Expression of Glucokinase in Glucose-Unresponsive Human Fetal Pancreatic Islet-Like Cell Clusters1

Abstract Glucokinase (GK) is the glucose sensor in the adult β-cell, resulting in fuel for insulin synthesis and secretion. Defects in this enzyme in the β-cell are responsible for the genetic disorder maturity-onset diabetes of the young, with the β-cell being unable to secrete insulin appropriately when challenged with glucose. The human fetalβ -cell is also unable to secrete insulin when exposed to glucose, but whether GK is present and functional in this developing cell is unknown. To determine the expression of GK in human fetal pancreatic tissue, cytosolic protein was extracted from human fetal islet-like cell clusters (ICCs) at 17–19 weeks gestation and examined for protein content and enzyme activity. On Western blots, a single band corresponding to GK was seen at 52 kDa, and this was similar to that obtained from human adult islets. The maximal velocity (Vmax) of GK was less in fetal ICCs than that in adult islets (8.7 vs. 20.7 nmol/mg protein·h); similar Km values were found in both ICCs and islets. No attempt was made to determine which cells in an ICC contained GK. Glucose utilization was determined radiometrically; the Vmax of the high Km component was less in ICCs than in islets (31.3 pmol/ICC·h vs. 101.4 pmol/islet·h). Culture of ICCs for 3–7 days in medium containing 11.2 mmol/L glucose resulted in a 3.7-fold increase in the Vmax of GK and a 1.8-fold increase in glucose utilization. These enhanced activities of glucose phosphorylation and glycolysis, however, did not lead to the β-cell being able to secrete insulin when exposed to glucose. In conclusion, glucokinase is present and functional in human fetal ICCs, but the inability of the human fetal β-cell to secrete insulin in response to an acute glucose challenge is not due to immaturity of this enzyme.

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CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line

Frontiers in Genetics ◽

10.3389/fgene.2021.664799 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xue Zhang ◽

Lihua Zhao ◽

Runbing Jin ◽

Min Li ◽

Mei-Shuang Li ◽

...

Keyword(s):

Cell Line ◽

Gene Knockout ◽

Pancreatic Tissue ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Min6 Cells ◽

Insulin Synthesis ◽

Guide Rna

Many ion channels participate in controlling insulin synthesis and secretion of pancreatic β-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic β-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic β-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC−/−) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC−/− MIN6 cells. Therefore, α-ENaC−/− MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic β-cells.

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Insulin synthesis, secretory competence, and glucose utilization are sensitized by transgenic yeast hexokinase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)40753-8 ◽

1994 ◽

Vol 269 (22) ◽

pp. 15814-15818

Author(s):

M.E. Voss-McCowan ◽

B. Xu ◽

P.N. Epstein

Keyword(s):

Glucose Utilization ◽

Insulin Synthesis ◽

Transgenic Yeast ◽

Yeast Hexokinase

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Thermostable glucose isomerase from psychrotolerant Streptomyces species

International Journal of Life Sciences ◽

10.3126/ijls.v1i0.2300 ◽

1970 ◽

Vol 1 ◽

pp. 6-10 ◽

Cited By ~ 3

Author(s):

Bidur Dhungel ◽

Manoj Subedi ◽

Kiran Babu Tiwari ◽

Upendra Thapa Shrestha ◽

Subarna Pokhrel ◽

...

Keyword(s):

Specific Activity ◽

Fold Increase ◽

Glucose Isomerase ◽

Enzyme Concentration ◽

Maximal Velocity ◽

Ph Optimum ◽

Streptomyces Spp ◽

Optimum Ph ◽

Optimum Reaction ◽

Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

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An investigation of arterial insufficiency in the rat hindlimb Correlation of skeletal muscle bloodflow and glucose utilization in vivo

Biochemical Journal ◽

10.1042/bj2400395 ◽

1986 ◽

Vol 240 (2) ◽

pp. 395-401 ◽

Cited By ~ 8

Author(s):

R A Challiss ◽

D J Hayes ◽

G K Radda

Keyword(s):

Skeletal Muscle ◽

Sciatic Nerve ◽

Glucose Uptake ◽

Nerve Stimulation ◽

Linear Correlation ◽

Glucose Utilization ◽

Fold Increase ◽

Artery Ligation ◽

Sciatic Nerve Stimulation

Muscle bloodflow and the rate of glucose uptake and phosphorylation were measured in vivo in rats 7 days after unilateral femoral artery ligation and section. Bloodflow was determined by using radiolabelled microspheres. At rest, bloodflow to the gastrocnemius, plantaris and soleus muscles of the ligated limb was similar to their respective mean contralateral control values; however, bilateral sciatic nerve stimulation at 1 Hz caused a less pronounced hyperaemic response in the muscles of the ligated limb, being 59, 63 and 49% of their mean control values in the gastrocnemius, plantaris and soleus muscles respectively. The rate of glucose utilization was determined by using the 2-deoxy[3H]glucose method [Ferré, Leturque, Burnol, Penicaud & Girard (1985) Biochem. J. 228, 103-110]. At rest, the rate of glucose uptake and phosphorylation was statistically significantly increased in the gastrocnemius and soleus muscles of the ligated limb, being 126 and 140% of the mean control values respectively. Bilateral sciatic nerve stimulation at 1 Hz caused a 3-5-fold increase in the rate of glucose utilization by the ligated and contralateral control limbs; furthermore, the rate of glucose utilization was significantly increased in the muscles of the ligated limb, being 140, 129 and 207% of their mean control values respectively. For the range of bloodflow to normally perfused skeletal muscle at rest or during isometric contraction determined in the present study, a linear correlation between the rate of glucose utilization and bloodflow can be demonstrated. Applying similar methods of regression analysis to glucose utilization and bloodflow to muscles of the ligated limb reveals a similar linear correlation. However, the rate of glucose utilization at a given bloodflow is increased in muscles of the ligated limb, indicating an adaptation of skeletal muscle to hypoperfusion.

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β-Cell adaptation in 60% pancreatectomy rats that preserves normoinsulinemia and normoglycemia

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.1.e68 ◽

2000 ◽

Vol 279 (1) ◽

pp. E68-E73 ◽

Cited By ~ 30

Author(s):

Ye Qi Liu ◽

Peter W. Nevin ◽

Jack L. Leahy

Keyword(s):

Insulin Secretion ◽

Regulatory Element ◽

Cell Mass ◽

Maximal Velocity ◽

Β Cells ◽

Β Cell ◽

Biochemical Basis ◽

Adaptive Mechanisms ◽

Sense And Respond ◽

Glucose Responsiveness

Islet β-cells are the regulatory element of the glucose homeostasis system. When functioning normally, they precisely counterbalance changes in insulin sensitivity or β-cell mass to preserve normoglycemia. This understanding seems counter to the dogma that β-cells are regulated by glycemia. We studied 60% pancreatectomy rats (Px) 4 wk postsurgery to elucidate the β-cell adaptive mechanisms. Nonfasting glycemia and insulinemia were identical in Px and sham-operated controls. There was partial regeneration of the excised β-cells in the Px rats, but it was limited in scope, with the pancreas β-cell mass reaching 55% of the shams (40% increase from the time of surgery). More consequential was a heightened glucose responsiveness of Px islets so that glucose utilization and insulin secretion per milligram of islet protein were both 80% augmented at normal levels of glycemia. Investigation of the biochemical basis showed a doubled glucokinase maximal velocity in Px islets, with no change in the glucokinase protein concentration after adjustment for the different β-cell mass in Px and sham islets. Hexokinase activity measured in islet extracts was also minimally increased, but the glucose 6-phosphate concentration and basal glucose usage of Px islets were not different from those in islets from sham-operated rats. The dominant β-cell adaptive response in the 60% Px rats was an increased catalytic activity of glucokinase. The remaining β-cells thus sense, and respond to, perceived hyperglycemia despite glycemia actually being normal. β-Cell mass and insulin secretion are both augmented so that whole pancreas insulin output, and consequently glycemia, are maintained at normal levels.

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The agouti gene product stimulates pancreatic β-cell Ca2+ signaling and insulin release

Physiological Genomics ◽

10.1152/physiolgenomics.1999.1.1.11 ◽

1999 ◽

Vol 1 (1) ◽

pp. 11-19 ◽

Cited By ~ 28

Author(s):

B. Z. XUE ◽

W. O. WILKISON ◽

R. L. MYNATT ◽

N. MOUSTAID ◽

M. GOLDMAN ◽

...

Keyword(s):

Gene Product ◽

Insulin Release ◽

Fold Increase ◽

Cell Types ◽

Human Pancreas ◽

Pancreatic Β Cell ◽

Β Cell ◽

Agouti Protein ◽

Intracellular Ca ◽

Agouti Gene

Xue, B. Z., W. O. Wilkison, R. L. Mynatt, N. Moustaid, M. Goldman, and M. B. Zemel. The agouti gene product stimulates pancreatic β-cell Ca2+ signaling and insulin release. Physiol. Genomics 1: 11-19, 1999.—Ubiquitous expression of the mouse agouti gene results in obesity and hyperinsulinemia. Human agouti is expressed in adipose tissue, and we found recombinant agouti protein to stimulate lipogenesis in adipocytes in a Ca2+-dependent fashion. However, adipocyte-specific agouti transgenic mice only became obese in the presence of hyperinsulinemia. Because intracellular Ca2+ concentration ([Ca2+]i) is a primary signal for insulin release, and we have shown agouti protein to increase [Ca2+]i in several cell types, we examined the effects of agouti on [Ca2+]i and insulin release. We demonstrated the expression of agouti in human pancreas and generated recombinant agouti to study its effects on Ca2+ signaling and insulin release. Agouti (100 nM) stimulated Ca2+ influx, [Ca2+]i increase, and a marked stimulation of insulin release in two β-cell lines (RIN-5F and HIT-T15; P < 0.05). Agouti exerted comparable effects in isolated human pancreatic islets and β-cells, with a 5-fold increase in Ca2+ influx ( P < 0.001) and a 2.2-fold increase in insulin release ( P < 0.01). These data suggest a potential role for agouti in the development of hyperinsulinemia in humans.

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The ontogeny of the endocrine pancreas in the fetal/newborn baboon

Journal of Endocrinology ◽

10.1530/joe-12-0070 ◽

2012 ◽

Vol 214 (3) ◽

pp. 289-299 ◽

Cited By ~ 16

Author(s):

Amy R Quinn ◽

Cynthia L Blanco ◽

Carla Perego ◽

Giovanna Finzi ◽

Stefano La Rosa ◽

...

Keyword(s):

Endocrine Cells ◽

Endocrine Pancreas ◽

Fold Increase ◽

Pancreatic Tissue ◽

Developmental Differences ◽

Confocal Imaging ◽

Cell Type ◽

Pancreas Islet ◽

Pancreas Weight

Erratic regulation of glucose metabolism including hyperglycemia is a common condition in premature infants and is associated with increased morbidity and mortality. The objective of this study was to examine histological and ultrastructural differences in the endocrine pancreas in fetal (throughout gestation) and neonatal baboons. Twelve fetal baboons were delivered at 125 days (d) gestational age (GA), 140d GA, or 175d GA. Eight animals were delivered at term (185d GA); half were fed for 5 days. Seventy-three nondiabetic adult baboons were used for comparison. Pancreatic tissue was studied using light microscopy, confocal imaging, and electron microscopy. The fetal and neonatal endocrine pancreas islet architecture became more organized as GA advanced. The percent areas of α-β-δ-cell type were similar within each fetal and newborn GA (NS) but were higher than the adults (P<0.05) regardless of GA. The ratio of β cells within the islet (whole and core) increased with gestation (P<0.01). Neonatal baboons, which survived for 5 days (feeding), had a 2.5-fold increase in pancreas weight compared with their counterparts killed at birth (P=0.01). Endocrine cells were also found in exocrine ductal and acinar cells in 125, 140 and 175d GA fetuses. Subpopulation of tissue that coexpressed trypsin and glucagon/insulin shows the presence of cells with mixed endo–exocrine lineage in fetuses. In summary, the fetal endocrine pancreas has no prevalence of a α-β-δ-cell type with larger endocrine cell percent areas than adults. Cells with mixed endocrine/exocrine phenotype occur during fetal development. Developmental differences may play a role in glucose homeostasis during the neonatal period and may have long-term implications.

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SERCA1a can functionally substitute for SERCA2a in the heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.276.1.h89 ◽

1999 ◽

Vol 276 (1) ◽

pp. H89-H97 ◽

Cited By ~ 8

Author(s):

Yong Ji ◽

Evgeny Loukianov ◽

Tanya Loukianova ◽

Larry R. Jones ◽

Muthu Periasamy

Keyword(s):

Turnover Rate ◽

Ph Dependence ◽

Fold Increase ◽

Enzymatic Properties ◽

Hill Coefficient ◽

Maximal Velocity ◽

Apparent Affinity ◽

Serca Pump ◽

Fast Twitch ◽

The Hill

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an ∼2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889–897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased (∼1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.

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