The Polycystin-1, Lipoxygenase,and α-Toxin Domain Regulates Polycystin-1 Trafficking

Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and l-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits β-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.     

Glucose- and hormone-induced cAMP oscillations in α- and β-cells within intact pancreatic islets

OBJECTIVE

cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown.

RESEARCH DESIGN AND METHODS

The subplasma-membrane cAMP concentration ([cAMP]_pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells.

RESULTS

In islets exposed to 3 mmol/L glucose, [cAMP]_pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]_pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]_pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]_pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in the β-cells but not caused by the changes in [Ca²⁺]_i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]_pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively.

CONCLUSIONS

Oscillatory [cAMP]_pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]_pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.Cyclic AMP and Ca²⁺ are key messengers in the regulation of insulin and glucagon secretion from pancreatic β- and α-cells, respectively, by nutrients, hormones, and neural factors. Glucose stimulation of insulin secretion involves uptake and metabolism of the sugar in the β-cells, closure of ATP-sensitive K⁺ channels, and depolarization-induced Ca²⁺ entry generating oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that trigger periodic exocytosis of secretory granules (1,2). This process is amplified by mechanism(s) acting distal to the elevation of Ca²⁺ (3). cAMP promotes exocytosis by facilitating the generation of Ca²⁺ signals (4,5), by sensitizing the secretory machinery to Ca²⁺ (4,6), and by stimulating mobilization and priming of granules via protein kinase A– and Epac-dependent pathways (7,8).Measurements of the cAMP concentration beneath the plasma membrane ([cAMP]_pm) in individual INS-1 β-cells showed that glucagon-like peptide-1(7-36)-amide (GLP-1) induces [cAMP]_pm elevation, often manifested as oscillations (9). Glucose has been regarded to only modestly raise islet cAMP, supposedly by amplifying the effect of glucagon (10), but single-cell cAMP recordings have recently shown that glucose alone induces marked elevation of cAMP in MIN6 β-cells (11,12) and primary mouse β-cells (12,13). Although one study reported that the glucose-induced cAMP response depends on elevation of [Ca²⁺]_i (11), other studies show only partial or no Ca²⁺-dependence of the cAMP signal (12,13). Like hormone stimulation, glucose induces oscillations of [cAMP]_pm, and coordination of the [cAMP]_pm and [Ca²⁺]_i elevations generates pulsatile insulin release (12,14).There are 10 isoforms of cAMP-generating adenylyl cyclases (ACs) with different regulatory properties, nine of which are transmembrane (tm) proteins stimulated by Gα_s and the plant diterpene forskolin. Such tmACs mediate the cAMP-elevating action of glucagon and incretin hormones (15). β-cells express multiple tmACs (16), and the Ca²⁺-stimulated AC8 has been proposed to be particularly important for integrating hormone- and depolarization-evoked signals (17). Soluble AC (sAC) is the only isoform that lacks transmembrane domains. It is insensitive to forskolin and G-proteins but stimulated by bicarbonate (18) and Ca²⁺ (19). Although sAC was first found in the testis, it also seems to be expressed in other tissues and was recently proposed to be involved in glucose-induced cAMP production in insulin-secreting cells (20).Like insulin secretion, exocytosis of glucagon from the α-cells is triggered by an increase of [Ca²⁺]_i (21). Glucagon release is stimulated by absence of glucose and is maximally inhibited when the sugar concentration approaches the threshold for stimulation of insulin secretion (22). Under hypoglycemic conditions, glucagon secretion is also stimulated by adrenaline, which raises [Ca²⁺]_i and [cAMP]_pm via α₁- and β-adrenergic mechanisms (23,24). There are fundamentally different ideas about the mechanisms underlying glucose inhibition of glucagon secretion, including paracrine influences from β- and δ-cells (25–29) and direct actions of glucose on the α-cells, resulting in depolarization- (30) or hyperpolarization-mediated (22) inhibition of exocytosis. Apart from the inhibitory effect of glucose, we observed that very high glucose concentrations unexpectedly stimulate glucagon secretion (31). The stimulatory component may be important under physiological conditions because the hormone is released in pulses from rat (32) and human (33) islets. Glucose thus causes alternating periods of stimulation and inhibition resulting in time-average reduction of glucagon secretion. Ca²⁺ is probably not the only messenger in glucose-regulated glucagon release (29). Like for insulin secretion, cAMP is believed to promote glucagon release by enhancing intracellular Ca²⁺ mobilization, Ca²⁺ influx through the plasma membrane, and mobilization of secretory granules (23,24,34,35). However, it has also been suggested that cAMP-mediated reduction of N-type Ca²⁺ currents can explain the inhibitory effect of GLP-1 on glucagon secretion (36).Until now, nothing was known about cAMP kinetics in α-cells and all information on primary β-cells was based on studies of dispersed islet cells. However, as a result of gap junctional coupling and paracrine influences, the electrophysiological characteristics and [Ca²⁺]_i signaling in intact islets differ considerably from those in dispersed β-cells (2). Therefore, the aim of the current study was to clarify how glucose, glucagon, and GLP-1 affect cAMP signaling in α- and β-cells within intact islets of Langerhans.     

Expression of α5β1 and α6β1 integrins in IgA nephropathy (IgAN) with mild and severe proteinuria. An immunohistochemical study

Immunoperoxidase staining was carried out using monoclonal antibodies against integrin α5β1 and integrin α6β1 on renal biopsy specimens from patients with IgA nephropathy with mild proteinuria (n = 15) and with severe proteinuria (n = 10). Our study revealed increase in glomerular immunoexpression of α5β1 in renal biopsies in IgA nephropathy with severe proteinuria. There were no statistical differences between interstitial α5β1 immunostaining, as well astubular α6β1 immunoexpression in renal tissue between patients with mild and severe proteinuria. The intensity of interstitial α5β1 integrin immunoexpression was positively correlated with the degree of interstitial fibrosis in both studied groups, meanwhile the intensity of tubular α6β1 integrin immunoexpression was not related to the degree of renal interstitial fibrosis in patients with mild and severe proteinuria. Our results suggest that elevated immunoexpression of α5β1 integrin on endothelial glomerular cells in the renal tissue in patients with severe proteinuria may point to the role of this integrin in the mechanism of glomerular injury in these cases of IgA nephropathy. The positive association between the interstitial expression of α5β1 integrin and the relative interstitial cortical volume in renal biopsies in patients with mild and severe proteinuria suggests that α5β1 integrin may play a role in the pathogenesis of chronic progressive renal disease in both studied group. Lack of positive correlation between tubular α6β1 integrin immunoexpression and the relative interstitial cortical volumen may indicate that this molecule play no role in the patomechanism of interstitial fibrosis in IgA nephropathy with mild and severe proteinuria. This revised version was published online in August 2006 with corrections to the Cover Date.     

AMP-Activated Protein Kinase α1 Protects Against Diet-Induced Insulin Resistance and Obesity

AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1^−/−) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1^−/− mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1^−/− mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1^−/− mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1^−/− mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.AMP-activated protein kinase (AMPK) is a major cellular energy sensor and plays a major role in regulating metabolic homeostasis (1,2). In mammals, AMPK is a heterotrimeric complex with a catalytic subunit (α1 or α2) and two regulatory subunits (β1 or β1 and γ1, γ2, or γ3) (1,2). AMPKα2 is the predominant catalytic form of AMPK in the liver, muscle, and hypothalamus. There is evidence that AMPKα2 is important for the regulation of systemic insulin sensitivity and metabolic homeostasis. In the hypothalamus, AMPKα2 signals regulate food intake and body weight gain (3). Mice globally deficient in AMPKα2 display different metabolic phenotypes when fed different diets (4,5). A lack of AMPKα2 activity in skeletal muscle exacerbates glucose intolerance and the insulin resistance that is caused by high-fat diets (HFDs) (6). In addition, AMPKα2 is required for the effects of many physiologic regulators or pharmaceutical modalities that maintain insulin sensitivity and metabolic homeostasis (7–10).Mice deficient in AMPKα1 had an increased inflammatory response in an experimental autoimmune encephalomyelitis model (11). AMPKα1 deficiency elevated the levels of reactive oxygen species and oxidized proteins, thereafter shortening the erythrocyte life span in mice (12). Macrophage AMPKα1 has been characterized as a key regulator of inflammatory function (13,14). Its role in protecting against diet-induced metabolic disorders has been hypothesized but not demonstrated (14). The activation of AMPK in adipocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) suppresses adipocyte differentiation and diet-induced obesity (15). However, the activation of AMPK is able to reduce isoproterenol-induced lipolysis; this result is supported by a decrease in adipocyte size and adipose mass in globally deficient in AMPKα1 (AMPKα1^−/−) mice (16). To define the physiologic role of AMPKα1 in energy homeostasis, we administered an HFD to AMPKα1^−/− mice and then evaluated diet-induced obesity and insulin resistance. We also used bone marrow (BM) transplantation (BMT) to characterize the specific roles of AMPKα1 in macrophages and adipocytes in the regulation of the diet-induced inflammatory response, adiposity, and systemic insulin resistance.     

Up-regulation of α1-adrenoceptors in burn and keloid scars

Stimulation of α₁-adrenoceptors evokes inflammatory cytokine production, boosts neurogenic inflammation and pain, and influences cellular migration and proliferation. Hence, these receptors may play a role both in normal and abnormal wound healing. To investigate this, the distribution of α₁-adrenoceptors in skin biopsies of burn scars (N = 17), keloid scars (N = 12) and unscarred skin (N = 17) was assessed using immunohistochemistry. Staining intensity was greater on vascular smooth muscle in burn scars than in unscarred tissue, consistent with heightened expression of α₁-adrenoceptors. In addition, expression of α₁-adrenoceptors was greater on dermal nerve fibres, blood vessels and fibroblasts in keloid scars than in either burn scars or unscarred skin. These findings suggest that increased vascular expression of α₁-adrenoceptors could alter circulatory dynamics both in burn and keloid scars. In addition, the augmented expression of α₁-adrenoceptors in keloid tissue may contribute to processes that produce or maintain keloid scars, and might be a source of the uncomfortable sensations often associated with these scars.     

Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

The urea transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC-α regulates UT-A1 urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC-α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC-α–deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC-α–induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.     

Renal α-adrenergic receptors and genetic hypertension

The hypothesis has been proposed that an increase in the number of renal -adrenergic receptors may contribute to the pathogenesis of genetic hypertension. Herein we review recent findings regarding expression of renal ₁ (_{1 a},_{1 b})- and ₂ (_{2 a},_{2 b})-adregenergic subtypes and we provide an updated revision of the above-stated hypothesis. Enhancement in receptor number or in post-receptor components responsible for ₁- and ₂-adregenergic mediated sodium reabsorption in proximal tubule may contribute to sodium retention and an elevation in blood pressure. Perhaps such changes contribute to the increase in blood pressure in genetically determined hypertension in humans, although direct tests of this notion have not yet been performed.     

Distribution of α- and δ-tocopherols in seminal plasma and sperm fractions of men with normal and abnormal semen parameters

The aim of this study was to investigate the presence of the different isoforms of tocopherol (T) in seminal plasma (P) and in the sperm fractions of individuals with abnormal (group 1) and normal (group 2) sperm parameters; the relationships between these isoforms and conventional sperm parameters were also explored. Two vitamin E homologues, α-T and δ-T, were identified in the semen of all participants. Although α-T and δ-T concentrations were similar in the semen of the 2 groups, group 1 showed a lower α-T ratio (S/P) (0.90 vs. 1.20, P < .001) and δ-T ratio (0.86 vs 1.13, P = .007) than group 2. In addition, both T ratios were correlated with the percentage of viable cells, detected by eosin staining. These results suggested that α-T and δ-T are not homogeneously distributed in the semen fractions; in normal semen they are more concentrated in the sperm membrane, whereas in abnormal semen the damaged sperm cells may release both Ts in the plasma. To verify whether sperm membrane breakage could alter α-T and δ-T distribution between the seminal plasma and the spermatozoa, normal sperm samples were sonicated; after sonication a consistent sperm plasma membrane fragmentation, highlighted by transmission electron microscopy, and a concomitant release of α-T and δ-T were observed. In conclusion, the Ts coupled directly with the sperm membrane seem to play the main protective role in the semen, and the release of α-T and δ-T in the P fraction is probably an index of lower antioxidant power and sperm quality.     

Assessment of β-Cell Mass and α- and β-Cell Survival and Function by Arginine Stimulation in Human Autologous Islet Recipients

We used intravenous arginine with measurements of insulin, C-peptide, and glucagon to examine β-cell and α-cell survival and function in a group of 10 chronic pancreatitis recipients 1–8 years after total pancreatectomy and autoislet transplantation. Insulin and C-peptide responses correlated robustly with the number of islets transplanted (correlation coefficients range 0.81–0.91; P < 0.01–0.001). Since a wide range of islets were transplanted, we normalized the insulin and C-peptide responses to the number of islets transplanted in each recipient for comparison with responses in normal subjects. No significant differences were observed in terms of magnitude and timing of hormone release in the two groups. Three recipients had a portion of the autoislets placed within their peritoneal cavities, which appeared to be functioning normally up to 7 years posttransplant. Glucagon responses to arginine were normally timed and normally suppressed by intravenous glucose infusion. These findings indicate that arginine stimulation testing may be a means of assessing the numbers of native islets available in autologous islet transplant candidates and is a means of following posttransplant α- and β-cell function and survival.     

Retinol and α-Tocopherol in Morbid Obesity and Nonalcoholic Fatty Liver Disease

Background  

We aimed to study serum retinol and α-tocopherol in a cohort of obese patients and their possible association with several obesity-related conditions, given that the former may be implicated in a diminished capacity of anti-inflammatory and antioxidant potential in obese patients.