Insulin detemir attenuates food intake, body weight gain and fat mass gain in diet-induced obese Sprague-Dawley rats.

OBJECTIVE: Initiation and intensification of insulin therapy commonly causes weight gain, a barrier to therapy. A contrasting body of evidence indicates that insulin functions as an adiposity negative feedback signal and reduces food intake, weight gain and adiposity via action in the central nervous system. Basal insulin analogs, detemir (Det) and glargine (Glar), have been associated with less hypoglycemia compared with neutral protamine hagedorn insulin, and Det with less weight gain, especially in patients with higher body mass index (BMI). We sought to determine whether insulin therapy per se causes body weight and fat mass gain when delivered via a clinically relevant subcutaneous (SC) route in the absence of hypoglycemia and glycosuria in non-diabetic lean and diet-induced obese rats. MATERIALS AND METHODS: Rats were exposed to either a low-fat diet (LFD; 13.5% fat) or high-fat diet (HFD; 60% fat), and received Det (0.5 U kg(-1)), Glar (0.2 U kg(-1)) or vehicle (Veh) SC once daily for 4 weeks. These dosages of insulin were equipotent in rats with respect to blood-glucose concentration and did not induce hypoglycemia. RESULTS: As predicted by current models of energy homeostasis, neither insulin Det nor Glar therapy affected food intake and weight gain in LFD rats. Det treatment significantly attenuated food intake, body weight gain and fat mass gain relative to the Glar and Veh in high-fat fed animals, mirroring observations in humans. CONCLUSIONS: That neither insulin group gained excess weight, suggests weight gain with SC basal insulin therapy may not be inevitable. Our data further suggest that Det possesses a unique property to attenuate the development of obesity associated with a HFD.

Glucokinase activator PSN-GK1 displays enhanced antihyperglycaemic and insulinotropic actions.

AIMS/HYPOTHESIS: We evaluated the insulinotropic and antihyperglycaemic actions of glucokinase activators (GKAs), especially through acute and subchronic studies in rodent diabetes models with (2R)-2-(4-cyclopropanesulphonylphenyl)-N-(5-fluorothiazol-2-yl)-3-(tetrahydropyran-4-yl)propionamide (PSN-GK1), a novel and potent GKA. MATERIALS AND METHODS: The action of PSN-GK1 on or in the following were investigated: (1) on human liver glucokinase, insulin secretion from MIN6 cells and 2-deoxy-D: -[(3)H]glucose (2-DG) uptake into rat hepatocytes; and (2) in Zucker diabetic fatty rats and in non-diabetic C57Bl/6, diabetic db/db and ob/ob mice. RESULTS: At 5 mmol/l glucose, PSN-GK1 activated glucokinase (4.3-fold, median effective concentration [EC(50)] 130 nmol/l), increased MIN6 insulin secretion (26-fold, EC(50) 267 nmol/l) and 2-DG hepatocytic uptake (threefold, EC(50) 1 micromol/l); at higher glucose concentrations, EC(50)s and fold-effectiveness were both lower. In C57Bl/6 mice, PSN-GK1 reduced blood glucose at 1 and 10 mg/kg (by mouth), but insulin was increased significantly at only the higher dose. In hyperinsulinaemic 10-mmol/l glucose clamps, PSN-GK1 increased 2-DG incorporation into liver glycogen sixfold, directly demonstrating liver effects. PSN-GK1 improved glycaemic profiles in db/db mice and Zucker diabetic fatty rats, diabetic animal models in which GKA efficacy has not previously been described, without causing hypoglycaemia. In ob/ob mice, it dose-dependently reduced excursions in OGTTs. Moreover, after subchronic administration, no tachyphylaxis was evident and glycaemia was improved without alterations to lipid levels, liver weight, glycogen content or body weight. CONCLUSIONS/INTERPRETATION: PSN-GK1 was potently antihyperglycaemic through its effects on insulin release and hepatic glucose metabolism. It is one of the most potent GKAs described in the literature and is active in diabetic animal models where GKAs have not been reported to show efficacy to date. Ongoing human trials are investigating the potential of this novel therapeutic approach.

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.

Human ZHX1: cloning, chromosomal location, and interaction with transcription factor NF-Y.

NF-YA, B, and C comprise the heterotrimeric transcription factor known as nuclear factor Y (NF-Y) or CCAAT-binding protein (CBF). NF-Y binds many CCAAT and Y box (an inverted CCAAT box, ATTGG) elements. Mutations of these elements that disrupt the binding of NF-Y result in decreased transcription from various tissue-specific and inducible promoters. We employed a yeast two-hybrid system to screen a human liver cDNA library in an effort to isolate proteins that interact with NF-Y and that may play a role in tissue-specific or hormone-inducible promoter activity. Using a fragment of the NF-YA subunit as bait we isolated a cDNA that encodes most of the open reading frame of the human zinc fingers and homeobox 1 (ZHX1) protein. The complete open reading frame was subsequently isolated and found to encode a protein of 873 amino acids that contains two zinc fingers and five homeodomain motifs. Northern blot analysis of poly(A)(+) RNA isolated from various tissues revealed two major ZHX1 transcripts of about 4.5 and 5 kilobases. Both transcripts were expressed ubiquitously, although the 5-kilobase transcript is of greater abundance in most tissues examined. The human ZHX1 gene is located on chromosome 8q, between markers CHCL.GATA50B06 and CHLC. GATA7G07.

Functional interaction between the N- and C-terminal halves of human hexokinase II.

Mammalian hexokinases (HKs) I-III are composed of two highly homologous approximately 50-kDa halves. Studies of HKI indicate that the C-terminal half of the molecule is active and is sensitive to inhibition by glucose 6-phosphate (G6P), whereas the N-terminal half binds G6P but is devoid of catalytic activity. In contrast, both the N- and C-terminal halves of HKII (N-HKII and C-HKII, respectively) are catalytically active, and when expressed as discrete proteins both are inhibited by G6P. However, C-HKII has a significantly higher Ki for G6P (KiG6P) than N-HKII. We here address the question of whether the high KiG6P of the C-terminal half (C-half) of HKII is decreased by interaction with the N-terminal half (N-half) in the context of the intact enzyme. A chimeric protein consisting of the N-half of HKI and the C-half of HKII was prepared. Because the N-half of HKI is unable to phosphorylate glucose, the catalytic activity of this chimeric enzyme depends entirely on the C-HKII component. The KiG6P of this chimeric enzyme is similar to that of HKI and is significantly lower than that of C-HKII. When a conserved amino acid (Asp209) required for glucose binding is mutated in the N-half of this chimeric protein, a significantly higher KiG6P (similar to that of C-HKII) is observed. However, mutation of a second conserved amino acid (Ser155), also involved in catalysis but not required for glucose binding, does not increase the KiG6P of the chimeric enzyme. This resembles the behavior of HKII, in which a D209A mutation results in an increase in the KiG6P of the enzyme, whereas a S155A mutation does not. These results suggest an interaction in which glucose binding by the N-half causes the activity of the C-half to be regulated by significantly lower concentrations of G6P.

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites.

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

Analysis of the signaling pathway involved in the regulation of hexokinase II gene transcription by insulin.

The hexokinases, by converting glucose to glucose 6-phosphate, help maintain the glucose concentration gradient that results in the movement of glucose into cells through the facilitative glucose transporters. Hexokinase II (HKII) is the major hexokinase isoform in skeletal muscle, heart, and adipose tissue. Insulin induces HKII gene transcription in L6 myotubes, and this, in turn, increases HKII mRNA and the rates of HKII protein synthesis and glucose phosphorylation in these cells. Inhibitors of distinct insulin signaling pathways were used to dissect the molecular mechanism by which HKII gene expression is induced by insulin in L6 myotubes. Treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), or with rapamycin, an inhibitor of the pathway from the insulin receptor to p70/p85 ribosomal S6 protein kinase (p70(s6k)), prevented the induction of HKII mRNA by insulin. In contrast, treatment with PD98059, an inhibitor of mitogen-activated protein kinase activation, had no effect on insulin-induced HKII mRNA. In addition, rapamycin blocked the insulin-induced expression of an HKII promoter-chloramphenicol acetyltransferase fusion gene transiently transfected into L6 myotubes, whereas PD98059 had no such effect. These results suggest that a phosphatidylinositol 3-kinase/p70(s6k)-dependent pathway is required for regulation of HKII gene transcription by insulin and that the Ras-mitogen-activated protein kinase-dependent pathway is probably not involved.

Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat hexokinase II gene.

Hexokinases catalyze the phosphorylation of glucose and initiate cellular glucose metabolism. Hexokinase II (HKII) is the principal hexokinase isoform in skeletal muscle, heart, and adipose tissue. Isoproterenol and exogenous cyclic AMP (cAMP) increase HKII gene transcription in L6 myotubes. Various segments of the HKII promoter that direct the expression of the chloramphenicol acetyltransferase reporter gene were transfected into L6 myotubes to identify basal and cAMP response elements. The 5'-flanking region that extends 90 base pairs upstream of the transcription start site includes a CCAAT box and a cAMP response element (CRE); both contribute to basal promoter activity and each provides an independent, maximal response to cAMP. An inverted CCAAT motif, or Y box, located just upstream of the CCAAT box, contributes to basal promoter activity but is not involved in the cAMP response. Homo- and heterodimers composed of the CRE-binding protein and activating transcription factor-1 bind specifically to the CRE. The Y box and the CCAAT box specifically bind the factor NF-Y (also known as CBF).

Overexpression of hexokinase II in transgenic mice. Evidence that increased phosphorylation augments muscle glucose uptake.

Hexokinase II (HKII) is the predominant isozyme expressed in peripheral insulin-responsive tissues. To explore the role of HKII in muscle glucose metabolism, two lines of transgenic mice were generated where overexpression was restricted to striated muscle; HKII protein levels and activity were increased by 3-8-fold. Oral glucose tolerance, intravenous insulin tolerance, and insulin and lactate levels were unaffected in transgenic mice. There was a trend toward increased levels of muscle glycogen; however, glucose-6-phosphate levels were increased by 43% in transgenic skeletal muscle following in vivo glucose and insulin administration. Using 2-[3H]deoxyglucose as a tracer, in vitro basal and insulin-stimulated glucose uptake were determined in extensor digitorum longus, soleus, and epitrochlearis muscles. Maximal insulin-stimulated glucose uptake was increased by 17% (extensor digitorum longus), 34% (soleus), and 90% (epitrochlearis) in transgenic muscles; basal and submaximal glucose uptake was also modestly increased in soleus and epitrochlearis. These data suggest that increased muscle HKII (corresponding to the upper end of the physiologic range) may not be sufficient to augment net in vivo glucose homeostasis. However, glucose phosphorylation can represent a rate-limiting step for skeletal muscle glucose utilization since muscle glucose-6-phosphate levels are increased during in vivo hyperinsulinemia and hyperglycemia; furthermore, basal and insulin-mediated muscle glucose uptake can be increased by a selective increase in HKII expression.

A novel (TA)n polymorphism in the hexokinase II gene: application to noninsulin-dependent diabetes mellitus in the Pima Indians.

Hexokinase II, one member of a family of structurally similar enzymes that catalyze the phosphorylation of glucose in the 6-position, has been suggested to play a role in the pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM). The gene for hexokinase II, HK2, has been previously mapped to human chromosome 2p13 by fluorescence in situ hybridization, and two-point linkage analysis has placed it near the locus for transforming growth factor alpha, TGFA. We now report the characterization of a (TA)n polymorphism in intron 12 of HK2. Using multipoint analysis of CEPH family genotypes, we have determined the most likely locus order to be cen-D2S169-[D2S286-HK2]-[D2S145-D2S291]-[+ ++D2S45-D2S101-TGFA]-tel. As HKII is a candidate gene that could contribute to the manifestation of insulin resistance and NIDDM, we genotyped 1152 Pima Indians, a Native American tribe that has the highest reported prevalence of NIDDM in the world. Although we did not detect any linkage or association of HK2 with insulin resistance or NIDDM in the Pima Indians, the polymorphism and detailed mapping of HK2 described in this report should prove useful in the assessment of the role of this gene in the predisposition to NIDDM in other populations.

Variant sequences of the Hexokinase II gene in familial NIDDM.

UNLABELLED: Hexokinase II (HKII) plays a central role in the intracellular metabolism of glucose in skeletal muscle, catalysing the phosphorylation of glucose to glucose 6-phosphate. It is therefore considered to be a potentially important candidate gene in the development of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM). The aim of this study was to screen the HKII gene for mutations in NIDDM subjects from insulin-resistant families. Insulin sensitivity was assessed in unaffected first degree relatives from families with two or more living NIDDM subjects, and 15 families were identified as being insulin resistant. In 15 NIDDM subjects (one from each family) and 4 normoglycaemic control subjects, all 18 exons of the HKII gene were amplified by the polymerase chain reaction, and the products screened for mutations using a combination of single-stranded conformational polymorphism analysis and direct sequencing. Six sequence variations were detected in the NIDDM subjects; four silent polymorphisms [GAT vs GAC at codon 251 in exon 7, AAT vs AAC at codon 692 in exon 15, CCG vs CCC at codon 736 in exon 15, and CTG vs CTA at codon 766 in exon 16]; a single base change [T-->C], 22 base pairs distal to the exon-intron junction of exon 17 in the 5'-splice donor; and a single amino acid substitution [Gln142-->His] in exon 4, which was identified in 6 of the 15 NIDDM subjects. The frequency of the mutated codon 142 allele however, was comparable between NIDDM subjects with familial NIDDM (n = 56) and normoglycaemic control subjects (n = 48) (18.8% and 14.6% for NIDDM subjects and control subjects respectively; chi 2 = 0.6, p > 0.25). In addition, measures of insulin sensitivity were comparable in normal glucose tolerant subjects with (n = 20) and without (n = 40) the codon 142 polymorphism. IN CONCLUSION: (1) mutations in the coding regions of the HKII gene are unlikely to be major determinants in the development of insulin resistance and familial NIDDM; although (2) the influence of the codon 142 mutation in combination with other abnormalities of the insulin-signalling pathway on insulin action remain to be addressed.

Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus.

Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed.

Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.

The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.

Regulation of hexokinase II gene transcription and glucose phosphorylation by catecholamines, cyclic AMP, and insulin.

The hexokinases, by converting glucose to glucose-6-phosphate, help maintain the downhill gradient that results in movement of glucose into cells through the facilitative glucose transporters. GLUT4 and hexokinase (HK) II are the major transporter and hexokinase isoforms in skeletal muscle, heart, and adipose tissue, wherein insulin promotes glucose utilization. To understand whether hormones influence the contribution of phosphorylation to cellular glucose utilization, we investigated the effects that catecholamines, cyclic AMP (cAMP), and insulin have on HKII gene expression in cells representative of muscle (L6 cells) and brown (BFC-1B cells) and white (3T3-F442A cells) adipose tissues. Isoproterenol or the cAMP analog 8-chlorophenylthio-cAMP selectively increase HKII gene transcription in L6 cells, as does insulin (Printz RL, Koch S, Potter LP, O'Doherty RM, Tiesinga JJ, Moritz S, Granner DK: Hexokinase II mRNA and gene structure, regulation by insulin, and evolution. J Biol Chem 268:5209-5219, 1993), and cause a concentration- and time-dependent increase of HKII mRNA in both muscle and fat cell lines without changing HKI mRNA. Isoproterenol and insulin also increase the rate of synthesis of HKII protein and increase glucose phosphorylation and glucose utilization in L6 cells.

Structural and functional analysis of the human phosphoenolpyruvate carboxykinase gene promoter.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the rate limiting step in hepatic and renal gluconeogenesis. Glucagon (acting via cyclic AMP (cAMP)) and glucocorticoids stimulate PEPCK gene transcription, whereas insulin has the opposite effect. Since these are the major regulatory hormones controlling glucose homeostasis, and because increased hepatic glucose production is one of the characteristics of non-insulin dependent diabetes mellitus (NIDDM), investigators have speculated that the regulation of PEPCK gene expression may be defective in patients with NIDDM. To begin to investigate this possibility we have isolated and sequenced the human PEPCK gene promoter. In addition, we have constructed and analyzed a human PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion gene in an effort to correlate differences between the rat and human promoter sequences and the hormonal regulation of transcription.

Expression cloning of a zinc-finger cyclic AMP-response-element-binding protein.

In response to specific extracellular signals, intracellular cyclic AMP levels increase, leading to a variety of responses including the alteration of transcription of many eukaryotic genes. This transcriptional effect is frequently mediated through the cyclic AMP-response element (CRE) motif T(T/G)ACGTCA. Using an expression screening approach we have cloned a yeast gene, MSN2, that encodes a 78 kDa protein that recognizes this consensus CRE motif. Phosphorylation of the MSN2 protein by the catalytic subunit of protein kinase A stimulates DNA binding in vitro. Two putative Cys2His2-type zinc fingers present in the C-terminal 79 amino acids of the MSN2 protein are sufficient to confer CRE-binding specificity. Therefore, MSN2 represents a novel CRE-binding protein distinct from the multiple previously characterized basic region-leucine zipper repeat CRE-binding proteins.

Isolation, characterization and chromosomal localization of a human pseudogene for hexokinase II.

A processed pseudogene for hexokinase II (HKII), the first such reported for a member of the hexokinase gene family, was isolated from a human genomic library by using a rat HKII cDNA as a probe. The pseudogene contains a region that is identical to the open reading frame of the human HKII cDNA at 97% of the nucleotide positions, but it contains several frameshift mutations, small deletions and insertions, and several stop codons. The human HKII pseudogene is located on the X chromosome and is integrated into a long interspersed nuclear repetitive DNA element (LINE). We estimate that this integration event occurred approximately 14-16 Myr (million years) ago.