Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity,leading to PKC-dependent insulin exocytosis

Hypoglycemic sulfonylureas such as glibenclamide have been widely used to treat type 2 diabetic patients for 40 yr, but controversy remains about their mode of action. The widely held view is that they promote rapid insulin exocytosis by binding to and blocking pancreatic beta-cell ATP-dependent K+ (KATP) channels in the plasma membrane. This event stimulates Ca2+ influx and sets in motion the exocytotic release of insulin. However, recent reports show that >90% of glibenclamide-binding sites are localized intracellularly and that the drug can stimulate insulin release independently of changes in KATP channels and cytoplasmic free Ca2+. Also, glibenclamide specifically and progressively accumulates in islets in association with secretory granules and mitochondria and causes long-lasting insulin secretion. It has been proposed that nutrient insulin secretagogues stimulate insulin release by increasing formation of malonyl-CoA, which, by blocking carnitine palmitoyltransferase 1 (CPT-1), switches fatty acid (FA) catabolism to synthesis of PKC-activating lipids. We show that glibenclamide dose-dependently inhibits beta-cell CPT-1 activity, consequently suppressing FA oxidation to the same extent as glucose in cultured fetal rat islets. This is associated with enhanced diacylglycerol (DAG) formation, PKC activation, and KATP-independent glibenclamide-stimulated insulin exocytosis. The fat oxidation inhibitor etomoxir stimulated KATP-independent insulin secretion to the same extent as glibenclamide, and the action of both drugs was not additive. We propose a mechanism in which inhibition of CPT-1 activity by glibenclamide switches beta-cell FA metabolism to DAG synthesis and subsequent PKC-dependent and KATP-independent insulin exocytosis. We suggest that chronic CPT inhibition, through the progressive islet accumulation of glibenclamide, may explain the prolonged stimulation of insulin secretion in some diabetic patients even after drug removal that contributes to the sustained hypoglycemia of the sulfonylurea.     

Poly-acid properties of biosynthetic hyaluronan studied by titration

In this study the poly-acid properties of biosynthetic hyaluronan produced by fermentation of Bacillus subtilis have been investigated. Potentiometric titration as well as ¹H NMR titration have been used to determine the dissociation constants of the carboxylic group on hyaluronic acid. The intrinsic pK_a and pK_{a, α=0.5} were determined in the presence of 0.1 M salt to be 2.99 and 3.37, respectively. The pK_{a, α=0.5} was found to be unaffected by variations in the ionic strength which is in good agreement with the fact that at α = 0.5, 50% of the carboxylic moieties on the hyaluronan are charged. On the other hand the intrinsic pK_a was found to be dependent on the ionic strength until the Debye-Hückel screening length approaches the length of repeating disaccharide unit of hyaluronic acid.Our findings are in good agreement with previously determined dissociation constants for other sources of hyaluronan. We have also shown that ¹H NMR spectroscopy is the preferred method for polyelectrolyte titration because of the ability to isolate the contribution of several ionisable groups on a polymer on molecular level.     

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

Hiding in the Shadows: Philip Morris and the Use of Third Parties to Oppose Ingredient Disclosure Regulations

Background

In 1996 Massachusetts proposed regulations that would require tobacco companies to disclose information about the ingredients in their products on a by-brand basis. This paper examines the strategies employed by Philip Morris to stop these regulations from being implemented.

Methods and Finding

We used previously secret tobacco industry documents and published literature to examine the activities of the tobacco companies after the regulations were proposed. Philip Morris hired a public relations firm to establish a coalition that was instructed to oppose the regulations by linking them to other industrial sectors (the slippery slope) and stating they would damage the state''s economy. Philip Morris also retained a polling firm to test the popularity of specific arguments against ingredient disclosure and developed a strategic plan for opposing similar regulations in Vermont.

Conclusion

Tobacco companies have historically used third parties to form coalitions to oppose ingredient disclosure regulations. These coalitions have had success preventing regulations from being implemented after they are initially proposed by creating the appearance of local opposition. With countries around the world currently implementing ingredient disclosure regulations in the WHO Framework Convention on Tobacco, governments and regulatory agencies should be aware of the political strategies that the tobacco companies have used to create the impression of popular opposition to these measures.     

Effect of smoke-free legislation on adult smoking behaviour in England in the 18 months following implementation

Background

Comprehensive smoke-free legislation covering all enclosed public places and workplaces was implemented in England on 1 July 2007. This study examines the impact of this legislation on smoking prevalence, number of cigarettes smoked and location of smoking, controlling for secular trends through the end of 2008.

Method and Findings

Repeat cross sectional survey using nationally representative data from the Health Survey for England (HSE). In total there are 54,333 respondents from 2003–2008. Logit and linear regression models were used to examine the effect of the legislation on smoking prevalence and the number of cigarettes smoked daily among continuing smokers which took the underlying trend into account. Our finding suggest that smoking prevalence (current smoker) decreased from 25% in 2003 to 21% in 2008 (AOR = 0.96 per year, 95% CI = 0.95–0.98, P<0.01) and the mean number of cigarettes consumed daily by smokers decreased from 14.1 in 2003 to 13.1 in 2008 (coefficient for time trend = −0.28±0.06 SE cig/day per year, P<0.01). After adjusting for these trends the introduction of smoke-free legislation was not associated with additional reductions in smoking prevalence (AOR = 1.02, 95% CI = 0.94–1.11, P = 0.596) or daily cigarette use in smokers (0.42±0.28 SE; P = 0.142). The percentage of respondents reporting smoking ‘at work’ and ‘inside pubs or bars’ decreased significantly from 14% to 2% (p<0.001) and from 34% to 2% (p<0.001), respectively, after the legislation. The percentage reporting smoking ‘inside restaurants, cafes, or canteens’ decreased significantly from 9% to 1% (p<0.001) and ‘inside their home’ decreased significantly from 65% to 55% (p<0.01).

Conclusion

There is widespread compliance with the smoke-free legislation in England, which has led to large drops in indoor smoking in all venues, including at home. Declines in smoking prevalence and consumption continued along existing trends; they did not accelerate during the 18 months immediately following implementation.     

On the botanic model of plant growth with intermediate vegetative-reproductive stage

The application of dynamic optimization to mathematical models of ontogenic biological growth has been the subject of much research [see e.g. . J. Theor. Biol. 33, 299-307]. Koz?owsky and Zió?ko [1988. Thor. Popul. Biol. 34, 118-129] and Zió?ko and Koz?owski [1995. IEEE Trans. Automat. Contr. 40(10), 1779-1783] presented a model with gradual transition from vegetative to reproductive growth. The central point of their model is a mixed state-control constraint on the rate of reproductive growth, which leads to a mixed vegetative-reproductive growth period. Their model is modified here in order to take into account the difference of photosynthesis use efficiency when energy is accumulated in the vegetative and in the reproductive organs of a plant, respectively. The simple assumption on correlation between photosynthesis and temperature permits us to modify the model in a form that is useful for changing climate. Unfortunately, the mathematical solution of the optimal control problem in Koz?owsky and Zió?ko (1988) and Zió?ko and Koz?owski (1995) is incorrect. The strict mathematical solution is presented here, the numerical example from is solved, and the results are compared. The influence of the length of the season and the relative photosynthesis use efficiency, as well as of the potential sink demand of the reproductive organs, on the location and duration of the mixed vegetative-reproduction period of growth is investigated numerically. The results show that the mixed growth period is increased and shifted toward the end of the season when the lengths of the season is increased. Additional details of the sensitivity analysis are also presented.     

Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.     

Monomeric G-protein, Rhes, is not an imidazoline-regulated protein in pancreatic beta-cells

The monomeric G-protein, Rhes, is a candidate imidazoline-regulated molecule involved in mediating the insulin secretory response to efaroxan [S.L. Chan, L.K. Monks, H. Gao, P. Deaville, N.G. Morgan, Identification of the monomeric G-protein, Rhes, as an efaroxan-regulated protein in the pancreatic beta-cell, Br. J. Pharmacol. 136 (1) (2002) 31-36]. This suggestion was based on observations regarding changes in Rhes mRNA expression in rat islets and pancreatic beta-cells after prolonged culture with efaroxan, leading to desensitization of the insulin response to the compound. To verify this report, we have evaluated the effects of the imidazoline compounds efaroxan and BL11282 on Rhes mRNA expression in isolated rat pancreatic islets maintained in conditions identical to those used by Chan et al. The results demonstrate that desensitization of the insulin response to efaroxan, or to another imidazoline, BL11282, does not change Rhes mRNA expression levels. Transfection of MIN6 cells with plasmids containing Rhes or Rhes-antisense also does not alter efaroxan- or BL11282-induced insulin secretion. Together, these data do not support the hypothesis that Rhes is an imidazoline-regulated protein.     

Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.