A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents.

AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

Effects of rectal administration of taurocholic acid on glucagon-like peptide-1 and peptide YY secretion in healthy humans.

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), secreted by enteroendocrine L-cells located most densely in the colon and rectum, are of fundamental importance in blood glucose and appetite regulation. In animal models, colonic administration of bile acids can stimulate GLP-1 and PYY by TGR5 receptor activation. We evaluated the effects of taurocholic acid (TCA), administered as an enema, on plasma GLP-1 and PYY, as well as gastrointestinal sensations in 10 healthy male subjects, and observed that rectal administration of TCA promptly stimulated secretion of both GLP-1 and PYY, and increased fullness, in a dose-dependent manner. These observations confirm that topical application of bile acids to the distal gut may have potential for the management of type 2 diabetes and obesity.

Rectal taurocholate increases L cell and insulin secretion, and decreases blood glucose and food intake in obese type 2 diabetic volunteers.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are secreted from enteroendocrine L cells in response to numerous stimuli, including bile salts. Both have multiple effects that are potentially useful in treating diabetes and obesity. L cell number and hormone content in the intestine are highest in the rectum in humans. We investigated the effects of intrarectal sodium taurocholate on plasma GLP-1, PYY, insulin and glucose concentrations, and on food intake of a subsequent meal. METHODS: Ten obese type 2 diabetic volunteers were each studied on five separate occasions after an overnight fast and oral administration of 100 mg sitagliptin 10 h before the study. They then received an intrarectal infusion of either one of four doses of taurocholate (0.66, 2, 6.66 or 20 mmol, each in 20 ml of vehicle) or vehicle alone (1% carboxymethyl cellulose) single-blind over 1 min. Hormone and glucose measurements were made prior to, and for 1 h following, the infusion. The consumption of a previously selected favourite meal eaten to satiety was measured 75 min after the infusion. RESULTS: Taurocholate dose-dependently increased GLP-1, PYY and insulin, with 20 mmol doses resulting in peak concentrations 7.2-, 4.2- and 2.6-fold higher, respectively, than those achieved with placebo (p < 0.0001 for each). Plasma glucose decreased by up to 3.8 mmol/l (p < 0.001). Energy intake was decreased dose-dependently by up to 47% (p < 0.0001). The ED(50) values for effects on integrated GLP-1, insulin, PYY, food intake and glucose-lowering responses were 8.1, 10.5, 18.5, 24.2 and 25.1 mmol, respectively. CONCLUSIONS/INTERPRETATION: Therapies that increase bile salts (or their mimics) in the distal bowel may be valuable in the treatment of type 2 diabetes and obesity.

Davalintide (AC2307), a novel amylin-mimetic peptide: enhanced pharmacological properties over native amylin to reduce food intake and body weight.

OBJECTIVE: The current set of studies describe the in vivo metabolic actions of the novel amylin-mimetic peptide davalintide (AC2307) in rodents and compares these effects with those of the native peptide. RESEARCH DESIGN AND METHODS: The anti-obesity effects of davalintide were examined after intraperitoneal injection or sustained peripheral infusion through subcutaneously implanted osmotic pumps. The effect of davalintide on food intake after lesioning of the area postrema (AP) and neuronal activation as measured by c-Fos, were also investigated. RESULTS: Similar to amylin, davalintide bound with high affinity to amylin, calcitonin and calcitonin gene-related peptide receptors. Acutely, davalintide displayed greater suppression of dark-cycle feeding and an extended duration of action compared with amylin (23 versus 6 h). Davalintide had no effect on locomotor activity or kaolin consumption at doses that decreased food intake. Davalintide-induced weight loss through infusion was dose dependent, durable up to 8 weeks, fat-specific and lean-sparing, and was associated with a shift in food preference away from high-fat (palatable) chow. Metabolic rate was maintained during active weight loss. Both davalintide and amylin failed to suppress food intake after lesioning of the AP and activated similar brain nuclei, with davalintide displaying an extended duration of c-Fos expression compared with amylin (8 versus 2 h). CONCLUSION: Davalintide displayed enhanced in vivo metabolic activity over amylin while retaining the beneficial properties possessed by the native molecule. In vitro receptor binding, c-Fos expression and AP lesion studies suggest that the metabolic actions of davalintide and amylin occur through activation of similar neuronal pathways.

Dose-response for glycaemic and metabolic changes 28 days after single injection of long-acting release exenatide in diabetic fatty Zucker rats.

AIMS/HYPOTHESIS: Exenatide (exendin-4) injected subcutaneously twice daily reduces glycaemic deterioration in diabetic fatty Zucker (ZDF) rats and reduces HbA1c in humans with type 2 diabetes. Because tachyphylaxis may develop with continuous peptide exposure, we examined the activity of a long-acting-release (LAR) formulation of exenatide on HbA1c, insulin sensitivity and beta cell secretion in ZDF rats. METHODS: Single subcutaneous injections of a poly-lactide-glycolide microsphere suspension (3% peptide) containing 0, 1, 10, 100, 1,000, 3,000 or 9,000 mug exenatide were administered to 9-week-old ZDF rats with matched initial HbA1c values (n=7 rats/group). RESULTS: In contrast to the progressive 3.22+/-0.42% increase in HbA1c in control ZDF rats observed over 28 days, single exenatide-LAR injections dose-proportionally prevented such glycaemic deterioration (median effective dose 74 microg+/-0.1 log per rat; median effective concentration 52 pmol/l+/-0.06 log). Hyperinsulinaemic-euglycaemic clamp procedures incorporating an intraclamp glucose challenge performed 28 days after treatment revealed increases in beta cell response to the glucose challenge at lower exenatide-LAR doses, and up to a 2.1-fold increase in insulin sensitivity at higher exenatide-LAR doses. CONCLUSIONS/INTERPRETATION: The finding that a single dose of exenatide-LAR enhanced glucose control for 28 days in the ZDF rat model of type 2 diabetes suggests that tachyphylaxis is unlikely to be a feature of exenatide-LAR preparations, and supports further clinical exploration.

Effects of PYY[3-36] in rodent models of diabetes and obesity.

BACKGROUND: Peptide YY (PYY) is a 36 amino-acid peptide secreted from ileal L cells following meals. The cleaved subpeptide PYY[3-36] is biologically active and may constitute the majority of circulating PYY-like immunoreactivity. The peptide family that includes PYY, pancreatic peptide and neuropeptide Y is noted for its orexigenic effect following intracerebroventricular administration. OBJECTIVE: To investigate the effects of peripheral (intraperitoneal and chronic subcutaneous) infusions of PYY[3-36] on food intake, body weight and glycemic indices. DESIGN/RESULTS: Food intake was measured in normal mice and in several rodent models of obesity and type II diabetes. In marked contrast to the reported central orexigenic effects, in the present study, PYY[3-36] acutely inhibited food intake by up to 45%, with an ED(50) of 12.5 microg/kg in fasted female NIH/Swiss mice. A 4-week infusion reduced weight gain in female ob/ob mice, without affecting the cumulative food intake. In diet-induced obese male mice, PYY[3-36] infusion reduced cumulative food intake, weight gain and epididymal fat weight (as a fraction of carcass) with similar ED(50)'s (466, 297 and 201 microg/kg/day, respectively) and prevented a diet-induced increase in HbA1c. Infusion at 100 microg/kg/day for 8 weeks in male fa/fa rats reduced the weight gain (288+/-11 vs 326+/-12 g in saline-infused controls; P<0.05), similar to effects in a pair-fed group. In female ob/ob and db/db mice, there was no acute effect of PYY[3-36] on plasma glucose concentrations. In male diabetic fatty Zucker rats, PYY[3-36] infused for 4 weeks reduced HbA1c and fructosamine (ED(50)'s 30 and 44 microg/kg/day). CONCLUSION: Peripheral PYY[3-36] administration reduced the food intake, body weight gain and glycemic indices in diverse rodent models of metabolic disease of both sexes. These findings justify further exploration of the potential physiologic and therapeutic roles of PYY[3-36].

Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta).

Exendin-4 is a 39 amino acid peptide isolated from the salivary secretions of the Gila monster (Heloderma suspectum). It shows 53% sequence similarity to glucagon-like peptide (GLP)-1. Unlike GLP-1, exendin-4 has a prolonged glucose-lowering action in vivo. We compared the potency and duration of glucose-lowering effects of exendin-4 and GLP-1 in hyperglycemic db/db and ob/ob mice. Whereas reductions in plasma glucose of up to 35% vanished within 1 h with most doses of GLP-1, the same doses of exendin-4 resulted in a similar glucose-lowering effect that persisted for >4 h. Exendin-4 was 5,530-fold more potent than GLP-1 in db/db mice (effective doses, 50% [ED50s] of 0.059 microg/kg +/-0.15 log and 329 microg/kg+/-0.22 log, respectively) and was 5,480-fold more potent in ob/ob mice (ED50s of 0.136 microg/kg+/-0.10 log and 744 microg/kg+/-0.21 log, respectively) when the percentage fall in plasma glucose at 1 h was used as the indicator response. Exendin-4 dose-dependently accelerated glucose lowering in diabetic rhesus monkeys by up to 37% with an ED50 of 0.25 microg/kg +/-0.09 log. In two experiments in which diabetic fatty Zucker rats were injected subcutaneously twice daily for 5-6 weeks with doses of exendin-4 up to 100 microg x rat(-1) x day(-1) (approximately 250 microg/kg), HbA1c was reduced relative to saline-injected control rats. Exendin-4 treatment was also associated in each of these experiments with weight loss and improved insulin sensitivity, as demonstrated by increases of up to 32 and 49%, respectively, in the glucose infusion rate (GIR) in the hyperinsulinemic euglycemic clamp. ED50s for weight loss and the increase in clamp GIR were 1.0 microg/kg+/-0.15 log and 2.4 microg/kg+/-0.41 log, respectively. In conclusion, acute and chronic administration of exendin-4 has demonstrated an antidiabetic effect in several animal models of type 2 diabetes.

Hypoglycemia overrides amylin-mediated regulation of gastric emptying in rats.

Amylin, a 37-amino acid peptide hormone co-secreted with insulin, potently governs the rate of gastric emptying. Hypoglycemia, in the absence of agents such as amylin, is reported to accelerate gastric emptying. We asked whether hypoglycemia had a similar action on gastric emptying in the presence of amylin. In preliminary experiments using a phenol red gavage technique in fasted SD rats, we showed that insulin administration accelerated gastric emptying in a dosage-dependent manner. This acceleration was totally prevented by coadministration of glucose in dosages that prevented a change in plasma glucose, indicating that insulin per se did not affect gastric emptying. The effect on gastric emptying of hypoglycemia induced by a 5 mU/min insulin infusion (t = 5-90 min) was assessed in conscious rats continuously infused with amylin (50 pmol x kg-1 x min-1; t = -30 to 90 min). Gastric emptying was indicated by the appearance in plasma of label from 3-O-methyl-[3H]glucose gavaged at t = 0 min. Label appearance was markedly inhibited in rats preinfused with amylin (84% reduced vs. saline controls at t = 30 min), indicating amylin inhibition of gastric emptying. In amylin-treated rats that were subsequently infused with insulin, gavaged label abruptly appeared in plasma when plasma glucose had fallen to 2.1 +/- 0.1 mmol/l (at t approximately 45 min), consistent with a reversal by hypoglycemia of amylin's inhibition of gastric emptying. These data support the idea of a central "fail-safe" mechanism whereby hypoglycemia can override the slowing of gastric emptying by amylin.

Dose-response for glucagonostatic effect of amylin in rats.

Glucagon secretion from pancreatic alpha cells is inhibited by insulin from beta cells. Amylin is a partner hormone to insulin cosecreted in response to nutrient stimuli, which, like insulin, inhibits beta-cell secretion. We investigated whether amylin also inhibits alpha-cell secretion of glucagon in response to infused L-arginine. Rat amylin (1.2, 3.6, 12, 36, or 120 pmol/kg/min; calculated plasma concentration, 13, 47, 195, 713, and 2,950 pmol/L, respectively; n = 7, 8, 6, 4, and 7) or saline (n = 23) was infused into anesthetized male Harlan-Sprague-Dawley rats during hyperinsulinemic-euglycemic clamps, which were used to equalize the influences of glucose and insulin on glucagon secretion. Plasma glucose and insulin concentrations and mean arterial pressures were not different between amylin- and saline-treated rats during a 10-minute 2-mmol L-arginine infusion delivered during the clamps. Plasma glucagon measurements taken during and after the arginine challenge showed that compared with saline infusions, amylin administration dose-dependently suppressed the glucagon response to arginine by a maximum of 62% (incremental area under the curve [AUC] 0 to 60 minutes) with a plasma amylin EC50 of 18 pmol/L +/- 0.3 log units. These data indicate that amylin potently inhibits arginine-stimulated glucagon secretion.

Comparison of the in vitro and in vivo pharmacology of adrenomedullin, calcitonin gene-related peptide and amylin in rats.

Adrenomedullin has been reported to be structurally similar to a group of peptides that includes amylin, calcitonin and calcitonin gene-related peptide (CGRP). Human and rat adrenomedullin displaced [125I]CGRP from membranes of SK-N-MC cells (CGRP receptors) with affinities intermediate between those of rat amylin and rat CGRP alpha (Ki values 0.12 +/- 0.06, 0.017 +/- 0.007, 3.83 +/- 1.14 and 0.007 +/- 0.001 nM, respectively). In contrast Ki values for displacement of [125I]rat amylin from accumbens membranes (amylin receptors), and [125I]salmon calcitonin from T47D cells (calcitonin receptors) were lower than with rat amylin or rat CGRP alpha in these preparations (51 +/- 5, 34 +/- 2, 0.024 +/- 0.002, 0.31 +/- 0.07 nM, respectively, at amylin receptors; 33 +/- 5, 69 +/- 29, 2.7 +/- 1.5 and 13 +/- 3 nM, respectively, at calcitonin receptors). In anesthetized rats, the hypotensive potency of adrenomedullin was between that of amylin and CGRP alpha. In contrast, for amylin or calcitonin agonist actions (inhibition of [14C]glycogen formation in soleus muscle, hyperlactemia, hypocalcemia and inhibition of gastric emptying), human adrenomedullin was without measurable effect. Thus, in its binding behaviour and in its biological actions, adrenomedullin appeared to behave as a potent CGRP agonist, but as a poor amylin or calcitonin agonist.

Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus transcription factor IIIA.

Zinc finger proteins of the Cys2His2 class are DNA sequence-specific transcription factors. Previous structural studies of zinc finger protein-DNA complexes have shown that amino acids in the finger tip and alpha-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein transcription factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that a polypeptide containing zinc fingers 1-5 (zf1-5) binds oligonucleotides with modifications in the major groove of the finger 4 binding site with wild-type affinity. Mutagenesis and binding site selection studies were performed to determine whether high affinity DNA binding by zf1-5 requires a particular sequence in the binding site for finger 4. Several mutations in this region of the 5S gene reduced the DNA-binding affinity of zf1-5; however, selection and amplification binding assays did not recover the wild-type finger 4 binding site sequence from a pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on the top strand was highly selected within the finger 4 binding site. We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure rather than by a unique DNA sequence. Deletion of finger 4 from zf1-5 results in a protein with poor binding affinity, demonstrating the importance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions between fingers.

Dose-responses for the slowing of gastric emptying in a rodent model by glucagon-like peptide (7-36) NH2, amylin, cholecystokinin, and other possible regulators of nutrient uptake.

Several peptides have been proposed as regulators of nutrient release from the stomach and subsequent uptake from the gut. Using a phenol red gavage method, we compared the potencies of subcutaneously preinjected amylin, glucagon-like peptide-1 (7-36)amide (GLP-1), cholecystokinin octapeptide (CCK-8), gastric inhibitory peptide (GIP), glucagon, and pancreatic peptide on slowing the release of an acaloric gel from rat stomach. The latter three peptides did not fully inhibit gastric emptying at subcutaneous doses up to 100 micrograms. Amylin, GLP-1, and CCK-8 fully inhibited gastric emptying, with ED50s of 0.42 +/- 0.07, 6.1 +/- 0.12, and 8.5 +/- 0.20 nmol/kg +/- SE of log, respectively.

Diabetogenic effects of salmon calcitonin are attributable to amylin-like activity.

During the development of synthetic calcitonins for therapeutic use in bone disease, a "diabetogenic" (hyperglycemic) effect was observed, particularly with salmon calcitonin. The effect was attributed by some to inhibition of insulin secretion. We have recently reported high-affinity (28 pmol/L) amylin-binding sites in certain areas of rat brain, and found that these sites also bind salmon but not rat calcitonin with comparable high affinity. Rat amylin and salmon calcitonin have been determined to have significant structural homology. In vitro and in vivo studies indicate that rat amylin can exert calcitonin-like effects on osteoclasts and on plasma calcium. Here we report that salmon calcitonin mimics the actions of rat amylin on skeletal muscle glycogen metabolism in vitro; it stimulates glycogenolysis and inhibits incorporation of radiolabeled glucose into glycogen (50% effective concentration [EC50], 0.4 +/- 0.11 nmol/L log and 8.4 +/- 0.05 nmol/L log, respectively). In anesthetized rats, salmon calcitonin, like rat amylin, rapidly increases plasma lactate concentration, followed by a slower increase in glucose concentration. Like amylin, salmon calcitonin also inhibits the insulin response to 2 mmol infused glucose (insulin increments suppressed by 52% and 57% at 10 minutes for salmon calcitonin and amylin). Other shared actions, such as suppression of appetite, stimulation of renin secretion, inhibition of gastric acid secretion, and inhibition of gastric emptying, further affirm our proposal that the exogenous peptide, salmon calcitonin, is a mimic of endogenous amylin in the rat.

Mineral induced formation of sugar phosphates.

Glycolaldehyde phosphate, sorbed from highly dilute, weakly alkaline solution into the interlayer of common expanding sheet structure metal hydroxide minerals, condenses extensively to racemic aldotetrose-2,4-diphosphates and aldohexose-2,4,6-triphosphates. The reaction proceeds mainly through racemic erythrose-2,4-phosphate, and terminates with a large fraction of racemic altrose-2,4,6-phosphate. In the absence of an inductive mineral phase, no detectable homogeneous reaction takes place in the concentration- and pH range used. The reactant glycolaldehyde phosphate is practically completely sorbed within an hour from solutions with concentrations as low as 50 micrometers; the half-time for conversion to hexose phosphates is of the order of two days at room temperature and pH 9.5. Total production of sugar phosphates in the mineral interlayer is largely independent of the glycolaldehyde phosphate concentration in the external solution, but is determined by the total amount of GAP offered for sorption up to the capacity of the mineral. In the presence of equimolar amounts of rac-glyceraldehyde-2-phosphate, but under otherwise similar conditions, aldopentose-2,4,-diphosphates also form, but only as a small fraction of the hexose-2,4,6-phosphates.

Gastric emptying is accelerated in diabetic BB rats and is slowed by subcutaneous injections of amylin.

Gastric emptying was measured in normal and insulin-treated spontaneously diabetic BB rats using the retention of an acaloric methylcellulose gel containing phenol red delivered by gavage. Dye content in stomachs removed after killing 20 min later was determined spectroscopically, and was compared to that in rats killed immediately after gavage to assess emptying. Diabetic rats had a markedly greater gastric emptying (90.3 +/- 1.7% passed) compared to normal Harlan Sprague Dawley rats (49.1 +/- 4.7% passed; p < 0.001) and non-diabetic BB rats (61.1 +/- 9.2% passed; p < 0.001). The pancreatic beta-cell peptide, amylin, which is deficient in insulin-dependent diabetes mellitus, dose-dependently inhibited gastric emptying in both normal and diabetic rats. The ED50 of the response in normal rats measured by phenol red and novel [3-3H]glucose gavage techniques was approximately 0.4 microgram. This dose was estimated to increase plasma amylin concentration by a mean of approximately 20 pmol/l to concentrations within the range observed in vivo. It is proposed that amylin could participate in the physiological control of nutrient entry into the duodenum and that the accelerated gastric emptying seen in BB rats could be related to their lack of amylin secretion.

Amylin regulation of carbohydrate metabolism.

This review describes how amylin may work in the control of carbohydrate metabolism by actions on gastric emptying and on muscle glycogen metabolism. Amylin, which is co-secreted with insulin from pancreatic beta-cells in response to nutrient stimuli, affects both carbohydrate absorption and carbohydrate disposal. Amylin appears to regulate carbohydrate metabolism as a partner to insulin. Defending fuel stores tends to be hierarchical; plasma glucose is defended first, then muscle glycogen, then liver glycogen, then fat. Fuel stores are replenished by both incorporating ingested nutrient and by translocating nutrient stores among body sites. Lactate may better be regarded as a vector of fuel transfer rather than a 'dead end' in metabolism. Amylin can promote the translocation of lactate from muscle to liver. The amylin effect, illustrated by the simultaneous decrease in muscle glycogen and increase in liver glycogen [53, 56], is similar to the catecholamine effect observed by Cori et al. [57]. Amylin thus may be important in maintaining liver glycogen stores via the Cori cycle and the 'indirect' glycogen synthesis pathway [58,59]. Unlike catecholamines, amylin does not mobilize fat or impede insulin action in adipose tissue [30,35]. It can supply lactate to the liver, and because lactate is a preferred lipogenic substrate [60], may thereby favour fat storage. Amylin may also help to control carbohydrate absorption via an 'entero-insular loop' to ensure that absorption from the gut remains within the regulatory limits for carbohydrate disposal by peripheral tissues. This regulatory system is essential for normal control of plasma glucose and appears to be disrupted in type-1 diabetes, an amylin-deficient state.

Interaction of the RNA binding fingers of Xenopus transcription factor IIIA with specific regions of 5 S ribosomal RNA.

Zinc fingers 4 to 7 of Xenopus transcription factor IIIA (TFIIIA) represent the minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for their effects on the binding affinity of full-length TFIIIA and a polypeptide consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease footprinting was used to compare the binding sites of TFIIIA and zf4-7 on 5 S RNA. The consistency between the data obtained from these two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA bind to a central core region on the 5 S RNA molecule consisting of loop B/helix II/loop A/helix V/region E. This information was used to design a truncated 75-nucleotide-long RNA molecule that retains high affinity for zf4-7. Therefore, we conclude that the specific interaction of TFIIIA with 5 S RNA can be represented by a complex formed between a four zinc finger polypeptide and a truncated 5 S RNA molecule.

Crystal structures of synthetic 7 angstrom and 10 angstrom manganates substituted by mono- and divalent cations.

The crystal structures of synthetic 7 angstrom and 10 angstrom manganates, synthetic birnessite and buserite, substituted by mono- and divalent cations were investigated by X-ray and electron diffractions. The monoclinic unit cell parameters of the subcell of lithium 7 angstrom manganate, which is one of the best ordered manganates, were obtained by computing the X-ray powder diffraction data: a = 5.152 angstroms, b = 2.845 angstroms, c = 7.196 angstroms, beta = 103.08 degrees. On the basis of the indices obtained by computing the X-ray diffraction data of Li 7 angstrom manganate, monovalent Na, K and Cs and divalent Be, Sr and Ba 7 angstrom manganates were interpreted as the same monoclinic structure with beta = 100-103 degrees as that of Li 7 angstrom manganate, from their X-ray diffraction data. In addition, divalent Mg, Ca and Ni 10 angstrom manganates were also interpreted as the same monoclinic crystal system with beta=90-94 degrees. The unit cell parameters, especially a, c and beta change possibly with the type of substituent cation probably because of the different ionic radius, hydration energy and molar ratio of substituent cation to manganese. However, these diffraction data, except for those of Sr and Ba 7 angstrom and Ca and Ni 10 angstrom manganates, reveal only some parts of the host manganese structure with the edge-shared [MnO6] octahedral layer. On the other hand, one of the superlattice reflections observed in the electron diffractions was found in the X-ray diffraction lines for heavier divalent cations Sr and Ba 7 angstrom and Ca and Ni 10 angtrom manganates. The reflection presumably results from the substituent cation position in the interlayer which is associated with the vacancies in the edge-shared [MnO6] layer and indicates that the essential vacancies are linearly arranged parallel to the b-axis. Furthermore, the characteristic superlattice reflection patterns for several cations, Li, Mg, Ca, Sr, Ba and Ni, manganates were interpreted that the substituent cations are regularly distributed in the interlayer according to the exchange percentage of substituent cation to Na+. In contrast, the streaking in the a-direction observed strongly in the electron diffractions for heavier monovalent cations, K and Cs, manganates probably results from the disordering of their cations in the a-direction in the interlayer.

Selective amylin antagonist suppresses rise in plasma lactate after intravenous glucose in the rat. Evidence for a metabolic role of endogenous amylin.

Data presented here provide the first demonstration that circulating amylin regulates metabolism in vivo, and support an endocrine hormonal role that is distinct from its autocrine action at pancreatic islets. When rats were pre-treated with the potent amylin antagonist AC187 (n = 18), and then administered a 2 mmol glucose load, the rise in plasma lactate was less than in rats administered glucose only (n = 27; P < 0.02). When rats were treated so that plasma glucose and insulin profiles were similar (n = 8), the increase in plasma lactate in the presence of AC187 was only 50.3% as high as the increase when AC187 was absent (P < 0.001). These experimental results fit with the view that some of the lactate appearing in plasma after a glucose load comes from insulin-sensitive tissues. The experiments also support the view that an important fraction of the increase in lactate depends on processes inhibited by a selective amylin antagonist, most likely amylin action in muscle.

Amylin and insulin in rat soleus muscle: dose responses for cosecreted noncompetitive antagonists.

Increasing concentrations of amylin progressively depressed the maximal insulin-stimulated radioglucose incorporation into soleus muscle glycogen, but did not substantively change the EC50 (range 0.78 to 1.52 nM); these findings show noncompetitive, insurmountable antagonism of insulin action by amylin. The results from 36 combinations of different insulin and amylin concentrations were used to construct a response surface that can be used to predict the response for any combination of insulin and amylin concentration. The predicted response to a constant ratio of insulin and amylin concentration is a bell-shaped curve. The experimentally determined response to increasing amounts of an amylin-insulin mixture (molar ratio of 0.14:1, within the range measured for pancreatic secretion and plasma levels) gave a bell-shaped response rather than the sigmoidal response seen with insulin alone. The amylin dose-response relation in the soleus system provides a useful bioassay for amylin agonists. The dose response for highly purified, synthetic human amylin obtained by measuring amylin concentrations by radioimmunoassay in the incubation medium gave an EC50 of 456 pM (+/- 0.18 log units). Human amylin had a potency greater than or equal to that of human insulin in this highly insulin-sensitive preparation.