N-Hydroxyethyl-1-Deoxynojirimycin (Miglitol) Restores the Counterregulatory Response to Hypoglycemia Following Antecedent Hypoglycemia.

Antecedent hypoglycemia suppresses the counterregulatory responses to subsequent hypoglycemic episodes, which can be prevented by normalizing portal-mesenteric vein (PMV) glycemia alone during the antecedent bout. Since the sodium-glucose transporter 3 receptor has been implicated in PMV glucosensing, we hypothesized that PMV infusion of the sodium-glucose cotransporter 3 receptor agonist N-hydroxyethyl-1-deoxynojirimycin (miglitol) would rescue the sympathoadrenal response to subsequent hypoglycemia. Rats underwent hyperinsulinemic-hypoglycemic clamps on 2 consecutive days without miglitol infusion (antecedent hypoglycemia without miglitol [HYPO]) or with miglitol infused upstream in the PMV, perfusing the glucosensors, or adjacent to the liver, bypassing PMV glucosensors, on day 1 or day 2. Control animals underwent day 1 euglycemic clamps, followed by hypoglycemic clamps on day 2. Peak epinephrine (EPI) responses for HYPO on day 2 were significantly blunted when compared with controls. Miglitol infusion on day 1 proved ineffective in restoring the EPI response following antecedent hypoglycemia, but day 2 miglitol infusion restored EPI responses to control levels. As norepinephrine and glucagon demonstrated similar responses, day 2 administration of miglitol effectively restored the counterregulatory response following antecedent hypoglycemia. In subsequent experiments, we demonstrate similar results with reduced miglitol infusion doses, approaching those currently prescribed for type 2 diabetes (correcting for rodent size), as well as the efficacy of oral miglitol administration in restoring the counterregulatory responses following antecedent hypoglycemia.

Catecholaminergic projections into an interconnected forebrain network control the sensitivity of male rats to diet-induced obesity.

Hindbrain catecholamine neurons convey gut-derived metabolic signals to an interconnected neuronal network in the hypothalamus and adjacent forebrain. These neurons are critical for short-term glycemic control, glucocorticoid and glucoprivic feeding responses, and glucagon-like peptide 1 (GLP-1) signaling. Here we investigate whether these pathways also contribute to long-term energy homeostasis by controlling obesogenic sensitivity to a high-fat/high-sucrose choice (HFSC) diet. We ablated hindbrain-originating catecholaminergic projections by injecting anti-dopamine-ß-hydroxylase-conjugated saporin (DSAP) into the paraventricular nucleus of the hypothalamus (PVH) of male rats fed a chow diet for up to 12 wk or a HFSC diet for 8 wk. We measured the effects of DSAP lesions on food choices; visceral adiposity; plasma glucose, insulin, and leptin; and indicators of long-term ACTH and corticosterone secretion. We also determined lesion effects on the number of carbohydrate or fat calories required to increase visceral fat. Finally, we examined corticotropin-releasing hormone levels in the PVH and arcuate nucleus expression of neuropeptide Y ( Npy), agouti-related peptide ( Agrp), and proopiomelanocortin ( Pomc). DSAP-injected chow-fed rats slowly increase visceral adiposity but quickly develop mild insulin resistance and elevated blood glucose. DSAP-injected HFSC-fed rats, however, dramatically increase food intake, body weight, and visceral adiposity beyond the level in control HFSC-fed rats. These changes are concomitant with 1) a reduction in the number of carbohydrate calories required to generate visceral fat, 2) abnormal Npy, Agrp, and Pomc expression, and 3) aberrant control of insulin secretion and glucocorticoid negative feedback. Long-term metabolic adaptations to high-carbohydrate diets, therefore, require intact forebrain catecholamine projections. Without them, animals cannot alter forebrain mechanisms to restrain increased visceral adiposity.

The rate of fall of blood glucose determines the necessity of forebrain-projecting catecholaminergic neurons for male rat sympathoadrenal responses.

Different onset rates of insulin-induced hypoglycemia use distinct glucosensors to activate sympathoadrenal counterregulatory responses (CRRs). Glucosensory elements in the portal-mesenteric veins are dispensable with faster rates when brain elements predominate, but are essential for responses to the slower-onset hypoglycemia that is common with insulin therapy. Whether a similar rate-associated divergence exists within more expansive brain networks is unknown. Hindbrain catecholamine neurons distribute glycemia-related information throughout the forebrain. We tested in male rats whether catecholaminergic neurons that project to the medial and ventromedial hypothalamus are required for sympathoadrenal CRRs to rapid- and slow-onset hypoglycemia and whether these neurons are differentially engaged as onset rates change. Using a catecholamine-specific neurotoxin and hyperinsulinemic-hypoglycemic clamps, we found that sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia require hypothalamus-projecting catecholaminergic neurons, the majority of which originate in the ventrolateral medulla. As determined with Fos, these neurons are differentially activated by the two onset rates. We conclude that 1) catecholaminergic projections to the hypothalamus provide essential information for activating sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia, 2) hypoglycemia onset rates have a major impact on the hypothalamic mechanisms that enable sympathoadrenal CRRs, and 3) hypoglycemia-related sensory information activates hindbrain catecholaminergic neurons in a rate-dependent manner.

Continuous 24-h nicotinic acid infusion in rats causes FFA rebound and insulin resistance by altering gene expression and basal lipolysis in adipose tissue.

Nicotinic acid (NA) has been used as a lipid drug for five decades. The lipid-lowering effects of NA are attributed to its ability to suppress lipolysis in adipocytes and lower plasma FFA levels. However, plasma FFA levels often rebound during NA treatment, offsetting some of the lipid-lowering effects of NA and/or causing insulin resistance, but the underlying mechanisms are unclear. The present study was designed to determine whether a prolonged, continuous NA infusion in rats produces a FFA rebound and/or insulin resistance. NA infusion rapidly lowered plasma FFA levels (>60%, P < 0.01), and this effect was maintained for ≥5 h. However, when this infusion was extended to 24 h, plasma FFA levels rebounded to the levels of saline-infused control rats. This was not due to a downregulation of NA action, because when the NA infusion was stopped, plasma FFA levels rapidly increased more than twofold (P < 0.01), indicating that basal lipolysis was increased. Microarray analysis revealed many changes in gene expression in adipose tissue, which would contribute to the increase in basal lipolysis. In particular, phosphodiesterase-3B gene expression decreased significantly, which would increase cAMP levels and thus lipolysis. Hyperinsulinemic glucose clamps showed that insulin's action on glucose metabolism was improved during 24-h NA infusion but became impaired with increased plasma FFA levels after cessation of NA infusion. In conclusion, a 24-h continuous NA infusion in rats resulted in an FFA rebound, which appeared to be due to altered gene expression and increased basal lipolysis in adipose tissue. In addition, our data support a previous suggestion that insulin resistance develops as a result of FFA rebound during NA treatment. Thus, the present study provides an animal model and potential molecular mechanisms of FFA rebound and insulin resistance, observed in clinical studies with chronic NA treatment.

USF1 contributes to high serum lipid levels in Dutch FCHL families and U.S. whites with coronary artery disease.

OBJECTIVE: Familial combined hyperlipidemia (FCHL) characterized by high serum total cholesterol and/or triglycerides (TGs) is a common dyslipidemia predisposing to coronary artery disease (CAD). Recently, the upstream transcription factor 1 (USF1) was linked and associated with FCHL and TGs in Finnish FCHL families. Here we examined the previously associated rs3737787 SNP in extended Dutch FCHL families (n=532) and in a cohort of US subjects who underwent diagnostic coronary angiography (n=1533). METHODS AND RESULTS: In males of the Dutch FCHL families, we observed significant sex-dependent associations between the common allele of rs3737787 and FCHL, TGs, and related metabolic traits (P=0.02 to 0.006). In the U.S. Whites, sex-dependent associations with TGs and related metabolic traits were observed for the common allele of rs3737787 in males (P=0.04 to 0.02) and rare allele in females (P=0.05 to 0.002). This intriguing relationship was further supported by the highly significant genotype x sex interactions observed for TGs in the Dutch and TGs and body mass index (BMI) in U.S. White subjects with CAD (P=0.0005 to 0.00004). CONCLUSIONS: These data show that USF1 influences several cardiovascular risk factors in a sex-dependent manner in Dutch FCHL families and U.S. Whites with CAD. A significant interaction between sex and genotype was shown to affect TGs and BMI.

PRKCA and multiple sclerosis: association in two independent populations.

Multiple sclerosis (MS) is a chronic disease of the central nervous system responsible for a large portion of neurological disabilities in young adults. Similar to what occurs in numerous complex diseases, both unknown environmental factors and genetic predisposition are required to generate MS. We ascertained a set of 63 Finnish MS families, originating from a high-risk region of the country, to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24. Initial single nucleotide polymorphism (SNP)-based association implicated PRKCA (protein kinase C alpha) gene, and this association was replicated in an independent set of 148 Finnish MS families (p = 0.0004; remaining significant after correction for multiple testing). Further, a dense set of 211 SNPs evenly covering the PRKCA gene and the flanking regions was selected from the dbSNP database and analyzed in two large, independent MS cohorts: in 211 Finnish and 554 Canadian MS families. A multipoint SNP analysis indicated linkage to PRKCA and its telomeric flanking region in both populations, and SNP haplotype and genotype combination analyses revealed an allelic variant of PRKCA, which covers the region between introns 3 and 8, to be over-represented in Finnish MS cases (odds ratio = 1.34, 95% confidence interval 1.07-1.68). A second allelic variant, covering the same region of the PRKCA gene, showed somewhat stronger evidence for association in the Canadian families (odds ratio = 1.64, 95% confidence interval 1.39-1.94). Initial functional relevance for disease predisposition was suggested by the expression analysis: The transcript levels of PRKCA showed correlation with the copy number of the Finnish and Canadian "risk" haplotypes in CD4-negative mononuclear cells of five Finnish multiplex families and in lymphoblast cell lines of 11 Centre d'Etude du Polymorphisme Humain (CEPH) individuals of European origin.

Fine mapping of the multiple sclerosis susceptibility locus on 5p14-p12.

Linkage analyses have identified four major MS susceptibility loci in Finns. Here we have fine mapped the region on chromosome 5p in 28 Finnish MS families. Marker D5S416 provided the highest pairwise LOD score, and multipoint and haplotype analyses restrict the critical region to about 5.3 Mb on 5p15 between markers D5S1987 and D5S416. Ascertaining for HLA type and geographical origin indicated that families with and without the HLA DR15 risk haplotype, as well as families within and outside an internal high-risk region, contributed to the linkage to 5p, implying the general significance for this locus in Finnish MS families.

Familial combined hyperlipidemia in Mexicans: association with upstream transcription factor 1 and linkage on chromosome 16q24.1.

OBJECTIVE: To investigate the largely unknown genetic component of the common lipid disorder, familial combined hyperlipidemia (FCHL) in Mexicans, we analyzed the upstream transcription factor 1 (USF1) gene that was recently associated with FCHL and high triglycerides (TG) in Finns. We also analyzed the Mexican FCHL families for 26 microsatellite markers residing in the seven chromosomal regions on 2p25.1, 9p23, 10q11.23, 11q13, 16q24.1, 19q13, and 21q21, previously linked to FCHL in whites. METHODS AND RESULTS: We genotyped 314 individuals in 24 Mexican families for 13 SNPs spanning an 88-kb region, including USF1. The FCHL and TG traits showed significant evidence for association with 3 SNPs, hCV1459766, rs3737787, and rs2073658, and haplotype analyses further supported these findings (probability values of 0.05 to 0.0009 for SNPs and their haplotypes). Of these SNPs, hCV1459766 is located in the F11 receptor (F11R) gene, located next to USF1, making it difficult to exclude. Importantly, the association was restricted to a considerably smaller region than in the Finns (14 kb versus 46 kb), possibly because of a different underlying linkage disequilibrium structure. In addition, 1 of the 7 regions, 16q24.1, showed suggestive evidence for linkage (a lod score of 2.6) for total cholesterol in Mexicans. CONCLUSIONS: This study, the first to extensively investigate the genetic component of the common FCHL disorder in Mexicans, provides independent evidence for the role of USF1 in FCHL in an outbred population and links the 16q24.1 region to an FCHL-component trait in Mexicans.

Comparison of GenFlex Tag array and Pyrosequencing in SNP genotyping.

With the completion of the Human Genome Project, over 2 million sequence-verified single nucleotide polymorphisms (SNPs) have been deposited in public databases. The challenge has shifted from SNP identification to high-throughput SNP genotyping. Although this has had little impact on molecular diagnostics, it provides the potential for future molecular diagnostics of complex traits to include SNP profiling. Accordingly, efficient, accurate, and flexible SNP genotyping are needed. In addition, the drive for low cost has pushed genotyping reactions toward multiplexing capability. We compared two SNP genotyping techniques: Affymetrix GenFlex Tag array and Pyrosequencing. The reference method was a well-established, solid-phase, single nucleotide extension reaction technique based on tritium detection. Fourteen SNPs were selected from the fine mapping project of a multiple sclerosis locus on chromosome 17q. Using all three techniques and the reference method, the SNPs were analyzed in 96 related individuals. Without extensive optimization, we successfully genotyped 11 of 14 SNPs with both GenFlex and Pyrosequencing. Our study suggests that the Pyrosequencing technique provides higher accuracy between the two systems which is most likely due to the single-stranded template in the extension reaction. Thus, Pyrosequencing has potential for diagnostic applications. Pyrosequencing, however, is not optimal for large SNP profiling analyses wherein multiplexing potential is an advantage.