Rapamycin/IL-2 Combination Therapy in Patients With Type 1 Diabetes Augments Tregs yet Transiently Impairs β-Cell Function

Rapamycin/interleukin-2 (IL-2) combination treatment of NOD mice effectively treats autoimmune diabetes. We performed a phase 1 clinical trial to test the safety and immunologic effects of rapamycin/IL-2 combination therapy in type 1 diabetic (T1D) patients. Nine T1D subjects were treated with 2-4 mg/day rapamycin orally for 3 months and 4.5 × 10(6) IU IL-2 s.c. three times per week for 1 month. β-Cell function was monitored by measuring C-peptide. Immunologic changes were monitored using flow cytometry and serum analyses. Regulatory T cells (Tregs) increased within the first month of therapy, yet clinical and metabolic data demonstrated a transient worsening in all subjects. The increase in Tregs was transient, paralleling IL-2 treatment, whereas the response of Tregs to IL-2, as measured by STAT5 phosphorylation, increased and persisted after treatment. No differences were observed in effector T-cell subset frequencies, but an increase in natural killer cells and eosinophils occurred with IL-2 therapy. Rapamycin/IL-2 therapy, as given in this phase 1 study, resulted in transient β-cell dysfunction despite an increase in Tregs. Such results highlight the difficulties in translating therapies to the clinic and emphasize the importance of broadly interrogating the immune system to evaluate the effects of therapy.     

Gene Variants in the Novel Type 2 Diabetes Loci CDC123/CAMK1D, THADA, ADAMTS9, BCL11A, and MTNR1B Affect Different Aspects of Pancreatic ��-Cell Function

OBJECTIVE

Recently, results from a meta-analysis of genome-wide association studies have yielded a number of novel type 2 diabetes loci. However, conflicting results have been published regarding their effects on insulin secretion and insulin sensitivity. In this study we used hyperglycemic clamps with three different stimuli to test associations between these novel loci and various measures of β-cell function.

RESEARCH DESIGN AND METHODS

For this study, 336 participants, 180 normal glucose tolerant and 156 impaired glucose tolerant, underwent a 2-h hyperglycemic clamp. In a subset we also assessed the response to glucagon-like peptide (GLP)-1 and arginine during an extended clamp (n = 123). All subjects were genotyped for gene variants in JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2/ADAMS30, DCD, VEGFA, BCL11A, HNF1B, WFS1, and MTNR1B.

RESULTS

Gene variants in CDC123/CAMK1D, ADAMTS9, BCL11A, and MTNR1B affected various aspects of the insulin response to glucose (all P < 6.9 × 10⁻³). The THADA gene variant was associated with lower β-cell response to GLP-1 and arginine (both P < 1.6 × 10⁻³), suggesting lower β-cell mass as a possible pathogenic mechanism. Remarkably, we also noted a trend toward an increased insulin response to GLP-1 in carriers of MTNR1B (P = 0.03), which may offer new therapeutic possibilities. The other seven loci were not detectably associated with β-cell function.

CONCLUSIONS

Diabetes risk alleles in CDC123/CAMK1D, THADA, ADAMTS9, BCL11A, and MTNR1B are associated with various specific aspects of β-cell function. These findings point to a clear diversity in the impact that these various gene variants may have on (dys)function of pancreatic β-cells.Genome-wide association (GWA) studies have revealed a large number of novel type 2 diabetes susceptibility loci (1–4). Most of the genes identified during the first wave of GWA study results are shown to affect β-cell function, indicated by lower insulin responses to oral (OGTTs) or intravenous (IVGTTs) glucose tolerance tests (5). By applying the hyperglycemic clamp methodology, considered the gold standard for measurements of β-cell function, we further refined the observed β-cell defects to defects in first- but not second-phase glucose-stimulated insulin secretion (GSIS) (6) or incretin-stimulated secretion (7). This differentiation is of importance to help resolve the pathogenic mechanism of the diabetes loci identified by GWA studies.More recently the Diabetes Genetics Replication And Meta-analysis (DIAGRAM) Consortium published at least six additional susceptibility loci, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, and NOTCH2/ADAM30 (8), and three putative susceptibility loci, DCD, VEGFA, and BCL11A. Studies using OGTTs have yielded conflicting results on the effects of these new loci on β-cell function and insulin sensitivity. Grarup et al. (9) reported β-cell dysfunction associated with gene variants in JAZF1, TSPAN8/LGR5, and CDC123/CAMK1D. The results for CDC123/CAMK1D have only been replicated by Sanghera et al. (10) in Asian Indians but not by three other studies in Caucasians. All of the other three studies also failed to replicate the results for JAZF1 and TSPAN8/LGR5 (11–13). Furthermore, gene variants in three other loci have been established as true type 2 diabetes susceptibility loci, HNF1B, WFS1, and MTNR1B (14–19). Although mutations in HNF1B are associated with β-cell defects in maturity-onset diabetes of the young, it is unknown whether the type 2 diabetes–associated common single nucleotide polymorphism (SNP) is also associated with reduced β-cell function (14,15). It has been shown that WFS1 associates with reduced oral (11,13,20–22) but not intravenous glucose-stimulated insulin secretion (22). Schäfer et al. (22) further demonstrated that the WFS1 gene affects glucagon-like peptide (GLP)-1–stimulated insulin secretion during clamps. For the MTNR1B locus, several studies have shown reduced insulin secretion in response to glucose (17–19,23,24).In this study 180 normal (NGT) and 156 impaired (IGT) glucose tolerant subjects originating from three independent studies in the Netherlands were genotyped for variants in JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2/ADAMS30, DCD, VEGFA, BCL11A, HNF1B, WFS1, and MTNR1B. We tested whether these loci are associated with alterations in β-cell function as assessed by hyperglycemic clamp methodology with, in a subset, two additional secretagogues, namely GLP-1 and arginine. Arginine stimulation during hyperglycemia is a test of (near) maximal insulin secretion and has been proposed as a proxy for β-cell mass (25).     

Improvement in Insulin Sensitivity and Β-Cell Function Following Ileal Interposition with Sleeve Gastrectomy in Type 2 Diabetic Patients: Potential Mechanisms

Introduction  

Bariatric surgery in morbidly obese type 2 diabetic (T2DM) patients is associated with high rates of diabetes remission. We investigated the mechanisms of the anti-diabetic effect of the laparoscopic ileal interposition with sleeve gastrectomy (LII-SG) in normal weight (NW), overweight (OW) and obese (OB) T2DM patients.     

Deletion of Pten in Pancreatic ��-Cells Protects Against Deficient ��-Cell Mass and Function in Mouse Models of Type 2 Diabetes

OBJECTIVE

Type 2 diabetes is characterized by diminished pancreatic β-cell mass and function. Insulin signaling within the β-cells has been shown to play a critical role in maintaining the essential function of the β-cells. Under basal conditions, enhanced insulin-PI3K signaling via deletion of phosphatase with tensin homology (PTEN), a negative regulator of this pathway, leads to increased β-cell mass and function. In this study, we investigated the effects of prolonged β-cell–specific PTEN deletion in models of type 2 diabetes.

RESEARCH DESIGN AND METHODS

Two models of type 2 diabetes were employed: a high-fat diet (HFD) model and a db/db model that harbors a global leptin-signaling defect. A Cre-loxP system driven by the rat insulin promoter (RIP) was employed to obtain mice with β-cell–specific PTEN deletion (RIPcre⁺ Pten^fl/fl).

RESULTS

PTEN expression in islets was upregulated in both models of type 2 diabetes. RIPcre⁺ Pten^fl/fl mice were completely protected against diabetes in both models of type 2 diabetes. The islets of RIPcre⁺ Pten^fl/fl mice already exhibited increased β-cell mass under basal conditions, and there was no further increase under diabetic conditions. Their β-cell function and islet PI3K signaling remained intact, in contrast to HFD-fed wild-type and db/db islets that exhibited diminished β-cell function and attenuated PI3K signaling. These protective effects in β-cells occurred in the absence of compromised response to DNA-damaging stimuli.

CONCLUSIONS

PTEN exerts a critical negative effect on both β-cell mass and function. Thus PTEN inhibition in β-cells can be a novel therapeutic intervention to prevent the decline of β-cell mass and function in type 2 diabetes.The quintessential defects in type 2 diabetes are the development of peripheral insulin resistance and β-cell dysfunction (1–3). In fact, the loss of insulin secretion in β-cells in response to glucose occurs before the emergence of insulin resistance and hyperglycemia (4–6). Once insulin resistance develops, hyperglycemia, high-circulating free fatty acids, and inflammatory cytokines further abrogate glucose-stimulated insulin secretion (2,7–9). It is becoming increasingly clear that insulin/insulin-like growth factor 1 (IGF-1) signaling plays an important role in the maintenance of β-cell function under both basal and diabetic conditions. Mice with β-cell–specific deletion of IGF-1 receptor exhibit a defect in glucose-stimulated insulin secretion (10,11), whereas insulin receptor deletion in β-cells results in both attenuated insulin secretion in response to glucose and reduced β-cell mass with aging (12,13). Thus, β-cells are not only an essential source of the hormone insulin, but are also a critical target of insulin action in the maintenance of β-cell function.Phosphoinositide 3-kinase (PI3K) signaling cascade is one of the major intracellular signaling pathways through which insulin and IGF-1 mediate their effects (14). Phosphatase with tensin homology (PTEN) is a dual-specific phosphatase and a potent negative regulator of this pathway by its ability to dephosphorylate phosphatidylinosotol-3,4,5-triphosphate (PIP3) to phosphatidylinosotol-4,5-bisphosphate (PIP2), thereby effectively removing the critical secondary messenger of this signaling cascade (15,16). Although PTEN was first discovered as a tumor suppressor, recent studies have highlighted the important physiologic role of PTEN in metabolism (16–18). Tissue-targeted deletion of PTEN in liver, fat, or muscle lead to improved insulin sensitivity in these insulin-responsive tissues and protects mice from HFD-induced diabetes (19–22). Additionally, we and others have reported that mice with PTEN deletion in pancreatic β-cells show increased β-cell mass because of both increased proliferation and reduced apoptosis without compromising β-cell function under the basal condition (23,24).PTEN has been shown to be upregulated in models of insulin resistance, including a genetic model of combined ablation of insulin/IGF-1 signaling in β-cells (25–27). Furthermore, in vitro overexpression of PTEN in pancreatic β-cell lines showed impaired insulin secretion in response to ambient glucose (28). However, the regulation of PTEN expression in β-cells in models of type 2 diabetes in vivo was unknown. We show here that PTEN expression was increased in islets of both high-fat diet (HFD)-fed and db/db mice, which was accompanied by attenuation in PI3K signaling, suggesting the potential causal role of PTEN in the pathogenesis of β-cell dysfunction in type 2 diabetes. In this report, we investigated the essential role of PTEN in β-cells in the context of type 2 diabetes models. We used the rat insulin promoter (RIP) to drive deletion of PTEN in the Cre-loxP system (RIPcre⁺ Pten^fl/fl). RIPcre⁺ Pten^fl/fl mice were protected from HFD-induced type 2 diabetes because of their increased islet mass and proliferation with intact β-cell function. β-Cells from RIPcre⁺ Pten^fl/fl mice were protected against HFD-induced β-cell dysfunction both in vitro and in vivo, which can be attributed to the constitutively active PI3K signaling in their islets. Furthermore, RIPcre⁺ Pten^fl/fl mice in the db/db background still remained euglycemic, despite being severely insulin resistant. Interestingly, their β-cell mass was not significantly different from db/db littermates. However, their β-cell function and islet PI3K signaling remained intact. Together, our data highlight the critical role of β-cell PTEN in the development of β-cell dysfunction in type 2 diabetes and support PTEN as a potential therapeutic target for β-cell growth and the preservation of its function.     

NF-κB and COX-2 repression with topical application of hesperidin and naringin hydrogels augments repair and regeneration of deep dermal wounds

IntroductionWound injury is common and causes serious complications if not treated properly. The moist dressing heals wounds faster than other dressings. Therefore, we sought to study the effect of hesperidin/naringin hydrogel wound dressing or their combinations on the deep dermal wounds in mice.MethodsA rectangular full thickness skin flap of 2.5 × 1.5 cm was excised from depilated mice dorsum and the wound was fully covered with 5% hesperidin/5% naringin hydrogel or both in the ratio of 1:1, 2:1, or 1:2, respectively once daily until complete healing of the wound. Data were collected on wound contraction, mean wound healing time, collagen, DNA, and nitric oxide syntheses, glutathione concentration, superoxide dismutase activity, and lipid peroxidation throughout healing. Expression of NF-κB and COX-2 were also estimated in the regenerating granulation tissue using Western blot.FindingsDressing of wounds with 5% hesperidin hydrogel led to a higher and early wound contraction and significantly reduced mean wound healing time by 5.7 days than 5% naringin or combination of hesperidin and naringin hydrogels in the ratio of 1:1, 2:1, or 1:2. Hesperidin hydrogel wound dressing caused higher collagen and DNA syntheses than other groups at all times after injury. Glutathione concentration and superoxide dismutase activity increased followed by a decline in lipid peroxidation in regenerating wounds after hesperidin/naringin hydrogel application and a maximum effect was observed for hesperidin alone. The hesperidin/naringin hydrogel suppressed NF-κB and COX-2 expression on days 6 and 12.ConclusionsApplication of 5% hesperidin hydrogel was more effective than 5% naringin or combination of hesperidin and naringin gels (1:1, 2:1 or 1:2) indicated by a greater wound contraction, reduced mean wound healing time, elevated collagen and DNA syntheses, rise in glutathione concentration, and superoxide dismutase activity followed by reduced lipid peroxidation, and NF-κB, and COX-2 expression.     

Circulating TGF-β1–Regulated miRNAs and the Risk of Rapid Progression to ESRD in Type 1 Diabetes

We investigated whether circulating TGF-β1–regulated miRNAs detectable in plasma are associated with the risk of rapid progression to end-stage renal disease (ESRD) in a cohort of proteinuric patients with type 1 diabetes (T1D) and normal eGFR. Plasma specimens obtained at entry to the study were examined in two prospective subgroups that were followed for 7–20 years (rapid progressors and nonprogressors), as well as a reference panel of normoalbuminuric T1D patients. Of the five miRNAs examined in this study, let-7c-5p and miR-29a-3p were significantly associated with protection against rapid progression and let-7b-5p and miR-21-5p were significantly associated with the increased risk of ESRD. In logistic analysis, controlling for HbA_1c and other covariates, let-7c-5p and miR-29a-3p were associated with more than a 50% reduction in the risk of rapid progression (P ≤ 0.001), while let-7b-5p and miR-21-5p were associated with a >2.5-fold increase in the risk of ESRD (P ≤ 0.005). This study is the first prospective study to demonstrate that circulating TGF-β1–regulated miRNAs are deregulated early in T1D patients who are at risk for rapid progression to ESRD.     

Mechanisms of vasodilation to PTH 1–84, PTH 1–34, and PTHrP 1–34 in rat bone resistance arteries

Summary

Parathyroid hormone (PTH) augments bone metabolism and bone mass when given intermittently. Enhanced blood flow is requisite to support high tissue metabolism. The bone arteries are responsive to all three PTH analogs, which may serve to augment skeletal blood flow during intermittent PTH administration.

Introduction

PTH augments bone metabolism. Yet, mechanisms by which PTH regulates bone blood vessels are unknown. We deciphered (1) endothelium-dependent and endothelium-independent vasodilation to PTH 1–84, PTH 1–34, and PTHrP 1–34, (2) the signaling pathways (i.e., endothelial nitric oxide synthase [eNOS], cyclooxygenase [COX], protein kinase C [PKC], and protein kinase A [PKA]), and (3) receptor activation.

Methods

Femoral principal nutrient arteries (PNAs) were given cumulative doses (10^?13–10^?8 M) of PTH 1–84, PTH 1–34, and PTHrP 1–34 with and without signaling pathway blockade. Vasodilation was also determined following endothelial cell removal (i.e., denudation), PTH 1 receptor (PTH1R) inhibition and to sodium nitroprusside (SNP; a nitric oxide [NO] donor).

Results

Vasodilation was lowest to PTH 1–34, and maximal dilation was highest to PTHrP 1–34. Inhibition of eNOS reduced vasodilation to PTH 1–84 (?80 %), PTH 1–34 (?66 %), and PTHrP 1–34 (?48 %), evidencing the contribution of NO. Vasodilation following denudation was eliminated (PTH 1–84 and PTHrP 1–34) and impaired (PTH 1–34, 17 % of maximum), highlighting the importance of endothelial cells for PTH signaling. Denuded and intact PNAs responded similarly to SNP. Both PKA and PKC inhibition diminished vasodilation in all three analogs to varying degrees. PTH1R blockade reduced vasodilation to 1, 12, and 12 % to PTH 1–84, PTH 1–34, and PTHrP 1–34, respectively.

Conclusions

Vasodilation of femoral PNAs to the PTH analogs occurred via activation of the endothelial cell PTH1R for NO-mediated events. PTH 1–84 and PTHrP 1–34 primarily stimulated PKA signaling, and PTH 1–34 equally stimulated PKA and PKC signaling.

     

Effects of Common Genetic Variants Associated With Type 2 Diabetes and Glycemic Traits on α- and β-Cell Function and Insulin Action in Humans

Although meta-analyses of genome-wide association studies have identified >60 single nucleotide polymorphisms (SNPs) associated with type 2 diabetes and/or glycemic traits, there is little information on whether these variants also affect α-cell function. The aim of the current study was to evaluate the effects of glycemia-associated genetic loci on islet function in vivo and in vitro. We studied 43 SNPs in 4,654 normoglycemic participants from the Finnish population-based Prevalence, Prediction, and Prevention of Diabetes-Botnia (PPP-Botnia) Study. Islet function was assessed, in vivo, by measuring insulin and glucagon concentrations during oral glucose tolerance test, and, in vitro, by measuring glucose-stimulated insulin and glucagon secretion from human pancreatic islets. Carriers of risk variants in BCL11A, HHEX, ZBED3, HNF1A, IGF1, and NOTCH2 showed elevated whereas those in CRY2, IGF2BP2, TSPAN8, and KCNJ11 showed decreased fasting and/or 2-h glucagon concentrations in vivo. Variants in BCL11A, TSPAN8, and NOTCH2 affected glucagon secretion both in vivo and in vitro. The MTNR1B variant was a clear outlier in the relationship analysis between insulin secretion and action, as well as between insulin, glucose, and glucagon. Many of the genetic variants shown to be associated with type 2 diabetes or glycemic traits also exert pleiotropic in vivo and in vitro effects on islet function.In the last few years, genome-wide association studies have substantially increased the knowledge of genetic variants predisposing to type 2 diabetes. Although the majority of these single nucleotide polymorphisms (SNPs) seem to influence insulin secretion, few, if any, studies have assessed their simultaneous effects on β- and α-cell function in vivo and in vitro. The aim of the current study was to provide a comprehensive evaluation of the effects of genetic loci associated with type 2 diabetes (1) and/or glucose and insulin levels (2) on islet function in vivo, in a large well-characterized population-based study from the western coast of Finland (the Prevalence, Prediction, and Prevention of Diabetes-Botnia [PPP-Botnia] Study), and in vitro, in human pancreatic islets. Islet function was assessed by measuring insulin and glucagon concentrations during an oral glucose tolerance test (OGTT).     

β-Cell Function,Incretin Effect,and Incretin Hormones in Obese Youth Along the Span of Glucose Tolerance From Normal to Prediabetes to Type 2 Diabetes

Using the hyperglycemic and euglycemic clamp, we demonstrated impaired β-cell function in obese youth with increasing dysglycemia. Herein we describe oral glucose tolerance test (OGTT)-modeled β-cell function and incretin effect in obese adolescents spanning the range of glucose tolerance. β-Cell function parameters were derived from established mathematical models yielding β-cell glucose sensitivity (βCGS), rate sensitivity, and insulin sensitivity in 255 obese adolescents (173 with normal glucose tolerance [NGT], 48 with impaired glucose tolerance [IGT], and 34 with type 2 diabetes [T2D]). The incretin effect was calculated as the ratio of the OGTT-βCGS to the 2-h hyperglycemic clamp-βCGS. Incretin and glucagon concentrations were measured during the OGTT. Compared with NGT, βCGS was 30 and 65% lower in youth with IGT and T2D, respectively; rate sensitivity was 40% lower in T2D. Youth with IGT or T2D had 32 and 38% reduced incretin effect compared with NGT in the face of similar changes in GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) in response to oral glucose. We conclude that glucose sensitivity deteriorates progressively in obese youth across the spectrum of glucose tolerance in association with impairment in incretin effect without reduction in GLP-1 or GIP, similar to that seen in adult dysglycemia.