Two dose-ranging studies with PF-04937319, a systemic partial activator of glucokinase, as add-on therapy to metformin in adults with type 2 diabetes.

AIM: To assess the efficacy and safety of a range of doses of a systemic, partial, glucokinase activator, PF-04937319, as add-on therapy to metformin, in patients with type 2 diabetes mellitus (T2DM). METHODS: Patients were randomized to once-daily PF-04937319 doses of 10, 50, 100 mg, or matching placebo (Study B1621002); or PF-04937319 doses of 3, 20, 50, 100 mg, or matching placebo (Study B1621007). Titrated glimepiride (Study B1621002) or sitagliptin (Study B1621007) were included in a double-dummy manner. The primary measure was change from baseline in glycated haemoglobin (HbA1c) at week 12. Key secondary measures included other glycaemic variables and safety and tolerability. RESULTS: In the 639 patients randomized, the minimally efficacious PF-04937319 dose was identified as 50 mg once daily. At the highest PF-04937319 dose tested (100 mg), on average, a clinically significant reduction in HbA1c [-4.94 or -5.11 mmol/mol (-0.45 or -0.47%), placebo-adjusted], which was similar to that achieved with sitagliptin [-4.69 mmol/mol (-0.43%)] but lower than that achieved with titrated glimepiride [-9.07 mmol/mol (-0.83%)], was observed. At this dose, the effect on fasting plasma glucose was not consistent between the two studies (Study B1621002 vs Study B1621007: placebo-adjusted mean change of -0.83 vs +0.50 mmol/l). PF-04937319 was well tolerated at doses up to 100 mg. Hypoglycaemia was reported in 2.5% of patients (on placebo), 5.1% of patients (on PF-04937319 100 mg), 1.8% of patients (on sitagliptin) and 34.4% of patients (on titrated glimepiride). CONCLUSIONS: In patients on metformin monotherapy, the addition of a 100-mg dose of PF-04937319 improved glycaemic control and was well tolerated.

A gene variant near ATM is significantly associated with metformin treatment response in type 2 diabetes: a replication and meta-analysis of five cohorts.

AIMS/HYPOTHESIS: In this study we aimed to replicate the previously reported association between the glycaemic response to metformin and the SNP rs11212617 at a locus that includes the ataxia telangiectasia mutated (ATM) gene in multiple additional populations. METHODS: Incident users of metformin selected from the Diabetes Care System West-Friesland (DCS, n = 929) and the Rotterdam Study (n = 182) from the Netherlands, and the CARDS Trial (n = 254) from the UK were genotyped for rs11212617 and tested for an association with both HbA(1c) reduction and treatment success, defined as the ability to reach the treatment target of an HbA(1c) ≤ 7 % (53 mmol/mol). Finally, a meta-analysis including data from literature was performed. RESULTS: In the DCS cohort, we observed an association between rs11212617 genotype and treatment success on metformin (OR 1.27, 95% CI 1.03, 1.58, p = 0.028); in the smaller Rotterdam Study cohort, a numerically similar but non-significant trend was observed (OR 1.45, 95% CI 0.87, 2.39, p = 0.15); while in the CARDS cohort there was no significant association. In meta-analyses of these three cohorts separately or combined with the previously published cohorts, rs11212617 genotype is associated with metformin treatment success (OR 1.24, 95% CI 1.04, 1.49, p = 0.016 and OR 1.25, 95% CI 1.33, 1.38, p = 7.8 × 10(-6), respectively). CONCLUSIONS/INTERPRETATION: A gene variant near ATM is significantly associated with metformin treatment response in type 2 diabetic patients from the Netherlands and the UK. This is the first robustly replicated common susceptibility locus found to be associated with metformin treatment response.

Effects of a long-acting GLP-1 mimetic (PF-04603629) on pulse rate and diastolic blood pressure in patients with type 2 diabetes mellitus.

PF-04603629, an exendin-transferrin fusion protein, is a long-acting glucagon-like peptide-1 (GLP-1) mimetic. This randomized, double-blind study characterized the safety and pharmacodynamics of a single dose of PF-04603629 (n = 57; 1-70 mg) or placebo (n = 14) in subjects with type 2 diabetes mellitus (T2DM). There were dose-dependent decreases from baseline in day 6 glucose area under the curve following a mixed meal test (-27 ± 12% with 70 mg). Most treatment-related adverse events were gastrointestinal, with nausea and vomiting most frequent at 70 mg. Pulse rate (PR) and diastolic blood pressure (DBP) increased dose dependently within the normal range. At 24 h postdose mean PR increased 23 ± 9 bpm and mean DBP increased 10 ± 5 mmHg with 70 mg. In conclusion, PF-04603629 exhibited efficacy and tolerability consistent with its mechanism of action; however, PR and DBP increased. Similar effects have been reported occasionally with other GLP-1 mimetics. These data underscore the importance of careful assessments of haemodynamic effects in GLP-1 analogues.

A Dose-Ranging Study of the DPP-IV Inhibitor PF-734200 Added to Metformin in Subjects With Type 2 Diabetes*.

The purpose of this phase 2, multicentre, randomized, double-blind, placebo-controlled, 12-week dose-ranging study was to assess the efficacy, safety, and tolerability of the dipeptidyl peptidase-IV (DPP-IV) inhibitor PF-734200 in adult subjects with type 2 diabetes who were on a stable dose of metformin. Men and women with inadequate glycaemic control with metformin as their sole diabetes medication were randomized to placebo or PF-734200 2 mg, 5 mg, 10 mg, or 20 mg every day. A population subset underwent mixed meal tolerance tests (MMTT) at baseline and week 12. A total of 301 subjects were treated. At week 12, PF-734200 doses of ≥5 mg produced a statistically significant reduction in haemoglobin A (1C) (HbA (1c)) compared with placebo. The mean (95% confidence interval) placebo-adjusted changes in HbA (1c) were -0.31% (-0.70 to 0.08), -0.74% (-1.12 to -0.36), -0.70% (-1.02 to -0.38), and -0.75% (-1.07 to -0.43) for the 2 mg, 5 mg, 10 mg, and 20 mg doses, respectively. PF-734200 20 mg significantly reduced glucose area under the curve following MMTT (-12.8% [-22.9 to -2.7]; p=0.003) compared with placebo. The reductions observed with other doses were not statistically significant. PF-734200 was safe and well tolerated at all doses tested when added to metformin. PF-734200 safely and effectively lowered HbA (1c) in subjects receiving metformin. The 20 mg dose provided the greatest improvements in post-prandial glucose.

Pancreatic perfusion of healthy individuals and type 1 diabetic patients as assessed by magnetic resonance perfusion imaging.

AIMS/HYPOTHESIS: Loss of pancreatic beta cell mass and function leads to the development of diabetes mellitus. Currently there is no technical way to non-invasively image islet function and mass. Murine models suggest that islets are highly vascularised organs that make a significant contribution to the total pancreatic blood flow. The current study was undertaken to test with arterial spin labelling (ASL) magnetic resonance imaging if islet mass and/or stimulation of human pancreatic islets by hyperglycaemia can differentially increase whole-pancreas perfusion, thereby distinguishing non-diabetic from type 1 diabetic patients. METHODS: We assessed pancreatic blood flow using ASL at baseline, during a hyperglycaemia clamp study (glucose at 11 mmol/l) and during recovery to euglycaemia. RESULTS: Seventeen healthy volunteers and seven type 1 diabetic patients were studied. In healthy volunteers we observed no change in pancreatic blood flow during the three phases of the study. A trend for an increase in blood flow was observed in the two control tissues, the liver and kidney. Similarly, there was no significant difference in blood flow during the three stages (baseline, hyperglycaemia and recovery) in diabetic patients and there was no significant difference observed between diabetic patients and normal volunteers. CONCLUSIONS/INTERPRETATION: Our data suggest that in humans neither increased demand nor islet mass has a substantial influence on pancreatic perfusion. It is possible, however, that the current state-of-the art imaging technology employed in this study might not be sensitive enough to distinguish between a true effect and noise. TRIAL REGISTRATION: ClinicalTrials.gov NCT00280085.

Elevated K(+) induces myristoylated alanine-rich C-kinase substrate phosphorylation and phospholipase D activation in glomerulosa cells.

Elevated extracellular potassium concentrations ([K(+)](e)) are known to stimulate aldosterone secretion from adrenal glomerulosa cells in vivo and in vitro. The mechanism is thought to involve depolarization-elicited activation of voltage-dependent calcium channels and an increase in calcium influx. Until now protein kinase C (PKC) was thought not to play a role in the steroidogenic response to elevated [K(+)](e). In this report, we provide evidence in bovine adrenal glomerulosa cells to suggest that elevated [K(+)](e) increases PKC activity, as shown by an enhancement in the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS). Elevated [K(+)](e)-induced MARCKS phosphorylation was delayed and transient and was not the result of a local production of angiotensin II (AngII). MARCKS phosphorylation in response to elevated [K(+)](e) was not accompanied by phosphoinositide hydrolysis but was inhibited by a selective PKC inhibitor. Elevated [K(+)](e) also activated phospholipase D (PLD) in a delayed but sustained manner. We propose that the observed PLD activation mediates the elevated [K(+)](e)-induced MARCKS phosphorylation via PKC, although other factors may modulate this phosphorylation event.

Angiotensin II priming of aldosterone secretion with agents that enhance Ca(2+) influx.

We have previously shown that angiotensin II (AngII) is able to prime, or sensitize, the secretory response of cultured bovine adrenal glomerulosa cells to the Ca(2+) channel agonist, BAY K8644. We examined the ability of AngII to prime glomerulosa cells to an elevated extracellular K(+) level, a physiological agonist that also triggers Ca(2+) influx. K(+) (9 mM) elicited enhanced secretion in AngII-primed cells compared to those with no prior exposure to the hormone, suggesting that AngII can sensitize glomerulosa cells to respond to increases in extracellular K(+). The potential involvement of protein kinase C (PKC) in priming was investigated by determining whether enhanced Ca(2+) influx could maintain the AngII-induced phosphorylation of the endogenous PKC substrate, myristoylated, alanine-rich C kinase substrate (MARCKS). Incubation with the AngII antagonist, saralasin, for 30 min following an AngII exposure reduced the AngII-induced increase in MARCKS phosphorylation. 100 nM BAY K8644 together with saralasin was unable to maintain AngII-stimulated MARCKS phosphorylation. On the other hand, phosphorylation of the steroidogenic acute regulatory (StAR) protein was sustained with saralasin exposure, both in the presence and absence of BAY K8644. This observation suggests that persistent StAR phosphorylation/activation may play a role in priming.

ANPs effect on MARCKS and StAR phosphorylation in agonist-stimulated glomerulosa cells.

Atrial natriuretic peptide (ANP) is a cardiac hormone that inhibits aldosterone secretion induced by all physiologic agonists. The purpose of this study is to explore ANP-induced changes in the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and the steroidogenic acute regulatory protein (StAR), in AngII or K(+)-stimulated glomerulosa cells. The data show that ANP completely inhibits the phosphorylation of MARCKS and partially inhibits that of StAR in cells stimulated with K(+). ANP also partially inhibits MARCKS phosphorylation but does not affect StAR phosphorylation in cells stimulated with AngII. These effects appear to be cGMP-independent and at least partially dependent on inhibition of protein kinase C (PKC). To our knowledge, this is the first report of ANP modulating either MARCKS or StAR phosphorylation in [(32)P]-labeled cells. The data also support the hypothesis that ANP inhibits aldosterone secretion acting as a step involved in cholesterol transport to the mitochondria.

Differential effects of agonists of aldosterone secretion on steroidogenic acute regulatory phosphorylation.

The steroidogenic acute regulatory (StAR) protein mediates cholesterol transport within the mitochondria, and its phosphorylation is believed to be required for steroidogenesis. Increased extracellular potassium concentrations (K(+)), angiotensin II (AngII), and adrenocorticotropic hormone (ACTH) induce aldosterone secretion from bovine adrenal glomerulosa cells. We hypothesized that, although these agonists act via different signaling pathways, StAR phosphorylation should be common to their action. We studied the effects of K(+), AngII, and ACTH, at concentrations that yield comparable secretory responses, on StAR phosphorylation. All three agents induced significant increases in StAR phosphorylation although the response to ACTH was less than that of AngII and K(+). In cells stimulated with the protein kinase C (PKC) agonist 12-tetradecanoylphorbol 13-acetate (TPA), the Ca(2+) channel agonist BAY K8644, and the adenylate cyclase agonist forskolin, TPA caused a small but statistically significant increase in StAR phosphorylation while BAY K8644 and forskolin had no significant effect. Interestingly, the combination of TPA and BAY K8644 produced a larger increase in StAR phosphorylation than the agents alone. We conclude that in cultured bovine adrenal glomerulosa cells the PKC signaling pathway is most effective at inducing StAR phosphorylation but that there is no simple correlation between this event and aldosterone production.

Effects of angiotensin II and adrenocorticotropic hormone on myristoylated alanine-rich C-kinase substrate phosphorylation in glomerulosa cells.

Angiotensin II (AngII) is thought to stimulate aldosterone secretion from bovine adrenal glomerulosa cells in part via activation of protein kinase C (PKC), while adrenocorticotropic hormone (ACTH) functions through increases in intracellular cAMP levels and calcium influx. Rather than using invasive homogenization techniques as in previous studies, we chose to monitor PKC activity in intact glomerulosa cells in situ by measuring the phosphorylation of the endogenous PKC substrate, myristoylated alanine-rich C-kinase substrate (MARCKS). AngII enhanced MARCKS phosphorylation in a rapid, sustained manner; whereas ACTH induced a rapid and sustained inhibition of MARCKS phosphorylation. Studies using pharmacological agents to mimic various signals indicated that the AngII-induced MARCKS phosphorylation was due to PKC activation, and the ACTH-elicited decrease was mediated by increases in calcium influx rather than cAMP production. We propose that changes in the phosphorylation state of MARCKS, an actin-binding protein, may contribute to cytoskeletal rearrangements involved in steroidogenesis.

Stimulation of atrial natriuretic peptide receptor/guanylyl cyclase- A signaling pathway antagonizes the activation of protein kinase C-alpha in murine Leydig cells.

Atrial natriuretic peptide (ANP) regulates diverse physiological responses by binding to its specific guanylyl cyclase-A receptor (Npra) which synthesizes the intracellular second messenger cGMP. To understand the molecular mechanisms of cellular signaling of ANP, we have studied its effect on the enzymatic activity of overexpressed protein kinase C (PKC) in murine Leydig tumor (MA-10) cells which were transfected with PKC-alpha cDNA. Treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (ANG II) and endothelin-1 (ET-1) stimulated the PKC activity by 4-5-fold in PKC-alpha cDNA transfected MA-10 cells. The pretreatment of PKC-alpha transfected cells with ANP significantly inhibited the TPA-, ANG II- and ET-1-stimulated PKC activity. The agonist-stimulated PKC activity was also inhibited in the presence of 8-bromo-cGMP, however, cAMP had no effect on stimulatory PKC activity. The exposure of cells to Npra- antagonist A71915, which blocks the production of cGMP, significantly reduced the inhibitory effect of ANP on agonist-stimulated PKC activity and accumulation of intracellular cGMP in MA-10 cells. Similarly, inhibition of cGMP-dependent protein kinase by KT5823, restored the stimulatory levels of PKC activity in the presence of ANP. These results provide direct evidence that ANP antagonizes the agonist-stimulated PKC activity in MA-10 cells, involving the specific receptor Npra, its second messenger cGMP and cGMP-dependent protein kinase. Together, these findings implicate that ANP may act as a negative mediator of 'cross-talk' between PKC-alpha and Npra signaling pathway in MA-10 cells.