Clinical importance of insulin secretion and its interaction with insulin resistance in the treatment of type 2 diabetes mellitus and its complications.

Type 2 diabetes primarily develops from pathogenic defects in the mechanisms of insulin secretion and hepatic and peripheral insulin action. The consequent disruption of normal glucose metabolism involves a number of organ systems and is ultimately manifested in fasting and daytime hyperglycemia. Chronically elevated blood glucose concentrations determine the progression of the disease by further exacerbating insulin resistance and causing beta-cell exhaustion in addition to decreasing their responsiveness to glucose. The beta-cell secretory dysfunction is characterized by the lack of the early phase of glucose-induced insulin secretion and the insufficient and delayed late phase of secretion. Glycemic levels in patients with type 2 diabetes are directly related to the risk of developing microvascular and macrovascular complications, the main cause of the morbidity and mortality associated with this disease. The goal of treatment is to decrease the risk and delay the progression of these complications by improving glycemic control. Current oral antidiabetic agents, used as monotherapy or in combination, include traditional insulin secretagogues, insulin sensitizers and inhibitors of carbohydrate absorption. A greater understanding of the pathophysiology of type 2 diabetes and recent findings on the significance of meal-related glycemia to overall glycemic control are expanding the therapeutic options for treating this disease.

AP-1 and Cbfa/runt physically interact and regulate parathyroid hormone-dependent MMP13 expression in osteoblasts through a new osteoblast-specific element 2/AP-1 composite element.

The expression of MMP13 (collagenase-3), a member of the matrix metalloproteinase family, is increased in vivo as well as in cultured osteosarcoma cell lines by parathyroid hormone (PTH), a major regulator of calcium homeostasis. Binding sites for AP-1 and Cbfa/Runt transcription factors in close proximity have been identified as cis-acting elements in the murine and rat mmp13 promoter required for PTH-induced expression. The cooperative function of these factors in response to PTH in osteoblastic cells suggests a direct interaction between AP-1 and Cbfa/Runt transcription factors. Here, we demonstrate interaction between c-Jun and c-Fos with Cbfa/Runt proteins. This interaction depends on the leucine zipper of c-Jun or c-Fos and the Runt domain of Cbfa/Runt proteins, respectively. Moreover, c-Fos interacts with the C-terminal part of Cbfa1 and Cbfa2, sharing a conserved transcriptional repression domain. In addition to the distal osteoblast-specific element 2 (OSE2) element in the murine and rat mmp13 promoter, we identified a new proximal OSE2 site overlapping with the TRE motif. Both interaction of Cbfa/Runt proteins with AP-1 and the presence of a functional proximal OSE2 site are required for enhanced transcriptional activity of the mmp13 promoter in transient transfected fibroblasts and in PTH-treated osteosarcoma cells.

beta-cell dysfunction and failure in type 2 diabetes: potential mechanisms.

Type 2 diabetes is characterized by a progressive loss of beta-cell function throughout the course of the disease. The pattern of loss is an initial defect in early or first-phase insulin secretion, followed by a decreasing maximal capacity of glucose to potentiate all nonglucose signals. Last, a defective steady-state and basal insulin secretion develops, leading to complete beta-cell failure requiring insulin treatment. This functional loss exceeds the expected impact of a 20-50% loss of beta-cells reported at autopsy, which has been associated with amyloid deposits. This review summarizes the nature of the amyloid deposition process and its association with disproportionate hyperproinsulinemia. It reviews recent studies in IAPP (islet-amyloid polypeptide, or amylin) transgenic mice developing islet amyloid deposits and hyperglycemia to suggest that the process of amyloid fibril formation impairs function early and leads to beta-cell failure and eventual death. Based on the known association of amyloid deposits and relative hyperproinsulinemia, it is hypothesized that fibril formation begins during impaired glucose tolerance after other factors cause the initial defects in early insulin secretion and insulin action. Thus, the process that leads to beta-cell loss is implicated in the deposition of amyloid and the late unrelenting progressive hyperglycemia now found in all patients despite current therapies.

Obesity, body fat distribution, insulin sensitivity and Islet beta-cell function as explanations for metabolic diversity.

Studies of metabolic processes have been enhanced by our understanding of the relationships among obesity, body fat distribution, insulin sensitivity and islet beta-cell function. Thus, we have learned that although insulin resistance is usually associated with obesity, even lean subjects can be insulin resistant due to the accumulation of visceral fat. Insulin sensitivity and beta-cell function are also intimately linked. The hyperbolic relationship between these two parameters explains why insulin-resistant individuals have markedly enhanced insulin responses, whereas subjects who are insulin sensitive exhibit very low responses. Failure to take into account this relationship will lead to erroneous conclusions. By accounting for this important interaction, it has been clearly demonstrated that subjects at high risk of developing type 2 diabetes (older individuals, women with a history of gestational diabetes or polycystic ovary syndrome, subjects with impaired glucose tolerance and first-degree relatives of individuals with type 2 diabetes) have impaired beta-cell function. Furthermore, the progression from normal glucose tolerance to impaired glucose tolerance and type 2 diabetes is associated with declining insulin secretion.

Expression of collagenase-3 (MMP-13) in c-fos-induced osteosarcomas and chondrosarcomas is restricted to a subset of cells of the osteo-/chondrogenic lineage.

The interstitial collagenases have been suggested to play a critical role in bone formation, remodeling, and cancerogenesis. We have previously shown that during mouse development expression of collagenase-3 (MMP-13) is restricted to bone and cartilage (Gack et al., 1995; Tuckermann et al., 2000) and is affected in mice with altered c-Fos and Cbfa-1 expression (Gack et al., 1994; Porte et al., 1999). In this study, using immunohistochemistry (IHC) and in situ hybridization (ISH) techniques, we have identified cells of the osteoblastic lineage to be the origin of strongly enhanced levels of MMP-13 transcripts in c-fos-induced osteosarcomas. Expression in these cells is further increased in c-fos/c-jun double transgenic mice and paralleled by Cbfa-1 expression. Similarly, in spontaneous and radiation-induced osteosarcomas, both c-Fos and MMP-13 proteins are detectable, suggesting that overexpression of both genes is a characteristic feature of osteosarcomas of different origin. We also observed high levels of MMP-13 in c-Fos-induced chondrosarcomas. In osteoblast-like cells and in cells of late chondrocyte differentiation such as hypertrophic chondrocytes, high levels of MMP-13 transcripts were found. In contrast, in anaplastic areas of the tumors representing highly proliferating chondrocytes, no MMP-13 expression is detectable, suggesting that in addition to Fos/AP-1, bone-specific transcription factors are responsible for restricted expression of collagenase-3/MMP-13 in a specific subset of cells of bone and cartilage in physiology and pathology.

Central nervous system control of food intake.

New information regarding neuronal circuits that control food intake and their hormonal regulation has extended our understanding of energy homeostasis, the process whereby energy intake is matched to energy expenditure over time. The profound obesity that results in rodents (and in the rare human case as well) from mutation of key signalling molecules involved in this regulatory system highlights its importance to human health. Although each new signalling pathway discovered in the hypothalamus is a potential target for drug development in the treatment of obesity, the growing number of such signalling molecules indicates that food intake is controlled by a highly complex process. To better understand how energy homeostasis can be achieved, we describe a model that delineates the roles of individual hormonal and neuropeptide signalling pathways in the control of food intake and the means by which obesity can arise from inherited or acquired defects in their function.

Insulin and leptin: dual adiposity signals to the brain for the regulation of food intake and body weight.

Insulin and leptin are hypothesized to be 'adiposity signals' for the long-term regulation of body weight by the brain. Accordingly, a change in the plasma levels of leptin or insulin indicates a state of altered energy homeostasis and adiposity, and the brain responds by adjusting food intake to restore adipose tissue mass to a regulated level. The candidate site for the brain's detection of leptin adiposity signaling is the hypothalamic arcuate nucleus, where leptin inhibits expression neuropeptide Y and increases expression of the pro-opiomelanocortin (POMC) precursor of alphaMSH. Insulin also inhibits arcuate nucleus expression of neuropeptide Y but its effects on other hypothalamic signaling systems are not known. Leptin-responsive neurons in the arcuate nucleus are hypothesized to project to the paraventricular nucleus and lateral hypothalamic area where they are proposed to influence the expression of peptides that regulate food intake. Future development of this model will incorporate brain pathways for integration of leptin and insulin adiposity signaling to the hypothalamus with meal-related signals that act in the caudal brainstem. Recent research showing that leptin and insulin enhance the satiety action of peripheral CCK, thereby causing meals to be terminated earlier and reducing cumulative food intake, suggests that hypothalamic pathways that are sensitive to leptin and insulin adiposity signals have anatomical connections with caudal brainstem neurons that respond to meal-related signals and regulate meal size. The recent findings that insulin alters the expression and function of neural transporters for dopamine and norepinephrine indicate that adiposity signals may influence food intake by acting on non-peptide neurotransmitter systems.

Reduced beta-cell function contributes to impaired glucose tolerance in dogs made obese by high-fat feeding.

The ability to increase beta-cell function in the face of reduced insulin sensitivity is essential for normal glucose tolerance. Because high-fat feeding reduces both insulin sensitivity and glucose tolerance, we hypothesized that it also reduces beta-cell compensation. To test this hypothesis, we used intravenous glucose tolerance testing with minimal model analysis to measure glucose tolerance (K(g)), insulin sensitivity (S(I)), and the acute insulin response to glucose (AIR(g)) in nine dogs fed a chow diet and again after 7 wk of high-fat feeding. Additionally, we measured the effect of consuming each diet on 24-h profiles of insulin and glucose. After high-fat feeding, S(I) decreased by 57% (P = 0.003) but AIR(g) was unchanged. This absence of beta-cell compensation to insulin resistance contributed to a 41% reduction of K(g) (P = 0.003) and abolished the normal hyperbolic relationship between AIR(g) and S(I) observed at baseline. High-fat feeding also elicited a 44% lower 24-h insulin level (P = 0.004) in association with an 8% reduction of glucose (P = 0.0003). We conclude that high-fat feeding causes insulin resistance that is not compensated for by increased insulin secretion and that this contributes to the development of glucose intolerance. These effects of high-fat feeding may be especially deleterious to individuals predisposed to type 2 diabetes mellitus.

Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone.

PTH is a major regulator of calcium homeostasis by mobilizing calcium through bone resorption. We show that the expression of collagenase-3 (MMP-13), a member of the family of matrix metalloproteinases, required for the cleavage of collagens in the bone, is increased upon PTH injection in mice. A cis-acting element in the collagenase-3 promoter was identified which, together with AP-1, is required for induction by PTH. This element contains CCACA motifs which are required for binding of the 65 kDa osteoblast-specific splice variant of Cbfal. Introduction of mutations in this binding site that interfere with protein interaction also eliminates PTH inducibility and transactivation by Cbfa/ Runt proteins. While DNA binding activity of AP-1 is increased upon PTH treatment, high basal level of Cbfa/Runt binding activity is detectable in untreated cells which is not further increased by PTH, suggesting that AP-1 and Cbfal contribute to transcriptional activation through different mechanisms. In agreement with the critical role of both proteins defined in tissue culture cells, expression of collagenase-3 is reduced in mice lacking c-fos and is completely absent in cbfa1-/-embryos. These data provide the first evidence for a critical role of Cbfal, a major regulator of bone development, in PTH-dependent processes such as bone resorption.

Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus.

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Mechanisms for hyperglycemia in the metabolic syndrome. The key role of beta-cell dysfunction.

Hyperglycemia in Type 2 diabetes represents a steady-state re-regulation of plasma glucose to a higher-than-normal level after an overnight fast. The underlying pathophysiology represents an interaction between impaired beta-cell function and peripheral and hepatic insulin resistance which leads to abnormal hepatic glucose production. Subjects with the Metabolic Syndrome are at an increased risk for Type 2 diabetes and often have one or both of these disorders present even when glucose tolerance is normal. Thus, sophisticated measures of beta-cell function and insulin sensitivity demonstrate a high frequency in populations characterized as having a high prevalence of atherosclerosis, central obesity, hypertension, and dyslipidemia with or without impaired glucose tolerance. Hyperglycemia compensates for the impairment of beta-cell function and therefore, in our view, the beta-cell is the critical factor in its development. Hyperinsulinemia, a curvilinear compensation for insulin resistance that is closely correlated with central adiposity, is another important predictor of hyperglycemia. In a Japanese-American population followed for five years, impaired beta-cell function was present at baseline and preceded the accumulation of intraabdominal fat in those who developed Type 2 diabetes five years later. This interaction between these two pathophysiologic abnormalities in this sequence supports the hypothesis that beta-cell dysfunction contributes to the development of central adiposity by reduced CNS insulin signaling.

NPY and food intake: discrepancies in the model.

The evidence that NPY is an endogenous neurotransmitter that modulates both sides of the energy equation is clear and compelling. While agreeing with this (and indeed contributing to the growing literature supporting the concept), we have found that the interpretation of the increased food intake stimulated by intraventricular (i.v.t.) NPY is more complex than first appears. We discuss evidence suggesting that NPY additionally (and presumably at other receptor populations in the brain) causes sensations that produce aversion or illness. Specifically, the i.v.t. administration of NPY at doses that stimulate eating also cause the formation of a conditioned taste aversion and the animal engages in a form of pica behavior (kaolin consumption). It also suppresses an otherwise robust increase of sodium consumption. We discuss evidence suggesting that whereas NPY activates feeding behavior by stimulating the complex sequence of behaviors beginning with the seeking and finding of food and ending with food ingestion, NPY does not stimulate increased eating in the absence of the anticipatory preliminary behaviors. Finally, we briefly review evidence suggesting that whatever sensation is aroused by i.v.t. NPY, it is not necessarily the same sensation that is aroused when animals are food-deprived. Hence, one must be cautious in interpreting NPY as solely an orexigen.

Signals that regulate food intake and energy homeostasis.

Feeding behavior is critical for survival. In addition to providing all of the body's macronutrients (carbohydrates, lipids, and proteins) and most micronutrients (minerals and vitamins), feeding behavior is a fundamental aspect of energy homeostasis, the process by which body fuel stored in the form of adipose tissue is held constant over long intervals. For this process to occur, the amount of energy consumed must match precisely the amount of energy expended. This review focuses on the molecular signals that modulate food intake while integrating the body's immediate and long-term energy needs.

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.

Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.

Effect of troglitazone on B cell function, insulin sensitivity, and glycemic control in subjects with type 2 diabetes mellitus.

We studied the effects of troglitazone (200-800 mg daily) or placebo on carbohydrate metabolism in 18 subjects with type 2 diabetes (mean age, 66 yr; body mass index, 27.7 kg/m2) at baseline and after taking medication for 12 weeks. We measured fasting proinsulin (PI) and immunoreactive insulin (IRI) levels in all subjects. Thirteen subjects underwent additional metabolic studies, including injection of arginine to determine the acute insulin response, and an i.v. glucose tolerance test to measure the insulin sensitivity index (SI) and glucose effectiveness at zero insulin using the minimal model, i.v. glucose tolerance, and acute insulin response to glucose. Troglitazone treatment resulted in a decrease in fasting plasma glucose from 11.2 +/- 0.7 to 9.6 +/- 0.9 mmol/L (P = 0.02). This was associated with a decrease in the fasting IRI concentration (111 +/- 20 to 82 +/- 13 pmol/L; P = 0.02) and a trend toward a decrease in the fasting PI concentration (43 +/- 11 to 25 +/- 4 pmol/L; P = 0.06). A significant decrease in PI/IRI was observed (38.3 +/- 3.6% to 32.6 +/- 3.2%; P = 0.04). Troglitazone therapy was also associated with a decrease in the acute insulin response to arginine (226 +/- 34 to 167 +/- 25 pmol/L; P = .01) and a near-significant percent increase in S(I) (75 +/- 35%; P = 0.06). Glucose effectiveness at zero insulin, i.v. glucose tolerance, and acute insulin response to glucose did not change. Thus, we found that the decrease in plasma glucose during troglitazone therapy is associated with a dose-related decrease in PI/IRI and an increase in S(I), suggesting that changes in both B cell function and insulin sensitivity contribute to the improvement in metabolic status.

Disproportionately elevated proinsulin levels reflect the degree of impaired B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus.

An increased proportion of fasting proinsulin (PI) relative to immunoreactive insulin (IRI; increased PI/IRI) occurs in noninsulin-dependent diabetes mellitus (NIDDM). To determine whether the magnitude of the increase in PI/IRI is an indicator of the degree of reduced B cell secretory capacity, we measured fasting plasma glucose, PI, IRI, and PI/IRI and related them to maximal B cell secretory capacity (AIRmax) in 9 subjects with NIDDM [age, 61 +/- 3 yr; body mass index (BMI), 27.5 +/- 1.3 kg/m2; duration of NIDDM, 10.8 +/- 1.8 yr; mean +/- SEM] and in 10 healthy subjects matched for age and BMI (age, 61 +/- 6 yr; BMI, 27.9 +/- 1.5 kg/m2). AIRmax was quantified as the incremental insulin response to i.v. arginine at maximal glycemic potentiation (plasma glucose > 25 mmol/L). Mean fasting plasma glucose was 13.7 +/- 1.4 mmol/L (range, 7.5-18.3 mmol/L) in NIDDM subjects and 5.0 +/- 0.1 mmol/L in the controls. Fasting PI was higher in NIDDM (33.1 +/- 5.2) than in controls (9.4 +/- 2.5 pmol/L; P < 0.01), but IRI levels were similar (93.4 +/- 10.9 vs. 82.8 +/- 23.4 pmol/L; P = NS). The PI/IRI ratio was significantly elevated in NIDDM compared to control subjects (35.9 +/- 4.1% vs. 12.8 +/- 0.8%; P < 0.01). After elevation of the glucose level to 30.3 +/- 0.4 mmol/L (NIDDM) and 30.3 +/- 0.5 mmol/L (controls), AIRmax was quantified as 622 +/- 71 pmol/L in NIDDM and 1997 +/- 315 pmol/L in controls, (P < 0.001). The PI/IRI ratio correlated inversely with AIRmax in the NIDDM patients (r = -0.76; P < 0.01). We conclude that the magnitude of the elevation in fasting PI/IRI is related to the reduction in AIRmax. Thus, the fasting PI/IRI ratio appears to be a marker of the degree of reduced AIRmax in NIDDM.

Cerebrospinal fluid and plasma insulin levels in Alzheimer's disease: relationship to severity of dementia and apolipoprotein E genotype.

Patients with Alzheimer's disease (AD) have elevations of fasting plasma insulin that are hypothesized to be associated with disrupted brain insulin metabolism. We examined paired fasted plasma and CSF insulin levels in 25 patients with AD and 14 healthy age-matched adults and determined whether insulin levels were related to severity of dementia and apolipoprotein E-epsilon4 homozygosity, a known genetic risk factor for AD. The AD patients had lower CSF insulin, higher plasma insulin, and a reduced CSF-to-plasma insulin ratio when compared with healthy adults. The differences were greater for patients with more advanced AD. Patients who were not apolipoprotein E-epsilon4 homozygotes had higher plasma insulin levels and reduced CSF-to-plasma ratios, whereas epsilon4 homozygotes with AD had normal values. Both plasma and CSF insulin levels are abnormal in AD, and there are metabolic differences among apolipoprotein E genotypes.