Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus.

Type 2 or non-insulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes worldwide, affecting approximately 4% of the world's adult population. It is multifactorial in origin with both genetic and environmental factors contributing to its development. A genome-wide screen for type 2 diabetes genes carried out in Mexican Americans localized a susceptibility gene, designated NIDDM1, to chromosome 2. Here we describe the positional cloning of a gene located in the NIDDM1 region that shows association with type 2 diabetes in Mexican Americans and a Northern European population from the Botnia region of Finland. This putative diabetes-susceptibility gene encodes a ubiquitously expressed member of the calpain-like cysteine protease family, calpain-10 (CAPN10). This finding suggests a novel pathway that may contribute to the development of type 2 diabetes.

Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I.

Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factor-regulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular pools under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel.

Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells.

Rat pancreatic AR42J cells possess exocrine and neuroendocrine properties. Activin A induces morphological changes and converts them into neuron-like cells. In activin-treated cells, mRNA for pancreatic polypeptide (PP) but not that for either insulin or glucagon was detected by reverse transcription-PCR. About 25% of the cells were stained by anti-PP antibody. When AR42J cells were incubated with betacellulin, a small portion of the cells were stained positively with antiinsulin and anti-PP antibodies. The effect of betacellulin was dose dependent, being maximal at 2 nM. Approximately 4% of the cells became insulin positive at this concentration, and mRNAs for insulin and PP were detected. When AR42J cells were incubated with a combination of betacellulin and activin A, approximately 10% of the cells became insulin positive. Morphologically, the insulin-positive cells were composed of two types of cells: neuron-like and round-shaped cells. Immunoreactive PP was found in the latter type of cells. The mRNAs for insulin, PP, glucose transporter 2, and glucokinase, but not glucagon, were detected. Depolarizing concentration of potassium, tolbutamide, carbachol, and glucagon-like peptide-1 stimulated the release of immunoreactive insulin. These results indicate that betacellulin and activin A convert amylase-secreting AR42J cells into cells secreting insulin. AR42J cells provide a model system to study the formation of pancreatic endocrine cells.

Dynamin is involved in human epithelial cell vacuolation caused by the Helicobacter pylori-produced cytotoxin VacA.

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. To elucidate the molecular mechanism of vacuole formation by VacA, we examined the participation of dynamin, a GTPase functioning in intracellular vesicle formation, in human HeLa cells. Immunocytochemistry revealed that endogenous dynamin was localized to vacuoles induced by VacA. In cells transiently transfected with a GTPase-defective (dominant-negative) dynamin mutant, VacA failed to induce vacuolation. In contrast, VacA did induce vacuolation in cells transiently transfected with wild-type dynamin. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with the dominant-negative dynamin mutant. In contrast, uptake was markedly enhanced in cells stably transfected with wild-type dynamin. Moreover, VacA cytopathic effects on the viability of HeLa cells were inhibited in cells stably transfected with dominant-negative dynamin-1. Sequential immunocytochemical observation confirmed that expression of dominant-negative dynamin did not affect VacA attachment to or internalization into HeLa cells. We suggest that dynamin is involved in the intracellular vacuolation induced by VacA.

Conversion of amylase-secreting rat pancreatic AR42J cells to neuronlike cells by activin A.

When AR42J cells, an amylase-secreting pancreatic exocrine cell line, were treated with activin A, cells extended neuritelike processes, and, concomitantly, amylase-containing vesicles disappeared. Immunofluorescence and immunoelectron microscopy revealed that these processes had neurite-specific cytoskeletal architectures: neurofilaments and microtubule bundles with cross-bridges of microtubule-associated protein 2. In addition to such morphological changes, activin-treated cells exhibited a marked increase in cytoplasmic free calcium concentration in response to depolarizing concentration of potassium. Moreover, activin-treated AR42J cells expressed mRNA for alpha 1 subunit of the neuroendocrine/beta cell-type voltage-dependent calcium channel. In naive AR42J cells, a sulfonylurea compound, tolbutamide, did not affect free calcium concentration, while it induced a marked elevation of free calcium in activin-treated cells. Single channel recording of the membrane patch revealed the existence of ATP-sensitive potassium channel in activin-treated cells. These results indicate that activin A converts amylase-secreting AR42J cells to neuronlike cells. Given that pancreatic endocrine cells possess neuronlike properties and express ATP-sensitive potassium channel as well as neuroendocrine/beta cell-type voltage-dependent calcium channel, activin treatment of AR42J cells may provide an in vitro model system to study the conversion of pancreatic exocrine cells to endocrine cells in islets.

Changes in the expression of transcription factors in pancreatic AR42J cells during differentiation into insulin-producing cells.

Pancreatic AR42J cells possess both exocrine and neuroendocrine properties and convert to insulin-producing cells upon treatment with activin A and hepatocyte growth factor (HGF). We studied changes in the mRNA expression of various transcription factors during the course of differentiation. Among the transcription factors studied, expression levels of Pax4 and neurogenin3 changed significantly. These two factors were not detected in naive cells, whereas their mRNA levels were markedly increased after treatment with activin A and HGF. Thus, these two factors were induced by activin A. Transfection of Pax4 did not induce any changes in morphology or expression of pancreatic polypeptide (PP). Furthermore, introduction of antisense Pax4 did not affect the conversion into insulin-producing cells induced by activin A and HGF. In contrast, transfection of neurogenin3 induced morphological changes similar to those induced by activin A. In addition, transfection of neurogenin3 induced the expression of PP. Conversely, introduction of antisense neurogenin3 blocked the differentiation of AR42J cells induced by activin A and HGF. These results indicate that activin A regulates the expression of neurogenin3, which is critical for the differentiation of AR42J into endocrine cells.

Genes expressed during the differentiation of pancreatic AR42J cells into insulin-secreting cells.

Pancreatic AR42J cells have the feature of pluripotency of the common precursor cells of the pancreas. Dexamethasone (Dx) converts them to exocrine cells, whereas activin A (Act) converts them into endocrine cells expressing pancreatic polypeptide. A combination of Act and betacellulin (BTC) converts them further into insulin-secreting cells. The present study identifies some of the genes involved in the process of differentiation that is induced by these factors, using the mRNA differential display and screening of the cDNA expression array. The expression levels of 7 genes were increased by Act alone, and a combination of Act and BTC increased the expression of 25 more genes. Of these, 16 represented known genes or homologues of genes characterized previously. Nine of the identified genes were unrelated to any other sequences in the database. An inhibitor of the mitogen-activated protein kinase pathway, PD098059, which blocks the differentiation into insulin-secreting cells, inhibited the expression of 18 of the 25 genes, suggesting that the proteins encoded by these genes are associated with the differentiation into insulin-producing cells. These include known genes encoding extracellular signaling molecules, such as parathyroid hormone-related peptide, cytoskeletal proteins, and intracellular signaling molecules. Identification and characterization of these differentially expressed genes should help to clarify the molecular mechanism of differentiation of pancreatic cells and the gene products that enable the beta-cells to produce insulin.

Formation of insulin-producing cells from pancreatic acinar AR42J cells by hepatocyte growth factor.

Pancreatic AR42J cells are derived from acinar cells and express both exocrine and neuroendocrine properties. We have recently shown that these cells convert into insulin-producing cells in vitro after treatment with activin A and betacellulin. Here, we investigated the effect of hepatocyte growth factor (HGF) in those cells. When AR42J cells were incubated with HGF, DNA synthesis was attenuated, and the amylase content was reduced in a concentration-dependent manner. HGF-treated cells extended processes, but bundle formation was not observed using an antibody against tubulin. Reverse both insulin and pancreatic polypeptide (PP) were expressed in HGF-treated, but not naive, AR42J cells. Immunocytochemical analysis indicated that approximately 3% of the HGF-treated cells were stained with antiinsulin antibody, and some were also stained with anti-PP antibody. When AR42J cells were exposed to a combination of activin A and HGF, cells extended longer processes, and over 10% of them were stained with antiinsulin antibody. In these cells, messenger RNAs for insulin, PP, glucose transporter 2, and glucokinase, but not those for glucagon or somatostatin, were expressed. A subclone of AR42J cells, AR42J-B13, was obtained. Most of the AR42J-B13 cells converted to insulin-producing cells after the incubation with activin A and HGF. Insulin secretion was augmented by tolbutamide, depolarizing concentrations of potassium, carbachol, and glucagon-like peptide-1 in these cells. These results indicate that HGF reduces the acinar cell-like property of AR42J cells and converts them into insulin-producing cells. The effect of HGF was markedly enhanced by activin A.

A case of hepatocolic fistula after percutaneous drainage for a gas-containing pyogenic liver abscess.

We describe a rare case of gas-containing pyogenic liver abscess which penetrated the adjacent colon, forming a hepatocolic fistula, after percutaneous transhepatic abscess drainage (PTAD) had been performed. To the best of our knowledge, this is the first report of hepatocolic fistula associated with a gas-forming liver abscess in a diabetic patient, with radiological and surgical confirmation of the fistula.

Force-velocity relation and contractility in striated muscles.

For skeletal muscle, the physiological meaning of Hill's hyperbolic force-velocity equation and the factors affecting it, such as the active state, method of velocity measurement and mode of stimulation, have been discussed. After the development of the sliding filament theory, Hill's equation was generalized to all partially activated isometric tensions and the meaning of Hill's dynamic constants was interpreted from the kinetic analysis of the cross-bridge cycle. In cardiac muscle, determination of the precise force-velocity relation was almost impossible, but most difficulties were overcome by tetanizing the cardiac muscle. As a result, the force-velocity properties of cardiac muscle were confirmed to be very similar to those of skeletal muscle. The maximum shortening velocity under no load, v0, was once used as an index of myocardial contractility which is insensitive to muscle length, but it is now believed that at least at shorter lengths, v0 may vary with muscle length and degree of activation. As new approaches, studies on sarcomere dynamics by the laser diffraction method, observation of calcium transient and pressure-velocity measurement in whole ventricle have been introduced.

Force-load-velocity relation and the internal load of tetanized frog cardiac muscle.

A frog ventricular muscle strip could be fully tetanized by alternating current stimulation at 10 Hz and 20 V/cm in a solution containing 9 mM Ca2+. During isometric tetanus, the controlled release was made and the shortening velocities against various loads were measured. The isometric force was varied by reducing the stimulus intensity in K+-rich solution, or by reducing the external Ca2+ concentration. The force-load-velocity relation was described by a simple hyperbolic equation: (P+A)(v+b)=b(F+A), A=(F/Fm)a for shortening, and (2F-P+A')(-v+b')=b'(F+A'),A'=(F/Fm)a' for lengthening, where F is the isometric force, Fm is the maximum isometric force at the optimal muscle length, Lm, P is the load, v is the velocity, a, b, a' and b' are constants. The values of constants were a/Fm=0.51, b=0.75 Lm/sec for shortening and a'/Fm=0.39, b'=0.75 Lm/sec for lengthening at 20 degrees C. At muscle lengths shorter than 0.92 Lm, the internal load defined as the difference between the external load and calculated load at a given velocity increased in proportion to both the velocity and the decrease in muscle length.

Tetanic contraction and tension-length relation of frog ventricular muscle.

Complete tetanic contraction was generated in frog ventricular muscle by repetitive electrical stimulation. The maximum stimulus was a transverse alternating current at 10 Hz and 17-20 V/cm in peak to peak voltage in an external solution containing 9 mM Ca2+. The maximum isometric tension thus obtained was twice or more than that of the twitch tension at 20 degrees C. The tetanic tension and its rate of rise declined with decreasing external Ca2+ concentration and less than half of the maximum tension was generated at 1.8 mM Ca2+. Various tetanic tensions less than the maximum were obtained in the partially depolarized muscle in excess K+ solution by reducing the stimulus intensity. Adrenaline (5X10(-6)g/ml) potentiated the submaximal tetanic tension as well as the twitch tension, although no effect was observed for the maximum tetanic tension. The tension-length relation for the tetanic contraction of ventricular muscle was similar to that of the skeletal muscle, but the tension fell almost linearly at shorter lengths than 0.9 Lm, where Lm is the optimum length at which the maximum tension, Fm, was generated. Fm was 4.6 g/mm2, while the sarcomere length at Lm was 2.0-2.2 mum.

Contraction produced by intracellular injection of calcium, strontium, and barium in the single crayfish muscle fibers.

Ca ions were ionophoretically injected through an intracellular microelectrode into the single muscle fiber of a crayfish, and the resulting contraction sphere was observed under a microscope and photographed with a movie camera. The minimum contraction produced by the threshold current involved usually three or four, sometimes two, sarcomers on both sides of the injecting pipette but contraction involving only one sarcomere was not observered. The rheobase of the Ca-injecting current was 3.2 X 10(-9) A. The strength-duration curves were determined for Ca-, Sr-, and Ba-injecting currents; all fitted a similar hyperbolic equation. The threshold amount of Ca above rheobasic injection was 2.1 X 10(-15)mol, and the ratios between threshold amounts were Ca: Sr: Ba=1: 1.9: 3.0. The effects of Ca and Sr were additive for the contraction. More current was required for the Ca-injection to produce the contraction in the K-depolarized-or 15mM-procaine-treated muscle, although less current was sufficient for the muscle treated with 0.5-1.0 mM of caffeine. The participation of the Ca-induced Ca release mechanism in the contraction produced by Ca injection and the role of Sr or Ba as a substitute for Ca were discussed.

Force-velocity relation for the tetanic contraction of frog atrial muscle.

The hyperbolic force-velocity relation was obtained by afterload method for the tetanic contraction of frog atrial muscle. The dynamic constants (a/Fm and b), the maximum velocity and especially the maximum tension were considerably smaller than those of ventricular muscle.

The pressure-volume and stiffness-pressure relations in the isolated rabbit left ventricle.

A balloon was introduced into the isolated rabbit left ventricle with intraventricular volume being controlled by a feedback amplifier. Pressure-volume and stiffness-pressure relations were determined as intraventricular volume was changed. These ventricular relations were explained according to the theory of wall muscle mechanics.

Binding of 45Ca to intercalated discs of cardiac muscles studied by electron microscope autoradiography.

The binding sites of Ca2+ entering the injured cardiac muscle cell was investigated by 45Ca electron microscope autoradiography in guinea pig papillary muscle and in frog ventricular muscle. The muscle was injured in Ca-free Tyrode's solution, transfered into 45Ca-Tyrode's solution and, after 30 min, fixed and cut into thin sections. An autoradiogram was taken after exposure (4--7 weeks) the thin film of the section was developed. Fine, or sometimes filamentous, silver grains produced by radiation with 45Ca were frequently observed on the intercalated discs of injured cells where most of the nexal membranes were separated. The number of 45Ca grains on the discs of injured cells was eight times more than that found on intact cells. The concentration of 45Ca grains estimated by the number of grains per unit area was 3.2 times higher in the disc region than that in the other cytoplasmic regions in the injured cells, while in the intact cells it was only 1/3 of that found in the latter. The localization of 45Ca grains along the disc of the injured cell was also examined and the fine grains were seen to be located on the separated nexal membrane or at the cytoplasmic side of the desmosome and fascia adherens. It is likely that Ca2+ binds with the nexal membrane and the resulting structural change, such as nexal separation, is indispensable for intercellular uncoupling or healing-over of cardiac muscles.

Intracellular distribution of calcium in cardiac muscles studied by electron microscope autoradiography.

The intracellular distribution of calcium was investigated by 45Ca electron microscope autoradiography in the frog or rat ventricular muscle and guinea pig papillary muscle. The muscle was incubated for 30 min in 45Ca-Ringer's solution or high Ca solution in which 1/10--1/20 of Ca was replaced with 45Ca. The distribution of developed silver grains sensitized by the 45Ca (45Ca grains) was found to be 38% in the mitochondria, 43% in the myofibrils and 19% in the other regions (8% in the cytoplasma and 11% in the various membranous structures) in the frog muscle cell incubated in the 45Ca-Ringer's solution. After electrical stimulation (10 Hz, 5 min), the distribution was 22% in the mitochondria, 53% in the myofibrils and 25% in other regions. Similar results were obtained after high K stimulation in the frog ventricular muscles and after electrical stimulation in high Ca solution in guinea pig papillary muscles. Following the addition of 1 X 10(-6) g/ml adrenaline into the incubating solution, the grain distribution in guinea pig papillary muscles did not show significant change but the distribution in myofibril decreased after electrical stimulation, while it increased in the mitochondria. Longitudinal localization of 45Ca grains along the myofibril was also examined and it was a general tendency throughout all animals examined that the grain density at the A band and near the Z band decreased after electrical stimulation, while it increased at the I band and, especially, at the A-I junction.

The dynamics of contraction in the guinea pig taenia coli.

Tension-length, load-velocity and tension-extension relations were studied in the taenia coli muscle of the guinea pig weighing 0.25--0.5 kg at 36--37 degrees C. The muscle was relaxed by 10(-6) g/ml adrenaline and stimulated by a strong AC field. The tension-length diagram was far wider than that of the skeletal muscle and sufficient tension was generated at longer lengths than 1.8 Lm, where Lm is the optimal length at which the maximum tension, Fm, is generated. The developed force per unit cross-sectional area was almost unchanged between 1.2--2.0 Lm. Average of the maximum forces was 2.2 kg/cm2. Load-velocity curves obtained at various isometric forces at Lm coincided with each other, if the velocity was plotted against the relative force. All curves can be expressed by a single force-load-velocity equation, (P+A)(v+b)=b(F+A), A=(F/Fm)a, where P is the load, F the isometric force, v the velocity, a and b are constants. The maximum velocity per unit muscle length was constant, irrespective of the muscle length. The compliance of the series elastic component, that is, the slope of the tension-extension curve, did not depend on the isometric force but decreased with decreasing muscle length. The internal lengthening of the series elastic component by the full isometric tension was about 3% of the muscle length at Lm.

Ca binding of isolated cardiac nexus membranes related to intercellular uncoupling.

Nexus membranes were isolated from cardiac muscles of guinea pig or ox. It was confirmed by autoradiography that Ca2+ was actually bound with the nexus, and by atomic absorption spectroscopy that bound Ca increased extensively at [Ca2+] higher than 10(-6) to 10(-5) M and showed the dose-response relationship against [Ca2+].

A mechanochemical model for the steady and transient contractions of the skeletal muscle.

A mechanochemical model for muscle contraction was presented which consisted of three subsystems: the regulatory mechanism of contraction by Ca ion, the cross-bridge cycle coupled with actin-myosin interaction and the dynamics of contraction with an external load. It was assumed that both the rate constant of the cross-bridge cycle and the net force of the cross-bridge were linear functions of the sliding velocity. Most parameters in the model were determined from the experimental data, but several were estimated by simulation techniques. The model adequately described the force-load-velocity relation, the rates of energy and heat output as well as some basic mechanical properties of muscle. Not only the steady-state contraction but also the twitch response could be explained by the model. Time courses of tension and shortening during isometric and isotonic twitches were calculated by the model on a digital computer. The calculated curves agreed satisfactorily with the experimental ones obtained from the frog semitendinosus muscle. The rate of tension rise of the isometric twitch was shown to attain the peak at nearly the same time as does the calculated Ca concentration curve.