Mari Malkki

Isolated Low HDL Cholesterol: An Insulin-Resistant State

High levels of very-low-density lipoprotein (VLDL) triglycerides (TGs) and low levels of high-density lipoprotein (HDL) cholesterol have been found to be associated with insulin resistance. However, direct evidence that patients with isolated low HDL cholesterol are insulin resistant is still lacking. Therefore, we investigated the degree of insulin resistance and intracellular metabolism of glucose by the euglycemic glucose clamp technique and indirect calorimetry in three groups of subjects with normal glucose tolerance: 17 male control subjects with normolipidemia, 12 male patients with isolated low HDL cholesterol (low HDL group), and 10 male patients with low HDL cholesterol and hypertriglyceridemia (low HDL/high TG group). Fasting, 1-h, and 2-h glucose levels did not differ between the groups in an oral glucose tolerance test (OGTT). In contrast, insulin levels during an OGTT were significantly higher in the low HDL group than in the control group (fasting insulin: 85 ± 11 vs. 50 ± 6 pM, P = 0.005; 1-h insulin: 622 ± 92 vs. 394 ± 64 pM, P = 0.004; and 2-h insulin: 343 ± 73 vs. 194 ± 40 pM, P = 0.006). Similarly, insulin levels were also higher in the low HDL/high TG group than in the control group (fasting insulin: 82 ± 14 pM, P = 0.037; 1-h insulin: 795 ± 179 pM, P = 0.063; and 2-h insulin: 488 ± 145 pM, P = 0.040). During the euglycemic hyperinsulinemic clamp, the rates of whole body glucose uptake were similarly reduced in the low HDL group (46.5 ± 2.8 μmol · kg−1 · min−1, P = 0.003) and in the low HDL/high TG group (45.1 ± 5.4 μmol · kg−1 · min−1, P = 0.021) compared with those in the control group (58.8 ± 2.5 μmol · kg−1 · min−1). The rates of glucose oxidation during the euglycemic clamp were lower in the low HDL patients than in control subjects, but the differences were not statistically significant (low HDL group: 17.3 ± 0.9 μmol · kg−1 · min−1 P = 0.092; low HDL/high TG group: 17.5 ±1.9 μmol · kg−1 · min−1 P = 0.098; and control group: 21.0 ±1.5 junol · kg−1 · min−1). The rates of glucose nonoxidation were reduced in the low HDL group (29.3 ± 2.5 μmol · kg−1 · min−1, P = 0.042) and in the low HDL/high TG group (27.6 ± 5.0 μmol · kg−1 · min−1, P = 0.056) compared with those in the control group (37.7 ± 2.6 μmol · · kg−1 · min−1), although the latter difference was not statistically significant. We conclude that patients with low HDL cholesterol are insulin resistant independently of TG levels.

Amino Acid Substitutions in Hexokinase II Among Patients With NIDDM

Hexokinase (HK) II plays an important role in intracellular glucose metabolism by catalyzing the conversion of glucose to glucose-6-phosphate. HKII is considered to be a promising candidate gene for non-insulin-dependent diabetes mellitus (NIDDM) and insulin resistance. Therefore, we investigated the frequency of variants in the coding region of the HKII gene in patients with NIDDM. Initial screening included a population-based sample of 40 Finnish patients with typical NIDDM, and subsequent screening included an additional 72 patients with NIDDM. By applying single-strand conformation polymorphism analysis and direct sequencing, the following amino acid substitutions were found among the 112 NIDDM patients: Ala314Val in one patient (0.9%), Arg353Cys in three patients (2.7%), and Arg775Gln substitution in three patients (2.7%). We also screened 97 subjects with completely normal glucose tolerance and a negative family history of diabetes for these mutations. The Ala314Val and the Arg353Cys substitutions were not found in control subjects, but the Arg775Gln substitution was found in two (2.1%) control subjects. None of these mutations were located close to the glucose- and ATP-binding sites of HKII. We conclude that mutations of the HKII gene are not a major etiological factor for NIDDM in the Finnish population.

What Is the Role for Donor Natural Killer Cells after Nonmyeloablative Conditioning

We investigated the impacts of the tempo of early (days 14, 28, and 42) donor T cell and natural killer (NK) cell engraftment, missing recipient killer cell immunoglobulin-like receptor (KIR) ligands, and numbers of donor inhibitory and activating KIR genes on hematopoietic cell transplantation (HCT) outcomes in 282 patients with hematologic malignancies given nonmyeloablative conditioning. Modeling chimerism levels as a continuous linear variable, we found that high early donor T cell chimerism was significantly associated with acute graft-versus-host disease (aGVHD) (P = .01), whereas high donor NK cell chimerism levels had no such association (P = .38). Conversely, high donor NK cell chimerism levels were significantly associated with low relapse risk (P = .0009), whereas no significant association was seen with high donor T cell chimerism (P = .10). The qualitative associations between donor T cell and NK cell chimerism levels and GVHD and relapse did not change after adjustment for the presence of recipient KIR ligands or numbers of donor inhibitory or activating KIR genes. Our data indicate that prompt engraftment of donor NK cells correlated with lessened risks of relapse, but not with GVHD, whereas the converse was true for T cells.

Mouse hexokinase II gene: structure, cDNA, promoter analysis, and expression pattern

Abstract. In mammalian tissues, the phosphorylation of intracellular glucose to glucose-6-phosphate (Glu-6-P) is facilitated by four distinct hexokinase (HK) isoenzymes, designated as HKI-IV. Because of the role of HKII as a leading glycolytic enzyme in insulin-sensitive tissues such as skeletal muscle, heart, and adipose tissue, defects in HKII function could contribute to the development of insulin resistance and perhaps Type 2 diabetes. As a first step towards elucidation of the physiological role of HKII in insulin resistance and type 2 diabetes using mouse knock-out models, we determined the genomic structure, sequence of the cDNA and of 4.8 kb of the 5′ regulatory region, and tissue-specific expression of the mouse HKII gene. The gene comprises 18 exons that span approximately 50 kb of DNA. Nucleotide sequence of the proximal promoter revealed a number of conserved putative transcription factor binding motifs. We also found numerous repeat elements throughout the mouse HKII gene. The mouse HKII cDNA is approximately 5.5 kb in length and contains an open reading frome of 2751 bp encoding a protein of 917 amino acids. The mouse HKII gene is predominantly expressed in skeletal muscle, heart, and adipose tissue. The transcription initiation and polyadenylation sites for the mouse HKII mRNA were similar to those of the rat and human genes.

The human hexokinase II gene promoter: functional characterization and detection of variants among patients with NIDDM

Summary Hexokinase II (HKII) plays an important role in facilitating glucose uptake by skeletal muscle, heart, and adipose tissue in response to insulin. We have cloned and sequenced the proximal promoter region of the human HKII gene, determined the transcription start sites and screened the 2.0 kb of the proximal 5 ′ flanking region for variants in non-insulin-dependent diabetic patients and control subjects. We found three variants in this region, one in the 5 ′ untranslated region (G→C at + 217) and two in the promoter region (T→G at –1043 and G→A at –1159). The allele frequencies of these variants did not differ between the diabetic and control subjects and these variants are not associated with insulin resistance. Various segments of the human HKII promoter were tested for driving expression of the luciferase reporter gene. The proximal 500 bp and 400 bp of the promoter were sufficient to drive maximal activity in adipocyte (3T3F442A) and myocyte (C2C12F3) cell lines, respectively. This region of the promoter is GC-rich and contains eight consensus binding sites for the transcription factor Sp-1, five for AP-2, two putative response elements for each of insulin and cyclic AMP. The proximal 175 bases of the promoter retained only 7–15 % of maximal activity. Sequence elements located between positions –304 and –215 accounted for approximately 80 % of the basal HKII promoter. In addition, the region between –215 and –184 contains a negative regulatory element for expression in 3T3F442A but not in C2C12F3 cells. [Diabetologia (1997) 40: 1461–1469]

Polymorphisms of the human hexokinase II gene: lack of association with NIDDM and insulin resistance

SummarySkeletal muscle and adipose tissue hexokinase II is a promising candidate gene for non-insulin-dependent diabetes mellitus (NIDDM) and insulin resistance. Therefore, we investigated the association of alleles at four polymorphic loci in this gene with NIDDM and insulin resistance in 110 Finnish diabetic patients with NIDDM and in 97 Finnish control subjects with normal glucose tolerance and a negative family history of diabetes. The four polymorphic nucleotide substitutions (silent) in the coding region of the hexokinase II gene were: GAC 251 GAT (exon 7), AAC 692 AAT and CCG 736 CCC (exon 15), and CTG 766 CTA (exon 16). Allele frequencies of each of these polymorphisms did not differ between patients with NIDDM and control subjects. In addition, subjects who were homozygous for the less frequent allele of each of the four polymorphisms had a similar degree of insulin resistance, as determined by the euglycaemic clamp technique, as did the subjects who were homozygous for the common allele in both control subjects and in patients with NIDDM. In conclusion, polymorphisms in the hexokinase II gene are not associated with the risk of NIDDM or insulin resistance in the Finnish population.

Glucokinase Gene Variants in Subjects With Late-Onset NIDDM and Impaired Glucose Tolerance

OBJECTIVE To investigate the frequency of variants of the glucokinase (GCK) gene in subjects with late-onset non-insulin-dependent diabetes mellitus (NIDDM) and in subjects with late-onset impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS The study population included 36 Finnish patients with late-onset NIDDM who were treated with diet for >8 years or who were newly diagnosed and 40 subjects with late-onset IGT who had low or normal insulin levels when tested by an oral glucose tolerance test. All exons, exon-intron junctions, and islet and liver promotor regions of the GCK gene were amplified with the polymerase chain reaction and screened for mutations using single-strand conformation polymorphism analysis. RESULTS A silent third-base substitution (TAC→TAT) in codon 215 of exon 6 was found in 2.8% of NIDDM patients and in 5.0% of IGT subjects. Polymorphisms were found in islet exon 1 at nucleotide 403 (C→G) in 16.7% of NIDDM patients and in 17.5% of IGT subjects and in the noncoding region of the islet promotor at nucleotide -30 (G→A) in 13.9% of NIDDM patients and in 25.0% of IGT subjects. Furthermore, in liver intron 1 a variant (C→T), 12 base pairs upstream from the splice acceptor site, was found in 5.6% of NIDDM patients and in 7.5% of IGT subjects. CONCLUSIONS These results indicate that the mutations in the coding region of the GCK gene are not likely to play a major role in the pathogenesis of late-onset NIDDM or IGT in the Finnish population.

Molecular patterns and sequence polymorphisms in the red and green visual pigment genes of Japanese men

The red-green pigment gene arrays of 203 (101 from a previous study and 102 from this study) randomly selected men of Japanese ancestry from the Seattle area were screened for the abnormal molecular patterns (deletions and red/green or green/red hybrid genes) that are usually associated with defective color vision. Such molecular patterns were found in approximately 5% of these individuals, which is equivalent to the frequency of phenotypic color vision defects in Japanese males in Japan. Thus, the majority of hybrid genes carried by Japanese males appear to be associated with defective color vision. In contrast, the frequency of hybrid genes among Caucasians and African-Americans is approximately two and five times the frequency of color vision defects in these two ethnic groups, respectively. The coding sequences of 50 males of Japanese ancestry were determined. All the polymorphisms in the red and green pigment genes that were detected in the Japanese sample had been observed in Caucasians and African-Americans. The same polymorphisms of the red pigment gene were present in the green pigment gene, suggesting that gene conversion contributes to sequence homogenization between these pigment genes. As is the case for Caucasians, exon 3 of the red and green pigment genes was observed to be a hot spot for recombination and gene conversion. Fewer polymorphic sites (4 vs 11) and haplotypes (5 vs 14) of the red pigment gene were observed in Japanese than in Caucasians. The Japanese population was more uniform with respect to the red pigment gene, with 70% of individuals having the same haplotype, as compared with the 43% for the Caucasian population. This difference was largely due to the lower degree of polymorphism at position 180 of the red pigment gene in Japanese (84% Ser and 16% Ala vs 62% Ser and 38% Ala.) The number of polymorphic sites and haplotypes in the green pigment gene was similar in the two populations. Nevertheless, the Japanese population was more uniform with 65% having the same haplotype. The difference in the frequency of alleles at position 283 accounted for this difference in haplotype distribution.

Functional consequences of naturally occurring variants of human hexokinase II

Summary Hexokinase II (HKII) catalyses a key step in glucose metabolism and can be regarded as a candidate gene for insulin resistance and type 2 (non-insulin-dependent) diabetes mellitus. We observed previously four amino acid substitutions among Finnish type 2 diabetic patients: Gln142His, Ala314Val; 0.9 %, Arg353Cys; 2.7 % and Arg775Gln; 2.7 %. The Arg775Gln mutation was also observed in normal control subjects (2.1 %) and the Gln142His substitution was found in both Type II diabetic and normal subjects with similar frequencies ( ∼ 20 %). Since Gln at position 142, Ala at 314 and Arg at 775 are present in human and rat hexokinases and could be important for structure and function of the enzyme, we generated all four substitutions by site-directed mutagenesis and expressed them in E.coli. None of these substitutions had any effect on HKII catalytic activity, Km or Vmax for glucose values in vitro. Thus unless these substitutions have an impact on enzyme activity or regulation in vivo, it is unlikely that these substitutions contribute to the aetiology of Type II diabetes. [Diabetologia (1998) 41: 1205–1209]