Greb1 Transiently Accelerates Pancreatic ß-Cell Proliferation in Diabetic Mice Exposed to Estradiol.

Decrease of pancreatic ß cells leads to diabetes. In an inducible cAMP early suppressor (ICER-Iγ) transgenic mouse model of severe type 2 diabetes with reduced insulin production and depleted ß cells, supplementation with high concentrations of 17ß-estradiol (E2) markedly enhances ß-cell proliferation and normalizes glucose levels. The current study explored the underlying mechanisms leading to a dynamic increase of ß cells and pathologic changes in diabetic mice exposed to E2. Gene expression profiling of pancreatic islets of 6-month-old ICER-transgenic mice recovering from diabetes due to elevated E2 levels identified growth regulation by estrogen in breast cancer 1 (Greb1) as a gene significantly up-regulated during the recovery phase. To substantiate this, ß-cell-specific Greb1-deficient mice were generated, and Greb1 was shown to be essential for recovery of depleted ß cells in diabetic mice. Graft growth and glucose lowering were observed in 50 islets with increased Greb1 expression transplanted adjacent to E2 pellets beneath the kidney capsule of streptozotocin-induced diabetic mice. Greb1 expression due to a drastic increase in exogenous or endogenous E2 was transient and closely correlated with changes in E2-related and some cell cycle-related genes. These findings provide new insights into in vivo proliferation of deficient ß cells and suggest the possibility of new therapeutic approaches targeting pancreatic ß cells that could revolutionize the concept of diabetes treatment, which has been considered difficult to cure completely.

Differences in long-term effects of standard rodent diets on blood glucose and body weight of offspring.

Standard rodent diets are similar and contain well-balanced components, such as crude protein, fat, fiber, and ash. However, it is not clear whether there are differences in their long-term effects on metabolism. Here, we investigated the effects of long-term feeding of major standard diets, CRF-1, CE-2, and FR-1 to wild-type (WT) C57BL/6 mice on the blood glucose levels and body weight gain of their offspring, which were raised on the same diet and in the same environment as the mothers. The offspring have been influenced by the maternal diet during the fetal and neonatal stages, and were maintained on the same diet thereafter (until 60 weeks of age). In the CE-2 group, the offspring showed stable blood glucose levels and had the lowest body weight. The FR1 group showed the lowest blood glucose level, but body weight was increased significantly compared to the CE-2 group. In the CRF-1 group, higher blood glucose levels were seen from the neonatal stage and body weight increased more rapidly than in the other groups. Next, to determine the effects of blood glucose level and housing pattern on food and water consumption, severely diabetic (hyperglycemic) inducible cAMP early repressor (ICER) transgenic (Tg) mice and littermate WT mice (normoglycemic) were fed CE-2 diet and housed individually or in groups. Food and water consumption of WT mice was independent of housing pattern, whereas Tg mice showed significantly increased food and water consumption when housed individually, compared to group housing, and this did not change at different ages. Thus, the selection of standard diet and rearing method can have a marked impact on experimental outcomes in experiments using mice and presumably mouse cells, especially in studies of metabolism, diabetes, and obesity.

Amelioration of Murine Diabetic Nephropathy with a SGLT2 Inhibitor Is Associated with Suppressing Abnormal Expression of Hypoxia-Inducible Factors.

Diabetic nephropathy (DN), once manifested, is unlikely to completely recover. Factors that influence DN progression were explored by investigating the process of glomerulosclerosis and interstitial fibrosis and chronological changes in glucose, albuminuria, hyperfiltration, and expressions of sodium-glucose cotransporter 2 (SGLT2) and hypoxia-inducible factors (HIFs) up to 50 weeks in inducible cAMP early repressor transgenic mice, a model of severe DN. Long-term intervention with the SGLT2 inhibitor canagliflozin or islet transplantation or heminephrectomy was used. Inducible cAMP early repressor transgenic mice exhibited progressive diabetic glomerulosclerosis and mild interstitial fibrosis, and expressed extensive HIF-1α and HIF-2α in glomerulus and tubules, with sustained hyperfiltration up to 50 weeks. Canagliflozin ameliorated glomerulosclerosis/interstitial fibrosis gradually and reduced HIF overexpression. Islet-transplanted mice exhibited no amelioration. None of the heminephrectomized diabetic mice survived the hyperfiltration overload, but all of the canagliflozin-treated mice survived with re-expressions of HIF-1α and HIF-2α. These results suggest that persistent glomerular hyperfiltration might initiate glomerular injury, and persistent overexpression of HIFs could promote the development of glomerulosclerosis and interstitial fibrosis. Canagliflozin attenuated both changes. Oxidative stress or hypoxia was undetectable in this model. The abnormal expression of HIF-1α and HIF-2α may be a potential therapeutic target for preventing glomerulosclerosis and interstitial fibrosis.

Effects of 17ß-Estradiol and Androgen on Glucose Metabolism in Skeletal Muscle.

Diabetes develops predominantly in males in experimental models, and extensive evidence suggests that 17ß-estradiol (E2) modulates progression of diabetes in humans. We previously developed a severely diabetic transgenic (Tg) mouse model by ß-cell-specific overexpression of inducible cAMP early repressor (ICER) and found that male ICER-Tg mice exhibit sustained severe hyperglycemia, but female ICER-Tg mice gradually became normoglycemic with aging. This implies that differences in circulating androgen and E2 levels might influence skeletal muscle glucose uptake and glycemic status. Here we examined whether a decrease of androgen or E2 excess can improve muscle glucose uptake in hyperglycemic male ICER-Tg mice and, conversely, whether a decrease of E2 or androgen excess can elevate blood glucose levels and impair muscle glucose uptake in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX) or ORX+E2 pellet implantation and normoglycemic female ICER-Tg mice with ovariectomy (OVX) or OVX+5α-DHT pellet implantation to alter the androgen to E2 ratio. ORX+E2 treatment of male ICER-Tg mice caused a rapid drop in blood glucose via both a dramatic increase of ß-cells and significantly improved muscle glucose uptake due to the induction of glucose transporter type 4 (GLUT4) expression and translocation of GLUT4 to the cell membrane. In contrast, OVX+5α-DHT-treated female ICER-Tg mice showed an elevation of blood glucose without any decrease of ß-cells; instead, they showed decreased muscle glucose uptake due to decreased activation of serine/threonine-specific protein kinase AKT and GLUT4 expression. These findings suggest that androgen (5α-DHT) promotes insulin resistance in females, whereas E2 improves insulin sensitivity in severely diabetic male mice.

Adjusting the 17ß-Estradiol-to-Androgen Ratio Ameliorates Diabetic Nephropathy.

Diabetes is manifested predominantly in males in experimental models, and compelling evidence suggests that 17ß-estradiol (E2) supplementation improves hyperglycemia in humans. We previously generated a severely diabetic transgenic (Tg) mouse model by ß-cell­specific overexpression of inducible cAMP early repressor (ICER) and found that male but not female ICER-Tg mice exhibit sustained hyperglycemia and develop major clinical and pathologic features of human diabetic nephropathy (DN). Thus, we hypothesized that differences in circulating hormone levels have a key role in determining susceptibility to diabetes. Here, we examined whether DN in male ICER-Tg mice is rescued by adjusting the androgen-to-E2 ratio to approximate that in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX), E2 pellet implantation, or both. E2 pellet implantation at an early stage of DN with or without ORX caused a rapid drop in blood glucose and a dramatic increase in ß-cell number, and it markedly inhibited DN progression [namely, E2 reduced glomerulosclerosis, collagen 4 deposition and albuminuria, and prevented hyperfiltration]. Furthermore, E2 pellet implantation was more effective than ORX alone and induced a remarkable improvement, even when initiated at advanced-stage DN. In contrast, induction of normoglycemia by islet transplant in ICER-Tg mice eliminated albuminuria but was less effective than E2 + ORX in reducing glomerulosclerosis, collagen 4 deposition, and hyperfiltration. These findings indicate that E2 treatment is effective, even after establishment of DN, whereas glucose normalization alone does not improve sclerotic lesions. We propose that E2 intervention is a potential therapeutic option for DN.

ß-cell induction in vivo in severely diabetic male mice by changing the circulating levels and pattern of the ratios of estradiol to androgens.

Previously we have generated transgenic (Tg) mice developing severe diabetes early in life with a profound depletion of ß-cells with ß-cell-directed expression of inducible cAMP early repressor-Iγ. Only male mice continue to demonstrate hyperglycemia throughout life. To investigate this sexual dimorphism, we treated severely diabetic male Tg mice with orchiectomy (ORX) or 17ß-estradiol (E2) pellet implantation alone or in combination with ORX and E2-implantation to change the circulating levels and patterns of the ratio of estradiol to androgens. In the Tg-ORX group, the blood-glucose levels decreased to a certain level within several weeks but never reached the female Tg-control level. In contrast, the Tg-ORX+E2 or Tg-E2 group showed a more rapid drop in blood glucose to the basal level with a substantial increase in ß-cells, thus preventing the occurrence of severe diabetes in the male mice. The ß-cells, not only within islet but also in and adjacent to ducts and scattered ß-cell clusters, were strongly induced by 1 week after treatment, and the islet morphology dramatically changed. Enhanced ß-cell induction in the ducts occurred concomitantly with markedly increased levels of pancreatic duodenal homeobox-1 and related transcription factors. The glucose-lowering and ß-cell-increasing effects were independent of the age at which the treatment is started. These data provide evidence that the circulating level of E2 and the ratio of E2 to T greatly affect the blood glucose levels, the ß-cell induction, and the islet morphology in diabetic male Tg mice. This novel mechanism offers great potential for developing strategies to increase the number of ß-cells in vivo.