bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism by which metformin reduces glucose production in type 2 diabetes.
Authors: Hundal R S , Krssak M , Dufour S , Laurent D , Lebon V , Chandramouli V , Inzucchi S E , Schumann W C , Petersen K F , Landau B R , Shulman G I
Published: March 2007

To examine the mechanism by which metformin lowers endogenous glucose production in type 2 diabetic patients, we studied seven type 2 diabetic subjects, with fasting hyperglycemia (15.5 +/- 1.3 mmol/l), before and after 3 months of metformin treatment. Seven healthy subjects, matched for sex, age, and BMI, served as control subjects. Rates of net hepatic glycogenolysis, estimated by 13C nuclear magnetic resonance spectroscopy, were combined with estimates of contributions to glucose production of gluconeogenesis and glycogenolysis, measured by labeling of blood glucose by 2H from ingested 2H2O. Glucose production was measured using [6,6-2H2]glucose. The rate of glucose production was twice as high in the diabetic subjects as in control subjects (0.70 +/- 0.05 vs. 0.36 +/- 0.03 mmol x m(-2) min(-1), P < 0.0001). Metformin reduced that rate by 24% (to 0.53 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0009) and fasting plasma glucose concentration by 30% (to 10.8 +/- 0.9 mmol/l, P = 0.0002). The rate of gluconeogenesis was three times higher in the diabetic subjects than in the control subjects (0.59 +/- 0.03 vs. 0.18 +/- 0.03 mmol x m(-2) min(-1) and metformin reduced that rate by 36% (to 0.38 +/- 0.03 mmol x m(-2) x min(-1), P = 0.01). By the 2H2O method, there was a twofold increase in rates of gluconeogenesis in diabetic subjects (0.42 +/- 0.04 mmol m(-2) x min(-1), which decreased by 33% after metformin treatment (0.28 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0002). There was no glycogen cycling in the control subjects, but in the diabetic subjects, glycogen cycling contributed to 25% of glucose production and explains the differences between the two methods used. In conclusion, patients with poorly controlled type 2 diabetes have increased rates of endogenous glucose production, which can be attributed to increased rates of gluconeogenesis. Metformin lowered the rate of glucose production in these patients through a reduction in gluconeogenesis.
Longitudinal compensation for fat-induced insulin resistance includes reduced insulin clearance and enhanced beta-cell response.
Authors: Mittelman S D , Van Citters G W , Kim S P , Davis D A , Dea M K , Hamilton-Wessler M , Bergman R N
Published: March 2007

Central adiposity is highly correlated with insulin resistance, which is an important risk factor for type 2 diabetes and other chronic diseases. However, in normal individuals, central adiposity can be tolerated for many years without development of impaired glucose tolerance or diabetes. Here we examine longitudinally the mechanisms by which glucose tolerance can be maintained in the face of substantial insulin resistance. Normal dogs were fed a diet enriched with moderate amounts of fat (2 g x kg(-1) x day(-1)), similar to that seen in modern "cafeteria" diets, and the time course of metabolic changes in these animals was examined over 12 weeks. Trunk adiposity as assessed by magnetic resonance imaging increased from 12 to 19%, but body weight remained unchanged. Insulin sensitivity (SI) as determined by frequently sampled intravenous glucose tolerance tests was measured over a 12-week period. SI decreased 35% by week 1 and remained impaired for the entire 12 weeks. Intravenous glucose tolerance was reduced transiently for 1 week, recovered to baseline, and then again began to decline after 8 weeks. First-phase insulin response began to increase after week 2, peaked by week 6 (190% of basal), and then declined. The increase in insulin response was due partially to enhanced beta-cell function (22%) but due also to an approximately 50% reduction in insulin clearance. This compensation by insulin clearance was also confirmed with insulin clamps performed in fat-fed versus control dogs. The present study confirms the ability of the normal individual to compensate for fat-induced insulin resistance by enhanced insulin response, such that the product of insulin sensitivity x secretion is little changed. However, the compensation is due as much to reduced insulin clearance as increased beta-cell sensitivity to glucose. Reduced hepatic extraction of insulin may be the first line of defense providing a higher proportion of secreted insulin to the periphery and sparing the beta-cells during compensation for the insulin-resistant state.
Impaired in vivo stimulation of lipolysis in adipose tissue by selective beta2-adrenergic agonist in obese adolescent girls.
Authors: Enoksson S , Talbot M , Rife F , Tamborlane W V , Sherwin R S , Caprio S
Published: March 2007

Studies performed in adults with long-standing obesity suggest a reduced lipolytic sensitivity to catecholamines in subcutaneous abdominal adipose tissue (AT). We used microdialysis to study the in situ lipolytic effects of dobutamine (selective beta1-agonist) and terbutaline (selective beta2-agonist) on glycerol release (lipolytic index) in abdominal subcutaneous AT in 10 obese girls aged 13-17 years, BMI 38 +/- 2.1 kg/m2, and in 7 lean girls aged 11-17 years, BMI 21 +/- 1.1 kg/m2, and compared them with 10 obese women aged 21-39 years, BMI 36 +/- 1.6 kg/m2, and 10 lean women aged 18-42 years, BMI 21 +/- 0.4 kg/m2. Terbutaline at 10(-6) mol/l stimulated glycerol release more efficiently in lean girls than in obese girls (peak response approximately 350 vs. 150% of control, P < 0.01). At the lower concentration of agonist, no significant difference was seen. In women, terbutaline was more effective in lean than in obese women in stimulating glycerol release at both 10(-8) mol/l (peak response lean approximately 175% vs. obese 125% of control) and 10(-6) mol/l (approximately 300 vs. 150% of control, P < 0.05). No significant difference in glycerol release between obese and lean girls or women was detected with selective beta1-stimulation. Our data demonstrate a specific impairment in the capacity of beta2-adrenergic agonists to promote lipolysis in subcutaneous abdominal adipose tissue of obese adolescent girls and women. Thus, decreased mobilization of fat during activation of the adrenergic system might be present early in the development of adolescent obesity.
Exercise and thiazolidinedione therapy normalize insulin action in the obese Zucker fatty rat.
Authors: Hevener A L , Reichart D , Olefsky J
Published: March 2007

Thiazolidinediones and exercise are both known to improve insulin action independently. Therefore, we determined whether combined therapy could normalize insulin action in the Zucker fatty (ZF) rat. Rats were fed troglitazone as a 0.2% food admixture over a 3-week exercise training period (treadmill running 5 days/week, 20 m/min, 0% grade, 60 min/day). Subsequent to drug and/or exercise therapy, animals were chronically cannulated in the carotid artery (sampling) and jugular vein (infusion). After a 4-day recovery from surgery, animals were exposed to a hyperinsulinemic (40 mU x kg(-1) x min(-1)) euglycemic clamp (8.5 +/- 0.12 mmol/l; P = 0.45 between groups). Independently, exercise (n = 7) and troglitazone (n = 7) improved the glucose disposal rate 20% (P = 0.04) and 76% (P = 0.001), respectively, when compared with untreated ZF controls (n = 11). In combination, exercise and troglitazone therapy (n = 6) produced significant increments in the following: tracer-determined glucose disposal rate (combined therapy, 52.4 +/- 2.9 mg x kg(-1) x min(-1), vs. untreated ZF, 25.8 +/- 0.8 mg x kg(-1) x min(-1); P = 0.0001), total GLUT4 protein (twofold increase; P = 0.001), insulin receptor substrate (IRS)-1 protein (fourfold increase; P = 0.0001), and Akt phosphorylation (2.9-fold increase; P = 0.002). In conclusion, 1) exercise and troglitazone therapy each improved insulin action in the ZF rat, whereas the combination of the two led to complete normalization of insulin sensitivity, and 2) combination treatment also resulted in normalization of GLUT4 total protein, IRS-1 protein, and Akt phosphorylation compared with lean littermates.
Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation.
Authors: Sandu O A , Ragolia L , Begum N
Published: March 2007

Our laboratory has demonstrated that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity in vascular smooth muscle cells (VSMCs). In this study, we examined whether diabetes is accompanied by alterations in MBP activation and elucidated the components of the signaling pathway that mediate the effects of diabetes. VSMCs isolated from Goto-Kakizaki (GK) diabetic rats (a model for type 2 diabetes) exhibited marked impairment in MBP activation by insulin that was accompanied by failure of insulin to decrease the phosphorylation of a regulatory myosin-bound subunit (MBS) of MBP and inhibit Rho kinase activity resulting in increased myosin light-chain (MLC)20 phosphorylation and VSMC contraction. In VSMCs isolated from control rats, insulin inactivates Rho kinase and decreases MBS phosphorylation, leading to MBP activation. In addition to this pathway, insulin also appears to activate MBP by stimulating the phosphatidylinositol (PI) 3-kinase/nitric oxide (NO)/cGMP signaling pathway because treatment with the synthetic inhibitors of PI 3-kinase, NO synthase (NOS), and cGMP all blocked insulin's effect on MBP activation, whereas cGMP agonists and sodium nitroprusside (SNP) mimicked insulin's effect on MBP activation. VSMCs from diabetic GK rats exhibit reductions in insulin-mediated induction of inducible NOS protein expression and cGMP generation but normal MBP activation in response to SNP and cGMP agonist. This observation led us to examine the effect of diabetes on the activation status of the upstream insulin-signaling components. Although GK diabetes did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor or its content, insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was severely impaired. This was accompanied by marked reductions in IRS-1-associated PI 3-kinase activity. We conclude that insulin stimulates MBP via its regulatory subunit, MBS, by inactivating Rho kinase and stimulating NO/cGMP signaling via PI 3-kinase as part of a complex signaling network that controls MLC20 phosphorylation and VSMC contraction. Defective signaling via Rho kinase and the IRS-1/PI 3-kinase/NOS/cGMP pathway may mediate the inhibitory effects of hyperglycemia and diabetes on MBP activation in this experimental model.
A common promoter variant of the leptin gene is associated with changes in the relationship between serum leptin and fat mass in obese girls.
Authors: Le Stunff C , Le Bihan C , Schork N J , Bougnères P
Published: March 2007

Mutations in the leptin gene lead to rare obese syndromes of Mendelian inheritance in humans and rodents. However, no relevant mutations are found in the coding region of leptin gene DNA in patients with common multifactorial obesity. These obese patients tend to have an elevation of serum leptin proportional to their adiposity but with a rather wide dispersion of leptin levels for a given body fat content, which in part is attributable to sexual dimorphism. The current study, performed in two independent Caucasian cohorts of obese girls, shows that a frequent promoter variant of the leptin gene is associated with changes in the relationship between serum leptin and body fatness. Girls of comparable adiposity have different circulating leptin levels, depending on their genotype at this locus. Girls with the -/- Lep -2,549 genotype have 25% lower mean leptin levels than the girls with other genotypes, as reflected by differences in the regression slopes of leptin-to-fat mass. Therefore, genetic factors related to the leptin gene may be important in defining the set point of obese individuals (i.e., the circulating leptin level for a given degree of body fatness). This definition may be of both physiological and therapeutic relevance, although a phenotypic association with an individual single-nucleotide polymorphism is not sufficient to assign function to this particular nucleotide site.
The transferrin receptor defines two distinct contraction-responsive GLUT4 vesicle populations in skeletal muscle.
Authors: Lemieux K , Han X X , Dombrowski L , Bonen A , Marette A
Published: March 2007

Insulin and contraction increase glucose transport in an additive fashion in skeletal muscle. However, it is still unclear whether they do so by inducing the recruitment of GLUT4 transporters from the same or distinct intracellular compartments to the plasma membrane and the T-tubules. Using the transferrin receptor as a recognized marker of recycling endosomes, we have examined whether insulin and/or contraction recruit GLUT4 from this pool to either the plasma membranes or T-tubules, isolated by subcellular fractionation of perfused hindlimb muscles. Either stimulus independently increased GLUT4 translocation from an intracellular fraction to both the plasma membrane and T-tubules. The combination of insulin and contraction induced a marked (approximately threefold) and almost fully additive increase in GLUT4 content, but only in the plasma membrane. Insulin did not stimulate transferrin receptor recruitment from the GLUT4-containing intracellular fraction to either the plasma membrane or the T-tubules. In contrast, contraction stimulated the recruitment of the transferrin receptor from the same GLUT4-containing intracellular fraction to the plasma membrane but not to the T-tubules. Contraction-induced recruitment of the transferrin receptor was also observed from immunopurified GLUT4 vesicles. It is concluded that muscle contraction stimulates translocation of GLUT4 from two distinct intracellular compartments: 1) a population of recycling endosomes that is selectively recruited to the plasma membrane and 2) from GLUT4 storage vesicles that are also insulin-responsive and recruited to both the plasma membrane and the T-tubules. The lack of additive translocation of GLUT4 to the T-tubules may be linked to the failure of GLUT4-containing recycling endosomes to be recruited to these structures.
Insulin enhances the bradykinin response in L8 rat skeletal myoblasts.
Authors: Kudoh A , Dietze G J , Rabito S F
Published: March 2007

Inhibitors of ACE/kininase II enhance insulin sensitivity, an action that is mediated in part by bradykinin (BK). We investigated whether insulin interacts with the BK receptor signaling to modulate the inositol 1,4,5-trisphosphate (IP3) response to BK in L8 rat skeletal myoblasts. Stimulation of the cultures with BK (10 nmol/l) for 15 s increased IP3 from a basal level of 75.2 +/- 7.6 to 200.2 +/- 15.7 pmol/mg protein. Treatment of the cultures with 1, 2, and 20 nmol/l of insulin for 90 min before adding BK increased IP3 formation by the same BK dose to 328.2 +/- 19, 434.5 +/- 18, and 460.8 +/-21.3 pmol/mg protein, respectively. When wortmannin was administered to inhibit phosphatidylinositol (PI) 3-kinases at lower concentration (1 nmol/l), it increased IP3 formation stimulated by BK only when insulin was present. At a higher concentration (100 nmol/l), wortmannin significantly enhanced BK-induced IP3 formation in the absence of insulin. Genistein and tyrphostin A-23, tyrosine kinase inhibitors, completely reversed the elevated IP3 formation by BK and insulin. The IP3 response to 10 nmol/l BK was 223.3 +/- 11.8 pmol/mg protein in the absence of insulin and 402.2 +/- 12.0 pmol/mg protein in the presence of 2 nmol/l insulin. However, when exposing the cultures to 1 nmol/l genistein or tyrphostin A-23, the IP3 response to BK in the presence of insulin decreased to 211.8 +/- 46.7 and 187.7 +/- 19.9 pmol/mg protein. Tyrphostin A-1, the inactive analog, was ineffective. Exposing the cells to 1 micromol/ 3,4,5-trimethoxybenzoic acid 8-[diethylamino]octyl ester, an intracellular Ca2+ antagonist, did not change the potentiation by insulin. But, exposing them to 0.1 micromol/l n-[6-aminohexyl]-5-chloro-1-naphthalene-sulfonamide, a calmodulin antagonist, resulted in enhanced IP3 response to BK alone to 292.2 +/- 18.5 pmol/mg protein and to BK in the presence of 1, 2, and 20 nmol/l insulin to 488 +/- 22.2, 625.5 +/- 11.6, and 665.2 +/- 15.9 pmol/mg protein, respectively. In conclusion, insulin potentiates BK-induced IP3 production in L8 rat skeletal myoblasts, and this action of insulin involves a tyrosine kinase. Inhibition of PI 3-kinases potentiated BK-induced IP3 formation in the presence of insulin. Calmodulin blocked the action of insulin. These results support a modulatory effect of insulin on the BK signaling system via a tyrosine kinase in L8 rat skeletal myoblasts that results in increased IP3 formation. Because BK release from skeletal muscle increases during contractions, this action of insulin is likely to play a role in the modulation of the excitation-contraction coupling process of the skeletal muscle.
Early pattern of differentiation in the human pancreas.
Authors: Polak M , Bouchareb-Banaei L , Scharfmann R , Czernichow P
Published: March 2007

In the early human embryonic/fetal pancreas, we studied 1) the ontogenetic pattern of the endocrine cells and the evolution of the endocrine mass, and 2) the morphogenetic pattern of development and, more precisely, the complex relationship of the epithelial mass with the surrounding mesenchyme. We studied 15 pancreases between 7 and 11 weeks of development (WD) by double immunohistochemistry. Epithelial cells in these pancreatic anlage were detected by cytokeratin staining, and differentiated endocrine cells were detected by insulin, glucagon, somatostatin, and pancreatic polypeptide staining. Proliferation was quantified using a nuclear marker, the Ki-67 antibody. At this early stage, the pancreas is made up of an epithelial mass composed of central ducts intermingled with a loose mesenchyme and peripheral ducts surrounded by a dense peripancreatic mesenchyme. Hormone-containing cells appear in the epithelium at 8 WD. Newly differentiated endocrine cells coexpress insulin, glucagon, and somatostatin; endocrine differentiation starts within the central ducts of the epithelial mass, at a distance from the dense peripancreatic surrounding mesenchyme. The fraction of the primitive endocrine cells undergoing proliferation is low (5% of the insulin cells at 8 WD, 3% at 11 WD), which is in favor of massive differentiation as the major mechanism for increasing endocrine mass. By contrast, the nonendocrine epithelial cells have a higher rate of proliferation; the epithelial cells in contact with the dense peripancreatic surrounding mesenchyme show more proliferation activity than those within the central part of the epithelial mass (at 11 WD, labeling index: periphery 65% vs. center 15%, P < 0.001). In conclusion, the patterns of endocrine differentiation and epithelial proliferation observed within the human pancreas early in development suggest that the mesenchyme plays a role in these phenomena.
Beta-cell transcription factors and diabetes: no evidence for diabetes-associated mutations in the hepatocyte nuclear factor-3beta gene (HNF3B) in Japanese patients with maturity-onset diabetes of the young.
Authors: Hinokio Y , Horikawa Y , Furuta H , Cox N J , Iwasaki N , Honda M , Ogata M , Iwamoto Y , Bell G I
Published: March 2007

Mutations in the transcription factors hepatocyte nuclear factor (HNF)-4alpha and -1alpha, insulin promoter factor-1, and HNF-1beta are the causes of four forms of maturity-onset diabetes of the young (MODY1 and 3-5, respectively). The winged-helix transcription factor HNF-3beta has been implicated in the regulation of expression of each of these MODY genes, suggesting that mutations in the HNF-3beta gene (HNF3B) may also cause MODY. We have tested this hypothesis by screening a panel of 57 unrelated Japanese subjects with a clinical diagnosis of MODY for mutations in HNF3B. This analysis revealed four frequent polymorphisms that were not associated with MODY, including one in the promoter region (-213A/G), two silent mutations in the codons for Ala 97 (291C/T) and Gly 279 (837A/G), and one in the 3'-untranslated region (1424C/T). Two rare substitutions in the 5'-untranslated region, -156C/T and -67A/C, were found in a heterozygous state in two subjects, and two subjects were heterozygous for putative missense mutations, S109N (326G > A) and A328V (983C>T). The two missense mutations were not found in 106 normal chromosomes from nondiabetic subjects. It was not possible to test for co-segregation of these mutations with diabetes and thus, it is unclear whether or not these mutations can cause MODY. The results of our study suggest that mutations in HNF3B are not a common cause of MODY in Japanese subjects.
Genetic variation in the hepatocyte nuclear factor-3beta gene (HNF3B) does not contribute to maturity-onset diabetes of the young in French Caucasians.
Authors: Abderrahmani A , Chèvre J C , Otabe S , Chikri M , Hani E H , Vaxillaire M , Hinokio Y , Horikawa Y , Bell G I , Froguel P
Published: March 2007

Mutations in genes encoding hepatocyte nuclear factor (HNF) are responsible for three of the five subtypes of maturity-onset diabetes of the young (MODY). This observation and molecular studies indicate that the HNF network is required for normal function of pancreatic beta-cells. This suggests that transcription factors involved in this complex network are candidates for genetic defects in MODY. Because the HNF-3beta gene is implicated in this network, we screened it for mutations in 21 probands of French ancestry with clinical diagnosis of MODY and early-onset type 2 diabetes. All of the five known MODY genes, HNF-4alpha, glucokinase, HNF-1alpha, HNF-1beta, and IPF1, were previously excluded as being the cause of diabetes in these families. By direct sequencing, we identified two transitions, an A-to-G at position -213 and a C-to-T at position -63 in the promoter and exon 1, respectively, of the HNF-3beta gene. A G-to-C transversion at position +32 in the intron 1 and three transitions, C-to-T at position 291, A-to-G at position 837, and G-to-A at position 1188 in the exon 3, resulting in noncoding mutations Ala97Ala, Gly279Gly, and Gln396Gln, respectively, were also identified. The allele frequencies were not significantly different between a control group and MODY probands. Familial segregation studies and linkage analysis showed that genetic variation in the HNF-3beta gene is unlikely to be the cause of early-onset type 2 diabetes in these Caucasian families.
Interleukin-1beta-induced alteration in a beta-cell phenotype can reduce cellular sensitivity to conditions that cause necrosis but not to cytokine-induced apoptosis.
Authors: Ling Z , Van de Casteele M , Eizirik D L , Pipeleers D G
Published: March 2007

Previous work has shown that interleukin-1beta (IL-1beta) alters protein expression in beta-cells. This alteration is associated with cell death in isolated rat islets but not in isolated rat beta-cells. We examined whether IL-1beta pretreatment of isolated beta-cells influences their sensitivity to toxic agents. After a 24-h culture with IL-1beta (30 U/ml), beta-cells exhibited a lower expression of the beta-cell-specific protein transcription factor pancreatic and duodenal homeobox gene (PDX)-1, glucose transporter GLUT2, and proinsulin convertase PC2, with a marked reduction (60-70%) in glucose-induced insulin production and selective sensitivity to the toxins alloxan (ALX) and streptozotocin (STZ). On the other hand, the cells presented an increased expression of Mn-superoxide dismutase, heat shock protein 70, inducible heme oxygenase, and inducible nitrite oxide synthase. This IL-1beta-induced alteration in beta-cell phenotype resulted in a reduced cellular sensitivity to the beta-cell-specific toxins ALX and STZ; the production of nontoxic conditions of nitric oxide (NO) also rendered the cells less susceptible to radical-induced damage. Exposure to IL-1beta can thus protect beta-cells against conditions that cause necrosis; however, it did not protect against apoptosis induced by the additional presence of interferon-gamma or tumor necrosis factor-alpha. Release of IL-1beta in the endocrine pancreas is thus not necessarily the cause of massive NO-dependent beta-cell destruction. On the contrary, IL-1beta may protect these cells against necrosis, though with a loss of their characteristic phenotype and homeostatic functions.
Zinc as a paracrine effector in pancreatic islet cell death.
Authors: Kim B J , Kim Y H , Kim S , Kim J W , Koh J Y , Oh S H , Lee M K , Kim K W , Lee M S
Published: March 2007

Because of a huge amount of Zn2+ in secretory granules of pancreatic islet beta-cells, Zn2+ released in certain conditions might affect the function or survival of islet cells. We studied potential paracrine effects of endogenous Zn2+ on beta-cell death. Zn2+ induced insulinoma/islet cell death in a dose-dependent manner. Chelation of released endogenous Zn2+ by CaEDTA significantly decreased streptozotocin (STZ)-induced islet cell death in an in vitro culture system simulating in vivo circumstances but not in the conventional culture system. Zn2+ chelation in vivo by continuous CaEDTA infusion significantly decreased the incidence of diabetes after STZ administration. N-(6-methoxy-quinolyl)-para-toluene-sulfonamide staining revealed that Zn2+ was densely deposited in degenerating islet cells 24 h after STZ treatment, which was decreased by CaEDTA infusion. We show here that Zn2+ is not a passive element for insulin storage but an active participant in islet cell death in certain conditions, which in time might contribute to the development of diabetes in aged people.
Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets.
Authors: Iismaa T P , Kerr E A , Wilson J R , Carpenter L , Sims N , Biden T J
Published: March 2007

Expression of muscarinic receptors in rat islets, RINm5F cells, and INS-1 cells was established by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by RNase protection. Both methods indicated that m3 and m1 receptors were expressed approximately equally in the various cellular preparations and to a much greater extent than the m5 subtype. However, the cell lines, especially RINm5F cells, expressed less of a given receptor subtype than did islets. Immunohistochemistry indicated that m3 receptors were expressed throughout the islet core. Binding studies using the radiolabeled muscarinic receptor antagonist QNB demonstrated a maximal binding capacity of INS-1 cells of 23.0+/-2.9 fmol/mg protein. Functional analyses were undertaken using INS-1 cells stably transfected with either m1 or m3 receptor cDNAs. Overexpression of either receptor did not affect basal responses but markedly enhanced maximal responses to the muscarinic receptor agonist carbachol. Although maximal hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) was twofold greater in m1-transfectants as compared with m3-transfectants, cell lines overexpressing either receptor gave essentially equivalent secretory responses to a full range of carbachol doses. The results demonstrate that both m1 and m3 muscarinic receptors are well expressed in pancreatic beta-cells, functionally linked to signaling pathways, and capable of initiating insulin secretion with equal potencies.
Type 2 diabetes and low birth weight: the role of paternal inheritance in the association of low birth weight and diabetes.
Authors: Lindsay R S , Dabelea D , Roumain J , Hanson R L , Bennett P H , Knowler W C
Published: March 2007

Lower birth weight is associated with an increased occurrence of type 2 diabetes in later life. Whether this relationship is explained by environmental or genetic factors is unknown. We have examined the potential for genetic influences by determining whether parental diabetes is associated with lower birth weight in 1,608 children of known birth weight and gestational age born between 1941 and 1993 in the Gila River Indian Community in Arizona. The previously described relationships of maternal diabetes to increased birth weight and offspring diabetes were observed. In contrast to this we have determined novel relationships between low birth weight and paternal diabetes. The offspring of diabetic fathers were, on average, 78 g lighter than the offspring of nondiabetic fathers. For fathers, lower birth weight in their offspring was associated with an increased risk of later diabetes, i.e., fathers of offspring in the lowest quintile of birth weight, who were not diabetic at the time of birth of their child, had a 1.8-fold increased risk of developing diabetes later in life (95% CI 1.2-2.7; P = 0.004). For children, lower birth weight predicted diabetes in the offspring if paternal but not maternal diabetes was present, but it was not associated with higher plasma glucose if neither parent had diabetes. We conclude that the risk of diabetes associated with low birth weight is strongly related to the development of paternal diabetes, suggesting a genetic link between lower birth weight and later diabetes.
Early glomerular macrophage recruitment in streptozotocin-induced diabetic rats.
Authors: Sassy-Prigent C , Heudes D , Mandet C , Bélair M F , Michel O , Perdereau B , Bariéty J , Bruneval P
Published: March 2007

Diabetic glomerulosclerosis is defined by increased glomerular extracellular matrix (ECM) that is mainly synthesized by mesangial cells that underwent an activation mediated by cytokines and growth factors from various cellular origins. In this study, we tested whether macrophages could infiltrate the glomeruli and influence ECM synthesis in experimental diabetes. To test our hypothesis, we initially studied the dynamics of glomerular macrophage recruitment in streptozotocin-induced diabetic rats at days 1, 2, 4, 8, 15, and 30 by using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on isolated glomeruli and immunohistochemistry and morphometry. We then assessed the role of macrophages on the basis of the pharmacological modulation of their recruitment by insulin or ACE inhibitor treatments and by X-irradiation-induced macrophage depletion at days 8 and 30. Macrophages were recruited within the glomeruli at the very early phase of hyperglycemia by using RT-PCR CD14 detection from day 2 and by using ED1 immunohistochemistry from day 8. This glomerular macrophage infiltration was associated with an increase in alpha1-chain type IV collagen mRNA. In parallel, the diabetic glomeruli became hypertrophic with an increase in the mesangial area. Macrophage recruitment was preceded by or associated with an increased glomerular expression of vascular cell adhesion molecule 1, intracellular adhesion molecule 1, and monocyte chemoattractant protein 1, which contributes to monocyte diapedesis. Glomerular interleukin-1beta mRNA synthesis was also enhanced as early as day 1 and could be involved in the increase in ECM and adhesion molecule gene expressions. Insulin treatment and irradiation-induced macrophage depletion completely prevented the glomerular macrophage recruitment and decreased alpha1-chain type IV collagen mRNA and mesangial area in diabetic rats, whereas ACE inhibitor treatment had an incomplete effect. It can be concluded that in the streptozotocin model, hyperglycemia is followed by an early macrophage recruitment that contributes to the molecular and structural events that could lead to glomerulosclerosis. Therefore, besides direct stimulation of mesangial cells by hyperglycemia, macrophages recruited in the glomeruli during the early phase of hyperglycemia could secondarily act on mesangial cells.
Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism.
Authors: Hayashi T , Hirshman M F , Fujii N , Habinowski S A , Witters L A , Goodyear L J
Published: March 2007

5'AMP-activated protein kinase (AMPK) can be activated in response to cellular fuel depletion and leads to switching off ATP-consuming pathways and switching on ATP-regenerating pathways in many cell types. We have hypothesized that AMPK is a central mediator of insulin-independent glucose transport, which enables fuel-depleted muscle cells to take up glucose for ATP regeneration under conditions of metabolic stress. To test this hypothesis, rat epitrochlearis muscles were isolated and incubated in vitro under several conditions that evoke metabolic stress accompanied by intracellular fuel depletion. Rates of glucose transport in the isolated muscles were increased by all of these conditions, including contraction (5-fold above basal), hypoxia (8-fold), 2,4-dinotrophenol (11-fold), rotenone (7-fold), and hyperosmolarity (8-fold). All of these stimuli simultaneously increased both alpha1 and alpha2 isoform-specific AMPK activity. There was close correlation between alpha1 (r2 = 0.72) and alpha2 (r2 = 0.67) AMPK activities and the rate of glucose transport, irrespective of the metabolic stress used, all of which compromised muscle fuel status as judged by ATP, phosphocreatine, and glycogen content. 5-Aminoimidazole-4-carboxamide ribonucleoside, a pharmacological AMPK activator that is metabolized to an AMP-mimetic ZMP, also increased both glucose transport and AMPK activity but did not change fuel status. Insulin stimulated glucose transport by 6.5-fold above basal but did not affect AMPK activity. These results suggest that the activation of AMPK may be a common mechanism leading to insulin-independent glucose transport in skeletal muscle under conditions of metabolic stress.
Immunohistochemical and ultrastructural localization of leptin and leptin receptor in human white adipose tissue and differentiating human adipose cells in primary culture.
Authors: Bornstein S R , Abu-Asab M , Glasow A , Päth G , Hauner H , Tsokos M , Chrousos G P , Scherbaum W A
Published: March 2007

Leptin is mainly produced in white adipose tissue and acts both at distant sites and locally at the tissue from which it originates. The cellular and subcellular localization of leptin and its receptor (Ob-receptor [Ob-R]) and their relationship to various stages of fat cell maturation have not been characterized as yet. Therefore, we analyzed leptin and Ob-R by using reverse transcriptase-polymerase chain reaction, immunohistochemistry, and ultrastructural immunogold labeling in human white adipose tissue and in human adipocyte cell cultures at early and late stages of differentiation. Both leptin and its receptor were present in mature unilocular fat cells. The thin cytoplasmic rim of the adipocytes exhibited the strongest expression of both leptin and Ob-R. At early stages of differentiating human adipocytes, leptin was mainly expressed in multilocular preadipocytes, whereas the Ob-R was found predominantly on fibroblast-like cells. Other cellular components of human white adipose tissue were characterized by anti-CD31 for endothelial cells, anti-CD68 for macrophages, and antibodies specifically labeling B-cells and T-cells. In addition to fat cells, endothelial cells were immunopositive for the full-length leptin receptor. On the ultrastructural level, leptin was mainly found attached to cellular membranes and in small alveolate vesicle-like structures in the cytoplasm of adipocytes. Leptin was also present on the cell membranes of endothelial cells and macrophages. We conclude that the expression of the Ob-R in human white adipose tissue is not restricted to adipocytes but is present in resident endothelial and immune cells. Ultrastructural localization studies revealed an association of leptin with cell membranes and small vesicles. The cellular and subcellular distribution of leptin and its receptor suggests an important autocrine and paracrine role for leptin in human adipose tissue.
Differential activation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosiglitazone.
Authors: Camp H S , Li O , Wise S C , Hong Y H , Frankowski C L , Shen X , Vanbogelen R , Leff T
Published: March 2007

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma and exert their antihyperglycemic effects by regulation of PPAR-gamma-responsive genes. We report here that PPAR-gamma activation by troglitazone depends on the experimental setting. Troglitazone acts as a partial agonist for PPAR-gamma in transfected muscle (C2C12) and kidney (HEK 293T) cells, producing a submaximal transcriptional response (1.8- to 2.5-fold activation) compared with rosiglitazone (7.4- to 13-fold activation). Additionally, troglitazone antagonizes rosiglitazone-stimulated PPAR-gamma transcriptional activity. Limited protease digestion of PPAR-gamma suggests conformational differences in the receptor bound to troglitazone versus rosiglitazone. Consistent with this finding, an in vitro coactivator association assay demonstrated that troglitazone-bound PPAR-gamma recruited the transcriptional coactivators p300 and steroid receptor coactivator 1 less efficiently than rosiglitazone-bound receptor. In contrast to these observations, troglitazone behaves as a full agonist of PPAR-gamma in 3T3L1 adipocytes. Two-dimensional protein gel electrophoresis demonstrated that troglitazone and rosiglitazone regulated distinct but overlapping sets of genes in several cell types. Thus, troglitazone may behave as a partial agonist under certain physiological circumstances and as a full agonist in others. These differences could be caused by variations in the amount of specific cofactors, differences in PPAR response elements, or the presence of different isoforms of PPAR-gamma.
Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf-1.
Authors: Heimberg H , Bouwens L , Heremans Y , Van De Casteele M , Lefebvre V , Pipeleers D
Published: March 2007

The homeodomain transcription factor encoded by the pancreatic and duodenal homeobox gene-1 (Ipf-1) is essential for pancreatic ontogenesis. Whether Ipf-1 is also involved in the neogenesis of beta-cells in the adult pancreas is unknown. We examined whether Ipf-1 is expressed in adult human pancreatic ducts, which are thought to generate new beta-cells. In tissue sections, virtually all duct cells were immunopositive for Ipf-1, as were the islet beta-cells but not the acinar cells. After isolation and culture, both duct and islet cell preparations contained the Ipf-1 immunoreactive proteins p42 and p45 (42 and 45 kDa, respectively) in similar proportions, but the expression levels were twofold lower in duct cells. After 4 h of labeling, the endocrine cells exhibited a sevenfold higher phosphorylation of p42 than the duct cells, whereas p45 was phosphorylated only in endocrine cells. Homeobox binding transcription factor complexes with Ipf-1 in duct cells differed from those in endocrine cells in terms of gel mobility, sequence specificity, and affinity. The observed similarities in Ipf-1 expression by adult human pancreatic duct cells and endocrine cells may reflect their common ontogenic origin, whereas the differences in Ipf-1 phosphorylation and complex formation may correlate with their divergent differentiation.
Validation of methods for measurement of insulin secretion in humans in vivo.
Authors: Kjems L L , Christiansen E , Vølund A , Bergman R N , Madsbad S
Published: March 2007

To detect and understand the changes in beta-cell function in the pathogenesis of type 2 diabetes, an accurate and precise estimation of prehepatic insulin secretion rate (ISR) is essential. There are two common methods to assess ISR, the deconvolution method (by Eaton and Polonsky)-considered the "gold standard"-and the combined model (by Vølund et al.). The deconvolution method is a 2-day method, which generally requires separate assessment of C-peptide kinetics, whereas the combined model is a single-day method that uses insulin and C-peptide data from a single test of interest. The validity of these mathematical techniques for quantification of insulin secretion have been tested in dogs, but not in humans. In the present studies, we examined the validity of both methods to recover the known infusion rates of insulin and C-peptide mimicking ISR during an oral glucose tolerance test. ISR from both the combined model and the deconvolution method were accurate, i.e., recovery of true ISR was not significantly different from 100%. Furthermore, both maximal and total ISRs from the combined model were strongly correlated to those obtained by the deconvolution method (r = 0.89 and r = 0.82, respectively). These results indicate that both approaches provide accurate assessment of prehepatic ISRs in type 2 diabetic patients and control subjects. A simplified version of the deconvolution method based on standard kinetic parameters for C-peptide (Van Cauter et al.) was compared with the 2-day deconvolution method, and a close agreement was found for the results of an oral glucose tolerance test. We also studied whether C-peptide kinetics are influenced by somatostatin infusion. The decay curves after bolus injection of exogenous biosynthetic human C-peptide, the kinetic parameters, and the metabolic clearance rate were similar whether measured during constant peripheral somatostatin infusion or without somatostatin infusion. Assessment of C-peptide kinetics can be performed without infusion of somatostatin, because the endogenous insulin concentration remains constant. Assessment of C-peptide kinetics with and without infusion of somatostatin results in nearly identical secretion rates for insulin during an oral glucose tolerance test.
Genetic modifiers of the insulin resistance phenotype in mice.
Authors: Kido Y , Philippe N , Schäffer A A , Accili D
Published: March 2007

Insulin resistance can result from genetic interactions among susceptibility alleles. To identify genetic loci predisposing to insulin resistance, we used crosses between different strains of mice with a targeted null allele of the insulin receptor gene. On the genetic background of B6 mice, the insulin receptor gene mutation causes mild hyperinsulinemia. In contrast, on the genetic background of 129/Sv mice, the same mutation causes severe hyperinsulinemia, suggesting that the 129/Sv strain harbors alleles that interact with the insulin receptor mutation and predispose to insulin resistance. As a first step to identify these alleles, we generated an F2 intercross between insulin receptor heterozygous mutant mice on B6 and 129/Sv backgrounds (B6IR x 129IR) and performed a genome-wide scan with polymorphic markers at a 20-cM resolution. We report the identification of loci on chromosomes 2 (logarithm of odds [LOD] 5.58) and 10 (LOD 5.58) that show significant evidence for linkage to plasma insulin levels as a quantitative trait. These findings indicate that targeted mutations in knockout mice can be used to unravel the complex genetic interactions underlying insulin resistance.
Mechanical stretch-induced fibronectin and transforming growth factor-beta1 production in human mesangial cells is p38 mitogen-activated protein kinase-dependent.
Authors: Gruden G , Zonca S , Hayward A , Thomas S , Maestrini S , Gnudi L , Viberti G C
Published: March 2007

Hemodynamic abnormalities are important in the pathogenesis of the excess mesangial matrix deposition of diabetic and other glomerulopathies. p38-Mitogen-activated protein (MAP) kinase, an important intracellular signaling molecule, is activated in the glomeruli of diabetic rats. We studied, in human mesangial cells, the effect of stretch on p38 MAP kinase activation and the role of p38 MAP kinase in stretch-induced fibronectin and transforming growth factor-beta1 (TGF-beta1) accumulation. p38 MAP kinase was activated by stretch in a rapid (11-fold increase at 30 min, P < 0.001) and sustained manner (3-fold increase at 33 h, P < 0.001); this activation was mediated by protein kinase C (PKC). Stretch-induced fibronectin and TGF-beta1 protein levels were completely abolished (100% inhibition, P < 0.001; and 92% inhibition, P < 0.01, respectively) by SB203580, a specific p38 MAP kinase inhibitor. At 33 h, TGF-beta1 blockade did not affect stretch-induced fibronectin production, but partially prevented stretch-induced p38 MAP kinase activation (59% inhibition, P < 0.05). TGF-beta1 induced fibronectin accumulation after 72 h of exposure via a p38 MAP kinase-dependent mechanism (30% increase over control, P < 0.01). In human mesangial cells, stretch activates, via a PKC-dependent mechanism, p38 MAP kinase, which independently induces TGF-beta1 and fibronectin. In turn, TGF-beta1 contributes to maintaining late p38 MAP kinase activation, which perpetuates fibronectin accumulation.
Glucose-regulated anaplerosis and cataplerosis in pancreatic beta-cells: possible implication of a pyruvate/citrate shuttle in insulin secretion.
Authors: Farfari S , Schulz V , Corkey B , Prentki M
Published: March 2007

The hypothesis proposing that anaplerosis and cataplerosis play an important role in fuel signaling by providing mitochondrially derived coupling factors for stimulation of insulin secretion was tested. A rise in citrate coincided with the initiation of insulin secretion in response to glucose in INS-1 beta-cells. The dose dependence of glucose-stimulated insulin release correlated closely with those of the cellular contents of citrate, malate, and citrate-derived malonyl-CoA. The glucose-induced elevations in citrate, alpha-ketoglutarate, malonyl-CoA, and the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium reduction state, an index of beta-cell metabolic activity, were unaffected by the Ca2+ chelator EGTA. Glucose induced a rise in both mitochondrial and cytosolic citrate and promoted efflux of citrate from the cells. The latter amounted to approximately 20% of glucose carbons entering the glycolytic pathway. Phenylacetic acid, a pyruvate carboxylase inhibitor, reduced the glucose-induced rise in citrate in INS-1 cells and insulin secretion in both INS-1 cells and rat islets. The results indicate the feasibility of a pyruvate/citrate shuttle in INS-1 beta-cells, allowing the regeneration of NAD+ in the cytosol and the formation of cytosolic acetyl-CoA, malonyl-CoA, and NADPH. The data suggest that anaplerosis and cataplerosis are early signaling events in beta-cell activation that do not require a rise in Ca2+. It is proposed that citrate is a signal of fuel abundance that contributes to beta-cell activation in both the mitochondrial and cytosolic compartments and that a major fate of anaplerotic glucose carbons is external citrate.