Multiple Low Doses Of Streptozotocin Research Articles

Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators.MethodsA male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators.ResultsSaline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1β) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks.ConclusionIntravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators.

Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes.

Much focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells. We induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry. We found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35's protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells. In conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

The Effects of Resistance Exercise Training on Skeletal Muscle Metabolism and Insulin Resistance Development in Female Rodents with Type 1 Diabetes.

The etiology of insulin resistance (IR) development in type 1 diabetes mellitus (T1DM) remains unclear; however, impaired skeletal muscle metabolism may play a role. While IR development has been established in male T1DM rodents, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1DM compared to aerobic exercise. The purpose of this study was to investigate the effects of RT on IR development in female T1DM rodents. Forty Sprague Dawley eight-week-old female rats were divided into four groups: control sedentary (CS; n = 10), control trained (CT; n = 10), T1DM sedentary (DS; n = 10), and T1DM trained (DT; n = 10). Multiple low-dose streptozotocin injections were used to induce T1DM. Blood glucose levels were maintained in the 4-9 mmol/l range with intensive insulin therapy. CT and DT underwent weighted ladder climbing 5 days/week for six weeks. Intravenous glucose tolerance tests (IVGTT) were conducted on all animals following the six-week period. Results demonstrate that DS animals exhibited significantly increased weekly blood glucose measures compared to all groups including DT (p < 0.0001), despite similar insulin dosage levels. This was concomitant with a significant increase in insulin-adjusted area under the curve following IVGTT in DS (p < 0.05), indicative of a reduction in insulin sensitivity. Both DT and DS exhibited greater serum insulin concentrations compared to CT and CS (p < 0.05). DS animals also exhibited significantly greater glycogen content in white gastrocnemius muscle compared to CS and DT (p < 0.05), whereas DT and DS animals exhibited greater p-Akt: Akt ratio in the white vastus lateralis muscle and citrate synthase activity in the red vastus lateralis muscle compared to CS and CT (p < 0.05). These results indicate that female rodents with T1DM develop poor glycemic control and IR which can be attenuated with RT, possibly related to differences in intramyocellular glycogen content.

TIMP3 overexpression in myeloid lineage alleviates pancreatic damage and confers resistance to the development of type 1 diabetes in the MLDS -induced model.

Type 1 diabetes mellitus (T1DM) development involves a complex interplay of genetic, environmental, and immunological factors. By modulating the activity of proteases and receptors, the protein tissue inhibitor of metalloproteinase 3 (TIMP3) plays a role in limiting the expression and function of pro-inflammatory cytokines, which have been implicated in the advancement of T1DM. This study was aimed at examining the effect of TIMP3 overexpression in myeloid cells on the development of T1DM. Twelve weeks after multiple low doses of streptozotocin (MLDS) treatment, diabetic mice overexpressing TIMP3 specifically in myeloid cells under the CD68 promoter (MacT3 mice) showed improved insulin secretion, islet morphology and vascularization, antioxidant defense system, and regulatory factors of mitochondrial biosynthesis and function. To get mechanistic insights into the origin of this protection, the severity of insulitis and inflammatory parameters were evaluated in pancreatic tissues 11 days after MLSD treatment, showing significantly reduced insulitis and levels of the pro-inflammatory cytokine tumor necrosis factor-α, interleukin -1β, and interferon -γ in MacT3 mice. The results indicate that TIMP3 is involved in maintaining islet architecture and functions, at least in part, through modulation of pro-inflammatory cytokine production associated with insulitis and may represent a novel therapeutic strategy for T1DM.

SAT073 β Cell FFA2 Deficiency Suppresses Multiple Low Dose Streptozotocin (MLDS) Induced Diabetes

Abstract Disclosure: K. Xu: None. M. Priyadarshini: None. P. Kumar: None. H.M. Electricwala: None. B.S. Prabhakar: None. B.T. Layden: None. Gut microbiome is recently recognized environmental factor in pathogenesis of type 1 diabetes (T1D). Reduced short chain fatty acid (SCFA) producing gut bacteria and also SCFA levels correlate with T1D risk (TEDDY study). We have previously shown that FFA2 is one of the main SCFA receptors, which is expressed on the β cell and mediates gut microbiome-β cell crosstalk. Based on these data, we hypothesized that β cell FFA2 may play a role in T1D β cell pathology. To examine this, we followed our novel tamoxifen induced β cell FFA2 knockout mice (FFA2 fl/fl; MIP CreERT+, FFA2 BKO) and wildtype littermates (FFA2 fl/fl) after multiple low dose streptozotocin (MLDS) induced T1D. Comparing to wildtype littermates, FFA2 BKO were significantly protected from MLDS insult and exhibited normal glycemic control owing to higher β cell mass. To understand the mechanism of this protective effect, we mined islet transcriptome data from our FFA2 global knockout mice and identified markedly upregulated type 1 interferon pathway in FFA2 global knockout. Accordingly, wildtype islets treated with high dose IFNα (mimicking T1D like conditions) showed significant upregulation of IFNαtargets while FFA2 BKO islets were unresponsive. Using pharmacological inhibition in isolated islets, we identified FFA2 mediated inhibition of NF-κB activation as a critical event in FFA2 mediated regulation of IFNαsignaling. In vivo studies with IFNAR1 antibody showed partial rescue of wildtype mice in the early stages of MLDS induced T1D. Thus, we propose a key role of β cell FFA2 in preserving β cell mass from IFNα signaling mediated damage in T1D. Further studies are needed to test this model. Presentation: Saturday, June 17, 2023

SAT071 Inhibition Of 12 Lipoxygenase In A Humanized Mouse Model Of Type 1 Diabetes Delays Progression Of Hyperglycemia

Abstract Disclosure: T. Nargis: None. A. Chakraborty: None. K. Figatner: None. D. Maloney: None. M. Boxer: None. S.A. Tersey: None. R.G. Mirmira: None. Type 1 diabetes (T1D) is an autoimmune disorder characterized by islet inflammation (insulitis). We have recently shown that a key mediator of inflammatory signaling in the islet beta cell is the enzyme 12-lipoxygenase (12-LOX), which generates proinflammatory eicosanoids that augment dysfunctional insulin secretion and beta-cell visibility to the immune system. Whereas inhibition of 12-LOX offers an opportunity to modify T1D progression by altering how the beta cell responds to insulitis, a barrier to testing inhibitors in preclinical models is the specificity of next generation inhibitors toward the human enzyme. To generate a preclinical platform for the study of human-specific 12-LOX inhibitors, we developed a mouse model in which the mouse-equivalent Alox15 coding region was replaced with the human ALOX12 gene, while retaining the mouse upstream control region. These mice (hALOX12) were developed on both the C57BL/6J and NOD genetic backgrounds, then subjected to treatment with the human 12-LOX inhibitor VLX-1005 to evaluate diabetes progression. C57BL/6J-wildtype and C57BL/6J-hALOX12 mice were confirmed to express exclusively mouse Alox15 and human ALOX12, respectively, using RNA isolated from islets. To test susceptibility to toxin-induced diabetes, C57BL/6J-wildtype and C57BL/6J-hALOX12 were subjected to multiple low-dose streptozotocin (STZ) to induce diabetes. Both wildtype and humanized mice exhibited gradual and equivalent hyperglycemia (within 25 days) following STZ injection. Following intraperitoneal VLX-1005 therapy (30 mg/kg), whereas C57BL/6J-wildtype mice developed diabetes (with a delay of 10 days) following STZ, C57BL/6J-hALOX12 mice showed complete protection against STZ-induced diabetes and exhibited significantly better glucose tolerance compared to vehicle- or VLX-1005-injected wildtype controls and vehicle-injected C57BL/6J-hALOX12 mice. Next, hALOX12 mice were backcrossed onto the NOD.ShiltJ background model of autoimmune T1D. NOD-hALOX12 mice received either vehicle or VLX-1005 (30 mg/kg) orally during the prediabetic phase (6-10 weeks of age) and were followed for diabetes incidence. Mice receiving VLX-1005 showed a significant delay with only 30% of female and 7% of male mice developing diabetes over 25 weeks of age. Pancreatic sections from the mice treated with VLX-1005 showed reduced insulitis and increased beta cell mass compared to vehicle controls. In summary, the hALOX12 mouse model serves as a preclinical translational platform to interrogate effects of next generation 12-LOX inhibitors and demonstrates the potential of VLX-1005 to modify T1D disease progression. Presentation: Saturday, June 17, 2023

Dose-dependent progression of multiple low dose streptozotocin-induced diabetes in mice.

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.

Deletion of carboxypeptidase E in beta cells disrupts proinsulin processing but does not lead to spontaneous development of diabetes in mice.

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic beta cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic beta cell-specific Cpe knockout mice (βCpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in βCpeKO islets remained intact. High fat diet-fed βCpeKO mice showed comparable weight gain and glucose tolerance compared to Wt littermates. Notably, beta-cell area was increased in chow-fed βCpeKO mice and beta-cell replication was elevated in βCpeKO islets. Transcriptomic analysis of βCpeKO beta cells revealed elevated glycolysis and Hif1α-target gene expression. Upon high glucose challenge, beta cells from βCpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin treatment, βCpeKO mice had accelerated hyperglycemia with reduced beta-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining beta cell function during the development of hyperglycemia.

581-P: The Effects of Resistance Training on Insulin Resistance Development in Female Rodents with Type 1 Diabetes

The etiology of insulin resistance (IR) development in type 1 diabetes (T1D) remains unclear; however, impaired glucose metabolism in skeletal muscle may play a role. While IR development has been established in male rodents with T1D, female rodents have yet to be examined in this context. Resistance exercise training (RT) has been shown to improve IR and is associated with a lower risk of hypoglycemia onset in T1D compared to aerobic exercise. The purpose of this study was to investigate the effects of RT on IR development in female rodents with T1D. Forty Sprague-Dawley 8-week-old female rats were divided into four groups: control sedentary (CS; n=10), control trained (CT; n=10), T1D sedentary (DS; n=10), T1D trained (DT; n=10). Multiple low-dose Streptozotocin injections (20 mg/kg each day for 7 consecutive days) were used to induce T1D. Blood glucose levels were maintained in normal range (4-9mmol/L) with one implanted insulin pellet (2IU/day). CT and DT underwent weighted ladder climbing 5 days/week for 6 weeks. Intravenous glucose tolerance tests (IVGTT) were conducted on all animals during weeks 4 and 7. Results demonstrate that DS animals exhibited significantly increased weekly blood glucose measures compared to all groups including DT (p&amp;lt;0.05), despite similar insulin dosage levels. This was concomitant with a significant increase in area under the curve following IVGTT from week 4 to week 7 in DS (p&amp;lt;0.05), indicative of a reduction in insulin sensitivity. DS animals also exhibited significantly greater glycogen content in white gastrocnemius muscle compared to all groups (p&amp;lt;0.05). These results indicate that female rodents with T1D develop poor glycemic control and IR which can be attenuated with RT, possibly related to differences in glycogen content within muscle. This data supports the negative role of elevated muscle glycogen on insulin sensitivity in T1D and the potential role of RT in ameliorating these metabolic changes. Disclosure M.Sammut: None. D.Mcbey: None. C.Melling: None.

#5554 ROLE OF THE TUBULAR EPITHELIAL CELL NLRP3 INFLAMMASOME DURING HYPERGLYCEMIA AND GLUCOSURIA IN DIABETES

Abstract Background and Aims Diabetes is a prominent risk factor for the progression of CKD because hyperglycemia and glucose filtration increase the hemodynamic and metabolic workload to the nephrons, which imposes adaptive mechanisms. Tubules adapt to diabetes by enlargement and epithelial cell hypertrophy but an exceeding metabolic workload promotes oxidative stress, cytokine signaling and tubular epithelial (TEC) cell demise leading to tubular atrophy and interstitial fibrosis. Several studies propose a role of the NLRP3 inflammasome in tubulointerstitial inflammation in diabetic and non-diabetic kidney disease. The NLRP3 inflammasome is a sensor of cellular damage responsible for regulate the IL-1β and IL-18 production, playing important role during the progression of CKD. However, the role of NLRP3 in diabetes-induced tubular adaptation is unknown. Method scRNAseq analysis showed previously that the TECs do not express NLRP3 or inflammasome components. However, a group of patients diagnosed with Cryopyrin-Associated Autoinflammatory Syndromes, have a mutation that overexpress NLRP3, which is associated with chronic and systemic inflammation. To investigate the role of NLRP3 on tubular cells under diabetes conditions, we generated a mouse line by crossing mice bearing LoxP-target A350V allele with Pax8-driven Cre transgenic mice. Animals carrying the A350V allele have overexpression of NLRP3 in tubular cells. The combination of unilateral nephrectomy and treatment with multiple low doses of streptozotocin (60 mg/Kg) accelerate the kidney injury caused by hyperglycaemia. Kidney function, histology and gene expression were evaluated at the end of 16 weeks of diabetes. In vitro experiments carried out using TECs isolated from Pax8-A350V, and stimulated with LPS (1µg/ml) and/or ATP (20 mM). Results In silico analysis showed TECs do not express NLRP3 or inflammasome components. A350V-TECs under inflammasome activation did not produce IL-1β or IL-18. However, the Nlrp3 overexpression upregulated kidney injury markers, as well IL-6 and TNF. Moreover, the increase in kidney inflammation was associated with significative upregulation of fibrosis markers (Tgfb1, Asma, and Fn1) and extracellular molecules (Ctgf, AnxaII, and Chd2). Diabetic mice carrying the A350V mutation did not have significative differences in blood glucose, albuminuria, or body weight when compared with diabetic control mice. The overexpression of NLRP3 in tubular cells was associated with significative reduction in GFR and BUN when compared with controls mice, even though no differences in tubular injury markers observed. Furthermore, Pax8-A350V diabetic mice expressed significantly more Nlrp3 and Il1b than control group. Although, no differences observed in cryopyrin staining. Conclusion Our data suggest the NLRP3-associated damage in tubular epithelial cells occurs in a non-canonical manner.

Anti-IL-6R prevents experimental type 1 diabetes

Abstract Type 1 diabetes (T1D) is an autoimmune disease, where the insulin producing β cells are damaged by immune attacks leading to a failure of blood glucose control. The anti-inflammatory cytokine IL-35 has been shown to prevent and reverse T1D in mouse models. IL-6 signals through a homodimer of gp130 which is one of the receptors of IL-35. We thus hypothesized that blocking IL-6 signaling would facilitate IL-35 signaling and prevent T1D. We used multiple low dose streptozotocin (MLDSTZ) to induce T1D in male CD-1 mice and treated them with anti-IL-6R to study the immune response. We found that anti-IL-6R treatment prevented MLDSTZ mice from developing hyperglycemia, and preserved the ability of β cells to produce insulin. We then used flow cytometry to investigate the T cell and B cell response on day 13 after the first injection of MLDSTZ. The proportions of Treg cells were not altered by anti-IL-6R treatment in MLDSTZ treated mice, but the treatment elevated the proportions of IL-35 producing Treg cells in the spleen and thymus. It also decreased IFN-γ production in CD4, CD8, Treg and B cells in the spleen. Furthermore, anti-IL-6R treatment only tended to increase IL-17 production in CD4, CD8 and Treg cells. Altogether, our data demonstrate that anti-IL-6R treatment prevents T1D in MLDSTZ mice by maintaining the Treg cell phenotype and by reducing IFN-γ production by T and B cells.

The glucagon receptor antagonist desHis1Pro4Glu9-glucagon(Lys12PAL) alters alpha-cell turnover and lineage in mice, but does not cause alpha-cell hyperplasia

ObjectiveGlucagon receptor (GCGR) antagonism elicits antihyperglycemic effects in rodents and humans. The present study investigates whether the well characterised peptide-based GCGR antagonist, desHis1Pro4Glu9-glucagon (Lys12PAL), alters alpha-cell turnover or identity in mice. MethodsMultiple low-dose streptozotocin (STZ) treated (50 mg/kg bw, 5 days) transgenic GluCreERT2;ROSA26-eYFP mice were employed. STZ mice received twice daily administration of saline vehicle or desHis1Pro4Glu9-glucagon (Lys12PAL), at low- or high-dose (25 and 100 nmol/kg, respectively) for 11 days. ResultsNo GCGR antagonist induced changes in food or fluid intake, body weight or glucose homeostasis were observed. As expected, STZ dramatically reduced (P < 0.001) islet numbers and increased (P < 0.01) alpha-to beta-cell ratio, which was linked to elevated (P < 0.05) levels of beta-cell apoptosis. Whilst treatment with desHis1Pro4Glu9-glucagon (Lys12PAL) decreased (P < 0.05-P < 0.001) alpha- and beta-cell areas, it also helped restore the classic rodent islet alpha-cell mantle in STZ mice. Interestingly, low-dose desHis1Pro4Glu9-glucagon (Lys12PAL) increased (P < 0.05) alpha-cell apoptosis rates whilst high dose decreased (p < 0.05) this parameter. This difference reflects substantially increased (P < 0.001) alpha-to beta-cell transdifferentiation following high dose desHis1Pro4Glu9-glucagon (Lys12PAL) treatment, which was not fully manifest with low-dose therapy. ConclusionsTaken together, the present study indicates that peptidic GCGR antagonists can positively influence alpha-cell turnover and lineage in identity in multiple low-dose STZ mice, but that such effects are dose-related.

Development of Type 1 Diabetes in Mice Is Associated with a Decrease in IL-2-Producing ILC3 and FoxP3+ Treg in the Small Intestine.

Recent data indicate the link between the number and function of T regulatory cells (Treg) in the gut immune tissue and initiation and development of autoimmunity associated with type 1 diabetes (T1D). Since type 3 innate lymphoid cells (ILC3) in the small intestine are essential for maintaining FoxP3+ Treg and there are no data about the possible role of ILC3 in T1D pathogenesis, the aim of this study was to explore ILC3-Treg link during the development of T1D. Mature diabetic NOD mice had lower frequencies of IL-2-producing ILC3 and Treg in small intestine lamina propria (SILP) compared to prediabetic NOD mice. Similarly, in multiple low doses of streptozotocin (MLDS)-induced T1D in C57BL/6 mice, hyperglycemic mice exhibited lower numbers of ILC3, IL-2+ ILC3 and Treg in SILP compared to healthy controls. To boost T1D severity, mice were treated with broad-spectrum antibiotics (ABX) for 14 days prior to T1D induction by MLDS. The higher incidence of T1D in ABX-treated mice was associated with significantly lower frequencies of IL-2+ ILC3 and FoxP3+ Treg in SILP compared with mice without ABX treatment. The obtained findings show that the lower proportions of IL-2-expressing ILC3 and FoxP3+ Treg in SILP coincided with diabetes progression and severity.

Probiotics of Lacticaseibacillus paracasei SD1 and Lacticaseibacillus rhamnosus SD11 attenuate inflammation and β-cell death in streptozotocin-induced type 1 diabetic mice.

Probiotics provide health benefits in various aspects and are believed to modulate the immune system by balancing gut microbiota homeostasis, termed the "microbiota-immune axis". Recent evidence supports that several Lactobacillus strains possess glucose-lowering and anti-inflammatory effects in an animal model of type 1 diabetes (T1D). Although probiotics of Lacticaseibacillus paracasei SD1 (SD1) and Lacticaseibacillus rhamnosus SD11 (SD11) exert human oral health benefits by reducing harmful bacterial populations, their clinical application regarding hypoglycemic-related traits as well as the underlying mechanisms are still lacking. In this report, we used multiple low doses of streptozotocin (STZ)-induced diabetic BALB/c mice to explore the effects of SD1 and SD11 supplementation on the regulation of markers related to T1D. Experimental mice were randomly assigned into five groups, non-STZ + V, STZ + V, STZ + SD1, STZ + SD11, and STZ + SDM (mixture of SD1 and SD11), and physiological data were measured every week. Blood and pancreas samples were collected at 4- and 8-weeks. Our results indicate that supplementation with SD1, SD11, or SDM for 8 weeks significantly improved body weights, glycemic levels, glucose tolerance, insulin levels, and lipid profiles. Probiotic administration also preserved islet integrity and increased β-cell mass in STZ-injected mice, as well as prevented infiltration of macrophages, CD4+, and CD8+ T cells into the islets. Significantly, SD1 and SD11 suppressed the levels of IL1-β, TNF-α and IFN-γ and increased IL-10, which is concomitant with the inhibition of cleaved caspase 3, caspase 9, caspase 8, proapoptotic Bax, NF-κBp65, pSTAT1, and iNOS. Additionally, the survival ability of β-cells was mediated by upregulated anti-apoptotic Bcl2. We conclude that SD1 and SD11 attenuate STZ-induced diabetic mice by stabilizing glycemic levels and reducing inflammation, thereby protecting β-cells. Among the probiotic treatment groups, SD11 revealed the best results in almost all parameters, indicating its potential use for alleviating hyperglycemia-associated symptoms.

Effects of artemether on pancreatic islet morphology, islet cell turnover and α-cell transdifferentiation in insulin-deficient GluCreERT2;ROSA26-eYFP diabetic mice.

The antimalarial drug artemether is suggested to effect pancreatic islet cell transdifferentiation, presumably through activation γ-aminobutyric acid receptors, but this biological action is contested. We have investigated changes in α-cell lineage in response to 10-days treatment with artemether (100 mg/kg oral, once daily) on a background of β-cell stress induced by multiple low-dose streptozotocin (STZ) injection in GluCreERT2; ROSA26-eYFP transgenic mice. Artemether intervention did not affect the actions of STZ on body weight, food and fluid intake or blood glucose. Circulating insulin and glucagon were reduced by STZ treatment, with a corresponding decline in pancreatic insulin content, which were not altered by artemether. The detrimental changes to pancreatic islet morphology induced by STZ were also evident in artemether-treated mice. Tracing of α-cell lineage, through co-staining for glucagon and yellow fluorescent protein (YFP), revealed a significant decrease of the proportion of glucagon+YFP- cells in STZ-diabetic mice, which was reversed by artemether. However, artemether had no effect on transdifferentiation of α-cells into β-cells and failed to augment the number of bi-hormonal, insulin+glucagon+, islet cells. Our observations confirm that artemisinin derivatives do not impart meaningful benefits on islet cell lineage transition events or pancreatic islet morphology.

Assessment of executive function in a rodent model of Type 1 diabetes

This study examined the impact of Type 1 Diabetes Mellitus (T1DM) on executive function using a series of operant conditioning-based tasks in rats. Sprague Dawley rats were randomized to either non-diabetic (n = 12; 6 male) or diabetic (n = 14; 6 male) groups. Diabetes was induced using multiple low-dose streptozotocin injections. All diabetic rodents were insulin-treated using subcutaneous insulin pellet implants (9–15 mM). At week 14 of the study, rats were placed on a food restricted diet to induce 5–10 % weight loss. Rodents were familiarized and their set-shifting ability was tested on a series of tasks that required continuous adjustments to novel stimulus-reward paradigms in order to receive food rewards. Results showed no differences in the number of trials, nor number and type of errors made to successfully complete each task between groups. Therefore, we report no differences in executive function, or more specifically set-shifting abilities between non-diabetic and diabetic rodents that receive insulin.

Nova1 or Bim Deficiency in Pancreatic β-Cells Does Not Alter Multiple Low-Dose Streptozotocin-Induced Diabetes and Diet-Induced Obesity in Mice.

The loss of functional pancreatic β-cell mass is an important hallmark of both type 1 and type 2 diabetes. The RNA-binding protein NOVA1 is expressed in human and rodent pancreatic β-cells. Previous in vitro studies indicated that NOVA1 is necessary for glucose-stimulated insulin secretion and its deficiency-enhanced cytokine-induced apoptosis. Moreover, Bim, a proapoptotic protein, is differentially spliced and potentiates apoptosis in NOVA1-deficient β-cells in culture. We generated two novel mouse models by Cre-Lox technology lacking Nova1 (βNova1−/−) or Bim (βBim−/−) in β-cells. To test the impact of Nova1 or Bim deletion on β-cell function, mice were subjected to multiple low-dose streptozotocin (MLD-STZ)-induced diabetes or high-fat diet-induced insulin resistance. β-cell-specific Nova1 or Bim deficiency failed to affect diabetes development in response to MLD-STZ-induced β-cell dysfunction and death evidenced by unaltered blood glucose levels and pancreatic insulin content. In addition, body composition, glucose and insulin tolerance test, and pancreatic insulin content were indistinguishable between control and βNova1−/− or βBim−/− mice on a high fat diet. Thus, Nova1 or Bim deletion in β-cells does not impact on glucose homeostasis or diabetes development in mice. Together, these data argue against an in vivo role for the Nova1-Bim axis in β-cells.

Combined therapy of GABA and sitagliptin prevents high-fat diet impairment of beta-cell function

We recently demonstrated that combined therapy of GABA and sitagliptin promoted beta-cell proliferation, and decreased beta-cell apoptosis in a multiple low-dose streptozotocin (STZ)-induced beta-cell injury mouse model. In this study, we examined whether this combined therapy is effective in ameliorating the impairment of beta-cell function caused by high-fat diet (HFD) feeding in mice. Male C57BL/6J mice were fed normal chow diet, HFD, or HFD combined with GABA, sitagliptin, or both drugs. Oral drug daily administration was initiated one week before HFD and maintained for two weeks. After two weeks of intervention, we found that GABA or sitagliptin administration ameliorated the impairment of glucose tolerance induced by HFD. This was associated with improved insulin secretion in vivo. Notably, combined administration of GABA and sitagliptin significantly enhanced these effects as compared to each of the monotherapies. Combined GABA and sitagliptin was superior at increasing beta-cell mass, and associated Ki67+ and PDX-1+ beta-cell counts. In addition, we found that HFD-induced compensatory beta-cell proliferation was associated with increased activation of unfolded protein response (UPR), as indicated by BiP expression. This could be an important mechanism of compensatory beta-cell proliferation, and beta cells treated with GABA and sitagliptin showed greater UPR activation. Our results suggest that the combined use of these agents produces superior therapeutic outcomes.

Resistance Training Improves Beta Cell Glucose Sensing and Survival in Diabetic Models.

Resistance training increases insulin secretion and beta cell function in healthy mice. Here, we explored the effects of resistance training on beta cell glucose sensing and survival by using in vitro and in vivo diabetic models. A pancreatic beta cell line (INS-1E), incubated with serum from trained mice, displayed increased insulin secretion, which could be linked with increased expression of glucose transporter 2 (GLUT2) and glucokinase (GCK). When cells were exposed to pro-inflammatory cytokines (in vitro type 1 diabetes), trained serum preserved both insulin secretion and GCK expression, reduced expression of proteins related to apoptotic pathways, and also protected cells from cytokine-induced apoptosis. Using 8-week-old C57BL/6 mice, turned diabetic by multiple low doses of streptozotocin, we observed that resistance training increased muscle mass and fat deposition, reduced fasting and fed glycemia, and improved glucose tolerance. These findings may be explained by the increased fasting and fed insulinemia, along with increased beta cell mass and beta cell number per islet, observed in diabetic-trained mice compared to diabetic sedentary mice. In conclusion, we believe that resistance training stimulates the release of humoral factors which can turn beta cells more resistant to harmful conditions and improve their response to a glucose stimulus.

Interleukin-35 Prevents the Elevation of the M1/M2 Ratio of Macrophages in Experimental Type 1 Diabetes.

Macrophages play an important role in the early development of type 1 diabetes (T1D). Based on the phenotype, macrophages can be classified into pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. Despite intensive research in the field of macrophages and T1D, the kinetic response of M1/M2 ratio has not been studied in T1D. Thus, herein, we studied the M1 and M2 macrophages in the early development of T1D using the multiple low dose streptozotocin (MLDSTZ) mouse model. We determined the proportions of M1 and M2 macrophages in thymic glands, pancreatic lymph nodes and spleens on days 3, 7 and 10 after the first injection of STZ. In addition, we investigated the effect of IL-35 in vivo on the M1/M2 ratio and IL-35+ plasmacytoid dendritic cells in diabetic mice and in vitro on the sorted macrophages. Our results revealed that the M1/M2 ratio is higher in STZ-treated mice but this was lowered upon the treatment with IL-35. Furthermore, IL-35 treated mice had lower blood glucose levels and a higher proportion of IL-35+ cells among pDCs. Macrophages treated with IL-35 in vitro also had a higher proportion of M2 macrophages. Together, our data indicate that, under diabetic conditions, pro-inflammatory macrophages increased, but IL-35 treatment decreased the pro-inflammatory macrophages and increased anti-inflammatory macrophages, further suggesting that IL-35 prevents hyperglycemia by maintaining the anti-inflammatory phenotype of macrophages and other immune cells. Thus, IL-35 should be further investigated for the treatment of T1D and other autoimmune disorders.