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Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2(+/Akita) β-cells. We used T antigen-transformed Ins2(+/Akita) and control Ins2(+/+) β-cells established from Akita and wild-type littermate mice. In Ins2(+/Akita) β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2(+/Akita) β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2(+/Akita) β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes.

Epidemiological studies have linked intrauterine growth retardation (IUGR) to the metabolic diseases, consisting of insulin resistance, type 2 diabetes, obesity and coronary artery disease, during adult life. To determine the internal relationship between IUGR and islet β cell function and insulin sensitivity, we established the IUGR model by maternal nutrition restriction during mid- to late-gestation. Glucose tolerance test and insulin tolerance test (ITT) in vivo and glucose stimulated insulin secretion (GSIS) test in vitro were performed at different stages in IUGR and normal groups. Body weight, pancreas weight and pancreas/body weight of IUGR rats were much lower than those in normal group before 3 weeks of age. While the growth of IUGR rats accelerated after 3 weeks, pancreas weight and pancreas/body weight remained lower till 15 weeks of age. In the newborns, the fasting glucose and insulin levels of IUGR rats were both lower than those of controls, whereas glucose levels at 120 and 180 min after glucose load were significantly higher in IUGR group. Between 3 and 15 weeks of age, both the fasting glucose and insulin levels were elevated and the glucose tolerance was impaired with time in IUGR rats. At age 15 weeks, the area under curve of insulin (AUCi) after glucose load in IUGR rats elevated markedly. Meanwhile, the stimulating index of islets in IUGR group during GSIS test at age 15 weeks was significantly lower than that of controls. ITT showed no significant difference in two groups before 7 weeks of age. However, in 15-week-old IUGR rats, there was a markedly blunted glycemic response to insulin load compared with normal group. These findings demonstrate that IUGR rats had both impaired pancreatic development and deteriorated glucose tolerance and insulin sensitivity, which would be the internal causes why they were prone to develop type 2 diabetes.

The essential folding and maturation process of proinsulin in β-cells is largely uncharacterized. To analyze this process, we improved approaches to immunoblotting, metabolic labeling, and data analysis used to determine the proportion of monomers and non-monomers and changes in composition of proinsulin in cells. We found the natural occurrence of a large proportion of proinsulin in various non-monomer states, i.e., aggregates, in normal mouse and human β-cells and a striking increase in the proportion of proinsulin non-monomers in Ins2(+/Akita) mice in response to a mutation (C96Y) in the insulin 2 (Ins2) gene. Proinsulin emerges in monomer and abundant dual-fate non-monomer states during nascent protein synthesis and shows heavy and preferential ATP/redox-sensitive disposal among secretory proteins during early post-translational processes. These findings support the preservation of proinsulin's aggregation-prone nature and low relative folding rate that permits the plentiful production of non-monomer forms with incomplete folding. Thus, in normal mouse/human β-cells, proinsulin's integrated maturation and degradation processes maintain a balance of natively and non-natively folded states, i.e., proinsulin homeostasis (PIHO). Further analysis discovered the high susceptibility of PIHO to cellular energy and calcium changes, endoplasmic reticulum (ER) and reductive/oxidative stress, and insults by thiol reagent and cytokine. These results expose a direct correlation between various extra-/intracellular influences and (a)typical integrations of proinsulin maturation and disposal processes. Overall, our findings demonstrated that the control of precursor maturation and disposal acts as an early regulative mechanism in normal insulin production, and its disorder is crucially linked to β-cell failure and diabetes pathogenesis.

Our recent studies have uncovered that aggregation-prone proinsulin preserves a low relative folding rate and maintains a homeostatic balance of natively and non-natively folded states (i.e., proinsulin homeostasis, PIHO) in β-cells as a result of the integration of maturation and disposal processes. Control of precursor maturation and disposal is thus an early regulative mechanism in the insulin production of β-cells. Herein, we show pathways involved in the disposal of endogenous proinsulin at the early secretory pathway. We conducted metabolic-labeling, immunoblotting, and immunohistochemistry studies to examine the effects of selective proteasome and lysosome or autophagy inhibitors on the kinetics of proinsulin and control proteins in various post-translational courses. Our metabolic-labeling studies found that the main lysosomal and ancillary proteasomal pathways participate in the heavy clearance of insulin precursor in mouse islets/β-cells cultured at the mimic physiological glucose concentrations. Further immunoblotting and immunohistochemistry studies in cloned β-cells validated that among secretory proteins, insulin precursor is heavily and preferentially removed. The rapid disposal of a large amount of insulin precursor after translation is achieved mainly through lysosomal autophagy and the subsequent basal disposals are carried out by both lysosomal and proteasomal pathways within a 30 to 60-minute post-translational process. The findings provide the first clear demonstration that lysosomal and proteasomal pathways both play roles in the normal maintenance of PIHO for insulin production, and defined the physiological participation of lysosomal autophagy in the protein quality control at the early secretory pathway of pancreatic β-cells.