Defect In Glucose Uptake Research Articles

Contribution of defects in glucose uptake to carbohydrate intolerance in liver cirrhosis: assessment during physiological glucose and insulin concentrations

It is well established that subjects with liver cirrhosis are insulin resistant, but the contribution of defects in insulin secretion and/or action to glucose intolerance remains unresolved. Healthy individuals and subjects with liver cirrhosis were studied on two occasions: 1) an oral glucose tolerance test was performed, and 2) insulin secretion was inhibited and glucose was infused in a pattern and amount mimicking the systemic delivery rate of glucose after a carbohydrate meal. Insulin was concurrently infused to mimic a healthy postprandial insulin profile. Postabsorptive glucose concentrations were equal (5.36 +/- 0.12 vs. 5.40 +/- 0.25 mmol/l, P = 0.89), despite higher insulin (P < 0.01), C-peptide (P < 0.01), and free fatty acid (P = 0.05) concentrations in cirrhotic than in control subjects. Endogenous glucose release (EGR; 11.50 +/- 0.50 vs. 11.73 +/- 1.00 mumol.kg(-1).min(-1), P = 0.84) and the contribution of gluconeogenesis to EGR (6.60 +/- 0.47 vs. 6.28 +/- 0.64 mumol.kg(-1).min(-1), P = 0.70) were unaltered by cirrhosis. A minimal model recently developed for the oral glucose tolerance test demonstrated an impaired insulin sensitivity index (P < 0.05), whereas the beta-cell response to glucose was unaltered (P = 0.72). During prandial glucose and insulin infusions, the integrated glycemic response was greater in cirrhotic than in control subjects (P < 0.05). EGR decreased promptly and comparably in both groups, but glucose disappearance was insufficient at the prevailing glucose concentration (P < 0.05). Moreover, identical rates of [3-(3)H]glucose infusion produced higher tracer concentrations in cirrhotic than in control subjects (P < 0.05), implying a defect in glucose uptake. In conclusion, carbohydrate intolerance in liver cirrhosis is determined by insulin resistance and the ability of glucose to stimulate insulin secretion. During prandial glucose and insulin concentrations, EGR suppression was unaltered, but glucose uptake was impaired, which demonstrates that intolerance can be ascribed to a defect in glucose uptake, rather than abnormalities in glucose production or beta-cell function. Although insulin secretion ameliorates glucose intolerance, impaired glucose uptake during physiological glucose and insulin concentrations produces marked and sustained hyperglycemia, despite concurrent abnormalities in glucose production or insulin secretion.

Disruption of IFT results in both exocrine and endocrine abnormalities in the pancreas of Tg737orpk mutant mice

While relatively ignored for years as vestigial, cilia have recently become the focus of intense interest as organelles that result in severe pathologies when disrupted. Here, we further establish a connection between cilia dysfunction and disease by showing that loss of polaris (Tg737), an intraflagellar transport (IFT) protein required for ciliogenesis, causes abnormalities in the exocrine and endocrine pancreas of the Tg737orpk mouse. Pathology is evident late in gestation as dilatations of the pancreatic ducts that continue to expand postnatally. Shortly after birth, the acini become disorganized, undergo apoptosis, and are largely ablated in late stage pathology. In addition, serum amylase levels are elevated and carboxypeptidase is abnormally activated within the pancreas. Ultrastructural analysis reveals that the acini undergo extensive vacuolization and have numerous ‘halo-granules' similar to that seen in induced models of pancreatitis resulting from duct obstruction. Intriguingly, although the acini are severely affected in Tg737orpk mutants, cilia and Tg737 expression are restricted to the ducts and islets and are not detected on acinar cells. Analysis of the endocrine pancreas in Tg737orpk mutants revealed normal differentiation and distribution of cell types in the islets. However, after fasting, mutant blood glucose levels are significantly lower than controls and when challenged in glucose tolerance tests, Tg737orpk mutants exhibited defects in glucose uptake. These findings are interesting in light of the recently proposed role for polaris, the protein encoded by the Tg737 gene, in the hedgehog pathway and hedgehog signaling in insulin production and glucose homeostasis.

Role of Foxa-2 in adipocyte metabolism and differentiation

Hepatocyte nuclear factors-3 (Foxa-1–3) are winged forkhead transcription factors that regulate gene expression in the liver and pancreatic islets and are required for normal metabolism. Here we show that Foxa-2 is expressed in preadipocytes and induced de novo in adipocytes of genetic and diet-induced rodent models of obesity. In preadipocytes Foxa-2 inhibits adipocyte differentiation by activating transcription of the Pref-1 gene. Foxa-2 and Pref-1 expression can be enhanced in primary preadipocytes by growth hormone, suggesting that the antiadipogenic activity of growth hormone is mediated by Foxa-2. In differentiated adipocytes Foxa-2 expression leads to induction of gene expression involved in glucose and fat metabolism, including glucose transporter-4, hexokinase-2, muscle-pyruvate kinase, hormone-sensitive lipase, and uncoupling proteins-2 and -3. Diet-induced obese mice with haploinsufficiency in Foxa-2 (Foxa-2+/–) develop increased adiposity compared with wild-type littermates as a result of decreased energy expenditure. Furthermore, adipocytes of these Foxa-2+/– mice exhibit defects in glucose uptake and metabolism. These data suggest that Foxa-2 plays an important role as a physiological regulator of adipocyte differentiation and metabolism.

Role of Foxa-2 in adipocyte metabolism and differentiation.

Hepatocyte nuclear factors-3 (Foxa-1-3) are winged forkhead transcription factors that regulate gene expression in the liver and pancreatic islets and are required for normal metabolism. Here we show that Foxa-2 is expressed in preadipocytes and induced de novo in adipocytes of genetic and diet-induced rodent models of obesity. In preadipocytes Foxa-2 inhibits adipocyte differentiation by activating transcription of the Pref-1 gene. Foxa-2 and Pref-1 expression can be enhanced in primary preadipocytes by growth hormone, suggesting that the antiadipogenic activity of growth hormone is mediated by Foxa-2. In differentiated adipocytes Foxa-2 expression leads to induction of gene expression involved in glucose and fat metabolism, including glucose transporter-4, hexokinase-2, muscle-pyruvate kinase, hormone-sensitive lipase, and uncoupling proteins-2 and -3. Diet-induced obese mice with haploinsufficiency in Foxa-2 (Foxa-2+/-) develop increased adiposity compared with wild-type littermates as a result of decreased energy expenditure. Furthermore, adipocytes of these Foxa-2+/- mice exhibit defects in glucose uptake and metabolism. These data suggest that Foxa-2 plays an important role as a physiological regulator of adipocyte differentiation and metabolism.

Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects.

Insulin resistance plays an important role in the pathogenesis of type 2 diabetes; however, the multiple mechanisms causing insulin resistance are not yet fully understood. The aim of this study was to explore the possible contribution of intramyocellular lipid content in the pathogenesis of skeletal muscle insulin resistance. We compared insulin-resistant and insulin-sensitive subjects. To meet stringent matching criteria for other known confounders of insulin resistance, these individuals were selected from an extensively metabolically characterized group of 280 first-degree relatives of type 2 diabetic subjects. Some 13 lean insulin-resistant and 13 lean insulin-sensitive subjects were matched for sex, age, BMI, percent body fat, physical fitness, and waist-to-hip ratio. Insulin sensitivity was determined by the hyperinsulinemic-euglycemic clamp method (for insulin-resistant subjects, glucose metabolic clearance rate [MCR] was 5.77+/-0.28 ml x kg(-1) x min(-1) [mean +/- SE]; for insulin-sensitive subjects, MCR was 10.15+/-0.7 ml x kg(-1) x min(-1); P<0.002). Proton magnetic resonance spectroscopy (MRS) was used to measure intramyocellular lipid content (IMCL) in both groups. MRS studies demonstrated that in soleus muscle, IMCL was increased by 84% (11.8+/-1.6 vs. 6.4+/-0.59 arbitrary units; P = 0.008 ), and in tibialis anterior muscle, IMCL was increased by 57% (3.26+/-0.36 vs. 2.08+/-0.3 arbitrary units; P = 0.017) in the insulin-resistant offspring, whereas the extramyocellular lipid content and total muscle lipid content were not statistically different between the two groups. These data demonstrate that in these well-matched groups of lean subjects, IMCL is increased in insulin-resistant offspring of type 2 diabetic subjects when compared with an insulin-sensitive group matched for age, BMI, body fat distribution, percent body fat, and degree of physical fitness. These results indicate that increased IMCL represents an early abnormality in the pathogenesis of insulin resistance and suggest that increased IMCL may contribute to the defective glucose uptake in skeletal muscle in insulin-resistant subjects.

Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione.

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg x min were 39-90% lower in such transgenic mice, compared with their control littermates (P < or = 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 +/- 17.8 vs. 166.8 +/- 15.1 pmol deoxyglucose/g x min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5'-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Mechanism of Hexosamine-Induced Insulin Resistance in Transgenic Mice Overexpressing Glutamine:Fructose-6-Phosphate Amidotransferase: Decreased Glucose Transporter GLUT4 Translocation and Reversal by Treatment with Thiazolidinedione

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg·min were 39–90% lower in such transgenic mice, compared with their control littermates (P ≤ 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 ± 17.8 vs. 166.8 ± 15.1 pmol deoxyglucose/g·min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5′-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Primary prevention of coronary heart disease: guidance from Framingham: a statement for healthcare professionals from the AHA Task Force on Risk Reduction. American Heart Association.

The Framingham Heart Study has contributed importantly to understanding of the causes of coronary heart disease (CHD), stroke, and other cardiovascular diseases. Framingham research has helped define the quantitative and additive nature of these causes or, as they are now called, “cardiovascular risk factors.”1 The National Cholesterol Education Program (NCEP)2 3 has made extensive use of Framingham data in developing its strategy for preventing CHD by controlling high cholesterol levels. The NCEP guidelines2 3 adjust the intensity of cholesterol-lowering therapy with absolute risk as determined by summation of risk factors. The National High Blood Pressure Education Program (NHBPEP) has set forth a parallel approach for blood pressure control. In contrast to the NCEP,2 however, earlier NHBPEP reports issued through the Joint National Committee4 did not match the intensity of therapy to absolute risk for CHD. “Normalization” of blood pressure is the essential goal of therapy regardless of risk status. Blood pressure–lowering therapy is carried out as much for prevention of stroke and other cardiovascular complications as for reduction of CHD risk. Nonetheless, risk assessment could be important for making decisions about type and intensity of therapy for hypertension. Thus, the most recent Joint National Committee report5 gives more attention to risk stratification for adjustment of therapy for hypertension. Although Framingham data have already been influential in the development of national guidelines for risk factor management, the opportunity may exist for both cholesterol and blood pressure programs to draw more extensively from Framingham results when formulating improved risk assessment guidelines and recommending more specific strategies for risk factor modification. The American Heart Association has previously used Framingham risk factor data to prepare charts for estimating CHD risk. Framingham investigators of the National Heart, Lung, and Blood Institute prepared the original charts and have now revised …

Mechanisms of Insulin-Resistant Glucose Utilization in Rat Skeletal Muscle

Defects in glucose uptake are among the primary defects associated with peripheral insulin resistance, but fundamental mechanisms leading to this state are poorly understood. In order to elucidate mechanisms leading toward defects in glucose transport, we have used a partially pancreatectomized infusion (PxI) animal model with infusions of saline, glucose, or insulin to examine individual and combined effects of hyperglycemia and hyperinsulinemia on skeletal muscle glucose utilization. Moderate hyperglycemia induced by pancreatectomy reduced basal hindlimb muscle glucose utilization by 57% without affecting maximal insulin-stimulated glucose utilization; insulin administered in an amount sufficient to correct this hyperglycemia did not alter basal glucose utilization, but maximal insulin-stimulated glucose utilization was sharply diminished (75%); hyperglycemia with hyperinsulinemia similarly reduced basal and maximal insulin-stimulated glucose utilization. In order to establish the role of the glucose transporter protein in these insulin-resistant states, we quantified GLUT 4 content by immunoblotting and GLUT 4 mRNA by solution hybridization/RNAse protection assays. Hyperglycemia (2 weeks) reduced total muscle GLUT 4 protein content (53%) and mRNA (46%), while subsequent hyperinsulinemia (72 h) with either normo- or hyperglycemia partially restored both total GLUT 4 protein and mRNA levels. As insulin-stimulated GLUT 4 content in plasma membranes was not diminished by combined hyperglycemia/hyperinsulinemia, these results indicate functional GLUT 4 translocation in this model and suggest suppression of GLUT 4 transporter activity.

The Cdc25 protein of Saccharomyces cerevisiae is required for normal glucose transport

The essential CDC25 gene product of Saccharomyces cerevisiae is the most upstream known component of the RAS/adenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its cAMP metabolism, it is concluded that this effect on glucose transport is independent of cAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased cAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the glucose transporters.

Insulin induces translocation of GLUT-4 glucose transporters in human skeletal muscle.

Understanding the molecular mechanisms involved in the regulation of glucose transport into human muscle is necessary to unravel possible defects in glucose uptake associated with insulin resistance in humans. Here we report a strategy to subfractionate human skeletal muscle biopsies (0.5 g) removed from vastus lateralis during a euglycemic insulinemic clamp procedure. A sucrose gradient separated total membranes into five fractions. Fraction 25 (25% sucrose) contained the plasma membrane markers alpha 1- and alpha 2-subunits of the Na(+)-K(+)-adenosinetriphosphatase and the GLUT-5 hexose transporter, recently immunolocalized to the cell surface of human skeletal muscle. The dihydropyridine receptor, a transverse tubule marker, was present exclusively in this fraction. The GLUT-4 glucose transporter was more concentrated in fraction 27.5 (27.5% sucrose) and largely diminished in plasma membrane markers. Open skeletal muscle biopsies were removed before and 30 min after clamping insulin to 550 pM. This increased GLUT-4 protein by 1.61-fold in fraction 25 and lowered it by 50% in fraction 27.5. Thus physiological concentrations of insulin induce translocation of glucose transporters from an internal membrane pool to surface membranes in human skeletal muscle.

Human hexokinase II: localization of the polymorphic gene to chromosome 2

Type 2 (non-insulin-dependent) diabetes mellitus is characterized by decreased levels of glucose 6-phosphate in skeletal muscle. It has been suggested that the lower concentrations of glucose 6-phosphate contribute to the defect in glucose metabolism noted in muscle tissue of subjects with Type 2 diabetes or subjects at increased risk of developing Type 2 diabetes. Lower levels of glucose 6-phosphate could be due to a defect in glucose uptake, or phosphorylation, or both. Hexokinase II is the isozyme of hexokinase that is expressed in skeletal muscle and is responsible for catalysing the phosphorylation of glucose in this tissue. The recent demonstration that mutations in another member of this family of glucose phosphorylating enzymes, glucokinase, can lead to the development of Type 2 diabetes prompted us to begin to examine the possible role of hexokinase II in the development of this genetically heterogeneous disorder. As a first step, we have cloned the human hexokinase II gene (HK2) and mapped it to human chromosome 2, band p13.1, by fluorescence in situ hybridization to metaphase chromosomes. In addition, we have identified and characterized a simple tandem repeat DNA polymorphism in HK2 and used this DNA polymorphism to localize this gene within the genetic linkage map of chromosome 2.

Demonstration of defective glucose uptake and storage in erythrocytes from non-insulin dependent diabetic patients and effects of metformin.

1. Red blood cells can store glucose and may thus participate in blood glucose homeostasis. We investigated if a defect in this process exists in non-insulin dependent diabetes (NIDD). 2. Blood was obtained in fasting conditions from 10 normal and 10 newly diagnosed NIDD patients (before and after 4 weeks Metformin therapy). Washed erythrocytes were resuspended in media containing various glucose concentrations (4.4, 6.6, 8.8 and 13.2 mmol/L). Total glucose uptake was calculated as the sum of the measurements of lactate as well as free glucose, the latter being determined before and after addition of amyloglucosidase to the pellet. 3. Cells from diabetics showed a pronounced reduction in glucose uptake, particularly in their capacity to store glucose as glycogen (reactive to amyloglucosidase). Metformin treatment almost normalized glycogen levels, whereas lactate declined concomitantly in the pellet. 4. Our data demonstrate that a defect in glucose uptake exists in erythrocytes from NIDD patients, affecting both free and stored glucose, and that this defect is reversed by Metformin treatment, indicating that this drug can increase glycogen levels even in insulin-insensitive cells. 5. Thus, in view of their total mass, erythrocytes may be important in the impaired glucose homeostasis in NIDD, in particular in marked hyperglycaemia such as after a meal.

Role of impaired intracellular glucose metabolism in the insulin resistance of aging

The insulin resistance of aging is characterized by both reduced glucose uptake and impaired intracellular glucose metabolism. The aim of this study was to determine whether impaired intracellular glucose metabolism contributes to insulin resistance in the elderly independent of reduced glucose uptake. To address this question, glucose uptake in non-obese elderly males was matched to controls using the glucose clamp technique, and intracellular glucose mtabolism was assessed in vivo by indirect calorimetry and in vitro by skeletal muscle biopsy for glycogen synthase activity. When elderly subjects were compared with controls at an equivalent basal glucose uptake of approximately 2.5 mg/kg fat-free mass (FFM)/min, muscle glycogen synthase activity was similar (fractional velocity of glycogen synthase at 0.1 mmol/L glucose-6-phosphate [FV 0.1], 0.06 ± 0.1 and 0.07 ± 0.1), but whole-body rates of glucose oxidation were reduced (1.36 ± 0.12 v 1.90 ± 0.11 mg/kg FFM/min, P < .05). During 40-mU/m 2/min hyperinsulinemic clamps at matched rates of glucose uptake (∼10.7 mg/kg FFM/min in both groups), glycogen synthase activity was again similar (FV 0.1, 0.15 ± 0.02 and 0.14 ± 0.02), and glucose oxidation remained reduced in the elderly (4.18 ± 0.25 v 4.77 ± 0.17 mg/kg FFM/min, P < .05). Only during clamps in the maximal range of glucose uptake (∼29.5 mg/kg FFM/min) was glucose oxidation between the groups comparable (5.97 ± 0.50 and 5.75 ± 0.31 mg/kg FFM/min). Plasma free fatty acid (FFA) concentrations, fat oxidation, and protein oxidation were similar under all study conditions. In summary, when glucose uptake in the elderly was normalized, (1) glucose oxidation remained reduced in the submaximal but not maximal range of glucose uptake, and (2) skeletal muscle glycogen synthase activity was similar to that of controls. In conclusion, impaired intracellular glucose metabolism contributes to the insulin resistance of aging independent of the defect in glucose uptake through decreased sensitivity of whole-body glucose oxidation to insulin stimulation. In contrast, skeletal muscle glycogen synthase is normal and does not contribute to the insulin resistance of aging.

Hexose Uptake and Transport in Polymorphonuclear Leukocytes from Patients with Glycogen Storage Disease Ib

Neutrophil functions and glucose metabolism are known to be impaired in glycogen storage disease (GSD) Ib patients. The uptake of nonmetabolizing glucose analogues into polymorphonuclear leukocytes (PMN) of GSD Ib patients was studied. 2-Deoxyglucose (2-DOG) and 3-O-methylglucose are transported across the cell membrane by facilitated diffusion mediated by the glucose transporter. Because 2-DOG is phosphorylated within the cell, its uptake rate reflects hexose transport as long as phosphorylation is not rate-limiting. These conditions prevail only at low 2-DOG concentrations. Transport of 5 microM DOG into GSD Ib patient PMN was found to be similar to controls (4.3 +/- 0.5 and 4.65 +/- 1.77 pmol/min X 10(6), respectively). In contrast, 2-DOG uptake at high concentrations (2 mM) decreased by 70% in patient PMN compared with control cells (0.17 +/- 0.06 and 0.51 +/- 0.11 nmol/min X 10(6), for patients and controls, respectively). Transport of 3-O-methylglucose (a glucose analogue that does not undergo intracellular phosphorylation) was not different in patient PMN compared with controls (1.86 +/- 0.53 and 2.19 +/- 0.30 nmol/min X 10(6), respectively). Hexose monophosphate shunt activity in PMN of GSD Ib patients at a glucose concentration of 2 mM was 43% of control values, whereas at 10 microM it was similar to controls. Taken together, these results suggest that the defect in glucose uptake and metabolism found in GSD Ib patient PMN is due to an impairment in hexose phosphorylation rather than in a reduction in the transmembrane glucose transport activity.

Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.

Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.