Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle

Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5′-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism

Acute Overactive Endocannabinoid Signaling Induces Glucose Intolerance, Hepatic Steatosis, and Novel Cannabinoid Receptor 1 Responsive Genes

Endocannabinoids regulate energy balance and lipid metabolism by stimulating the cannabinoid receptor type 1 (CB1). Genetic deletion and pharmacological antagonism have shown that CB1 signaling is necessary for the development of obesity and related metabolic disturbances. However, the sufficiency of endogenously produced endocannabinoids to cause hepatic lipid accumulation and insulin resistance, independent of food intake, has not been demonstrated. Here, we show that a single administration of isopropyl dodecylfluorophosphonate (IDFP), perhaps the most potent pharmacological inhibitor of endocannabinoid degradation, increases hepatic triglycerides (TG) and induces insulin resistance in mice. These effects involve increased CB1 signaling, as they are mitigated by pre-administration of a CB1 antagonist (AM251) and in CB1 knockout mice. Despite the strong physiological effects of CB1 on hepatic lipid and glucose metabolism, little is known about the downstream targets responsible for these effects. To elucidate transcriptional targets of CB1 signaling, we performed microarrays on hepatic RNA isolated from DMSO (control), IDFP and AM251/IDFP-treated mice. The gene for the secreted glycoprotein lipocalin 2 (lcn2), which has been implicated in obesity and insulin resistance, was among those most responsive to alterations in CB1 signaling. The expression pattern of IDFP mice segregated from DMSO mice in hierarchal cluster analysis and AM251 pre-administration reduced (>50%) the majority (303 of 533) of the IDFP induced alterations. Pathway analysis revealed that IDFP altered expression of genes involved in lipid, fatty acid and steroid metabolism, the acute phase response, and amino acid metabolism in a CB1-dependent manner. PCR confirmed array results of key target genes in multiple independent experiments. Overall, we show that acute IDFP treatment induces hepatic TG accumulation and insulin resistance, at least in part through the CB1 receptor, and identify novel cannabinoid responsive genes

Metabolic Regulation by Lipid Activated Receptors

Obesity and related metabolic disorders have reached epidemic levels with dire public health consequences. Efforts to stem the tide focus on behavioral and pharmacological interventions. Several hypolipidemic pharmaceutical agents target endogenous lipid receptors, including the peroxisomal proliferator activated receptor alpha (PPAR alpha) and cannabinoid receptor 1 (CB1). To further the understanding of these clinically relevant receptors, we elucidated the biochemical basis of PPAR alpha activation by lipoprotein lipolysis products and the metabolic and transcriptional responses to elevated endocannabinoid signaling. PPAR alpha is activated by fatty acids and their derivatives in vitro. While several specific pathways have been implicated in the generation of PPAR alpha ligands, we focused on lipoprotein lipase mediated lipolysis of triglyceride rich lipoproteins. Fatty acids activated PPAR alpha similarly to VLDL lipolytic products. Unbound fatty acid concentration determined the extent of PPAR alpha activation. Lipolysis of VLDL, but not physiological unbound fatty acid concentrations, created the fatty acid uptake necessary to stimulate PPAR alpha. Consistent with a role for vascular lipases in the activation of PPAR alpha, administration of a lipase inhibitor (p-407) prevented PPAR alpha dependent induction of target genes in fasted mice. Apolipoprotein CIII, an endogenous inhibitor of lipoprotein lipase, regulated access to the lipoprotein pool of PPAR alpha ligands. Our results support a role for the local generation of PPAR alpha ligands by lipase activity. The endocannabinoid system regulates diverse physiological functions, including energy balance. While loss of CB1 signaling has more pronounced effects on lipid parameters than expected based on weight loss alone, direct demonstration of endocannabinoid regulation of lipid metabolism is lacking. To test the effects of endogenously produced cannabinoids on lipid metabolism, independent of alterations in food intake, we analyzed tissues from mice treated with IDFP, an organophosphorus inhibitor of endocannabinoid breakdown. IDFP administration inhibited hepatic monoacylglycerol lipase leading to elevated levels of 2-arachidonylglycerol. We found that IDFP administration caused accumulation of apoE depleted VLDL. HDL particles accumulated apoE and failed to transfer it to VLDL in vitro. Importantly, these effects were prevented by pharmacological inhibition of CB1 and absent in plasma from CB1 mice. IDFP also caused a CB1-dependent increase in hepatic triglycerides. Thus, endocannabinoids inhibit the transfer of apoE from HDL to VLDL leading to apoE depletion of triglyceride rich lipoproteins. Microarray analysis allowed us to determine the effects of IDFP on expression of genes involved in lipid metabolism and to discover novel cannabinoid responsive genes. IDFP increased expression of lipogenic and SREBP2 target genes in a CB1-dependent fashion. On a global scale, pre-administration of a CB1 antagonist prevented many of the IDFP induced alterations in gene expression. IDFP decreased expression of genes involved in amino acid metabolism and inflammation. PCR analysis of selected mRNAs confirmed several of the key array findings. Our work indicates that endocannabinoids exert a large influence on hepatic lipid metabolism independent of food intake and suggest that peripherally restricted CB1 antagonists may be of therapeutic value. Overall, these findings shed light on the endogenous mechanisms of PPAR alpha activation and the hepatic responses to Cb1 activation. This information may help guide the continuing effort to develop treatments for metabolic disease

Long-Acting Bupivacaine for Pain Control After Cesarean Birth

Objective: To evaluate women\u27s postcesarean pain levels and total opioid use for standard opioid pain management compared with local anesthetic with patient-requested opioids. Design: Retrospective cohort study. Setting/local problem: Rural southeast Ohio. Ohio had a higher rate of opioid use disorder (1.4%) than both the regional average (0.8%) and the national average (0.7%). Participants: We performed a retrospective study of 402 medical records of women who gave birth by cesarean. Intervention: Women were provided one of three types of perioperative anesthesia: routine spinal (standard of care group), wound infiltration with liposomal bupivacaine (LB INF), and transversus abdominis plane (TAP) block with liposomal bupivacaine (LB TAP). Data were collected on the amount of opioids taken postoperatively (measured as morphine milligram equivalents [MME]), pain scores, and history of opioid use. Results: The LB INF and LB TAP groups had significantly lower total and average MME per day than the standard of care group (p \u3c .001). Pain scores for the LB INF group were lower on postoperative day (POD) 0 and POD1, whereas LB TAP pain scores were lower than standard of care pain scores on POD1 (p \u3c .004). Women with a prior history of substance use disorders reported higher pain scores, took more total opioids. and stayed in the hospital longer regardless of type of anesthesia received (p \u3c .001). Conclusion: LB INF and LB TAP were associated with lower amounts of opioids used and with lower postcesarean pain scores compared with the standard of care

Incidence and Visual Outcomes of Culture-Proven Endophthalmitis Following Dexamethasone Intravitreal Implant.

IMPORTANCE: The rate of endophthalmitis following dexamethasone intravitreal implant (DEX) has varied in large clinical trials. Furthermore, to our knowledge, the optimal management of eyes with endophthalmitis associated with DEX has not been established. OBJECTIVE: To report the incidence of culture-proven endophthalmitis in a single vitreoretinal practice over the course of 3 years and describe the clinical outcomes associated with each case of endophthalmitis. DESIGN, SETTING, AND PARTICIPANTS: All patients who received DEX between January 14, 2013, and August 31, 2016, were included in this retrospective single-center case series at a private vitreoretinal practice. The patients were identified during a search of the billing records over the period of interest. Cases of endophthalmitis associated with DEX were also identified. EXPOSURES: Treatment with DEX. MAIN OUTCOMES AND MEASURES: Development of endophthalmitis following DEX and the clinical management and outcomes of each case of endophthalmitis. RESULTS: Of the 1051 participants who collectively received 3593 injections of DEX, 4 patients developed endophthalmitis; all 4 patients were white, female, and 60 years or older (mean [SD] age, 75.6 [13] years). Two patients had culture-proven bacterial endophthalmitis after DEX monoinjections (0.06% of injections and 0.2% of patients). Three other cases of endophthalmitis developed after coinjection with bevacizumab (aggregate rate: 0.14% of injections and 0.38% of patients), of which 2 were culture positive. One patient developed endophthalmitis on 2 separate occasions. Vitrectomy was performed in 2 patients, and in 1 of these patients, the implant was removed. All 4 patients were treated with injection of intravitreous vancomycin and ceftazidime. CONCLUSIONS AND RELEVANCE: These data suggest that endophthalmitis is a rare event following injection of DEX. However, given the rarity of endophthalmitis following DEX and the heterogeneity among our reported cases, it remains unclear whether the DEX endophthalmitis rate approximates that of intravitreous anti-vascular endothelial growth factor therapy. These data also suggest that vitrectomy with removal of DEX may not be necessary in all cases of DEX-associated endophthalmitis

Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates

Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However, at metabolic steady state, FA oxidation must match FA intake plus synthesis; moreover, FA intake is low, not high, during CR. Therefore, it is not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole body fuel selection, endogenous FA synthesis, and gene expression in mice on CR. Within 2 days of CR being started, a shift to a cyclic, diurnal pattern of whole body FA metabolism occurred, with an initial phase of elevated endogenous FA synthesis [respiratory exchange ratio (RER) >1.10, lasting 4-6 h after food provision], followed by a prolonged phase of FA oxidation (RER = 0.70, lasting 18-20 h). CR mice oxidized four times as much fat per day as ad libitum (AL)-fed controls (367 +/- 19 vs. 97 +/- 14 mg/day, P < 0.001) despite reduced energy intake from fat. This increase in FA oxidation was balanced by a threefold increase in adipose tissue FA synthesis compared with AL. Expression of FA synthase and acetyl-CoA carboxylase mRNA were increased in adipose and liver in a time-dependent manner. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for oxidative damage and disease risk

Large-Scale Functional Genomics Screen to Identify Modulators of Human β-Cell Insulin Secretion

Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic β-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-βH1 cells, a human β-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-βH1 cells for large-scale siRNA screening to identify candidate genes regulating β-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional β-cell mass occurring in T2D