Arthrogryposis multiplex congenita (AMC) is a group of disorders characterized by congenital joint contractures caused by reduced fetal movements. AMC has an incidence of 1 in 3000 newborns and is genetically heterogeneous. We describe an autosomal recessive form of myogenic AMC in a large consanguineous family. The disease is characterized by bilateral clubfoot, decreased fetal movements, delay in motor milestones, then progressive motor decline after the first decade. Genome-wide linkage analysis revealed a single locus on chromosome 6q25 with Z(max) = 3.55 at theta = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Homozygous A to G nucleotide substitution of the conserved AG splice acceptor site at the junction of intron 136 and exon 137 of the SYNE-1 gene was found in patients. This mutation results in an aberrant retention of intron 136 of SYNE-1 RNA leading to premature stop codons and the lack of the C-terminal transmembrane domain KASH of nesprin-1, the SYNE-1 gene product. Mice lacking the KASH domain of nesprin-1 display a myopathic phenotype similar to that observed in patients. Altogether, these data strongly suggest that the splice site mutation of SYNE-1 gene found in the family is responsible for AMC. Recent reports have shown that mutations of the SYNE-1 gene might be responsible for autosomal recessive adult onset cerebellar ataxia. These data indicate that mutations of nesprin-1 which interacts with lamin A/C may lead to at least two distinct human disease phenotypes, myopathic or neurological, a feature similar to that found in laminopathies.

A full biphasic insulin response is the most sensitive index for well-coupled ␤-cell signal transduction. While first-phase insulin response is extremely sensitive to potentiating and inhibiting modulations, full expression of second-phase response requires near maximally activated ␤-cell fuel metabolism. In the isolated rat pancreas, accelerated calcium entry or activation of protein kinase (PK)-A or PKC result in no insulin response in the absence of fuel metabolism. At submaximal levels of ␤-cell fuel secretagogue, arginine (which promotes calcium entry) or glucagon (which activates PKA) produces a small first-phase insulin response but minimal or no second-phase response; carbachol (which activates PKC and promotes calcium entry) generates biphasic insulin response in the presence of minimal fuel (3.3 mmol/l glucose). Glucagon produces full biphasic response in the presence of 10.0 mmol/l glucose, whereas arginine requires near-maximal stimulatory glucose (16.7 mmol) to produce full biphasic insulin response. Thus, PKA and PKC signal pathways potentiate primary signals generated by fuel secretagogues to induce full biphasic insulin response, while calcium recruitment alone is insufficient to potentiate primary signals generated at low levels of fuel secretagogue. We suggest that three families of PKs (calmodulin-dependent PK [CaMK], PKA, and PKC) function as distal amplifiers for stimulus-secretion coupling signals originating from fuel metabolism, as well as from incretins acting through membrane receptors, adenylate cyclase, and phospholipase C. Several isoenzymes of PKA and PKC are present in pancreatic ␤-cells, but the specific function of most is still undefined. Each PK isoenzyme is activated and subsequently phosphorylates its specific effector protein by binding to a highly specific anchoring protein. Some diabetes-related ␤-cell derangements may be linked to abnormal function of one or more PK isoenzymes. Identification and characterization of the specific function of the individual PK isoenzymes may provide the tool to improve the insulin response of the diabetic patient. Diabetes 51 (Suppl. 1):S68 -S73, 2002

Glucose metabolism affects most major signal pathways in pancreatic ␤-cells. Multiple protein kinases, including protein kinase C (PKC) isoenzymes, are involved in these effects; however, their role is poorly defined. Moreover, the dynamics of kinase isoenzyme activation in reference to the biphasic insulin secretion is unknown. In perfused pancreas of Wistar rats, PKC␣ staining was strongly associated with insulin staining, jointly accumulating in the vicinity of the plasma membrane during early first-phase insulin response. The signal declined before the onset of second phase and reappeared during second-phase insulin release as foci, only weekly associated with insulin staining; this signal persisted for at least 15 min after glucose stimulation. In the GK rat, glucose had minimal effect on ␤-cell PKC␣. In control ␤-cells, PKC␦ stained as granulated foci with partial association with insulin staining; however, no glucose-dependent translocation was observed. In the GK rat, only minimal staining for PKC␦ was observed, increasing exclusively during early first-phase secretion. In Wistar ␤-cells, PKC concentrated near the nucleus, strongly associated with insulin staining, with dynamics resembling that of biphasic insulin response, but persisting for 15 min after cessation of stimulation. In GK rats, PKC staining lacked glucose-dependent changes or association with insulin. PKC exhibited bimodal dynamics in control ␤-cells: during early first phase, accumulation near the cell membrane was observed, dispersing thereafter. This was followed by a gradual accumulation near the nucleus; 15 min after glucose stimulus, clear PKC staining was observed within the nucleus. In the GK rat, a similar response was only occasionally observed. In control ␤-cells, glucose stimulation led to a transient recruitment of PKC, associated with first-phase insulin release, not seen in GK ␤-cell. Data from this and related studies support a role for PKC␣ in glucose-induced insulin granule recruitment for exocytosis; a role for PKC in activation of insulin granules for exocytosis and/or in the glucose-generated time-dependent potentiation signal for insulin release; and a dual function for PKC in initiating insulin release and in a regulatory role in the transcriptional machinery. Furthermore, diminished levels and/or activation of PKC␣, PKC, PKC, and PKC could be part of the defective signals downstream to glucose metabolism responsible for the deranged insulin secretion in the GK rat. Diabetes 55: 590 -599, 2006 G lucose metabolism is the primary initiator and regulator of most pancreatic ␤-cell functions: multiple ␤-cell genes are controlled by signals originating from glucose metabolism, as are all functions related to production, storage, and exocytosis of insulin. While the sequence of events involved in initiating glucose-induced insulin response have been well studied, those of extended release (second phase; see below) or insulin biosynthesis are considerably less clear.Multiple protein kinases are known to participate in relayi...

cAMP activates multiple signal pathways, crucial for the pancreatic beta-cells function and survival and is a major potentiator of insulin release. A family of phosphodiesterases (PDEs) terminate the cAMP signals. We examined the expression of PDEs in rat beta-cells and their role in the regulation of insulin response. Using RT-PCR and Western blot analyses, we identified PDE3A, PDE3B, PDE4B, PDE4D, and PDE8B in rat islets and in INS-1E cells and several possible splice variants of these PDEs. Specific depletion of PDE3A with small interfering (si) RNA (siPDE3A) led to a small (67%) increase in the insulin response to glucose in INS-1E cells but not rat islets. siPDE3A had no effect on the glucagon-like peptide-1 (10 nmol/liter) potentiated insulin response in rat islets. Depletion in PDE8B levels in rat islets using similar technology (siPDE8B) increased insulin response to glucose by 70%, the potentiation being of similar magnitude during the first and second phase insulin release. The siPDE8B-potentiated insulin response was further increased by 23% when glucagon-like peptide-1 was included during the glucose stimulus. In conclusion, PDE8B is expressed in a small number of tissues unrelated to glucose or fat metabolism. We propose that PDE8B, an 3-isobutyl-1-methylxanthine-insensitive cAMP-specific phosphodiesterase, could prove a novel target for enhanced insulin response, affecting a specific pool of cAMP involved in the control of insulin granule trafficking and exocytosis. Finally, we discuss evidence for functional compartmentation of cAMP in pancreatic beta-cells.

Psammomys obesus is a model of type 2 diabetes that displays resistance to insulin and deranged ␤-cell response to glucose. We examined the major signaling pathways for insulin release in P. obesus islets. Islets from hyperglycemic animals utilized twice as much glucose as islets from normoglycemic diabetes-prone or diabetes-resistant controls but exhibited similar rates of glucose oxidation. Fractional oxidation of glucose was constant in control islets over a range of concentrations, whereas islets from hyperglycemic P. obesus showed a decline at high glucose. The mitochondrial substrates ␣-ketoisocaproate and monomethyl succinate had no effect on insulin secretion in P. obesus islets. Basal insulin release in islets from diabetesresistant P. obesus was unaffected by glucagon-like peptide 1 (GLP-1) or forskolin, whereas that of islets of the diabetic line was augmented by the drugs. GLP-1 and forskolin potentiated the insulin response to maximal (11.1 mmol/l) glucose in islets from all groups. The phorbol ester phorbol myristic acid (PMA) potentiated basal insulin release in islets from prediabetic animals, but not those from hyperglycemic or diabetes-resistant P. obesus. At the maximal stimulatory glucose concentration, PMA potentiated insulin response in islets from normoglycemic prediabetic and diabetes-resistant P. obesus but had no effect on islets from hyperglycemic P. obesus. Maintenance of islets from hyperglycemic P. obesus for 18 h in low (3.3 mmol/l) glucose in the presence of diazoxide (375 µmol/l) dramatically improved the insulin response to glucose and restored the responsiveness to PMA. Immunohistochemical analysis indicated that hyperglycemia was associated with reduced expression of ␣-protein kinase C (PKC) and diminished translocation of -PKC. In summary, we found that 1) P. obesus islets have low oxidative capacity, probably resulting in limited ability to generate ATP to initiate and drive the insulin secretion; 2) insulin response potentiated by cyclic AMP-dependent protein kinase is intact in P. obesus islets, and increased sensitivity to GLP-1 or forskolin in the diabetic line may be secondary to increased sensitivity to glucose; and 3) islets of hyperglycemic P. obesus display reduced expression of ␣-PKC and diminished translocation of -PKC associated with impaired response to PMA. We conclude that low ␤-cell oxidative capacity coupled with impaired PKC-dependent signaling may contribute to the animals' poor adaptation to a highenergy diet. Diabetes 50:308-314, 2001T he gerbil Psammomys obesus seems an excellent natural model of type 2 diabetes: normoglycemic in its natural habitat, feeding on the low-energy salt bush (Atriplex halimus), it shows a high tendency to develop diabetes when fed an energy-rich laboratory diet (1-3). Diet-induced hyperglycemia in P. obesus is initially associated with hyperinsulinemia, a significant fraction of which consists of insulin precursor molecules (4,5). ␤-Cell insulin content is depleted and, with continued hyperglycemia, ␤-cell mass also is decrea...

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCepsilon in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCepsilon) or expression of inactive PKCepsilon (PKCepsilon-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCepsilon in the perinuclear region, an area known to be rich in ER-Golgi systems, associated with insulin-containing structures. ss'COP1 (RACK2) is the anchoring protein for PKCepsilon. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCepsilon-KD, and 60% in islets expressing RACK2 binding protein (epsilonV1-2); total protein biosynthesis was not affected. In islets expressing epsilonV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCepsilon plays a specific role in mediating the glucose-signal into insulin production: binding to ss'COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.

Spinal muscular atrophies (SMA) are frequent autosomal recessive disorders characterized by degeneration of lower motor neurons. SMA are caused by mutations of the survival of motor neuron gene (SMN1) leading to a reduction of the SMN protein amount. The identification of SMN interacting proteins involved in the formation of the spliceosome and splicing changes in SMN-deficient tissues of mutant mice strongly support the view that SMN is involved in the splicing reaction. However, the molecular pathway linking SMN defect to the SMA phenotype remains unclear. From a better knowledge of the genetic basis of SMA and the defects resulting from the mutations of SMN1 in cellular or animal models, several therapeutics strategies have been selected aiming at targeting SMN2, a partially functional copy of SMN1 gene which remains present in patients, or to prevent neurons from death. Refined characterization of the degenerative process in SMA and the identification of the defective molecular pathway downstream from the SMN defect will provide further exciting insight into this disease in the near future. They should contribute to clarify the pathophysiology of SMA, the function of SMN and should help in designing potential targeted or non-targeted therapeutic molecules.