Gen-Sheng Wang

PCSK9‐deficient mice exhibit impaired glucose tolerance and pancreatic islet abnormalities

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a liver‐secreted plasma enzyme, restricts hepatic uptake of low‐density lipoprotein (LDL) cholesterol by promoting the degradation of LDL receptors (LDLR). PCSK9 and LDLR are also expressed in insulin‐producing pancreatic islet β cells, possibly affecting the function of these cells. Here we show that, compared to control mice, PCSK9‐null male mice over 4 months of age carried more LDLR and less insulin in their pancreas; they were hypoinsulinemic, hyperglycemic and glucose‐intolerant; their islets exhibited signs of malformation, apoptosis and inflammation. Collectively, these observations suggest that PCSK9 may be necessary for the normal function of pancreatic islets.

Loss of Lkb1 in Adult β Cells Increases β Cell Mass and Enhances Glucose Tolerance in Mice

The Lkb1 tumor suppressor exerts its biological effects through phosphorylation and consequent activation of the AMP kinase (AMPK) family. Extensive genetic and biochemical evidence supports a role for Lkb1 in cell cycle arrest, establishment of cell polarity, and cellular energy metabolism. However, the role of Lkb1 and the AMPK family in beta cell function in vivo has not been established. We generated conditional knockout mice with a deletion of the Lkb1 gene in the beta cell compartment of pancreatic islets; these mice display improved glucose tolerance and protection against diet-induced hyperglycemia. Lkb1(-/-) beta cells are hypertrophic because of elevated mTOR activity; they also proliferate more and secrete more insulin in response to glucose. These data indicate that inhibiting Lkb1 activity in beta cells may facilitate beta cell expansion and glucose tolerance in vivo.

Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes

Aims/hypothesisResearch on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation.MethodsWe compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data.ResultsAfter type 1 diabetes manifestation CD8+ T cells, CD68+ macrophages and CD4+ T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed.Conclusions/interpretationWith the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Promotion of Autoimmune Diabetes by Cereal Diet in the Presence or Absence of Microbes Associated With Gut Immune Activation, Regulatory Imbalance, and Altered Cathelicidin Antimicrobial Peptide

We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3+ and CD8α+ lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3+ T cells expressing the Treg marker Foxp3+ was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163+ M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet–protected rats, and CAMP+ cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.

Pronounced cytosolic aggregation of cellular prion protein in pancreatic β -cells in response to hyperglycemia

Cellular prion protein (PrPC), an N-linked glycoprotein, is expressed in a variety of tissues, but its functions remain unclear. PrPC is abundantly expressed in the endocrine pancreas, which regulates blood glucose homeostasis. Therefore, we investigated whether the expression of PrPC was altered in islets of Langerhans in a model of spontaneous type 1 diabetes, the diabetes-prone BioBreeding (BBdp) rat and a model of β-cell adaptation to hyperglycemia, the chronic glucose-infused Sprague Dawley rat. Pancreatic sections from animals aged 7–100 days were stained immunohistochemically and evaluated using light, fluorescence and confocal microscopy. PrPC was ubiquitously expressed in all four major endocrine cell types within islets. Surprisingly, cytosolic inclusions containing PrPC were identified exclusively in a subpopulation of insulin-producing β-cells. The inclusions exhibited different molecular characteristics from the PrP aggregates previously described in vitro in neurons. The frequency of β-cells with PrPC inclusions increased with age and was threefold greater in diabetes-prone rats than in controls at 100 days. Cytosolic PrPC expression in β-cells was suppressed whereas the number and size of PrPC inclusions markedly increased in response to hyperglycemia during the first 2 days of continuous glucose infusion in Sprague Dawley rats. In summary, this is the first report describing in vivo cytosolic PrPC aggregation. These unique PrPC inclusions were β-cell specific, more frequent in diabetes-prone animals, and responded to hyperglycemia in glucose-infused Sprague Dawley rats. These data suggest a potential dysfunction in β-cells of diabetes-prone rats, and point to new avenues for the study of diabetes pathogenesis.

Increased islet neogenesis without increased islet mass precedes autoimmune attack in diabetes-prone rats

We reported previously that young BioBreeding diabetes-prone (BBdp) rats display increased neogenic extra-islet insulin+ clusters (EICs, <4 insulin+ cells) without an increase in β-cell mass. Therefore, we investigated the possibility that abnormal islet expansion occurs in BBdp rats before the appearance of islet inflammation. Islet expansion was analyzed in pancreata from 14 to 45 day BBdp and control (BioBreeding control, BBc) rats using immunohistochemistry, morphometry, laser capture microdissection and reverse transcriptase-PCR. mRNA expression for Neurogenin-3, a developmental marker of endocrine progenitors, was three-fold greater in EIC of weanling BBdp and BBc rats compared with islet cells. With increasing age (14–30 days), Neurogenin-3 expression decreased in EIC and increased in islets. In BBdp rats, EIC number and β-cell proliferation within EIC was greater compared with BBc animals; apoptosis did not differ. The area of small and medium islets in BBdp rats was greater than BBc rats between 14 and 30 days, but this did not result in increased total islet area or β-cell mass. In addition, the number and area of very large islets was low at 45 days. The frequency of proliferating β-cells decreased with increasing islet size in BBdp but was constant in BBc rats. Cell cycle analysis of islets revealed more G1 cells and fewer G2 cells in BBdp rats. The ratio of cyclinD2/Cdkn1a, genes that respectively promote or inhibit cell cycle progression, was decreased in BBdp islets. These results suggest that despite increased islet neogenesis, the capacity for islet expansion in diabetes-prone rats is compromised possibly due to decreased proliferative capacity with increasing islet size associated with a partial block at the G1/S cell cycle boundary in islet cells.

Impaired glucose tolerance in mice lacking cellular prion protein.

Objectives: We previously demonstrated that the expression of cellular prion protein (PrPC) in islet &bgr;-cells is suppressed in hyperglycemic rats suggesting a major role for PrPC in blood glucose regulation. To further characterize the function of PrPC in glucose homeostasis, we studied glucoregulation in PrPC knockout (PrPC KO) mice. Methods: Glucose tolerance, insulin secretion, and insulin sensitivity were analyzed to assess glucoregulation in Zrch I PrPC KO and the C57BL/6 (control) mice. Immunohistochemistry and morphometry were used to measure &bgr;-cell mass. Results: Male PrPC KO mice had significantly increased blood glucose concentration 60, 120, and 180 minutes after intraperitoneal injection of glucose compared with C57BL/6 mice. Female PrPC KO mice showed a less pronounced phenotype of glucose intolerance. Evaluation of &bgr;-cell mass, insulin and proinsulin deficiency, and insulin resistance in male mice revealed essentially no difference between PrPC KO and control mice. The only exception was an increase in serum insulin concentration in male PrPC KO mice 5 minutes after glucose injection. Conclusions: This report is the first to show that PrPC in &bgr;-cells is involved in glucoregulation. A further understanding of the role of PrPC in regulating &bgr;-cell function will provide valuable insight into the mechanisms of blood glucose regulation.

Cathelicidin antimicrobial peptide: a novel regulator of islet function, islet regeneration and selected gut bacteria

Cathelicidin antimicrobial peptide (CAMP) is a naturally occurring secreted peptide that is expressed in several organs with pleiotropic roles in immunomodulation, wound healing, and cell growth. We previously demonstrated that gut Camp expression is upregulated when type 1 diabetes–prone rats are protected from diabetes development. Unexpectedly, we have also identified novel CAMP expression in the pancreatic β-cells of rats, mice, and humans. CAMP was present even in sterile rat embryo islets, germ-free adult rat islets, and neogenic tubular complexes. Camp gene expression was downregulated in young BBdp rat islets before the onset of insulitis compared with control BBc rats. CAMP treatment of dispersed islets resulted in a significant increase in intracellular calcium mobilization, an effect that was both delayed and blunted in the absence of extracellular calcium. Additionally, CAMP treatment promoted insulin and glucagon secretion from isolated rat islets. Thus, CAMP is a promoter of islet paracrine signaling that enhances islet function and glucoregulation. Finally, daily treatment with the CAMP/LL-37 peptide in vivo in BBdp rats resulted in enhanced β-cell neogenesis and upregulation of potentially beneficial gut microbes. In particular, CAMP/LL-37 treatment shifted the abundance of specific bacterial populations, mitigating the gut dysbiosis observed in the BBdp rat. Taken together, these findings indicate a novel functional role for CAMP/LL-37 in islet biology and modification of gut microbiota.

Enhanced Islet Expansion by β-Cell Proliferation in Young Diabetes-Prone Rats Fed a Protective Diet

Type 1 diabetes is inhibited in diabetes‐prone BioBreeding (BBdp) rats fed a low‐antigen hydrolyzed casein (HC) diet. In cereal‐fed BBdp rats, islet expansion is defective accompanied by a futile upregulation of islet neogenesis without increased islet mass, due to a subtle blockage in islet cell cycle. We hypothesized that islet growth is enhanced before insulitis in HC‐fed young BBdp rats and that islet neogenesis could be stimulated by a trophic factor, islet neogenesis‐associated protein (INGAP). β‐Cell homeostasis was analyzed using immunohistochemistry, morphometry, laser capture microdissection and RT‐PCR in BBdp rats fed HC or cereal diets. β‐cell proliferation in small and medium islets, and the number and area fraction of medium and large islets were increased in HC‐fed animals. In situ islet cell cycle analysis revealed an increased proportion of proliferating S + G2 cells in medium and large islets of 25–45 day HC‐fed rats. Expression of the cell cycle inhibitor, p16INK4a correlated with islet size and the percentage of p16INK4a+ β‐cells increased in HC‐fed BBdp rats, likely reflecting an increase in large islet area fraction. In HC‐fed rats, extra‐islet insulin+ clusters (EIC), insulin+ duct cells, large islet area fraction, and β‐cell mass were increased. Neurogenin‐3 and Pdx‐1, markers of β‐cell progenitors, were increased in EIC of weanling HC‐fed rats. Daily injection of INGAP (30–45 days) increased the number of small islets, total islets, and insulin+ cells in small ducts. Thus, in BBdp rats fed a protective HC diet, β‐cell expansion is enhanced through increased β‐cell proliferation and stimulation of islet neogenesis. J. Cell. Physiol. 224: 501–508, 2010.

Variable effects of gender and Western diet on lipid and glucose homeostasis in aged PCSK9-deficient C57BL/6 mice 性别与西方饮食对CSK9基因缺陷的PC57BL/6老龄小鼠的脂质以及葡萄糖内稳态的不同影响

Proprotein convertase subtilisin/kexin‐type 9 (PCSK9) downregulates clearance of plasma cholesterol by liver. Its inactivation increases this clearance, reducing cardiovascular risk. However, a lack of PCSK9 could also lead to cholesterol accumulation in pancreatic islet beta cells, impairing insulin secretion. We reported earlier that 4‐month‐old male PCSK9‐deficient (KO) C57BL/6 mice were hyperglycemic and insulin‐insufficient relative to their wild‐type (WT) counterparts. Here, we examined how gender and diet affect lipid and glucose homeostasis in these mice at 8 months of age.