marker is currently available for clinical studies and diagnosis. For animal models, optical techniques offer a sufficient spatial resolution to image individual islets with a high specificity provided by fluorescent markers. Optical Coherence Microscopy (OCM) is a novel interferometric imaging technique measuring the back-scattered light from a sample to reveal its structure in depth. OCM provides an intrinsic contrast depending on the spatial variation of the index of refraction. A Fourier transform of the acquired spectrum extracts the whole depth structure, thereby requiring only scans in two dimensions to obtain a three-dimensional image with a fast acquisition time. In this thesis, we exploited the label-free capabilities and fast acquisition rate of OCM to study islets of Langerhans. We demonstrated that OCM signal is specific to the beta-cell volume due to the dominant scattering of the zinc-insulin crystalline structures inside the secretory granules. Besides structural information, OCM reveals the vascularization of pancreatic islets in situ. These advantages were exploited to image the progression of autoimmune diabetes and for the characterization of beta-cell tracers. The intrinsic contrast of OCM does not require genetically modified mice, nor the use of exogenous agents to image islets and their vascularization. OCM enhanced with a confocal fluorescence channel can assess beta-cell tracers labeled with a fluorophore in vitro and in vivo in wild type mice. As a proof of principle, we assessed the specificity of Cy5.5-exendin-3, an analogue of the glucagon-like peptide-1 receptor (GLP1R) for beta-cells. Our results confirmed the co-localization of the fluorescence-tagged tracer with the OCM islet signal. The high resolution of OCM serves as a pre-clinical optical platform to facilitate the initial tests aiming to determine the specificity of beta-cell tracers in vivo in mice. Finally, in order to perform non-invasive longitudinal studies, islets of Langerhans were transplanted into the anterior chamber of the eye. This transplantation model allowed us to follow individual islets over time in a spontaneous mouse model of type I diabetes. We demonstrated that alterations of the islet microvasculature accompany the progression of diabetes with a strong correlation between the degree of insulitis and the density of the vascular network.

1. Oxygen free radicals have been suggested to be a contributory factor in complications of diabetes mellitus. There are many reports indicating the changes in parameters of oxidative stress in diabetes mellitus. In this study we aimed to identify whether oxidative stress occurs in the liver and pancreas in the initial stages of development of diabetes. 2. We therefore investigated the lipid peroxide level (thiobarbituric acid-reactive substances, TBARS) and activities of antioxidant enzymes [superoxide dismutase (SOD), catalase and glutathione peroxidase] in liver and pancreas of control and streptozotocin-induced diabetic rats at various stages of development of diabetes. 3. Male Sprague-Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups consisting of six rats each. Rats in these subgroups were studied at weekly intervals (0 to 6 weeks). Plasma glucose levels, TBARS levels and activities of antioxidant enzymes were measured in liver and pancreas at various time intervals. 4. There was a significant (P < 0.05) and progressive increase in TBARS levels of liver and pancreas in the diabetic group. Total SOD and Cu-Zn-SOD activity increased (P < 0.05) with progression of diabetes while Mn-SOD activity showed no significant change in either tissue. Catalase and glutathione peroxidase activities increased significantly (P < 0.05) in liver and pancreas. 5. Immunohistochemical study of pancreatic islet revealed a decrease in the expression of insulin with progression of diabetes. However, glucagon and somatostatin showed an increase in immunoreactivity and a difference in their distribution pattern. 6. The findings of the present study suggest that oxidative stress starts at early onset of diabetes mellitus and increases progressively. In conclusion, the structural damage to these tissues or complications of diabetes mellitus may be due to oxidative stress.

Angiotensin-converting enzyme 2 (ACE2) gene therapy aimed at counteracting pancreatic ACE2 depletion improves glucose regulation in two diabetic mouse models: db/db mice and angiotensin II-infused mice. A disintegrin and metalloproteinase 17 (ADAM17) can cause shedding of ACE2 from the cell membrane. The aim of our studies was to determine whether ADAM17 depletes ACE2 levels in pancreatic islets and β-cells. Dynamics of ADAM17-mediated ACE2 shedding were investigated in 832/13 insulinoma cells. Within a wide range of ACE2 expression levels, including the level observed in mouse pancreatic islets, overexpression of ADAM17 increases shed ACE2 and decreases cellular ACE2 levels. We provide a mathematical description of shed and cellular ACE2 activities as a function of the ADAM17 activity. The effect of ADAM17 on the cellular ACE2 content was relatively modest with an absolute control strength value less than 0.25 and approaching 0 at low ADAM17 activities. Although we found that ADAM17 and ACE2 are both expressed in pancreatic islets, the β-cell is not the major cell type expressing ACE2 in islets. During diabetes progression in 8-, 12-, and 15-week-old db/db mice, ACE2 mRNA and ACE2 activity levels in pancreatic islets were not decreased over time nor significantly decreased compared with nondiabetic db/m mice. Levels of ADAM17 mRNA and ADAM17 activity were also not significantly changed. Inhibiting basal ADAM17 activity in mouse islets failed to affect ACE2 levels. We conclude that whereas ADAM17 has the ability to shed ACE2, ADAM17 does not deplete ACE2 from pancreatic islets in diabetic db/db mice.

Diet-induced hyperglycemia is described as one major contributor to the formation of advanced glycation end products (AGEs) under inflammatory conditions, crucial in type 2 diabetes progression. Previous studies have indicated high postprandial plasma AGE-levels in diabetic patients and after long-term carbohydrate feeding in animal models. Pancreatic islets play a key role in glucose metabolism; thus, their susceptibility to glycation reactions due to high amounts of dietary carbohydrates is of special interest. Therefore, diabetes-prone New Zealand Obese (NZO) mice received either a carbohydrate-free, high-fat diet (CFD) for 11 weeks or were additionally fed with a carbohydrate-rich diet (CRD) for 7 days. In the CRD group, hyperglycemia and hyperinsulinemia were induced accompanied by increasing plasma 3-nitrotyrosine (3-NT) levels, higher amounts of 3-NT and inducible nitric oxide synthase (iNOS) within pancreatic islets. Furthermore, N-ε-carboxymethyllysine (CML) was increased in the plasma of CRD-fed NZO mice and substantially higher amounts of arg-pyrimidine, pentosidine and the receptor for advanced glycation end products (RAGE) were observed in pancreatic islets. These findings indicate that a short-term intervention with carbohydrates is sufficient to form endogenous AGEs in plasma and pancreatic islets of NZO mice under hyperglycemic and inflammatory conditions.

The increasing global incidence of diabetes and advancements in clinical pancreatic islet transplantation for the treatment of Type I diabetes have renewed the interest in understanding the variations of beta cell mass and function relative not only to transplant outcome but also to the onset and progression of diabetes. A deeper comprehension of the molecular and cellular processes involved in pancreatic islet inflammation and cytotoxicity is necessary to further improve efficacy of islet transplantation and to develop new therapies aimed at preserving beta cell function in pathological conditions. Available diagnostic methods based on metabolic response are unsuitable as they lack correlation to islet mass, viability and function. Great emphasis has been placed on developing noninvasive imaging technologies which enable the tracking of both endogenous and transplanted islet mass and potentially function overtime, the characterization of changes in islet vasculature and the degree of T-cell infiltration during insulitis. Among the more relevant modalities are magnetic resonance, positron emitted tomography, single photon emission computed tomography, bioluminescence and fluorescence optical imaging. This review focuses on the most recent advancements in magnetic resonance imaging (MRI) of pancreatic islets. In-vitro approaches aimed at characterizing the potency of isolated islets as well as in-vivo advancements in the assessment of transplanted beta cell mass are presented together with the significant progress made in the in-vivo imaging of the endocrine pancreas and islet vasculature and inflammation. Different experimental approaches are compared via their advantages and limitations with respect to their clinical implementation.

AAV8-mediated Gene Transfer of Interleukin-4 to Endogenous β-Cells Prevents the Onset of Diabetes in NOD Mice

Accumulating evidence supports the "glucagonocentric hypothesis", in which antecedent α-cell failure and inhibition of glucagon secretion are responsible for diabetes progression. Protein kinase C (PKC) is involved in glucagon secretion from α-cells, although which PKC isozyme is involved and the mechanism underlying this PKC-regulated glucagon secretion remains unknown. Here, the involvement of PKCδ in the onset and progression of diabetes was elucidated. Immunofluorescence studies revealed that PKCδ was expressed and activated in α-cells of STZ-induced diabetic model mice. Phorbol 12-myristate 13-acetate (PMA) stimulation significantly augmented glucagon secretion from isolated islets. Pre-treatment with quercetin and rottlerin, PKCδ signaling inhibitors, significantly suppressed the PMA-induced elevation of glucagon secretion. While Go6976, a Ca2+-dependent PKC selective inhibitor did not suppress glucagon secretion. Quercetin suppressed PMA-induced phosphorylation of Tyr311 of PKCδ in isolated islets. However, quercetin itself had no effect on either glucagon secretion or glucagon mRNA expression. Our data suggest that PKCδ signaling inhibitors suppressed glucagon secretion. Elucidation of detailed signaling pathways causing PKCδ activation in the onset and progression of diabetes followed by the augmentation of glucagon secretion could lead to the identification of novel therapeutic target molecules and the development of novel therapeutic drugs for diabetes. J. Med. Invest. 64: 122-128, February, 2017.

AimsPancreatic β-cell apoptosis may be involved in the onset and progression of type 2 diabetes mellitus, although its mechanism remains unclear. We previously demonstrated that macrophage-derived interferon (IFN) β induced X-linked inhibitor of apoptosis–associated factor 1 (XAF1) expression in β-cells and accelerated β-cell apoptosis in vitro. Here, we explored the effects of XAF1 on β-cell function and progression of diabetes in vivo.MethodsPancreatic β-cell-selective XAF1 overexpressing (Xaf1 Tg) mice were generated. Xaf1 Tg mice and their wild-type (WT) littermates were fed either a normal diet or a 40% or 60% high-fat diet (HFD). The effects of β-cell XAF1 on β-cell apoptosis and exacerbation of diabetes were investigated.ResultsPalmitic acid induced IFNβ expression in macrophages, and HFD intake promoted macrophage infiltration in pancreatic islets, both of which cooperatively upregulated XAF1 expression in mouse islets. Furthermore, HFD-fed Xaf1 Tg mice demonstrated increased β-cell apoptosis, lowered insulin expression, and impaired glucose tolerance compared with WT mice fed the same diet. These effects were more pronounced in the 60%HFD group than in the 40%HFD group.ConclusionsPancreatic β-cell XAF1 expression was enhanced via HFD-induced, macrophage-derived IFNβ, which promoted β-cell apoptosis and led to a reduction in insulin secretion and progression of diabetes. To our knowledge, this is the first report to demonstrate an association between pancreatic β-cell XAF1 overexpression and exacerbation of diabetes, thus providing insight into the mechanism of β-cell mass reduction in diabetes.

Glucagon-like peptide 1 (GLP-1) and glucagon share the same precursor molecule proglucagon, but each arises from a distinct posttranslational process in a tissue-specific manner. Recently, it has been shown that GLP-1 is co-expressed with glucagon in pancreatic islet cells. This study was aimed to investigate the progressive changes of GLP-1 versus glucagon production in pancreatic islets during the course of diabetes development. Both type 1 (non-obese diabetes mice) and type 2 (db/db mice) diabetes models were employed in this study. The mice were monitored closely for their diabetes progression and were sacrificed at different stages according to their blood glucose levels. GLP-1 and glucagon expression in the pancreatic islets was examined using immunohistochemistry assays. Quantitative analysis was performed to evaluate the significance of the changes. The ratio of GLP-1-expressing cells to glucagon-expressing cells in the islets showed significant, progressive increase with the development of diabetes in db/db mice. The increase of GLP-1 expression was in agreement with the upregulation of PC1/3 expression in these cells. Interestingly, intra-islet GLP-1 expression was not significantly changed during the development of type 1 diabetes in non-obese diabetes mice. The study demonstrated that GLP-1 was progressively upregulated in pancreatic islets during type 2 diabetes development. In addition, the data suggest clear differences in intra-islet GLP-1 production between type 1 and type 2 diabetes developments. These differences may have an effect on the clinical and pathophysiological processes of these diseases and may be a target for therapeutic approaches.

Mitochondria are crucial for cell survival and function, partly through peptides encoded by the mitochondrial genome. Although mitochondrial dysfunction is a hallmark of age-related diseases and senescence, the role of mitochondrial-genome-encoded peptides in pancreatic β-cell senescence during type 1 and type 2 diabetes pathogenesis is largely unexplored. Here we show that MOTS-c levels decrease with aging and senescence in pancreatic islet cells. Treating aged C57BL/6 mouse pancreatic islets with MOTS-c reduced pancreatic islet senescence by modulating nuclear gene expression and metabolites involved in β-cell senescence. MOTS-c treatment improved pancreatic islet senescence and glucose intolerance in S961-treated C57BL/6 and in nonobese diabetic mice. In humans, circulating MOTS-c levels are lower in type 2 diabetes patients compared with healthy controls. Our findings suggest that mitochondrial-encoded MOTS-c regulate pancreatic islet cell senescence and that MOTS-c could act as a senotherapeutic agent to prevent pancreatic islet cell senescence and diabetes progression.

Reduced pancreatic β-cell function or mass is the critical problem in developing diabetes. Insulin release from β-cells depends on Ca2+ influx through high voltage-gated Ca2+ channels (HVCCs). Ca2+ influx also regulates insulin synthesis and insulin granule priming and contributes to β-cell electrical activity. The HVCCs are multisubunit protein complexes composed of a pore-forming α1 and auxiliary β and α2δ subunits. α2δ is a key regulator of membrane incorporation and function of HVCCs. Here we show that genetic deletion of α2δ-1, the dominant α2δ subunit in pancreatic islets, results in glucose intolerance and diabetes without affecting insulin sensitivity. Lack of the α2δ-1 subunit reduces the Ca2+ currents through all HVCC isoforms expressed in β-cells equally in male and female mice. The reduced Ca2+ influx alters the kinetics and amplitude of the global Ca2+ response to glucose in pancreatic islets and significantly reduces insulin release in both sexes. The progression of diabetes in males is aggravated by a selective loss of β-cell mass, while a stronger basal insulin release alleviates the diabetes symptoms in most α2δ-1-/- female mice. Together, these findings demonstrate that the loss of the Ca2+ channel α2δ-1 subunit function increases the susceptibility for developing diabetes in a sex-dependent manner.

ABSTRACTThe pancreatic islets of Langerhans, which are composed of α, β, δ, ε, and PP cells, orchestrate systemic glucose homeostasis through tightly regulated hormone secretion. Although the precise mechanisms involving β cells in the onset and progression of diabetes have been elucidated and insulin replacement therapy remains the primary treatment modality, the regulatory processes, functions, and specific roles of other pancreatic islet hormones in diabetes continue to be the subject of ongoing investigation. At present, a comprehensive review of the secretion and regulation of pancreatic islet cell hormones as well as the related mechanisms of diabetes is lacking. This review synthesizes current knowledge on the secretion mechanisms of insulin, glucagon, somatostatin, ghrelin, and pancreatic polypeptides, emphasizing their functional crosstalk in diabetes. Emerging advances include CRISPR‐based β‐cell regeneration, bioengineered islet transplantation, and bioelectronic interventions aimed at restoring pancreatic function. Future research directions highlight artificial intelligence‐guided prediction of hormone dynamics, therapeutics targeting the gut microbiome–islet axis, and tissue‐engineered artificial islets. By integrating mechanistic insights, physiological roles, and translational innovations, this review outlines precision strategies for targeting islet hormone networks, offering a roadmap toward restoring metabolic equilibrium in diabetes.

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and while both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, whereas pathways that were upregulated in both doses were more upregulated in the 35 mg/kg treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and while both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, while pathways that were upregulated in both doses were more upregulated in the 35 mg/kg treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

Peroxiredoxin 4 (PRDX4) is one of a newly discovered family of antioxidative proteins. We generated human PRDX4 (hPRDX4) transgenic (Tg) mice, displaying a high level of hPRDX4 expression in the pancreatic islets, and then focused on the functions of PRDX4 in a type 1 diabetes mellitus (T1DM) model using a single high dose of streptozotocin (SHDS). After SHDS-injection, Tg mice showed significantly less hyperglycemia and hypoinsulinemia and a much faster response on glucose tolerance test than wild-type (WT) mice. Morphologic and immunohistochemical observation revealed that the pancreatic islet areas of Tg mice were larger along with less CD3-positive lymphocyte infiltration compared with WT mice. Upon comparison between these two mouse models, β-cell apoptosis was also repressed, and reversely, β-cell proliferation was enhanced in Tg mice. Real-time RT-PCR demonstrated that the expression of many inflammatory-related molecules and their receptors and transcription factors were significantly downregulated in Tg mice. These data indicate that PRDX4 can protect pancreatic islet β-cells against injury caused by SHDS-induced insulitis, which strongly suggests that oxidative stress plays an essential role in SHDS-induced diabetes. This study, for the first time, implicates that PRDX4 has a pivotal protective function against diabetes progression in this T1DM model.