Preferential deposition of visceral adipose tissue occurs due to physical inactivity.

We hypothesised that strict inactivity (bed rest) would lead to regional differences in fat deposition. Twenty-four male subjects underwent 60 d bed rest and remained inactive (n = 9), performed resistance exercise plus whole-body vibration (RVE; n = 7) or resistance exercise only (RE; n = 8). Fat mass was assessed via dual X-ray absorptiometry. In the inactive subjects, fat deposition differed between body regions (P = 0.0005) with android region visceral adipose tissue increasing the most (+29% at the end of bed rest), followed by remainder of the trunk (from chin to the iliac crest; +10%) and the arms and legs (both +7%). Insulin sensitivity reduced in the inactive subjects at the end of bed rest (P = 0.036). RE did not have a significant impact on regional fat mass changes (P ⩾ 0.055). In RVE, increases in visceral adipose tissue (-14%; P = 0.028 vs inactive subjects) and in the arms (arms -8%, P = 0.011 vs inactive) were not seen. We conclude that inactivity leads to a preferential increase in visceral adipose tissue.

Comparison of oral glucose tolerance test (OGTT) 100 g with OGTT 75 g for evaluation of acromegalic patients and the impact of gender on test reproducibility.

CONTEXT: Growth hormone (GH) measurements during an oral glucose tolerance test (OGTT) are essential for the diagnosis and follow-up management of acromegaly. However, both 100 g glucose (OGTT(100) ) and 75 g glucose (OGTT(75) ) test variants are in clinical use. Whether the tests are interchangeable concerning GH nadir and test interpretation is unclear. Furthermore, information on test reproducibility and the impact of gender is scarce. OBJECTIVE: To compare both tests in acromegalic patients and to evaluate test reproducibility with respect to gender. DESIGN, SUBJECTS AND METHODS: OGTT(100) and OGTT(75) were performed on two consecutive days in 54 acromegalic patients (46·9 ± 1·8 years, 30 women). OGTT(75) was repeated on three different occasions in 11 healthy men and 13 healthy women at different phases of the menstrual cycle. RESULTS: GH nadirs were comparable between tests [2·40 ± 0·52 (OGTT(100) ) and 2·46 ± 0·54 µg/l (OGTT(75) ); P = 0·356]. There were no differences at any time point in the mean values of GH, serum glucose or insulin between the two test variants. Test interpretation was highly consistent between the OGTT(100) and OGTT(75) [area under the receiver operated curve (ROC) = 0·995]. In men, GH, insulin and glucose measurements during OGTT(75) were highly reproducible. In women, however, basal and GH nadirs were significantly higher midcycle (P < 0·05). CONCLUSIONS: In acromegalic patients, there is no difference in GH nadirs and test interpretation after the ingestion of 100 g or 75 g glucose. The OGTT(75) is highly reproducible in men, but in women, it should be performed preferably in the early follicular phase.

T3/rT3-ratio is associated with insulin resistance independent of TSH.

Thyroid dysfunction has been shown to be associated with insulin resistance (IR). This may involve peripheral thyroid hormone metabolism, which is assumed to be reflected by the ratio triiodothyronine/reverse triiodothyronine (T3/rT3-ratio). To explore a potential association between the T3/rT3-ratio and IR we investigated pairs which differed in IR, but were matched by sex, age, body mass index (BMI), and thyroid stimulating hormone (TSH). For this purpose, matched pair analyses were embedded into a cross sectional study group. 22 pairs were matched from either the first or the third tertile of HOMA%S of a cohort of 353 euthyroid subjects with normal glucose metabolism who did not take any medication. The T3/rT3-ratio was compared in the matched pairs. The T3/rT3-ratio was significantly increased in the insulin resistant subjects compared to their insulin sensitive partners (8.78 ± 0.47 vs. 7.33 ± 0.33, p=0.019). Furthermore the T3/rT3-ratio was lower in men compared to women (p for the within-subject effect=0.046) both in the insulin sensitive and the insulin resistant subjects. Here we show that the T3/rT3-ratio, which is supposed to reflect the tissue thyroid hormone metabolism, is significantly increased in insulin resistant subjects. This further supports a link between thyroid function and IR.

Annual change in insulin sensitivity.

The incidence of both type 2 diabetes and cardiac events is reported to be higher during winter, indicating a putative annual periodic change in insulin sensitivity (IS). Annual differences in IS - quantified as HOMA-%S and Matsuda-Sensitivity Index - were analyzed using a cosine wave-fitting algorithm in a cross-sectional study group including 2 385 participants. Additionally, semi-annual differences in IS were compared. We found periodicity for HOMA-%S and Matsuda-Sensitivity Index (p=0.02 or 0.006), which was strengthened after restriction to participants without diabetes (p=0.009 or 0.004). The rhythm amplitude of 0.08 indicated moderate changes in IS throughout the year. IS was significantly higher when participants were enrolled during the second vs. the first half of the year (HOMA-%S 112.0±3.0% vs. 97.4±2.4%, p<0.001). The impact of the half-year on IS, which remained significant after adjustment for confounders, was again moderate and explained only 0.5% of the variation. IS showed a significant moderate annual periodicity, which may affect the interpretation of studies reporting small changes in IS.

Improved insulin sensitivity, preserved beta cell function and improved whole-body glucose metabolism after low-dose growth hormone replacement therapy in adults with severe growth hormone deficiency: a pilot study.

AIMS/HYPOTHESIS: Growth hormone-deficient patients show deterioration of insulin sensitivity and beta cell function. High-dose growth hormone treatment often induces further impairment of insulin sensitivity, leading to an increase in insulin and glucose levels or even, in cases of preexisting beta cell defect, to overt diabetes. However, low-dose treatment may improve insulin sensitivity, although data in humans with detailed metabolic phenotyping are as yet not available. We postulated that long-term low-dose growth hormone replacement, restoring IGF-1 to the low-normal range, might beneficially affect glucose metabolism. METHODS: We studied prospectively the metabolic responses to 24 and 48 weeks of growth hormone treatment in a small group of six adults with severe growth hormone deficiency (four men, two women; age 40-59 years; BMI 30.2 +/- 1 kg/m(2); mean growth hormone dose 0.3 +/- 0.04 mg/day). All participants underwent an oral glucose tolerance test, euglycaemic-hyperinsulinaemic clamp and hyperglycaemic-hyperinsulinaemic clamp plus i.v. L: -arginine on three occasions. Insulin sensitivity was measured by calculating the M value during the steady state of the euglycaemic-hyperinsulinaemic clamp. Insulin secretion and clearance were estimated from AUC(C-peptide), AUC(insulin) and their ratio at each phase of the hyperglycaemic-hyperinsulinaemic clamp. RESULTS: Growth hormone significantly improved insulin sensitivity (M value 13.8 +/- 2.6 [baseline] vs 19.6 +/- 2.6 [24 weeks] and 23.7 +/- 1.9 [48 weeks] micromol kg(-1) min(-1); p < 0.01). Although the insulin response to glucose and arginine decreased slightly, the disposition index, integrating insulin sensitivity and secretion, significantly increased (p < 0.01), indicating an improvement in whole-body glucose metabolism. Insulin clearance was not affected during treatment (p > 0.05). CONCLUSIONS/INTERPRETATION: Our data indicate that long-term low-dose growth hormone treatment may improve insulin sensitivity and whole-body glucose metabolism in adults with severe growth hormone-deficiency.

Metabolic effects of diets differing in glycaemic index depend on age and endogenous glucose-dependent insulinotrophic polypeptide in mice.

AIMS/HYPOTHESIS: High- vs low-glycaemic index (GI) diets unfavourably affect body fat mass and metabolic markers in rodents. Different effects of these diets could be age-dependent, as well as mediated, in part, by carbohydrate-induced stimulation of glucose-dependent insulinotrophic polypeptide (GIP) signalling. METHODS: Young-adult (16 weeks) and aged (44 weeks) male wild-type (C57BL/6J) and GIP-receptor knockout (Gipr ( -/- )) mice were exposed to otherwise identical high-carbohydrate diets differing only in GI (20-26 weeks of intervention, n = 8-10 per group). Diet-induced changes in body fat distribution, liver fat, locomotor activity, markers of insulin sensitivity and substrate oxidation were investigated, as well as changes in the gene expression of anorexigenic and orexigenic hypothalamic factors related to food intake. RESULTS: Body weight significantly increased in young-adult high- vs low-GI fed mice (two-way ANOVA, p < 0.001), regardless of the Gipr genotype. The high-GI diet in young-adult mice also led to significantly increased fat mass and changes in metabolic markers that indicate reduced insulin sensitivity. Even though body fat mass also slightly increased in high- vs low-GI fed aged wild-type mice (p < 0.05), there were no significant changes in body weight and estimated insulin sensitivity in these animals. However, aged Gipr ( -/- ) vs wild-type mice on high-GI diet showed significantly lower cumulative net energy intake, increased locomotor activity and improved markers of insulin sensitivity. CONCLUSIONS/INTERPRETATION: The metabolic benefits of a low-GI diet appear to be more pronounced in younger animals, regardless of the Gipr genotype. Inactivation of GIP signalling in aged animals on a high-GI diet, however, could be beneficial.

Effects of euglycemic hyperinsulinemia and lipid infusion on circulating cholecystokinin.

AIMS: Functions of the gut hormone cholecystokinin (CCK) include an important role in the regulation of gastric emptying, postprandial glucose homeostasis, and postmeal satiety. Postprandial CCK responses are significantly blunted in type 2 diabetic patients by unknown mechanisms. We hypothesized that hyperinsulinemia and lipid infusion influence circulating levels of biologically active CCK. METHODS: Eleven healthy subjects were studied in a cross-over design after 10-h overnight fasts, using euglycemic-hyperinsulinemic clamps for 443 min, with an additional infusion of lipid-heparin (1.25 ml.min(-1)) or saline (1.25 ml.min(-1)) for the last 300 min after constant plasma glucose levels were achieved. RESULTS: Euglycemic-hyperinsulinemia resulted in a sustained, up to 5-fold increase of plasma CCK (P < 0.001). When adding lipid infusion instead of saline, CCK concentrations rapidly declined and returned to baseline levels (CCK(300 min) 1.1 +/- 0.2 vs. 3.3 +/- 0.3 pmol/liter, P < 0.001). Partial intraclass correlation showed an independent correlation of plasma CCK with free fatty acids (r(ic) = -0.377, P < 0.001) but not with serum insulin (r(ic) = 0.077, P = 0.32). Whole-body insulin sensitivity decreased in lipid-exposed subjects (M value 7.1 +/- 0.7 vs. 5.6 +/- 0.9 mg.kg.min(-1), P = 0.017) but was not independently correlated with CCK (r(ic) = 0.040, P = 0.61). CONCLUSIONS: We report novel findings showing that circulating CCK markedly increased in the euglycemic-hyperinsulinemic state, possibly as a result of near-complete suppression of circulating free fatty acids. Moreover, raising blood lipids even moderately by lipid infusion rapidly and significantly interfered with this effect, suggesting that a negative feedback mechanism of blood lipids on circulating CCK might exist.

Intravenous lipid and heparin infusion-induced elevation in free fatty acids and triglycerides modifies circulating androgen levels in women: a randomized, controlled trial.

BACKGROUND: The polycystic ovarian syndrome (PCOS) is characterized by hyperandrogenism and associated with obesity and impaired glucose metabolism. Despite the high prevalence of PCOS and the considerable clinical impact, the precise interplay between metabolism and hyperandrogenemia is not entirely clear. OBJECTIVE: The objective of the study was to analyze the effects of iv lipid and heparin infusion on circulating androgen levels in healthy women. DESIGN: This was a randomized, controlled, crossover trial. SETTING: The study was conducted at an endocrinology center. PATIENTS: Patients included 12 healthy young women during the early follicular phase of two subsequent cycles. INTERVENTION: After an overnight fast, a 20% lipid/heparin or a saline/heparin infusion was administered in random order for 330 min. MAIN OUTCOME MEASURES: A detailed characterization of androgen metabolism was performed. RESULTS: Elevations in free fatty acids and triglycerides, induced by lipid/heparin infusion, elevates the levels of androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone, 5alpha-dihydrotestosterone, estrone, and 17beta-estradiol. Urinary excretion of DHEA, DHEAS, 5-androstene-3beta,17beta-diol, and the sum of urinary excreted DHEA and its 16-hydroxylated downstream metabolites, 16alpha-hydroxy-DHEA and 5-androstene-3beta,16alpha,17beta-triol, were reduced. CONCLUSION: The mechanism of iv lipid and heparin infusion-induced elevation of circulating androgens described here might contribute to the development of hyperandrogenism in women with PCOS and suggests that lowering of hyperlipidemia might be a potential therapeutic target in patients with PCOS to treat hyperandrogenemia.

Euglycemic hyperinsulinemia differentially modulates circulating total and acylated-ghrelin in humans.

Ghrelin is a powerful orexigenic gut hormone. Circulating concentrations of total ghrelin are downregulated by food intake in both acute and chronic hyperinsulinemic states. However, in blood des-acylated (des-acyl) ghrelin is the predominant form that has no orexigenic effects in humans. Circulating acyl-ghrelin has been shown to be suppressed post-prandially and by pharmacological hyperinsulinemia. However, up to now responses of circulating acyl-ghrelin to moderate hyperinsulinemic and hyperinsulinemic-hyperlipidemic clamp conditions have not been reported. Fourteen healthy subjects were investigated using two-stepped euglycemic-hyperinsulinemic clamps (40 mU insulin/ m2/min; mean 148+/-7 min till steady state, followed by 300 min lipid/heparin infusion). Responses of total ghrelin and acyl-ghrelin were measured at timed intervals throughout the clamps. Des-acyl-ghrelin concentrations were calculated by subtraction. Total ghrelin significantly decreased vs baseline concentrations (819+/-92 vs 564+/-58 pg/ml, p<0.001), thereby confirming previous observations. Des-acyl ghrelin closely followed total ghrelin concentrations and significantly decreased vs baseline (772+/-92 vs 517+/-56 pg/ml, p<0.001). In contrast, neither euglycemichyperinsulinemia nor euglycemic-hyperinsulinemic- hyperlipidemia suppressed acyl-ghrelin below baseline concentrations throughout the clamps (46+/-3 vs 47+/-8 pg/ml, p=0.90). In conclusion, moderate hyperinsulinemic and hyperinsulinemic- hyperlipidemic clamp conditions differentially modulated circulating total ghrelin and acylghrelin in humans. Factors other than changes in insulin and lipid concentrations are likely to contribute to the previously reported post-prandial reduction of circulating acyl-ghrelin.

Deficiency of phosphofructo-1-kinase/muscle subtype in humans impairs insulin secretion and causes insulin resistance.

Non-insulin-dependent diabetes mellitus (NIDDM) is caused by peripheral insulin resistance and impaired beta cell function. Phosphofructo-1-kinase (PFK1) is a rate-limiting enzyme in glycolysis, and its muscle subtype (PFK1-M) deficiency leads to the autosomal recessively inherited glycogenosis type VII Tarui's disease. It was evaluated whether PFK1-M deficiency leads to alterations in insulin action or secretion in humans. A core family of four members was evaluated for PFK1-M deficiency by DNA and enzyme-activity analyses. All members underwent oral and intravenous glucose tolerance tests (oGTT and ivGTT) and an insulin-sensitivity test (IST) using octreotide. Enzyme activity determinations in red blood cells showed that the father (46 yr, body mass index [BMI] 22. 4 kg/m2) and older son (19 yr, BMI 17.8 kg/m2) had a homozygous, while the mother (47 yr, BMI 28.4 kg/m2) and younger son (13 yr, BMI 16.5 kg/m2) had a heterozygous PFK1-M deficiency. DNA analyses revealed an exon 5 missense mutation causing missplicing of one allele in all four family members, and an exon 22 frameshift mutation of the other allele of the two homozygously affected individuals. The father showed impaired glucose tolerance, and the mother showed NIDDM. By ivGTT, both parents and the older son had decreased first-phase insulin secretion and a diminished glucose disappearance rate. The IST showed marked insulin resistance in both parents and the older, homozygous son, and moderate resistance in the younger son. PFK1-M deficiency causes impaired insulin secretion in response to glucose, demonstrating its participation in islet glucose metabolism, and peripheral insulin resistance. These combined metabolic sequelae of PFK-1 deficiency identify it as a candidate gene predisposing to NIDDM.

Arabinoxylan consumption decreases postprandial serum glucose, serum insulin and plasma total ghrelin response in subjects with impaired glucose tolerance.

OBJECTIVE: Arabinoxylan (AX) consumption is associated with metabolic improvement during diabetes and with modulation of ghrelin, an orexigenic gut hormone. The effect of AX consumption on ghrelin secretion in disturbed metabolic states is unknown. Therefore, we investigated the postprandial responses to AX consumption of serum glucose, insulin and triglycerides and plasma total and acylated ghrelin in subjects with impaired glucose tolerance (IGT). DESIGN: Randomized, single-blind, controlled, crossover intervention trial. SUBJECTS: Seven female and four male adults with IGT, aged 55.5 years, and body mass index (BMI) 30.1 kg/m(2). INTERVENTION: Subjects received either placebo or 15 g AX supplement for 6 weeks with a 6-week washout period in-between. MAIN OUTCOME MEASUREMENTS: Postprandial responses of serum glucose, insulin and triglycerides, and plasma total and acylated ghrelin after a liquid meal challenge test (LMCT) measured at the beginning and at the end of the dietary intervention at -20, -5, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min. RESULTS: After LMCT, AX consumption resulted in lower postprandial responses in serum glucose, insulin and triglycerides (P<0.05). Compared to placebo, total plasma ghrelin was also reduced by 42+/-8 pg/ml (P<0.001) after AX consumption with no difference in plasma acylated ghrelin. CONCLUSION: AX consumption improved postprandial metabolic responses after an LMCT in subjects with IGT and reduced total ghrelin response. However, acylated ghrelin responses were unchanged, suggesting that the acylated ghrelin-mediated orexigenic regulation is not improved as only total plasma ghrelin decreased.

Glucagon suppression of ghrelin secretion is exerted at hypothalamus-pituitary level.

CONTEXT: The mechanisms underlying the well-known glucagon-induced satiety effect are unclear. Recently, we showed that glucagon induces a remarkable decrease in the orexigenic hormone ghrelin that might be responsible for this effect. OBJECTIVE: The objective of this study was to evaluate the putative role of the hypothalamic pituitary axis in glucagon's suppressive effect on ghrelin secretion. DESIGN, SUBJECTS, AND METHODS: Prospectively, we studied the endocrine and metabolic responses to im glucagon administration in 22 patients (16 males; age, 21-68 yr; body mass index, 28.1 +/- 1.1 kg/m(2)) with a known hypothalamic-pituitary lesion and at least one pituitary hormone deficiency. Control experiments were performed in 27 healthy subjects (15 males; age, 19-65 yr; body mass index, 25.5 +/- 0.9 kg/m(2)). RESULTS: The suppression of ghrelin by glucagon measured as area under the curve(240 min) was significantly greater in controls when compared with patients (P < 0.01). Although there was a significant decrease in ghrelin in controls (P < 0.001), ghrelin was almost unchanged in patients (P = 0.359). Changes in glucagon, glucose, and insulin levels were comparable between both groups. CONCLUSIONS: We show that the hypothalamic-pituitary axis plays an essential role in the suppression of ghrelin induced by im glucagon administration. Glucagon significantly decreases ghrelin levels in healthy subjects. However, in the absence of an intact hypothalamic-pituitary axis, this effect was abolished. The mechanisms responsible for our observation are unlikely to include changes in glucose or insulin levels.

Free fatty acids increase androgen precursors in vivo.

CONTEXT: There is considerable evidence that metabolic factors such as insulin resistance may induce hyperandrogenemia in polycystic ovary syndrome. However, other metabolic factors such as free fatty acids (FFAs) may also contribute to androgen excess. OBJECTIVE: The objective was to study effects of FFAs on adrenal production of androgen precursors in vivo. DESIGN AND PARTICIPANTS: We investigated eight healthy young men, because male individuals produce the androgen precursors dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione predominantly in the adrenal gland. A randomized controlled crossover trial was performed. INTERVENTION: After a 10-h overnight fast, 20% lipid/heparin or saline/heparin infusion was given at a rate of 1.5 ml/min. Four hours after start of lipid infusion, a euglycemic hyperinsulinemic clamp was performed. MAIN OUTCOME MEASURES: DHEA, androstenedione, 17-OH-progesterone, testosterone, estrone, LH, FSH, ACTH, and cortisol were measured. RESULTS: The adrenal androgen precursors DHEA and androstenedione showed a circadian decline during saline/heparin infusion (P < 0.05 vs. baseline, respectively), whereas no significant changes were observed during lipid/heparin infusion (P = not significant vs. baseline, respectively). Correspondingly, DHEA and androstenedione values were significantly elevated during lipid compared with saline infusion (P < 0.05, respectively), and areas under curve of both androgen precursors were significantly increased with lipid compared with saline infusion. Notably, all changes were detected before induction of insulin resistance. CONCLUSIONS: This study demonstrates that FFAs increase production of androgen precursors in vivo in men. These data tentatively suggest that hyperandrogenemia in polycystic ovary syndrome may be induced, at least in part, by elevated FFAs.

Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease.

Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P < 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P < 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

Glucagon inhibits ghrelin secretion in humans.

OBJECTIVE: It is well known that i.m. glucagon administration stimulates GH and cortisol release in humans, although the mechanisms are unclear. These effects are similar to those described for ghrelin on somatotroph and corticotroph function. The aim of the present study was to investigate the role of ghrelin in mediating the stimulatory effects of glucagon and to evaluate the effect of glucagon on ghrelin secretion. DESIGN AND METHODS: We studied the endocrine and metabolic response to i.m. glucagon administration in 24 subjects (14 men, 10 women; age 19-65 years; body mass index, 25.3 +/- 1 kg/m(2)), who were shown to have an intact anterior pituitary function as evaluated before enclosure. RESULTS: Serum ghrelin concentrations fell significantly at 30, 60, 120 and 180 min after glucagon administration (means +/- s.e.m.; baseline, 377.9 +/- 34.5 pg/ml; nadir, 294.6 +/- 28.3 pg/ml (60 min); P < 0.01). Conversely, i.m. glucagon elicited an increase in GH (baseline, 1.5 +/- 0.4 microg/l; peak, 14.2 +/- 2.7 microg/l (180 min); P < 0.01) and cortisol concentrations (baseline, 452.6 +/- 35.2 nmol/l; peak, 622.1 +/- 44 nmol/l (180 min); P < 0.01). The changes in ghrelin concentration at both 120 and 180 min were still significant after correction for glucose and insulin (P < 0.05). CONCLUSIONS: We show that i.m. glucagon decreases ghrelin significantly. Therefore, the already known stimulatory effects of i.m. glucagon on cortisol and GH are not mediated by a change in ghrelin concentrations. The mechanisms underlying the ghrelin suppression after i.m. glucagon are unlikely to include glucose or insulin variations and need to be further elucidated.

Insulin resistance and impaired insulin secretion due to phosphofructo-1-kinase-deficiency in humans.

UNLABELLED: The etiology of non-insulin-dependent diabetes mellitus (NIDDM) is usually explained as a combination of peripheral insulin resistance and impaired beta-cell function. Phosphofructo-1-kinase (PFK1) is a rate limiting enzyme in glycolysis, and its muscle subtype (PFK1-M) deficiency leads to an autosomal recessively inherited disorder known as glycogenosis type VII or Tarui's disease. It was evaluated whether PFK1-M deficiency leads to NIDDM in humans. A core family of four was evaluated for PFK1-M deficiency by DNA- and enzyme-activity-analyses. All members underwent oral and intravenous glucose tolerance test (oGTT/ivgtt), as well as an insulin sensitivity test (IST) using octreotide. RESULTS: Father (46 years, BMI 22.4 kg/m2) and older son (19 years, BMI 17.8 kg/m5) showed homozygous PFK1-M deficiency, while mother (47 years, BMI 28.4 kg/m5) and younger son (13 years, BMI 16.5 kg/m5) were shown to be heterozygously PFK1-M-deficient on enzyme activity levels. DNA analysis revealed an exon 5-missense-mutation at one allele of all four members, and an exon 22-frameshift-mutation at the other allele of the two homozygously affected individuals. By oGTT the father showed impaired glucose tolerance, and the mother clinical diabetes. By ivGTT both parents and the older son had a decreased first phase insulin secretion, and a diminished glucose disappearance rate. The IST showed marked insulin resistance in both parents and the older son, and moderate resistance in the younger son, previously not described. CONCLUSION: PFK1-M-deficiency leads to a metabolic state typical for early NIDDM in homozygously affected humans, especially concerning insulin resistance and loss of first phase beta-cell insulin secretion, and may contribute to the manifestation of NIDDM in a subgroup of patients.

Ca2+/calmodulin-dependent protein kinase II delta2 regulates gene expression of insulin in INS-1 rat insulinoma cells.

Ca(2+)/calmodulin-dependent protein kinase II is a member of a broad family of ubiquitously expressed Ca(2+) sensing serine/threonine-kinases. Ca(2+)/calmodulin-dependent protein kinase II is highly expressed in insulin secreting cells and is associated with insulin secretory granules and has been proposed to play an important role in exocytosis or in insulin granule transport to release sites. To elucidate its function the antisense sequence of the major beta-cell subtype, Ca(2+)/calmodulin-dependent protein kinase II delta(2), was stably expressed in INS-1 rat insulinoma cells. This caused a loss of Ca(2+)/calmodulin-dependent protein kinase II delta(2) expression at the mRNA and protein level, while the expression of the 95% homologous Ca(2+)/calmodulin-dependent protein kinase II gamma and of beta-cell specific proteins such as the homeodomain factor pancreatic-duodenal homeobox factor-1 (PDX-1, also referred to as islet/duodenum homeobox-1, IDX-1, insulin promoter factor-1, IPF-1 and somatostatin transactivating factor-1, STF-1), the glucagon-like peptide-1 (GLP-1) receptor and K(ATP)-channels K(IR)6.2/SUR-1 (sulfonylurea receptor-1) was not altered. Unexpectedly, the cells showed a large reduction of insulin gene expression, which was due to reduced insulin gene transcription. Electrophoretic mobility shift assays of PDX-1 binding to the insulin promoter A1 and E2/A3A4 elements showed additional bands indicating alterations of PDX-1 complex formation. Stable over expression of Ca(2+)/calmodulin-dependent protein kinase II delta(2), by contrast, was associated with elevated expression of insulin mRNA. Therefore, we conclude that Ca(2+)/calmodulin-dependent protein kinase II delta(2) links fuel-dependent increases in intracellular Ca(2+) concentrations to transcriptional regulation of genes related to the metabolic control of insulin secretion.

Insulinoma cells contain an isoform of Ca2+/calmodulin-dependent protein kinase II delta associated with insulin secretion vesicles.

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the beta3-, gamma-, delta2- and delta6-subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtypes was present. Antibodies generated against the bacterially expressed association domain of the delta2-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the delta2-subtype expressed in NIH3T3 fibroblasts was detected. The delta2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM kinase II delta2 thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion.

New isoforms of multifunctional calcium/calmodulin-dependent protein kinase II.

Calcium/calmodulin dependent protein kinase II (CaM kinase II) seems to act as an important regulator of intracellular signal transmission. Four subtypes, termed alpha to delta, have been cloned; some of them can exist as different splicing variants. All these isoforms share a great overall homology, and they contain 3 areas of low homology. We have identified 5 new variants of subtype delta so that the total number of different isoforms now adds up to 12. These variants are probably a result of different splicing and show several deletions in regard to subtype delta. The deletion sites do exactly match regions of low homology between the subtypes. This suggests a functional division of the CaM kinase II molecule into homologous and variable domains. The homologous domains are highly conserved. Therefore, it might be the case that the constitution of the variable domains is more significant for a certain isoform than its belonging to one of the 4 subtypes alpha to delta.

Effect of estradiol on insulin secreting INS-1 cells overexpressing estrogen receptors.

BACKGROUND: Estrogen has been shown to have profound effects on insulin and glucose metabolism in vivo. Indeed, estrogens were recently shown to modulate ion channel and secretory activities in endocrine cells. DESIGN AND METHODS: To investigate whether estrogenic influences are caused by direct effects on pancreatic beta-cells, we equipped INS-1 insulinoma cells with estrogen receptors and monitored insulin content and Ca(2+) fluxes as well as basal and stimulated insulin secretion upon different stimuli including glucose, the Ca(2+) ionophore ionomycin, the Ca(2+) channel agonist BayK8644, the protein kinase C activator TPA, and the adenylate cyclase activator forskolin. RESULTS AND CONCLUSION: Our data reveal that estradiol has no significant direct effect on proliferation rate, insulin content, basal and stimulated insulin output as well as Ca(2+) fluxes of insulin secreting cells in vitro, indicating that in vivo responses to estrogen on insulin and glucose metabolism result from indirect betacytotropic effects.