The objective of this dissertation research was to determine the specific actions of chronically elevated catecholamines on; 1.) fetal growth and ß-cell function during the third trimester in vivo in an ovine model of placental insufficiency-induced intrauterine growth restriction (PI-IUGR), and 2.) regulation of insulin secretion in vitro utilizing the mouse insulinoma cell line Min6.At 0.7-gestation, fetal weights were not different but PI fetuses had lower (P<0.05) basal blood oxygen content, plasma glucose, IGF-1, and insulin concentrations and greater norepinephrine concentrations (891±211 vs. 292±65 pg/ml; P<0.05) compared to controls. Glucose-stimulated insulin secretion (GSIS) was lower in PI than control fetuses (0.34±0.03 vs. 1.08±0.06 ng/ml; P<0.05). ADR-block increased GSIS in PI fetuses (1.19±0.11) but decreased GSIS in controls (0.86±0.02 ng/ml). Insulin content per islet was not different between PI and control fetuses. We concluded that elevated fetal plasma norepinephrine, in PI fetuses at 0.7 gestation, precedes growth restriction and suppresses insulin concentrations, and ADR-block revealed compensatory β-cells stimulus-secretion responsiveness. Therefore, to determine the effects of chronic hypercatecholamine exposure on fetal growth and β-cell function independent of hypoglycemia and hypoxemia, we performed surgical sham or adrenal demedullation (AD) at 0.65 gestation on control and IUGR fetuses (n= 5 Control-Sham, 5 Control-AD, 5 IUGR-Sham, 5 IUGR-AD fetuses). Studies commenced at 0.9 gestation under ambient conditions and steady-state reversal of arterial pO2 between IUGR and control fetuses. Plasma norepinephrine was 5-fold higher in IUGR-Sham vs. Control-Sham and reduced in IUGR-AD fetuses to concentrations not different from Control-Sham fetuses. Fetal mass was lower in IUGR vs. control fetuses but 92% greater in IUGR-AD compared to IUGR-Sham fetuses. Basal plasma glucose and arterial pO2 were lower in IUGR-Sham vs. Control-Sham, and IUGR-AD vs. Control-AD fetuses. Basal and glucose-stimulated insulin concentrations compared to Control-Sham were lower in IUGR-Sham and IUGR-AD and Control-AD fetuses. Oxygenation improved GSIS in IUGR-Sham and IUGR-AD fetuses. In conclusion, hypoglycemia, hypoxemia and norepinephrine interdependently and differentially regulate aspects of fetal growth and β-cell function in the IUGR fetus. In Min6 cells, we determined that GSIS responsiveness is enhanced and adrenergic receptor α2A is desensitized cells following chronic exposure to epinephrine.

In both Type 1 diabetes (T1D) and Type 2 diabetes (T2D), inadequate beta-cell mass and beta-cell dysfunction lead to impaired insulin secretion, and ultimately worsen glycemic control. Green tea has drawn wide attention due to its possible health-promoting properties, including enhancement of beta-cell function. We assessed the acute and relative long-term effects of epigallocatechin gallate (EGCG) on insulin secretion and synthesis from clonal beta-cells (INS1E cells), rat islets, and human islets, using 0.1, 1, or 5 µM. We determined if EGCG decreased blood glucose in healthy rats acutely, using 50 or 150 mg/kg body weight (BW), and after 12 days of supplementation in drinking water, using 0.1% and 0.5%. In the in vitro studies, EGCG significantly potentiated glucose-stimulated insulin secretion (GSIS) in rat islets (at 0.1, 1, and 5 µM) and human islets (at 1 µM), and elevated insulin content within INS1E cells (at 0.1, 1, and 5 µm) and human islets (at 1 µM), (P<0.05). Nutritional supplementation of EGCG (0.5% in drinking water) for 12 days in healthy rats significantly increased insulin synthesis, compared to that of controls, from 0.2 ± 0.02 to 1.4 ± 0.2 ng/mg protein, without alteration of insulin secretion in isolated islets (P<0.05). These findings demonstrate that EGCG may play a role in the regulation of pancreatic beta-cell function, thereby contributing to an anti-diabetic effect of this agent. / Master of Science

Diabetes is growing public health problem in the United States. Both in Type 1 and Type 2 diabetes, the deterioration of glycemic control over time is largely due to insulin secretory dysfunction and significant loss of functional β-cells. As such, the search for novel agents that promote β-cell survival and preserve functional β-cell mass are one of the essential strategies to prevent and treat the onset of diabetes. Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. It was recently shown that dietary intake of foods containing genistein improves diabetes in both experimental animals and humans. However, the potential anti-diabetic mechanisms of genistein are unclear. In the present study, we first investigated the effect of genistein on β-cell insulin secretion and proliferation and cellular signaling related to these effects in vitro and in vivo. We then determined its anti-diabetic potential in insulin-deficient and obese diabetic mouse models. The results in our study showed that exposure of clonal insulin secreting (INS1E) cells or isolated pancreatic islets to genistein at physiologically relevant concentrations (1-10 μM) enhanced glucose-stimulated insulin secretion (GSIS), whereas insulin content was not altered, suggesting that genistein-enhanced GSIS is not due to a modulation of insulin synthesis. This genistein's effect is protein tyrosine kinase- and KATP channel-independent. In addition, genistein had no effect on glucose transporter-2 expression or cellular ATP production, but similarly augmented pyruvate-stimulated insulin secretion in INS1E cells, indicating that genistein improvement of insulin secretion in β-cells is not related to an alternation in glucose uptake or the glycolytic pathway. Further, genistein (1-10 μM) induced both INS1 and human islet β-cell proliferation following 24 h of incubation, with 5 μM genistein inducing a maximal 27% increase. The effect of genistein on β-cell proliferation was neither dependent on estrogen receptors, nor shared by 17β-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of PKA or ERK1/2 completely abolished genistein-stimulated β-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for β-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in both insulin deficient type 1 and obese type 2 diabetic mice, concomitant with improved islet β-cell proliferation, survival, and mass. These changes were not due to alternations in animal body weight gain, food intake, fat deposit, plasma lipid profile, or peripheral insulin sensitivity. Collectively, these findings provide better understanding of the mechanism underlying the anti-diabetic effects of genistein. Loss of functional β-cell mass through apoptosis is central to the development of both T1D and T2D and islet β-cell preservation and regeneration are very important components of β-cell adaptation to increased apoptosis and insulin resistance and therefore holds promise as a treatment for this disease. In this context, these findings may potentially lead to the development of novel low-cost natural agents for prevention and treatment of diabetes. / Ph. D.

Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of ?-cells, leading to the impaired insulin secretion. Thus, the search for agents to protect b-cell and enhance its function is important for diabetes treatment. Studies have reported that baicalein, a flavone originally isolated from the roots of Chinese herb Scutellaria baicalensis, has various claimed beneficial effects on health, such as anti-oxidant, anti-viral, anti-thrombotic, and anti-inflammatory effects. However, it is unclear whether it exerts an anti-diabetic action. Here, we present evidence that baicalein may be a novel anti-diabetic agent. Specifically, dietary intake of baicalein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in high-fat diet (HFD)-fed middle-aged diabetic mice, which was associated with the improved isle t?-cell survival and mass. Baicalein treatment had no effect on food intake, body weight gain, circulating lipid profile, and insulin sensitivity in HFD-fed mice. In in-vitro studies, baicalein significantly augmented glucose-stimulated insulin secretion in insulin-secreting cells (INS1) and promotes viability of INS1 cells and human islets. These results demonstrate that baicalein may be a naturally occurring anti-diabetic agent by directly modulating pancreatic?-cell function. / Master of Science

Diabetes Mellitus (DM) is a metabolic disorder in which the body fails to achieve glucose homeostasis, due to either insulin resistance or reduced insulin secretion or both. This inadequate glucose control leads to hyperglycemia which, if left unchecked, leads to secondary complications like nephropathy, neuropathy, retinal degeneration and other serious conditions. In non-disease state, normal glucose level in the blood is maintained by pancreatic β-cells, which secrete insulin. However, during diabetes development, there is loss of β-cell mass and function; resulting in decreased insulin secretion which is the ultimate cause of hyperglycemia. The ability to non-invasively monitor changes in the β-cell mass during the development or treatment of diabetes would be a significant advance in diabetes management. However, a primary limitation for analysis of β-cell mass and developing dysfunction is the lack of specificity of β-cell targeting agents. Our novel approach for achieving the required specificity for a usable β-cell targeted contrast agent is to target a set of receptors on the cell surface that, as a combination, are unique to that cell. Through genetic screening, Glucagon Like Peptide-1 Receptor (GLP-1R) and α2Adrenergic Receptor (α2AR) were chosen as a potential molecular barcode for β-cells since their combination expression is relatively unique to the β-cells. GLP-1R and α2AR are both G-protein couple receptors (GPCRs) that, apart from being a β-cell specific combination, play an important role in regulating fundamental downstream signaling pathways in β-cells. To target these receptors effectively, we synthesized a multivalent ligand composed of Yohimbine (Yhb), an α2 adrenergic receptor (α2AR) antagonist, linked to an active Glucagon-like Peptide 1 analog (GLP-1₇₋₃₆). In this manuscript, I describe the synthesis and characterization of binding selectivity and signaling ability of GLP-1/Yhb at the cellular level. Using high throughput binding assays, we observed high affinity binding of GLP-1/Yhb to βTC3 cells, a β-cell mimetic line expressing both receptors, at a Kd of ~3 nM. Using microscopy, we observed significant Cy5-tagged GLP-1/Yhb binding and rapid internalization in cells expressing the complementary receptor pair at low concentrations, as low as 1 nM and 5 nM. When one of the receptors was made inaccessible due to presence of saturating quantities of a single unlabeled monomer, GLP-1/Yhb-Cy5 failed to bind to the cells at low concentrations (<10 nM). Similarly, in cells where either GLP-1R or α2AR were knocked down (using shRNA), binding of GLP-1/Yhb was significantly reduced (≤half of cells with both receptors), indicating strong selectivity of the ligand to cells expressing the combination of receptors. We also observed that GLP-1/Yhb construct modulates downstream signaling inβ TC3 cells resulting in enhanced Glucose Stimulated Insulin Secretion (GSIS). In presence of stimulatory glucose, GLP-1/Yhb significantly potentiated GSIS with a half-maximal effective dose of 2.6 nM. Compared to GLP-1₇₋₃₆ alone or GLP-1₇₋₃₆ and Yhb monomers added together, only GLP-1/Yhb could significantly potentiate GSIS at 1 nM, demonstrating that GLP-1/Yhb could translate high affinity binding to increased efficacy for GSIS potentiation. Unlike for insulin secretion, high affinity divalent binding did not translate to increased cAMP production at low concentrations, with significant increases above baseline seen only at 10 nM and higher. Nevertheless, these data show that GLP-1/Yhb binds selectively to β-cells and affects signaling, demonstrating its potential for targeted β-cell imaging and therapy. Overall, our work indicates that synthetic heterobivalent ligands, such as GLP-1/Yhb can be developed to increase cellular specificity and sensitivity making them a strong candidate for both noninvasive imaging and targeted therapy.

R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) pathway, but its function in the β-cell had not been explored. In Chapter 2, Rspo1 mRNA was found to be expressed in murine islets and the murine MIN6 and βTC β-cell lines, and Rspo1 protein was detected in MIN6 β-cells. Rspo1 activated cWnt signaling and induced insulin mRNA expression in MIN6 β-cells. Analysis of MIN6 and mouse β-cell proliferation revealed that Rspo1 stimulated cell growth and significantly abolished cytokine-induced cellular apoptosis. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. Chapter 2 further demonstrated that the glucagon-like peptide-1 receptor agonist, exendin-4 (EX4), stimulated Rspo1 mRNA transcript levels in MIN6 cells in a glucose-, time-, dose- and PI3-kinase-dependent fashion. Together, these studies demonstrate that Rspo1 is a novel β-cell growth factor and insulin secretagogue that is regulated by EX4. In Chapter 3, the role of Rspo1 in β-cells in vivo was explored using Rspo1 knock-out (Rspo1-/-) mice. Rspo1-/- mice had normal fasting glycemia but an improved glycemic control after an oral glucose challenge compared to Rspo1+/+ mice, with no difference in insulin sensitivity but an enhanced insulin response over 30 min; glucagon responses were normal. Rspo1 deficiency also resulted in an increase in β-cell mass in association with an increase in Ki67-positive β-cells, a marker of proliferation, relative to Rspo1+/+ mice. Rspo1-/- pancreatic tissues also demonstrated a significant increase in the number of insulin-positive ductal cells, suggestive of β-cell neogenesis. Rspo1-/- islets displayed no changes in glucose-induced insulin secretion but showed a complete absence of glucose-induced suppression glucagon secretion. Treatment of Rspo1-/- mice for 2 wk with EX4 resulted in a similar glycemic profile to EX4-treated Rspo1+/+ mice after an oral glucose challenge, with no changes in insulin sensitivity. Interestingly, EX4 administration to Rspo1-/- normalized β-cell mass to a level comparable to that in Rspo1+/+ mice. Although further studies are required, the findings in this thesis reveal a novel role for Rspo1 as a regulator of β-cell behaviour in vivo, and suggest novel roles for Rspo1 in both a- and ductal-cells.

R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) pathway, but its function in the β-cell had not been explored. In Chapter 2, Rspo1 mRNA was found to be expressed in murine islets and the murine MIN6 and βTC β-cell lines, and Rspo1 protein was detected in MIN6 β-cells. Rspo1 activated cWnt signaling and induced insulin mRNA expression in MIN6 β-cells. Analysis of MIN6 and mouse β-cell proliferation revealed that Rspo1 stimulated cell growth and significantly abolished cytokine-induced cellular apoptosis. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. Chapter 2 further demonstrated that the glucagon-like peptide-1 receptor agonist, exendin-4 (EX4), stimulated Rspo1 mRNA transcript levels in MIN6 cells in a glucose-, time-, dose- and PI3-kinase-dependent fashion. Together, these studies demonstrate that Rspo1 is a novel β-cell growth factor and insulin secretagogue that is regulated by EX4. In Chapter 3, the role of Rspo1 in β-cells in vivo was explored using Rspo1 knock-out (Rspo1-/-) mice. Rspo1-/- mice had normal fasting glycemia but an improved glycemic control after an oral glucose challenge compared to Rspo1+/+ mice, with no difference in insulin sensitivity but an enhanced insulin response over 30 min; glucagon responses were normal. Rspo1 deficiency also resulted in an increase in β-cell mass in association with an increase in Ki67-positive β-cells, a marker of proliferation, relative to Rspo1+/+ mice. Rspo1-/- pancreatic tissues also demonstrated a significant increase in the number of insulin-positive ductal cells, suggestive of β-cell neogenesis. Rspo1-/- islets displayed no changes in glucose-induced insulin secretion but showed a complete absence of glucose-induced suppression glucagon secretion. Treatment of Rspo1-/- mice for 2 wk with EX4 resulted in a similar glycemic profile to EX4-treated Rspo1+/+ mice after an oral glucose challenge, with no changes in insulin sensitivity. Interestingly, EX4 administration to Rspo1-/- normalized β-cell mass to a level comparable to that in Rspo1+/+ mice. Although further studies are required, the findings in this thesis reveal a novel role for Rspo1 as a regulator of β-cell behaviour in vivo, and suggest novel roles for Rspo1 in both a- and ductal-cells.

Glucose metabolism in pancreatic α- and β-cells is believed to regulate secretion of glucagon and insulin, respectively. In β-cells, ATP links glucose metabolism to electrical activity and insulin secretion. In α-cells, ATP has been attributed various roles in glucose-regulated glucagon release, but the underlying mechanisms are poorly understood. Despite its importance in insulin and glucagon secretion little is known about ATP kinetics in α- and β-cells. In this thesis, the novel fluorescent ATP biosensor Perceval was used to monitor physiologically relevant ATP concentrations with little influence of ADP. Glucose stimulation of β-cells within mouse and human pancreatic islets induced pronounced rise of ATP with superimposed oscillations. Simultaneous measurements of the sub-plasma membrane ATP and Ca2+ concentrations revealed glucose-induced oscillations in opposite phase. ATP increased further and the oscillations ceased when voltage-dependent Ca2+ influx was prevented. In contrast, ATP promptly decreased in response to K+-depolarization-induced elevation of Ca2+. Also mobilization of Ca2+ from intracellular stores lowered ATP, but the negative effect was not due to increased ATP consumption by the sarco/endoplasmic reticulum Ca2+-ATPase. Store-operated Ca2+ entry alone had little effect but markedly elevated ATP when combined with muscarinic receptor activation. When comparing ATP and Ca2+ responses in α- and β-cells within the same islet, glucose-induced ATP generation was much less pronounced and the dose-response relationship left-shifted in the α-cells. At basal glucose, individual α-cells showed Ca2+ and concomitant ATP oscillations in opposite-phase with variable frequency. These oscillations largely cancelled out when averaging data from several α-cells. At high glucose, the Ca2+ and ATP oscillations in α-cells tended to synchronize with the corresponding β-cell oscillations. Since β-cell Ca2+ oscillations drive pulsatile insulin secretion, which is antiparallel to pulsatile glucagon secretion, there seems to be an inverse relationship between changes in α-cell Ca2+ and glucagon release. This paradox is attributed to paracrine inhibition overriding Ca2+ stimulation, since somatostatin receptor blockade potently stimulated glucagon release with little effect on α-cell Ca2+ signalling. The data indicate that complex ATP-Ca2+ interactions in α- and β-cells underlie cell-intrinsic regulation of glucagon and insulin secretion and that paracrine inhibition of glucagon release becomes important in hyperglycaemia.

Type 2 diabetes accounts for 85-90% of all people with diabetes and is currently estimated to affect more than 180 million people worldwide, a figure estimated to double by the year 2030. Thus understanding the basic biology of glucose homeostasis and how it is altered during disease progression is crucial to the development of safe and effective treatment regimes. The link between high dietary fat and the development of type Il diabetes is well established. Chronic treatment of pancreatic islets with the lipid palmitate induces defects in glucose stimulated insulin secretion (GSIS) akin to those seen in the development of type Il diabetes. Previous studies from our group have identified the lipid-activated kinase protein kinase C epsilon (PKCε) as a potential mediator of some of these effects. Deletion of PKCε in mice results in complete protection from high-fat diet induced glucose intolerance. This protection is associated with enhanced circulating insulin suggesting that PKCε may be involved in the regulation of insulin release from the pancreatic β-Cell. The data presented here suggests that PKCs plays an important role in the regulation of insulin secretion under both physiological and pathophysiological conditions. We demonstrate that PKCε can be activated by chronic lipid treatment and acute cholinergic stimulation. Under these conditions insulin secretion is enhanced by PKCε deletion or inhibition suggesting that PKCε is a negative regulator of insulin secretion. Mechanistically the PKCs mediated inhibition of insulin release by acute or chronic PKCε activation appears to be distinct. The effect of PKCε induced by palmitate pre-treatment appears to be distal to calcium influx. The pool of pre-docked vesicles is enhanced in palmitate pre-treated β-cells lacking PKCε suggesting that PKCε may be involved in the regulation of vesicle dynamics. In contrast, calcium dynamics induced by cholinergic stimulation are altered by PKCε deletion, suggesting an effect on either the calcium channels themselves or on the upstream signalling. Given the ability of PKCε to inhibit insulin secretion, inhibition of PKCε in the β-cells of people suffering from insulin resistance and (or) type II diabetes represents a novel target for the treatment of type II diabetes.

Type 1 diabetes(T1D)is a disease that only gets more common. The etiology of the disease is not known but there are many existing theories about what the cause is. These different theories have been tested in vivoin rodents or invitro. The resultsfrom experiments done in those waysarenotall realistic because rodents differnotablyfrom humans,and when studies areperformed in vitrowith human isletsof Langerhans different hormones will accumulate. The aim of this studywas to establisha dynamic ex vivosystem in which stimulation of human islets of Langerhans can be performed in a more lifelike environment. To study islets in this system couldin the future lead to increased knowledge in the etiology of T1D.The perifusion system PERI-4.2 from Biorep Technologies together with an incubator with 37°Cand5% CO2were used to arrangethe ex vivosystem. An Insulin ELISA from Mercodia was performedto analyze the insulin secretion from the islets. Fourdifferent set ups for the system were tested and the last one showed the best results.In conclusion this study has shown that it is possible to preserve human islets of Langerhans in a dynamic ex vivosystem with a constant medium exchange if it is done under conditionswhere the islets are protected from shear forces from the supplying medium,together with a medium exchange rate which replaces the whole medium in at least one hour.