Increased islet mass is an adaptive mechanism that occurs to combat insulin resistance during pregnancy. Prolactin (PRL) can enhance beta-cell proliferation and insulin secretion in vitro, yet whether it is PRL or other pregnancy-related factors that mediate these adaptive changes during pregnancy is unknown. The objective of this study was to determine whether prolactin receptor (Prlr) is required for normal maternal glucose homeostasis during pregnancy. An ip glucose tolerance test was performed on timed-pregnant Prlr(+/+) and heterozygous null Prlr(+/-) mice on d 0, 15, and 18 of pregnancy. Compared with Prlr(+/+) mice, Prlr(+/-) mice had impaired glucose clearance, decreased glucose-stimulated insulin release, higher nonfasted blood glucose, and lower insulin levels during but not before pregnancy. There was no difference in their insulin tolerance. Prlr(+/+) mice show a significant incremental increase in islet density and beta-cell number and mass throughout pregnancy, which was attenuated in the Prlr(+/-) mice. Prlr(+/+) mice also had a more robust beta-cell proliferation rate during pregnancy, whereas there was no difference in apoptosis rate between the Prlr(+/+) and Prlr(+/-) mice before, during, or after pregnancy. Interestingly, genotype of the mothers had a significant impact on the offspring's phenotype, such that daughters derived from Prlr(+/-) mothers had a more severe phenotype than those derived from Prlr(+/+) mothers. In conclusion, this is the first in vivo demonstration that the action of pregnancy hormones, acting through Prlr, is required for normal maternal glucose tolerance during pregnancy by increasing beta-cell mass.

No abstract

Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular compartment to the cell surface; this phenomenon is defective in type 2 diabetes. Here we examine the involvement of actin filaments in GLUT4 translocation and their possible defects in insulin resistance, using L6 myotubes expressing myc-tagged GLUT4. Insulin caused membrane ruffling, a dynamic distortion of the myotube dorsal surface. Fluorescence microscopy and immunogold staining of surface GLUT4myc coupled to backscatter electron microscopy revealed a high density of this protein in membrane ruffles. The t-SNAREs syntaxin4 and SNAP-23 were also abundant in these regions. Below the membrane, GLUT4 and the vesicular protein VAMP2, but not VAMP3, colocalized with the actin structures supporting the membrane ruffles. GLUT4myc externalization and membrane ruffles were reduced by jasplakinolide and by swinholide-A, drugs that affect actin filament stability and prevent actin branching, respectively. Insulin resistance generated by prolonged (24 hours) exposure of myotubes to high glucose and insulin diminished the acute insulin-dependent remodeling of cortical actin and GLUT4myc translocation, reminiscent of the effect of swinholide-A. We propose that GLUT4 vesicle incorporation into the plasma membrane involves insulin-dependent cortical actin remodeling and that defective actin remodeling contributes to insulin resistance.

Hyperglycemia and hyperinsulinemia are cardinal features of acquired insulin resistance. In adipose cell cultures, high glucose and insulin cause insulin resistance of glucose uptake, but because of altered GLUT4 expression and contribution of GLUT1 to glucose uptake, the basis of insulin resistance could not be ascertained. Here we show that GLUT4 determines glucose uptake in L6 myotubes stably overexpressing myctagged GLUT4. Preincubation for 24 h with high glucose and insulin (high Glc/Ins) reduced insulin-stimulated GLUT4 translocation by 50%, without affecting GLUT4 expression. Insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and Akt phosphorylation also diminished, as did insulin-mediated glucose uptake. However, basal glucose uptake rose by 40% without any gain in surface GLUT4. High Glc/Ins elevated basal p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity, and a short inhibition of p38 MAPK with SB202190 corrected the rise in basal glucose uptake, suggesting that p38 MAPK activity contributes to this rise. We propose that in a cellular model of skeletal muscle, chronic exposure to high Glc/Ins reduced the acute, insulin-elicited GLUT4 translocation. In addition, basal state GLUT4 activity was augmented to partially compensate for the translocation defect, resulting in a more robust glucose uptake than what would be predicted from the amount of cell surface GLUT4 alone.

Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-à -vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (si-IRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70 -75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells. Insulin receptor substrates (IRSs)1 mediate diverse metabolic and mitogenic effects of insulin, and dysregulation of IRS expression and activation has been observed in both insulin resistance and diabetes (1-5). To date, six IRS proteins have been identified, but only IRS-1 and IRS-2 are thought to participate in regulation of glucose homeostasis (6 -9). Although IRS-1 and 2 have substantial amino acid similarity (10), there are significant structural differences between them. IRS-2 interacts with the insulin receptor via a phosphotyrosine-binding domain as well as a central domain located between amino acid 591 and 733 which is absent from IRS-1 (11, 12). Furthermore, IRS-1 and IRS-2 differ in their time course of insulin-stimulated tyrosine phosphorylation (13,14), interaction with the MAPK pathway (15), and intracellular localization (14).Defining the exact roles of IRS isoforms in insulin-stimulated glucose uptake is not without controversy. In rat adipocytes, overexpression of human IRS-1 increased, whereas elimination of IRS-1 via antisense ribozyme reduced, insulinstimulated GLUT4 translocation (16). However, other studies observed intact insulin-stimulated GLUT4 translocation when the interaction between insulin receptor and IRS was blocked, although insulin-mediated DNA synthesis and cell growth were reduced (17-19). Furthermore, IRS-1-null mice, although significan...

Indinavir is a useful tool to assess different functional contributions of GLUT4 to glucose uptake in common models of skeletal muscle and adipocytes.

Background: Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair.

Objective: The COVID-19 pandemic has led to significant public health measures that have resulted in decreased acute pediatric care utilization. We evaluated whether the rate of severe presentations of new onset type 1 diabetes (DM1), such as, diabetic ketoacidosis (DKA) has changed since the COVID-19 public health measures were enacted.Research Design and Methods: A retrospective chart review of children less than 18 years of age presenting with new onset DM1 during the pandemic period of March 17, 2020 to August 31, 2020 was conducted at two tertiary care pediatric hospitals in Alberta, Canada. Rates of DKA and severe DKA were compared to the same time period in the year 2019 (pre-pandemic control). Results:The number of children presenting with newly diagnosed DM1 was similar during the pandemic year of 2020 compared with 2019 (107 children in 2020 vs. 114 in 2019). The frequency of DKA at DM1 onset was significantly higher in the pandemic period (68.2% vs 45.6%; p < 0.001) and incidence of severe DKA was also higher (27.1% in 2020 vs 13.2% in 2019; p = 0.01).Conclusions: There was a significant increase in DKA and severe DKA in children presenting with new onset DM1 during the COVID-19 pandemic period. This emphasizes the need for educating health care professionals and families to be aware of the symptoms of hyperglycemia and the importance of early diagnosis and treatment even during public health measures for COVID-19.