ATP-Sensitive K+ Channel Signaling in Glucokinase-Deficient Diabetes
http://www.100md.com
糖尿病学杂志 2005年第10期
the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT
As the rate-limiting controller of glucose metabolism, glucokinase represents the primary -cell "glucose sensor." Inactivation of both glucokinase (GK) alleles results in permanent neonatal diabetes; inactivation of a single allele causes maturity-onset diabetes of the young type 2 (MODY-2). Similarly, mice lacking both alleles (GKeC/eC) exhibit severe neonatal diabetes and die within a week, whereas heterozygous GK+/eC mice exhibit markedly impaired glucose tolerance and diabetes, resembling MODY-2. Glucose metabolism increases the cytosolic [ATP]-to-[ADP] ratio, which closes ATP-sensitive K+ channels (KATP channels), leading to membrane depolarization, Ca2+ entry, and insulin exocytosis. Glucokinase insufficiency causes defective KATP channel regulation, which may underlie the impaired secretion. To test this prediction, we crossed mice lacking neuroendocrine glucokinase (nGK+/eC) with mice lacking KATP channels (Kir6.2eC/eC). Kir6.2 knockout rescues perinatal lethality of nGKeC/eC, although nGKeC/eCKir6.2eC/eC animals are postnatally diabetic and still die prematurely. nGK+/eC animals are diabetic on the Kir6.2+/+ background but only mildly glucose intolerant on the Kir6.2eC/eC background. In the presence of glutamine, isolated nGK+/eCKir6.2eC/eC islets show improved insulin secretion compared with nGK+/eCKir6.2+/+. The significant abrogation of nGKeC/eC and nGK+/eC phenotypes in the absence of KATP demonstrate that a major factor in glucokinase deficiency is indeed altered KATP signaling. The results have implications for understanding and therapy of glucokinase-related diabetes.
Glucose metabolism in pancreatic -cells is necessary to stimulate insulin secretion (1). Glucokinase, which phosphorylates glucose in the first, rate-limiting reaction, is a key component in the secretory pathway (2,3), and mutations of the GK gene are diabetogenic in humans (4). Heterozygous inactivating mutations cause maturity-onset diabetes of the young type 2 (MODY-2), an autosomal, dominantly inherited form of diabetes characterized by an early age of onset and pancreatic -cell dysfunction (5eC11). Homozygous inactivating GK mutations cause permanent neonatal diabetes in humans (12,13), which is characterized by perinatal hyperglycemia and low birth weight (14). The disease usually requires insulin treatment through the patient’s lifetime, starting within the first month of life. Targeted disruption of the neuroendocrine knockout of GK (nGK) gene in mice also causes diabetes (15). These mice lack glucokinase in pancreatic -cells and neurons but maintain normal liver glucokinase activity. Heterozygous null mice show early-onset mild diabetes caused by an impaired insulin-secretory response to glucose, resembling MODY-2. Importantly, homozygous nGK-deficient mice show severe perinatal diabetes, as in permanent neonatal diabetes, and die within a few days of birth (15).
ATP-sensitive K+ channels (KATP channels) couple glucose metabolism to cellular electrical activity and therefore play a critical role in excitation-secretion coupling in the pancreatic -cell. Glucose metabolism raises the cytosolic [ATP]-to-[ADP] ratio, which causes closure of the KATP channel and depolarization of the -cell membrane. Membrane depolarization, in turn, leads to opening of voltage-dependent Ca2+ channels and a rise in intracellular [Ca2+], which triggers insulin vesicle exocytosis (16) (Fig. 1). Glucose metabolism has additional downstream actions, including generation of GTP, which may activate "KATP-independent" pathways of insulin secretion. Naively, both electrical triggering of secretion through KATP channels and nonelectrical effects of glucose metabolism through ATP generation could be affected by glucokinase deficiency. The relative importance of each component is not clear, and it might be presumed that the consequences of failure to generate ATP should be much more devastating than simply blocking electrical signaling.
It has previously been shown that glucose fails to close KATP channels in nGKeC/eC -cells (17). Sulfonylureas act by closing the KATP channel independently of glucose metabolism, resulting in membrane depolarization and consequent stimulation of insulin secretion (18). Consistent with the idea that the primary downstream consequence of nGK deficiency is simply failure to close KATP channels, the sulfonylurea tolbutamide was effective at both inhibiting KATP channels and stimulating insulin secretion in nGKeC/eC -cells (17). These results imply that the defect in insulin secretion in glucokinase deficiency results essentially from the defective regulation of KATP channels, rather than from nonelectrical consequences of altered metabolism. Further indirect support for this hypothesis comes from: 1) the demonstration that mice with ATP-insensitive -cell KATP channels also die from neonatal diabetes (19) and 2) the recent dramatic demonstration that gain-of-function KATP mutations also cause permanent neonatal diabetes in humans (20eC24). A critical prediction is that the consequences of nGK deficiency should be abrogated in animals that lack KATP channels. Mice lacking Kir6.2-dependent KATP channel activity in all tissues have been generated by disruption of the pore-forming Kir6.2 subunit (25). KATP knockout mice (Kir6.2eC/eC) show a complex phenotype with transient hyperinsulinemia and hypoglycemia as neonates, which rapidly progress to hypoinsulinemia but euglycemia as adults (25eC28). By crossing nGK+/eC mice with Kir6.2eC/eC (KATP channel knockout) mice, we can test the critical prediction and separate electrical and metabolic consequences of nGK deletion. The results provide a striking demonstration of the importance of the electrical signal in transducing glucose metabolism to insulin secretion.
RESEARCH DESIGN AND METHODS
Previously, a global neuroendocrine knockout of GK (nGK) was achieved by removal of upstream promoter elements (15). This was expected to result in abolition of GK expression in -cells and also in gut and brain (15). Knockout of the Kir6.2 gene was achieved by targeted introduction of the neomycin-resistant gene into the coding region of Kir6.2 (25). Heterozygous nGK+/eC mice on a C57/BL6J background (gift from Dr. Yasuo Terauchi) (15) were bred to Kir6.2eC/eC on an outbred background (gift from Dr. Susumo Seino) (25) to generate double-heterozygous F1 mice. F1 mice were backcrossed to each other to generate F2 mice of expected genotype. nGKeC/eCKir6.2+/+ and nGKeC/eCKir6.2+/eC were not identified in the litters, probably because of perinatal lethality. For all experiments described, we backcrossed F2 heterozygous nGK+/eC mice (either on Kir6.2+/+ or Kir6.2eC/eC backgrounds) to the same genotypes, thus generating F3 litters of three genotypes (nGK+/+, nGK+/eC, and nGKeC/eC on Kir6.2+/+ or Kir6.2eC/eC backgrounds). Mice were typed for nGK and Kir6.2 genes as previously described by Terauchi et al. (15) and Miki et al. (25).
Previously, Kir6.2[AAA] mice expressing a dominant-negative Kir6.2 mutation (132GFG134AAA) mutation under control of the rat insulin 1 promoter were generated (29). For experiments shown in Fig. 7, these Kir6.2[AAA] mice (on C57BL6J background) were bred to nGK+/eC mice, also on C57BL6J background, to generate the relevant crosses.
To control for effects of genetic background, comparisons between genotypes were made with littermates in every experiment, and n values of at least three are reported in each paired genotype. Thus, it is reasonable to conclude that any effects of nonmutated chromosomes will be randomized within all genotypes.
Isolation of pancreatic islets and -cells.
Experiments using animals were performed in accordance with the relevant laws and institutional guidelines and were approved by the Washington University animal studies committee. Anesthetized mice were killed by cervical dislocation. Pancreata were removed and injected with Hank’s balanced salt solution containing type XI collagenase (0.5 mg/ml), pH 7.4. (Sigma, St. Louis, MO). Pancreata were digested for 7 min at 37°C then hand-shaken and washed three times in cold Hank’s solution (29). Islets were isolated by hand under a dissecting microscope and pooled. Islets were maintained overnight in CMRL-1640 (5.6 mmol/l glucose) supplemented with FCS (10%), penicillin (100 units/ml), and streptomycin (100 mg/ml).
Blood glucose and insulin levels.
Blood glucose was assayed using a glucose dehydrogenaseeCbased enzymatic assay and quantitated using a Glucometer Elite XL meter (Bayer, Elkhart, IN). Blood insulin levels were assayed on 15 e蘬 of mouse serum using an enzyme-linked immunosorbent assay kit with a rat insulin standard according to manufacturer’s procedure (Crystal Chem, Downers Grove, IL). Intraperitoneal glucose tolerance tests were performed on 5-week-old mice after a 16-h fast. Animals were injected intraperitoneally with glucose (1 g/kg). Blood was isolated from the tail vein at the times indicated and assayed for glucose as described above. Intraperitoneal insulin tolerance tests were performed on 4-month-old mice after a 6-h fast. Animals were injected intraperitoneally with insulin (0.5 unit/kg). Blood was isolated from the tail vein at the times indicated and assayed for glucose as described above.
Insulin release experiments.
After overnight incubation, pancreatic islets (10 per well in 12-well plates) were preincubated in glucose-free Dulbecco’s modified Eagle medium (DMEM) supplemented with 3 mmol/l of glucose for 2 h. The release assay was initiated by supplementing the DMEM glucose-free media with D+-glucose (1, 7, 16.7, or 23 mmol/l) as indicated. For amino acid stimulation of insulin release, Krebs-Ringer buffer supplemented with D-glucose and glutamine (10 mmol/l) was used in place of DMEM. Islets were incubated for 60 min at 37°C, and medium was removed and assayed for insulin content using a rat insulin radioimmunoassay (Linco, St. Charles, MO). Each experiment was repeated in triplicate.
Statistics.
Data are presented as means ± SE. Differences among the four genotypes were tested using ANOVA and post hoc Duncan’s test. A difference was defined as significant when P < 0.05. Nonsignificant differences are not indicated.
RESULTS
Neonatal survival of nGK knockout mice on the Kir6.2 knockout background.
Of 57 live births from cross-breeding of heterozygous nGK+/eC mice on the Kir6.2 wild-type background, only 2 mice were positively genotyped as nGKeC/eC (Fig. 2A). Both were found dead within the 1st week (genotyped postmortem) after birth (Fig. 2B). Conversely, on the Kir6.2 knockout background, nGKeC/eC pups were born at the expected ratio (1:2:1 for nGK+/+Kir6.2eC/eC, nGK+/eCKir6.2eC/eC, and nGKeC/eCKir6.2eC/eC, respectively) (Fig. 2A). These double-knockout mice survived much longer than nGKeC/eC on the Kir6.2 wild-type background, with a median survival time of 10 days, and 30% surviving past weaning (21 days) (Fig. 2B). As is shown in Fig. 2C, double-knockout nGKeC/eCKir6.2eC/eC mice were significantly smaller than both nGK+/eCKir6.2eC/eC and nGK+/+Kir6.2eC/eC mice at 4 weeks of age. By 5 weeks of age, nGKeC/eCKir6.2eC/eC mice weighed 50% (4.78 ± 0.17g) that of nGK+/+Kir6.2eC/eC (8.34 ± 0.24g) or nGK+/eCKir6.2eC/eC (9.02 ± 0.17g.) littermates. Nevertheless, it is striking that these double-knockout mice, which completely lack -cell glucokinase activity, can survive beyond weaning.
On this Kir6.2eC/eC background, fasting blood glucose was only slightly higher in nGKeC/eC than in nGK+/+ or nGK+/eC mice (Fig. 3A). However, fed glucose (Fig. 3B) was dramatically higher in nGKeC/eC mice, suggesting that these mice either still do not secrete enough insulin to lower blood glucose values or that they develop insulin resistance (see below).
nGK+/eC animals are diabetic on the Kir6.2+/+ background but not on the Kir6.2eC/eC background.
Heterozygous nGK+/eC mice were born at close to expected ratios on both the Kir6.2+/+ and Kir6.2eC/eC backgrounds (Fig. 2A) and developed apparently normally. As previously reported, on the wild-type Kir6.2+/+ background, heterozygous nGK+/eC mice show nonprogressive mild diabetes, as in human MODY-2. At birth, the blood glucose levels of nGK+/+ and nGK+/eC mice were similar (3.5 ± 0.4 and 3.9 ± 0.2 mmol/l, respectively), but by 12 weeks, nGK+/eCKir6.2+/+ mice showed a twofold increase in both fasting and fed blood glucose compared with nGK+/+Kir6.2+/+ mice (Fig. 3A and B).
By contrast, on the Kir6.2 knockout background, overt diabetes, as evidenced by elevated fasting glucose levels, is ameliorated in nGK+/eCKir6.2eC/eC mice, although fed glucose levels are elevated (Fig. 3A and B). Plasma insulin, measured under fed conditions, was slightly elevated in nGK+/eC mice versus nGK+/+ on both Kir6.2+/+ and Kir6.2eC/eC backgrounds (Fig. 3C).
Enhancement in glucose tolerance of heterozygous nGK-deficient mice in the absence of KATP channel activity.
To characterize physiological responses to glucose load, surviving mice underwent intraperitoneal glucose tolerance testing at 5 weeks of age. As previously reported, blood glucose before and after glucose load was significantly higher in nGK+/eC mice on the Kir6.2+/+ background, and Kir6.2eC/eC mice showed a very mild impairment in glucose tolerance with respect to Kir6.2+/+ mice (Fig. 4). Importantly, on the Kir6.2eC/eC background, there was dramatically improved glucose tolerance of nGK+/eC mice (Fig. 4), to the extent that they were not significantly different from nGK+/+ mice on the same background.
The glucose-lowering effect of intraperitoneal insulin injection (0.5 unit/kg) was assessed using insulin tolerance tests on 6-h fasted mice. nGK+/+Kir6.2eC/eC mice were slightly more sensitive to insulin than nGK+/+Kir6.2+/+ mice (Fig. 5), similar to a previous report (25). Insulin sensitivity was significantly enhanced in nGK+/eC mice on the Kir6.2+/+ background, whereas nGK+/eCKir6.2eC/eC mice showed intermediate sensitivity (Fig. 5). These results exclude enhanced insulin sensitivity as a possible mechanism for the improved glucose tolerance of nGK+/eC mice in the absence of KATP, and they point to enhanced insulin secretion as the more likely mechanism (see below).
Absence of KATP channel activity may restore insulin secretion from nGKeC/eC pancreatic islets.
To gain further insight into the cellular basis for differential phenotypes of double-knockout animals, islets were isolated from each of the different genotypes and phenotypically examined. Morphologically, islet appearance and size were not different between the various genotypes (for example, islet diameter was 175.5 ± 8.1 e蘭 for nGK+/eCKir6.2+/+ and 183.3 ± 7.3 e蘭 for nGK+/eCKir6.2eC/eC, n = 35 islets). Glucose-stimulated insulin secretion (GSIS) was assessed in isolated islets from all five available genotypes. As shown previously, the response of nGK+/eC islets was mildly reduced compared with nGK+/+, on the Kir6.2+/+ background (15). On the Kir6.2eC/eC background, GSIS was greatly reduced in the presence or absence of nGK (Fig. 6A), and there was no enhancement of release from nGK+/eC or nGKeC/eC, relative to the release from the same nGK genotypes on the Kir6.2+/+ background. These data thus reiterate the perplexing result that Kir6.2eC/eC (25) and SUR1eC/eC (26) mice are mildly glucose tolerant, yet have only minimal GSIS. Moreover, these results do not help to explain the enhanced glucose tolerance and survival of nGK-deficient mice on this Kir6.2eC/eC background. Recent studies have begun to shed light on the potential mechanisms in SUR1eC/eC mice (30eC32). In particular, glutamine has been shown to preferentially stimulate KATP-independent secretion from SUR1eC/eC islets (31). This effect has not been examined in Kir6.2eC/eC islets, but if the phenotypes of SUR1eC/eC and Kir6.2eC/eC indeed both result from loss of KATP, we would expect the same KATP-independent stimulation of Kir6.2eC/eC islets. As shown in Fig. 6B, glutamine markedly stimulates secretion from both nGK+/+ and nGK+/eC islets on the Kir6.2eC/eC background relative to the wild-type background. Given likely physiological levels of glutamine, this may well cause enhanced insulin secretion in vivo and explain the improved glucose tolerance of nGK+/eC on the Kir6.2eC/eC background (31) (see below).
-CelleCspecific reduction of KATP channel activity enhances glucose tolerance of nGK+/eC mice.
Although the above data are consistent with a specific loss of KATP channels in -cells being responsible for abrogation of the nGK-deficiency phenotype, it is a caveat that the Kir6.2 knockout is global. We previously generated -celleCspecific dominant-negative Kir6.2[AAA] mice using an insulin promoter to drive transgene expression (29). Kir6.2[AAA] mice, which lack KATP in 70% of -cells, hypersecrete insulin and have slightly enhanced glucose tolerance (29). In the current study, we bred these mice with nGK+/eCKir6.2+/+ mice to generate double-heterozygous (nGK+/eCKir6.2[AAA]) mice (see RESEARCH DESIGN AND METHODS). As shown in Fig. 7, these mice have a marked improvement in glucose tolerance compared to nGK+/eC alone. Although the improvement is not as dramatic as in the mice completely lacking KATP (Fig. 4), this result is consistent with -cell KATP channels indeed being the major players in rescuing the nGK-deficiency phenotype.
DISCUSSION
KATP-dependent versus KATP-independent pathways of insulin secretion: relative role in glucokinase-deficient diabetes.
Heterozygous loss-of-function mutations in the glucokinase (GK) gene are the most common cause of MODY-2, a relatively mild form of type 2 diabetes. Conversely, homozygous or compound heterozygous GK deficiency mutations underlie permanent neonatal diabetes, a more severe perinatal diabetes (13). Mimicking human permanent neonatal diabetes, homozygous neuroendocrine GK knockout mice die shortly after birth of severe diabetes (15,33). Consistent with a MODY-2 phenotype, mice lacking one allele of neuroendocrine GK exhibit mild hyperglycemia with normal basal glucose turnover rates, but they have impaired glucose tolerance and insulin secretion during hyperglycemia (2,15).
Glucokinase deficiency could affect insulin secretion by altering electrical and/or nonelectrical downstream consequences of glucose metabolism (Fig. 1). To date, however, the relative importance of electrical (KATP-dependent) versus nonelectrical (KATP-independent) consequences of neuroendocrine glucokinase deficiency remains unclear. KATP channels are under tight metabolic control, and any perturbation of metabolism that leads to decreased generation of ATP is expected to have pronounced inhibitory effects on excitation-secretion coupling because of enhanced KATP activity. Studies of the electrical activity of nGK-deficient mice indeed support the idea that reduced glycolytic metabolism specifically causes failure of KATP channels to close in response to glucose, contributing to the observed reduction in insulin secretion (17). Consistent with this conclusion, it was reported that oral sulfonylurea treatment could suppress perinatal death in nGKeC/eC mice (15), presumably by restoring insulin release. Glucokinase is critical for liver regulation of glucose production (34). This could contribute to the whole animal phenotype in complex ways, and for this reason, we examined a tissue-specific model in which the liver-specific isoform of glucokinase is expressed normally, and only the neuroendocrine-specific isoform is deleted (15). We specifically examined the consequences of nGK deficiency on the wild-type and KATP-null backgrounds to elucidate the relative importance of the two signaling pathways. Perinatal lethality of nGKeC/eC was absolute on the Kir6.2+/+ background; only 2 pups were detected of an expected 16 pups, from heterozygous nGK+/eCKir6.2+/+ backcrosses (Fig. 2), and both died within 5 days. In contrast, double-knockout mice (nGKeC/eCKir6.2eC/eC) were born at the expected ratio. They exhibited low body weight, severe diabetes, and died prematurely, but perinatal lethality is clearly abrogated (Fig. 2A and B). Amelioration of the nGKeC/eC phenotype on the Kir6.2eC/eC background can be explained by an increased, though still insufficient, level of circulating insulin. This striking demonstration of the nullifying effect of removal of the KATP "brake" in vivo is further supported by the results with heterozygous nGK+/eC mice. On the Kir6.2+/+ background, they have markedly elevated fed and fasting glucose levels, and they are markedly glucose intolerant. By contrast, on the Kir6.2eC/eC background, nGK+/eC mice have near-normal fasting glucose levels and similar glucose tolerance to nGK+/+Kir6.2eC/eC animals. The abolition of KATP significantly ameliorates the "MODY-2" phenotype of the nGK+/eC mice.
Complex phenotypes of Kir6.2-deficient x nGK-deficient mice.
The initial prediction that, by removing the electrical defect, the diabetic consequences of nGK deficiency should be significantly avoided on the Kir6.2eC/eC background was thus supported. However, the mechanistic consequences of the genetic alterations are not absolutely clear. The naive expectation of mice lacking KATP channels was a persistent hyperinsulinemic phenotype because of unregulated secretion, but several studies have now shown that this expectation is not met (25eC27). For unexplained reasons (but see below), Kir6.2eC/eC mice are normoglycemic, with slightly impaired glucose tolerance and reduced GSIS. Similarly, although glucose tolerance was greatly improved in nGK+/eC mice on the Kir6.2eC/eC background, GSIS in isolated islets was still notably reduced (Fig. 6A). However, as has recently been shown, the sensitizing effect of amino acids, in particular glutamine (30,31), is much greater in islets that lack KATP (SUR1eC/eC). Similarly, we find that Kir6.2eC/eC islets can show enhanced insulin secretion at physiological glucose concentration (compared with Kir6.2+/+ mice) in the presence of stimulatory glutamine (Fig. 6B). This enhancement is seen in both nGK+/+ and nGK+/eC islets on this background. We suggest that, in vivo, ambient glutamine levels may lead to such an enhancement of insulin secretion and that, in part, this underlies the abrogating effect of KATP deficiency on nGK-deficiency (31).
Because we are examining a global neuroendocrine glucokinase deficiency, it is conceivable that the effects of Kir6.2 knockout are mediated through extra-pancreatic consequences, although insulin tolerance tests (Fig. 5) show that nGK+/eCKir6.2eC/eC mice are no more insulin sensitive than nGK+/eCKir6.2+/+ mice, excluding enhanced insulin sensitivity as an explanation for the rescued phenotype. Further evidence for a pancreatic basis for the Kir6.2-deficient rescue of nGK deficiency would be a -celleCspecific KATP knockout. A tissue-targeted knockout has not been generated, although we have generated an insulin promotereCdriven dominant-negative Kir6.2[AAA] mouse (29) that lacks KATP in 70% of its -cells (29). The demonstration of marked improvement in glucose tolerance in double-heterozygous (nGK+/eCKir6.2[AAA]) mice, compared with nGK+/eC alone (Fig. 7), provides further support for -celleCspecific KATP channels indeed being the major players in rescuing the nGK-deficiency phenotype.
Relevance to glucokinase-deficiency diabetes in humans.
The significant reduction in fasting glucose, slight yet significant decrease in fed glucose concentration, and marked enhancement in glucose tolerance in nGK+/eCKir6.2eC/eC mice with respect to nGK+/eCKir6.2+/+ mice suggests that, at least in part, the MODY-2 phenotype is abrogated on the KATP knockout background, highlighting the importance of electrical signaling in the regulation of insulin secretion. Decreased insulin secretion in response to glucose from nGKeC/eCKir6.2eC/eC or nGK+/eCKir6.2eC/eC islets recapitulates the response observed in nGK+/+Kir6.2eC/eC islets, thus indicating that the impaired GSIS is due to the Kir6.2 knockout phenotype and not due to glucokinase deficiency. Most patients with heterozygous GK mutations do not require pharmacological treatment, and the majority of cases are managed with diet alone. However, during pregnancy, women with GK mutations are often treated with insulin to maintain normal blood sugar levels, resulting in babies that are large for gestational age because of the effect of insulin on fetal growth (35). In these cases, sulfonylurea treatment could presumably be a valid alternative treatment, given the normalizing effect of KATP deficiency that we now demonstrate, because nGK+/eCKir6.2+/+ transgenic mice do respond to glibenclamide treatment in vitro (15).
Taken together, the results from nGK-deficient and KATP-deficient crosses show a marked improvement in survivability and glucose tolerance of the former on the KATP null background. Neonatal lethality of nGKeC/eCKir6.2+/+ mice is avoided on the Kir6.2eC/eC background, demonstrating that this consequence is attributable to loss of metabolic coupling between glucose and insulin secretion, probably through electrical signaling. As with the now dramatically improved treatment options for KATP-induced permanent neonatal diabetes (21,24), we suggest that there may be a role for sulfonylurea therapy in permanent neonatal diabetes resulting from glucokinase deficiency (13). A significant number of nGKeC/eCKir6.2eC/eC mice survived beyond weaning. This should also allow more extensive studies of the nonelectrical consequences (which are still ultimately lethal) of total glucokinase deficiency. Such studies will have the potential to inform and further improve treatment options for diabetes resulting from glucokinase deficiency.
ACKNOWLEDGMENTS
This work was supported by a mentor-based minority postdoctoral fellowship (to M.S.R.) from the American Diabetes Association and Takeda Pharmaceuticals, by the National Institutes of Health Diabetes Research and Training Center (DK20579) at Washington University, and by National Institutes of Health grant DK69445 (to C.G.N.).
Kir6.2-deficient mice (25) and nGK-deficient mice (15) were kindly provided by Dr. Susumo Seino and Dr. Yasuo Terauchi, respectively.
DMEM, Dulbecco’s modified Eagle medium; GSIS, glucose-stimulated insulin secretion; KATP channel, ATP-sensitive K+ channel; MODY-2, maturity-onset diabetes of the young type 2
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ABSTRACT
As the rate-limiting controller of glucose metabolism, glucokinase represents the primary -cell "glucose sensor." Inactivation of both glucokinase (GK) alleles results in permanent neonatal diabetes; inactivation of a single allele causes maturity-onset diabetes of the young type 2 (MODY-2). Similarly, mice lacking both alleles (GKeC/eC) exhibit severe neonatal diabetes and die within a week, whereas heterozygous GK+/eC mice exhibit markedly impaired glucose tolerance and diabetes, resembling MODY-2. Glucose metabolism increases the cytosolic [ATP]-to-[ADP] ratio, which closes ATP-sensitive K+ channels (KATP channels), leading to membrane depolarization, Ca2+ entry, and insulin exocytosis. Glucokinase insufficiency causes defective KATP channel regulation, which may underlie the impaired secretion. To test this prediction, we crossed mice lacking neuroendocrine glucokinase (nGK+/eC) with mice lacking KATP channels (Kir6.2eC/eC). Kir6.2 knockout rescues perinatal lethality of nGKeC/eC, although nGKeC/eCKir6.2eC/eC animals are postnatally diabetic and still die prematurely. nGK+/eC animals are diabetic on the Kir6.2+/+ background but only mildly glucose intolerant on the Kir6.2eC/eC background. In the presence of glutamine, isolated nGK+/eCKir6.2eC/eC islets show improved insulin secretion compared with nGK+/eCKir6.2+/+. The significant abrogation of nGKeC/eC and nGK+/eC phenotypes in the absence of KATP demonstrate that a major factor in glucokinase deficiency is indeed altered KATP signaling. The results have implications for understanding and therapy of glucokinase-related diabetes.
Glucose metabolism in pancreatic -cells is necessary to stimulate insulin secretion (1). Glucokinase, which phosphorylates glucose in the first, rate-limiting reaction, is a key component in the secretory pathway (2,3), and mutations of the GK gene are diabetogenic in humans (4). Heterozygous inactivating mutations cause maturity-onset diabetes of the young type 2 (MODY-2), an autosomal, dominantly inherited form of diabetes characterized by an early age of onset and pancreatic -cell dysfunction (5eC11). Homozygous inactivating GK mutations cause permanent neonatal diabetes in humans (12,13), which is characterized by perinatal hyperglycemia and low birth weight (14). The disease usually requires insulin treatment through the patient’s lifetime, starting within the first month of life. Targeted disruption of the neuroendocrine knockout of GK (nGK) gene in mice also causes diabetes (15). These mice lack glucokinase in pancreatic -cells and neurons but maintain normal liver glucokinase activity. Heterozygous null mice show early-onset mild diabetes caused by an impaired insulin-secretory response to glucose, resembling MODY-2. Importantly, homozygous nGK-deficient mice show severe perinatal diabetes, as in permanent neonatal diabetes, and die within a few days of birth (15).
ATP-sensitive K+ channels (KATP channels) couple glucose metabolism to cellular electrical activity and therefore play a critical role in excitation-secretion coupling in the pancreatic -cell. Glucose metabolism raises the cytosolic [ATP]-to-[ADP] ratio, which causes closure of the KATP channel and depolarization of the -cell membrane. Membrane depolarization, in turn, leads to opening of voltage-dependent Ca2+ channels and a rise in intracellular [Ca2+], which triggers insulin vesicle exocytosis (16) (Fig. 1). Glucose metabolism has additional downstream actions, including generation of GTP, which may activate "KATP-independent" pathways of insulin secretion. Naively, both electrical triggering of secretion through KATP channels and nonelectrical effects of glucose metabolism through ATP generation could be affected by glucokinase deficiency. The relative importance of each component is not clear, and it might be presumed that the consequences of failure to generate ATP should be much more devastating than simply blocking electrical signaling.
It has previously been shown that glucose fails to close KATP channels in nGKeC/eC -cells (17). Sulfonylureas act by closing the KATP channel independently of glucose metabolism, resulting in membrane depolarization and consequent stimulation of insulin secretion (18). Consistent with the idea that the primary downstream consequence of nGK deficiency is simply failure to close KATP channels, the sulfonylurea tolbutamide was effective at both inhibiting KATP channels and stimulating insulin secretion in nGKeC/eC -cells (17). These results imply that the defect in insulin secretion in glucokinase deficiency results essentially from the defective regulation of KATP channels, rather than from nonelectrical consequences of altered metabolism. Further indirect support for this hypothesis comes from: 1) the demonstration that mice with ATP-insensitive -cell KATP channels also die from neonatal diabetes (19) and 2) the recent dramatic demonstration that gain-of-function KATP mutations also cause permanent neonatal diabetes in humans (20eC24). A critical prediction is that the consequences of nGK deficiency should be abrogated in animals that lack KATP channels. Mice lacking Kir6.2-dependent KATP channel activity in all tissues have been generated by disruption of the pore-forming Kir6.2 subunit (25). KATP knockout mice (Kir6.2eC/eC) show a complex phenotype with transient hyperinsulinemia and hypoglycemia as neonates, which rapidly progress to hypoinsulinemia but euglycemia as adults (25eC28). By crossing nGK+/eC mice with Kir6.2eC/eC (KATP channel knockout) mice, we can test the critical prediction and separate electrical and metabolic consequences of nGK deletion. The results provide a striking demonstration of the importance of the electrical signal in transducing glucose metabolism to insulin secretion.
RESEARCH DESIGN AND METHODS
Previously, a global neuroendocrine knockout of GK (nGK) was achieved by removal of upstream promoter elements (15). This was expected to result in abolition of GK expression in -cells and also in gut and brain (15). Knockout of the Kir6.2 gene was achieved by targeted introduction of the neomycin-resistant gene into the coding region of Kir6.2 (25). Heterozygous nGK+/eC mice on a C57/BL6J background (gift from Dr. Yasuo Terauchi) (15) were bred to Kir6.2eC/eC on an outbred background (gift from Dr. Susumo Seino) (25) to generate double-heterozygous F1 mice. F1 mice were backcrossed to each other to generate F2 mice of expected genotype. nGKeC/eCKir6.2+/+ and nGKeC/eCKir6.2+/eC were not identified in the litters, probably because of perinatal lethality. For all experiments described, we backcrossed F2 heterozygous nGK+/eC mice (either on Kir6.2+/+ or Kir6.2eC/eC backgrounds) to the same genotypes, thus generating F3 litters of three genotypes (nGK+/+, nGK+/eC, and nGKeC/eC on Kir6.2+/+ or Kir6.2eC/eC backgrounds). Mice were typed for nGK and Kir6.2 genes as previously described by Terauchi et al. (15) and Miki et al. (25).
Previously, Kir6.2[AAA] mice expressing a dominant-negative Kir6.2 mutation (132GFG134AAA) mutation under control of the rat insulin 1 promoter were generated (29). For experiments shown in Fig. 7, these Kir6.2[AAA] mice (on C57BL6J background) were bred to nGK+/eC mice, also on C57BL6J background, to generate the relevant crosses.
To control for effects of genetic background, comparisons between genotypes were made with littermates in every experiment, and n values of at least three are reported in each paired genotype. Thus, it is reasonable to conclude that any effects of nonmutated chromosomes will be randomized within all genotypes.
Isolation of pancreatic islets and -cells.
Experiments using animals were performed in accordance with the relevant laws and institutional guidelines and were approved by the Washington University animal studies committee. Anesthetized mice were killed by cervical dislocation. Pancreata were removed and injected with Hank’s balanced salt solution containing type XI collagenase (0.5 mg/ml), pH 7.4. (Sigma, St. Louis, MO). Pancreata were digested for 7 min at 37°C then hand-shaken and washed three times in cold Hank’s solution (29). Islets were isolated by hand under a dissecting microscope and pooled. Islets were maintained overnight in CMRL-1640 (5.6 mmol/l glucose) supplemented with FCS (10%), penicillin (100 units/ml), and streptomycin (100 mg/ml).
Blood glucose and insulin levels.
Blood glucose was assayed using a glucose dehydrogenaseeCbased enzymatic assay and quantitated using a Glucometer Elite XL meter (Bayer, Elkhart, IN). Blood insulin levels were assayed on 15 e蘬 of mouse serum using an enzyme-linked immunosorbent assay kit with a rat insulin standard according to manufacturer’s procedure (Crystal Chem, Downers Grove, IL). Intraperitoneal glucose tolerance tests were performed on 5-week-old mice after a 16-h fast. Animals were injected intraperitoneally with glucose (1 g/kg). Blood was isolated from the tail vein at the times indicated and assayed for glucose as described above. Intraperitoneal insulin tolerance tests were performed on 4-month-old mice after a 6-h fast. Animals were injected intraperitoneally with insulin (0.5 unit/kg). Blood was isolated from the tail vein at the times indicated and assayed for glucose as described above.
Insulin release experiments.
After overnight incubation, pancreatic islets (10 per well in 12-well plates) were preincubated in glucose-free Dulbecco’s modified Eagle medium (DMEM) supplemented with 3 mmol/l of glucose for 2 h. The release assay was initiated by supplementing the DMEM glucose-free media with D+-glucose (1, 7, 16.7, or 23 mmol/l) as indicated. For amino acid stimulation of insulin release, Krebs-Ringer buffer supplemented with D-glucose and glutamine (10 mmol/l) was used in place of DMEM. Islets were incubated for 60 min at 37°C, and medium was removed and assayed for insulin content using a rat insulin radioimmunoassay (Linco, St. Charles, MO). Each experiment was repeated in triplicate.
Statistics.
Data are presented as means ± SE. Differences among the four genotypes were tested using ANOVA and post hoc Duncan’s test. A difference was defined as significant when P < 0.05. Nonsignificant differences are not indicated.
RESULTS
Neonatal survival of nGK knockout mice on the Kir6.2 knockout background.
Of 57 live births from cross-breeding of heterozygous nGK+/eC mice on the Kir6.2 wild-type background, only 2 mice were positively genotyped as nGKeC/eC (Fig. 2A). Both were found dead within the 1st week (genotyped postmortem) after birth (Fig. 2B). Conversely, on the Kir6.2 knockout background, nGKeC/eC pups were born at the expected ratio (1:2:1 for nGK+/+Kir6.2eC/eC, nGK+/eCKir6.2eC/eC, and nGKeC/eCKir6.2eC/eC, respectively) (Fig. 2A). These double-knockout mice survived much longer than nGKeC/eC on the Kir6.2 wild-type background, with a median survival time of 10 days, and 30% surviving past weaning (21 days) (Fig. 2B). As is shown in Fig. 2C, double-knockout nGKeC/eCKir6.2eC/eC mice were significantly smaller than both nGK+/eCKir6.2eC/eC and nGK+/+Kir6.2eC/eC mice at 4 weeks of age. By 5 weeks of age, nGKeC/eCKir6.2eC/eC mice weighed 50% (4.78 ± 0.17g) that of nGK+/+Kir6.2eC/eC (8.34 ± 0.24g) or nGK+/eCKir6.2eC/eC (9.02 ± 0.17g.) littermates. Nevertheless, it is striking that these double-knockout mice, which completely lack -cell glucokinase activity, can survive beyond weaning.
On this Kir6.2eC/eC background, fasting blood glucose was only slightly higher in nGKeC/eC than in nGK+/+ or nGK+/eC mice (Fig. 3A). However, fed glucose (Fig. 3B) was dramatically higher in nGKeC/eC mice, suggesting that these mice either still do not secrete enough insulin to lower blood glucose values or that they develop insulin resistance (see below).
nGK+/eC animals are diabetic on the Kir6.2+/+ background but not on the Kir6.2eC/eC background.
Heterozygous nGK+/eC mice were born at close to expected ratios on both the Kir6.2+/+ and Kir6.2eC/eC backgrounds (Fig. 2A) and developed apparently normally. As previously reported, on the wild-type Kir6.2+/+ background, heterozygous nGK+/eC mice show nonprogressive mild diabetes, as in human MODY-2. At birth, the blood glucose levels of nGK+/+ and nGK+/eC mice were similar (3.5 ± 0.4 and 3.9 ± 0.2 mmol/l, respectively), but by 12 weeks, nGK+/eCKir6.2+/+ mice showed a twofold increase in both fasting and fed blood glucose compared with nGK+/+Kir6.2+/+ mice (Fig. 3A and B).
By contrast, on the Kir6.2 knockout background, overt diabetes, as evidenced by elevated fasting glucose levels, is ameliorated in nGK+/eCKir6.2eC/eC mice, although fed glucose levels are elevated (Fig. 3A and B). Plasma insulin, measured under fed conditions, was slightly elevated in nGK+/eC mice versus nGK+/+ on both Kir6.2+/+ and Kir6.2eC/eC backgrounds (Fig. 3C).
Enhancement in glucose tolerance of heterozygous nGK-deficient mice in the absence of KATP channel activity.
To characterize physiological responses to glucose load, surviving mice underwent intraperitoneal glucose tolerance testing at 5 weeks of age. As previously reported, blood glucose before and after glucose load was significantly higher in nGK+/eC mice on the Kir6.2+/+ background, and Kir6.2eC/eC mice showed a very mild impairment in glucose tolerance with respect to Kir6.2+/+ mice (Fig. 4). Importantly, on the Kir6.2eC/eC background, there was dramatically improved glucose tolerance of nGK+/eC mice (Fig. 4), to the extent that they were not significantly different from nGK+/+ mice on the same background.
The glucose-lowering effect of intraperitoneal insulin injection (0.5 unit/kg) was assessed using insulin tolerance tests on 6-h fasted mice. nGK+/+Kir6.2eC/eC mice were slightly more sensitive to insulin than nGK+/+Kir6.2+/+ mice (Fig. 5), similar to a previous report (25). Insulin sensitivity was significantly enhanced in nGK+/eC mice on the Kir6.2+/+ background, whereas nGK+/eCKir6.2eC/eC mice showed intermediate sensitivity (Fig. 5). These results exclude enhanced insulin sensitivity as a possible mechanism for the improved glucose tolerance of nGK+/eC mice in the absence of KATP, and they point to enhanced insulin secretion as the more likely mechanism (see below).
Absence of KATP channel activity may restore insulin secretion from nGKeC/eC pancreatic islets.
To gain further insight into the cellular basis for differential phenotypes of double-knockout animals, islets were isolated from each of the different genotypes and phenotypically examined. Morphologically, islet appearance and size were not different between the various genotypes (for example, islet diameter was 175.5 ± 8.1 e蘭 for nGK+/eCKir6.2+/+ and 183.3 ± 7.3 e蘭 for nGK+/eCKir6.2eC/eC, n = 35 islets). Glucose-stimulated insulin secretion (GSIS) was assessed in isolated islets from all five available genotypes. As shown previously, the response of nGK+/eC islets was mildly reduced compared with nGK+/+, on the Kir6.2+/+ background (15). On the Kir6.2eC/eC background, GSIS was greatly reduced in the presence or absence of nGK (Fig. 6A), and there was no enhancement of release from nGK+/eC or nGKeC/eC, relative to the release from the same nGK genotypes on the Kir6.2+/+ background. These data thus reiterate the perplexing result that Kir6.2eC/eC (25) and SUR1eC/eC (26) mice are mildly glucose tolerant, yet have only minimal GSIS. Moreover, these results do not help to explain the enhanced glucose tolerance and survival of nGK-deficient mice on this Kir6.2eC/eC background. Recent studies have begun to shed light on the potential mechanisms in SUR1eC/eC mice (30eC32). In particular, glutamine has been shown to preferentially stimulate KATP-independent secretion from SUR1eC/eC islets (31). This effect has not been examined in Kir6.2eC/eC islets, but if the phenotypes of SUR1eC/eC and Kir6.2eC/eC indeed both result from loss of KATP, we would expect the same KATP-independent stimulation of Kir6.2eC/eC islets. As shown in Fig. 6B, glutamine markedly stimulates secretion from both nGK+/+ and nGK+/eC islets on the Kir6.2eC/eC background relative to the wild-type background. Given likely physiological levels of glutamine, this may well cause enhanced insulin secretion in vivo and explain the improved glucose tolerance of nGK+/eC on the Kir6.2eC/eC background (31) (see below).
-CelleCspecific reduction of KATP channel activity enhances glucose tolerance of nGK+/eC mice.
Although the above data are consistent with a specific loss of KATP channels in -cells being responsible for abrogation of the nGK-deficiency phenotype, it is a caveat that the Kir6.2 knockout is global. We previously generated -celleCspecific dominant-negative Kir6.2[AAA] mice using an insulin promoter to drive transgene expression (29). Kir6.2[AAA] mice, which lack KATP in 70% of -cells, hypersecrete insulin and have slightly enhanced glucose tolerance (29). In the current study, we bred these mice with nGK+/eCKir6.2+/+ mice to generate double-heterozygous (nGK+/eCKir6.2[AAA]) mice (see RESEARCH DESIGN AND METHODS). As shown in Fig. 7, these mice have a marked improvement in glucose tolerance compared to nGK+/eC alone. Although the improvement is not as dramatic as in the mice completely lacking KATP (Fig. 4), this result is consistent with -cell KATP channels indeed being the major players in rescuing the nGK-deficiency phenotype.
DISCUSSION
KATP-dependent versus KATP-independent pathways of insulin secretion: relative role in glucokinase-deficient diabetes.
Heterozygous loss-of-function mutations in the glucokinase (GK) gene are the most common cause of MODY-2, a relatively mild form of type 2 diabetes. Conversely, homozygous or compound heterozygous GK deficiency mutations underlie permanent neonatal diabetes, a more severe perinatal diabetes (13). Mimicking human permanent neonatal diabetes, homozygous neuroendocrine GK knockout mice die shortly after birth of severe diabetes (15,33). Consistent with a MODY-2 phenotype, mice lacking one allele of neuroendocrine GK exhibit mild hyperglycemia with normal basal glucose turnover rates, but they have impaired glucose tolerance and insulin secretion during hyperglycemia (2,15).
Glucokinase deficiency could affect insulin secretion by altering electrical and/or nonelectrical downstream consequences of glucose metabolism (Fig. 1). To date, however, the relative importance of electrical (KATP-dependent) versus nonelectrical (KATP-independent) consequences of neuroendocrine glucokinase deficiency remains unclear. KATP channels are under tight metabolic control, and any perturbation of metabolism that leads to decreased generation of ATP is expected to have pronounced inhibitory effects on excitation-secretion coupling because of enhanced KATP activity. Studies of the electrical activity of nGK-deficient mice indeed support the idea that reduced glycolytic metabolism specifically causes failure of KATP channels to close in response to glucose, contributing to the observed reduction in insulin secretion (17). Consistent with this conclusion, it was reported that oral sulfonylurea treatment could suppress perinatal death in nGKeC/eC mice (15), presumably by restoring insulin release. Glucokinase is critical for liver regulation of glucose production (34). This could contribute to the whole animal phenotype in complex ways, and for this reason, we examined a tissue-specific model in which the liver-specific isoform of glucokinase is expressed normally, and only the neuroendocrine-specific isoform is deleted (15). We specifically examined the consequences of nGK deficiency on the wild-type and KATP-null backgrounds to elucidate the relative importance of the two signaling pathways. Perinatal lethality of nGKeC/eC was absolute on the Kir6.2+/+ background; only 2 pups were detected of an expected 16 pups, from heterozygous nGK+/eCKir6.2+/+ backcrosses (Fig. 2), and both died within 5 days. In contrast, double-knockout mice (nGKeC/eCKir6.2eC/eC) were born at the expected ratio. They exhibited low body weight, severe diabetes, and died prematurely, but perinatal lethality is clearly abrogated (Fig. 2A and B). Amelioration of the nGKeC/eC phenotype on the Kir6.2eC/eC background can be explained by an increased, though still insufficient, level of circulating insulin. This striking demonstration of the nullifying effect of removal of the KATP "brake" in vivo is further supported by the results with heterozygous nGK+/eC mice. On the Kir6.2+/+ background, they have markedly elevated fed and fasting glucose levels, and they are markedly glucose intolerant. By contrast, on the Kir6.2eC/eC background, nGK+/eC mice have near-normal fasting glucose levels and similar glucose tolerance to nGK+/+Kir6.2eC/eC animals. The abolition of KATP significantly ameliorates the "MODY-2" phenotype of the nGK+/eC mice.
Complex phenotypes of Kir6.2-deficient x nGK-deficient mice.
The initial prediction that, by removing the electrical defect, the diabetic consequences of nGK deficiency should be significantly avoided on the Kir6.2eC/eC background was thus supported. However, the mechanistic consequences of the genetic alterations are not absolutely clear. The naive expectation of mice lacking KATP channels was a persistent hyperinsulinemic phenotype because of unregulated secretion, but several studies have now shown that this expectation is not met (25eC27). For unexplained reasons (but see below), Kir6.2eC/eC mice are normoglycemic, with slightly impaired glucose tolerance and reduced GSIS. Similarly, although glucose tolerance was greatly improved in nGK+/eC mice on the Kir6.2eC/eC background, GSIS in isolated islets was still notably reduced (Fig. 6A). However, as has recently been shown, the sensitizing effect of amino acids, in particular glutamine (30,31), is much greater in islets that lack KATP (SUR1eC/eC). Similarly, we find that Kir6.2eC/eC islets can show enhanced insulin secretion at physiological glucose concentration (compared with Kir6.2+/+ mice) in the presence of stimulatory glutamine (Fig. 6B). This enhancement is seen in both nGK+/+ and nGK+/eC islets on this background. We suggest that, in vivo, ambient glutamine levels may lead to such an enhancement of insulin secretion and that, in part, this underlies the abrogating effect of KATP deficiency on nGK-deficiency (31).
Because we are examining a global neuroendocrine glucokinase deficiency, it is conceivable that the effects of Kir6.2 knockout are mediated through extra-pancreatic consequences, although insulin tolerance tests (Fig. 5) show that nGK+/eCKir6.2eC/eC mice are no more insulin sensitive than nGK+/eCKir6.2+/+ mice, excluding enhanced insulin sensitivity as an explanation for the rescued phenotype. Further evidence for a pancreatic basis for the Kir6.2-deficient rescue of nGK deficiency would be a -celleCspecific KATP knockout. A tissue-targeted knockout has not been generated, although we have generated an insulin promotereCdriven dominant-negative Kir6.2[AAA] mouse (29) that lacks KATP in 70% of its -cells (29). The demonstration of marked improvement in glucose tolerance in double-heterozygous (nGK+/eCKir6.2[AAA]) mice, compared with nGK+/eC alone (Fig. 7), provides further support for -celleCspecific KATP channels indeed being the major players in rescuing the nGK-deficiency phenotype.
Relevance to glucokinase-deficiency diabetes in humans.
The significant reduction in fasting glucose, slight yet significant decrease in fed glucose concentration, and marked enhancement in glucose tolerance in nGK+/eCKir6.2eC/eC mice with respect to nGK+/eCKir6.2+/+ mice suggests that, at least in part, the MODY-2 phenotype is abrogated on the KATP knockout background, highlighting the importance of electrical signaling in the regulation of insulin secretion. Decreased insulin secretion in response to glucose from nGKeC/eCKir6.2eC/eC or nGK+/eCKir6.2eC/eC islets recapitulates the response observed in nGK+/+Kir6.2eC/eC islets, thus indicating that the impaired GSIS is due to the Kir6.2 knockout phenotype and not due to glucokinase deficiency. Most patients with heterozygous GK mutations do not require pharmacological treatment, and the majority of cases are managed with diet alone. However, during pregnancy, women with GK mutations are often treated with insulin to maintain normal blood sugar levels, resulting in babies that are large for gestational age because of the effect of insulin on fetal growth (35). In these cases, sulfonylurea treatment could presumably be a valid alternative treatment, given the normalizing effect of KATP deficiency that we now demonstrate, because nGK+/eCKir6.2+/+ transgenic mice do respond to glibenclamide treatment in vitro (15).
Taken together, the results from nGK-deficient and KATP-deficient crosses show a marked improvement in survivability and glucose tolerance of the former on the KATP null background. Neonatal lethality of nGKeC/eCKir6.2+/+ mice is avoided on the Kir6.2eC/eC background, demonstrating that this consequence is attributable to loss of metabolic coupling between glucose and insulin secretion, probably through electrical signaling. As with the now dramatically improved treatment options for KATP-induced permanent neonatal diabetes (21,24), we suggest that there may be a role for sulfonylurea therapy in permanent neonatal diabetes resulting from glucokinase deficiency (13). A significant number of nGKeC/eCKir6.2eC/eC mice survived beyond weaning. This should also allow more extensive studies of the nonelectrical consequences (which are still ultimately lethal) of total glucokinase deficiency. Such studies will have the potential to inform and further improve treatment options for diabetes resulting from glucokinase deficiency.
ACKNOWLEDGMENTS
This work was supported by a mentor-based minority postdoctoral fellowship (to M.S.R.) from the American Diabetes Association and Takeda Pharmaceuticals, by the National Institutes of Health Diabetes Research and Training Center (DK20579) at Washington University, and by National Institutes of Health grant DK69445 (to C.G.N.).
Kir6.2-deficient mice (25) and nGK-deficient mice (15) were kindly provided by Dr. Susumo Seino and Dr. Yasuo Terauchi, respectively.
DMEM, Dulbecco’s modified Eagle medium; GSIS, glucose-stimulated insulin secretion; KATP channel, ATP-sensitive K+ channel; MODY-2, maturity-onset diabetes of the young type 2
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