Emily K. Sims

Elevations in the Fasting Serum Proinsulin-to-C-Peptide Ratio Precede the Onset of Type 1 Diabetes.

OBJECTIVE We tested whether an elevation in the serum proinsulin–to–C-peptide ratio (PI:C), a biomarker of β-cell endoplasmic reticulum (ER) dysfunction, was associated with progression to type 1 diabetes. RESEARCH DESIGN AND METHODS Fasting total PI and C levels were measured in banked serum samples obtained from TrialNet Pathway to Prevention (PTP) participants, a cohort of autoantibody-positive relatives without diabetes of individuals with type 1 diabetes. Samples were obtained ∼12 months before diabetes onset from PTP progressors in whom diabetes developed (n = 60), and were compared with age-, sex-, and BMI-matched nonprogressors who remained normoglycemic (n = 58). PI:C ratios were calculated as molar ratios and were multiplied by 100% to obtain PI levels as a percentage of C levels. RESULTS Although absolute PI levels did not differ between groups, PI:C ratios were significantly increased in antibody-positive subjects in whom there was progression to diabetes compared with nonprogressors (median 1.81% vs. 1.17%, P = 0.03). The difference between groups was most pronounced in subjects who were ≤10 years old, where the median progressor PI:C ratio was nearly triple that of nonprogressors; 90.0% of subjects in this age group within the upper PI:C quartile progressed to the development of diabetes. Logistic regression analysis, adjusted for age and BMI, demonstrated increased odds of progression for higher natural log PI:C ratio values (odds ratio 1.44, 95% CI 1.02, 2.05). CONCLUSIONS These data suggest that β-cell ER dysfunction precedes type 1 diabetes onset, especially in younger children. Elevations in the serum PI:C ratio may have utility in predicting the onset of type 1 diabetes in the presymptomatic phase.

Human Adipose‐Derived Stromal/Stem Cells Protect Against STZ‐Induced Hyperglycemia: Analysis of hASC‐Derived Paracrine Effectors

Adipose‐derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)‐derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in streptozotocin‐treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC‐derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP‐1) to be highly abundant factors secreted by hASCs. Notably, TIMP‐1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP‐1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP‐1 was increased in the serum of injected mice, while recombinant TIMP‐1 increased viability in INS‐1 cells treated with interleukin‐1beta, interferon‐gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP‐1, are able to mitigate against β cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects. Stem Cells 2014;32:1831–1842

Minireview: Emerging Roles for Extracellular Vesicles in Diabetes and Related Metabolic Disorders

Extracellular vesicles (EVs), membrane-contained vesicles released by most cell types, have attracted a large amount of research interest over the past decade. Because of their ability to transfer cargo via regulated processes, causing functional impacts on recipient cells, these structures may play important roles in cell-cell communication and have implications in the physiology of numerous organ systems. In addition, EVs have been described in most human biofluids and have wide potential as relatively noninvasive biomarkers of various pathologic conditions. Specifically, EVs produced by the pancreatic β-cell have been demonstrated to regulate physiologic and pathologic responses to β-cell stress, including β-cell proliferation and apoptosis. β-Cell EVs are also capable of interacting with immune cells and may contribute to the activation of autoimmune processes that trigger or propagate β-cell inflammation and destruction during the development of diabetes. EVs from adipose tissue have been shown to contribute to the development of the chronic inflammation and insulin resistance associated with obesity and metabolic syndrome via interactions with other adipose, liver, and muscle cells. Circulating EVs may also serve as biomarkers for metabolic derangements and complications associated with diabetes. This minireview describes the properties of EVs in general, followed by a more focused review of the literature describing EVs affecting the β-cell, β-cell autoimmunity, and the development of insulin resistance, which all have the potential to affect development of type 1 or type 2 diabetes.

Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β Cells via Activation of the Mammalian Target of Rapamycin Pathway

Saturated free fatty acids (FFAs) have complex effects on the islet β-cell, acutely promoting adaptive hyperplasia but chronically impairing insulin release. The acute effects of FFAs remain incompletely defined. To elucidate these early molecular events, we incubated mouse β-cells and islets with palmitate and then studied mRNA translation by polyribosomal profiling and analyzed signaling pathways by immunoblot analysis. We found that palmitate acutely increases polyribosome occupancy of total RNA, consistent with an increase in mRNA translation. This effect on translation was attributable to activation of mammalian target of rapamycin (mTOR) pathways via L-type Ca2+ channels but was independent of insulin signaling. Longer incubations led to depletion of polyribosome-associated RNA, consistent with activation of the unfolded protein response (UPR). Pharmacologic inhibition of mTOR suppressed both the acute effects of palmitate on mRNA translation and the chronic effects on the UPR. Islets from mice fed a high-fat diet for 7 days showed increases in polyribosome-associated RNA and phosphorylation of S6K, both consistent with activation of mTOR. Our results suggest that palmitate acutely activates mRNA translation and that this increase in protein load contributes to the later UPR.

Divergent compensatory responses to high-fat diet between C57BL6/J and C57BLKS/J inbred mouse strains

Impaired glucose tolerance (IGT) and type 2 diabetes (T2DM) are polygenic disorders with complex pathophysiologies; recapitulating them with mouse models is challenging. Despite 70% genetic homology, C57BL/6J (BL6) and C57BLKS/J (BLKS) inbred mouse strains differ in response to diet- and genetic-induced obesity. We hypothesized these differences would yield insight into IGT and T2DM susceptibility and response to pharmacological therapies. To this end, male 8-wk-old BL6 and BLKS mice were fed normal chow (18% kcal from fat), high-fat diet (HFD; 42% kcal from fat), or HFD supplemented with the PPARγ agonist pioglitazone (PIO; 140 mg PIO/kg diet) for 16 wk. Assessments of body composition, glucose homeostasis, insulin production, and energy metabolism, as well as histological analyses of pancreata were undertaken. BL6 mice gained weight and adiposity in response to HFD, leading to peripheral insulin resistance that was met with increased β-cell proliferation and insulin production. By contrast, BLKS mice responded to HFD by restricting food intake and increasing activity. These behavioral responses limited weight gain and protected against HFD-induced glucose intolerance, which in this strain was primarily due to β-cell dysfunction. PIO treatment did not affect HFD-induced weight gain in BL6 mice, and decreased visceral fat mass, whereas in BLKS mice PIO increased total fat mass without improving visceral fat mass. Differences in these responses to HFD and effects of PIO reflect divergent human responses to a Western lifestyle and underscore the careful consideration needed when choosing mouse models of diet-induced obesity and diabetes treatment.

Streptozotocin is equally diabetogenic whether administered to fed or fasted mice

Streptozotocin (STZ) is a selective pancreatic β cell toxin used to generate experimental hyperglycemia in rodent models. Several laboratory animal protocols suggest that STZ be administered to fasted rodents to minimize competition between STZ and glucose for low affinity GLUT2 transporters on β cells. However, whether the diabetogenic effects of multiple low dose (MLD)-STZ administration are enhanced by fasting has not been addressed. Given that repeated bouts of fasting can cause undue metabolic stress in mice, we compared the efficacy of MLD-STZ injections (50 mg/kg body weight daily for 5 days) to induce experimental hyperglycemia in both NOD/SCID/γchainnull and C57BL/6J mice that were either ad libitum fed (STZ-Fed) or that had been fasted for 6 h (STZ-Fasted) prior to the time of STZ administration. Both STZ-Fed and STZ-Fasted mice had significantly worse glucose tolerance than vehicle-treated control mice 10 days after initiation of the MLD-STZ regimen. In C57BL/6J mice, fasting glucose levels, serum insulin levels, β cell mass, and glucose disposal during intraperitoneal glucose tolerance tests (IPGTTs) were indistinguishable between STZ-Fed and STZ-Fasted mice 20 days after MLD-STZ. The glucose intolerant phenotypes persisted for 20 weeks thereafter, irrespective of whether C57BL/6J mice were fed or fasted at the time of STZ injections. However, STZ-Fasted C57BL/6J mice experienced significant weight loss during the repeated bouts of fasting/re-feeding that were required to complete the MLD-STZ protocol. In summary, induction of experimental hyperglycemia can be achieved using the MLD-STZ protocol without repeated bouts of fasting, which have the potential to cause metabolic stress in laboratory mice.

Fulvestrant treatment of precocious puberty in girls with McCune-Albright syndrome

BackgroundMcCune-Albright Syndrome (MAS) is usually characterized by the triad of precocious puberty (PP), fibrous dysplasia, and café au lait spots. Previous treatments investigated for PP have included aromatase inhibitors and the estrogen receptor modulator, tamoxifen. Although some agents have been partially effective, the optimal pharmacologic treatment of PP in girls with MAS has not been identified. The objective of this study was to evaluate the safety and efficacy of fulvestrant (FaslodexTM), a pure estrogen receptor antagonist, in girls with progressive precocious puberty (PP) associated with McCune-Albright Syndrome (MAS).MethodsIn this prospective international multicenter trial, thirty girls ≤ 10 years old with MAS and progressive PP received fulvestrant 4 mg/kg via monthly intramuscular injections for 12 months. Changes in vaginal bleeding, rates of bone age advancement, growth velocity, Tanner staging, predicted adult heights, and uterine and ovarian volumes were measured.ResultsMedian vaginal bleeding days decreased from 12.0 days per year to 1.0 day per year, with a median change in frequency of -3.6 days, (95% confidence interval (CI) -10.10, 0.00; p = 0.0146). Of patients with baseline bleeding, 74% experienced a ≥50% reduction in bleeding, and 35% experienced complete cessation during the study period (95% CI 51.6%, 89.8%; 16.4%, 57.3%, respectively). Average rates of bone age advancement (ΔBA/ΔCA) decreased from 1.99 pre-treatment to 1.06 on treatment (mean change -0.93, 95% CI -1.43, -0.43; p = 0.0007). No significant changes in uterine volumes or other endpoints or serious adverse events occurred.ConclusionsFulvestrant was well tolerated and moderately effective in decreasing vaginal bleeding and rates of skeletal maturation in girls with MAS. Longer-term studies aimed at further defining potential benefits and risks of this novel therapeutic approach in girls with MAS are needed.Trial registrationNCT00278915

Biomarkers of β-Cell Stress and Death in Type 1 Diabetes

The hallmark of type 1 diabetes (T1D) is a decline in functional β-cell mass arising as a result of autoimmunity. Immunomodulatory interventions at disease onset have resulted in partial stabilization of β-cell function, but full recovery of insulin secretion has remained elusive. Revised efforts have focused on disease prevention through interventions administered at earlier disease stages. To support this paradigm, there is a parallel effort ongoing to identify circulating biomarkers that have the potential to identify stress and death of the islet β-cells. Whereas no definitive biomarker(s) have been fully validated, several approaches hold promise that T1D can be reliably identified in the pre-symptomatic phase, such that either β-cell preservation or immunomodulatory agents might be employed in at-risk populations. This review summarizes the most promising protein- and nucleic acid-based biomarkers discovered to date and reviews the context in which they have been studied.

MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells

Aims/hypothesisThe role of beta cell microRNA (miR)-21 in the pathophysiology of type 1 diabetes has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in type 1 diabetes and the phenotype of beta cell miR-21 overexpression through target identification.MethodsIslets were isolated from NOD mice and mice treated with multiple low doses of streptozotocin, as a mouse model of diabetes. INS-1 832/13 beta cells and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the milieu of early type 1 diabetes. Cells and islets were transfected with miR-21 mimics or inhibitors. Luciferase assays and polyribosomal profiling (PRP) were performed to define miR-21–target interactions.ResultsBeta cell miR-21 was increased in in vivo models of type 1 diabetes and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase 3 levels, suggesting increased cell death. In silico prediction tools identified the antiapoptotic mRNA BCL2 as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and B cell lymphoma 2 (BCL2) protein production, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase 3 levels after cytokine treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress Bcl2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3′ untranslated region. With miR-21 overexpression, PRP revealed a shift of the Bcl2 message towards monosome-associated fractions, indicating inhibition of Bcl2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production.Conclusions/interpretationIn contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation.

Stem cells as a tool to improve outcomes of islet transplantation.

The publication of the promising results of the Edmonton protocol in 2000 generated optimism for islet transplantation as a potential cure for Type 1 Diabetes Mellitus. Unfortunately, follow-up data revealed that less than 10% of patients achieved long-term insulin independence. More recent data from other large trials like the Collaborative Islet Transplant Registry show incremental improvement with 44% of islet transplant recipients maintaining insulin independence at three years of follow-up. Multiple underlying issues have been identified that contribute to islet graft failure, and newer research has attempted to address these problems. Stem cells have been utilized not only as a functional replacement for β cells, but also as companion or supportive cells to address a variety of different obstacles that prevent ideal graft viability and function. In this paper, we outline the manners in which stem cells have been applied to address barriers to the achievement of long-term insulin independence following islet transplantation.