Background. Post-receptor insulin resistance (IR) is associated with hyperglycemia and hepatic steatosis. However, receptor-level IR (e.g. insulin receptor pathogenic variants, INSR) causes hyperglycemia without steatosis. We examined four pathologic conditions of IR in humans to examine pathways controlling lipid metabolism and gluconeogenesis. Methods. Cross-sectional study of severe, receptor IR (INSR, n=7), versus post-receptor IR that was severe (lipodystrophy, n=14), moderate (type 2 diabetes [T2D], n=9) or mild (obesity, n=8). Lipolysis (glycerol turnover), hepatic glucose production (HGP), gluconeogenesis (deuterium incorporation from body water into glucose), hepatic triglyceride (magnetic resonance spectroscopy), and hepatic fat oxidation (plasma β-hydroxybutyrate) were measured. Results. Lipolysis was 2-3-fold higher in INSR versus all other groups, and HGP 2-fold higher in INSR and lipodystrophy versus T2D and obesity (p<0.001) suggesting severe adipose and hepatic IR. INSR subjects had a higher contribution of gluconeogenesis to HGP, ~77%, versus 52-59% in other groups (p=0.0001). Despite high lipolysis, INSR subjects had low hepatic triglycerides (0.5 [0.1-0.5]), in contrast to lipodystrophy (10.6 [2.8-17.1], p<0.0001). β-hydroxybutyrate was 2-7-fold higher in INSR versus all other groups (p<0.0001) consistent with higher hepatic fat oxidation. Conclusion. These data support a key pathogenic role of adipose tissue IR to increase glycerol and FFA availability to the liver in both receptor and post-receptor IR. However, the fate of FFA diverges in these populations. In receptor-level IR, FFA oxidation drives gluconeogenesis rather than being reesterified to triglyceride. In contrast, in post-receptor IR, FFA contributes to both gluconeogenesis and hepatic steatosis. Trial registration. ClinicalTrials.gov NCT01778556; NCT00001987; NCT02457897 Funding. NIDDK, USDA ARS 58-3092-5-001
Hilal Sekizkardes, Stephanie T. Chung, Shaji Chacko, Morey Haymond, Megan Startzell, Mary Walter, Peter J. Walter, Marissa Lightbourne, Rebecca J. Brown
Meal ingestion increases body temperature in multiple species, an effect that is blunted by obesity. However, the mechanisms responsible for these phenomena remain incompletely understood. Here we show that refeeding increases plasma leptin concentrations approximately 8-fold in 48-hour-fasted lean rats, and this normalization of plasma leptin concentrations stimulates adrenomedullary catecholamine secretion. Increased adrenal medulla–derived plasma catecholamines were necessary and sufficient to increase body temperature postprandially, a process that required both fatty acids generated from adipose tissue lipolysis and β-adrenergic activation of brown adipose tissue (BAT). Diet-induced obese rats, which remained relatively hyperleptinemic while fasting, did not exhibit fasting-induced reductions in temperature. To examine the impact of feeding-induced increases in body temperature on energy balance, we compared rats fed chronically by either 2 carbohydrate-rich boluses daily or a continuous isocaloric intragastric infusion. Bolus feeding increased body temperature and reduced weight gain compared with continuous feeding, an effect abrogated by treatment with atenolol. In summary, these data demonstrate that leptin stimulates a hypothalamus–adrenal medulla–BAT axis, which is necessary and sufficient to induce lipolysis and, as a result, increase body temperature after refeeding.
Rachel J. Perry, Kun Lyu, Aviva Rabin-Court, Jianying Dong, Xiruo Li, Yunfan Yang, Hua Qing, Andrew Wang, Xiaoyong Yang, Gerald I. Shulman
To identify neurons that specifically increase blood glucose from among the diversely-functioning cell types in the ventromedial hypothalamic nucleus (VMN), we studied the cholecystokinin (CCK) receptor-B (CCKBR)-expressing VMN targets of glucose-elevating parabrachial nucleus neurons. Activating these VMNCCKBR neurons increased blood glucose. Furthermore, while silencing the broader VMN decreased energy expenditure and promoted weight gain without altering blood glucose, silencing VMNCCKBR neurons decreased hepatic glucose production (HGP), insulin-independently decreasing blood glucose without altering energy balance. Silencing VMNCCKBR neurons also impaired the counter-regulatory response (CRR) to insulin-induced hypoglycemia and glucoprivation and replicated hypoglycemia-associated autonomic failure (HAAF). Hence, VMNCCKBR cells represent a specialized subset of VMN cells that function to elevate glucose. These cells not only mediate the allostatic response to hypoglycemia, but also insulin-independently modulate the homeostatic setpoint for blood glucose, consistent with a role for the brain in the insulin-independent control of glucose homeostasis.
Jonathan N. Flak, Paulette Goforth, James Dell'Orco, Paul V. Sabatini, Chien Li, Nadejda Bozadjieva, Matthew J. Sorensen, Alec C. Valenta, Alan C. Rupp, Alison H. Affinati, Corentin Cras-Méneur, Ahsan Ansari, Jamie Sacksner, Nandan Kodur, Darleen A. Sandoval, Robert t. Kennedy, David Olson, Martin G. Myers Jr.
BACKGROUND. Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity. METHODS. We treated 14 healthy women of diverse ethnicity, 27.5 ± 1.1 y, BMI 25.4 ± 1.2 kg/m2, with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for four weeks, open-label. The primary endpoint was the change in BAT metabolic activity as measured by [18F]-2-fluoro-D-2-deoxy-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT). Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by frequently sampled intravenous glucose tolerance test. RESULTS. Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers high-density lipoprotein (HDL) and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has anti-diabetic and anti-inflammatory capabilities, increased with acute treatment and was 35% higher at study completion. Finally, an intravenous glucose tolerance test demonstrated higher insulin sensitivity, glucose effectiveness, and insulin secretion. CONCLUSION. These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease. TRIAL REGISTRATION. Clinicaltrials.gov NCT03049462. FUNDING. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), DK075112, DK075116, DK071013, and DK071014.
Alana E. O'Mara, James W. Johnson, Joyce D. Linderman, Robert J. Brychta, Suzanne McGehee, Laura A. Fletcher, Yael A. Fink, Devika Kapuria, Thomas M. Cassimatis, Nathan Kelsey, Cheryl Cero, Zahraa Abdul-Sater, Francesca Piccinini, Alison S. Baskin, Brooks P. Leitner, Hongyi Cai, Corina M. Millo, William Dieckmann, Mary Walter, Norman B. Javitt, Yaron Rotman, Peter J. Walter, Marilyn Ader, Richard N. Bergman, Peter Herscovitch, Kong Y. Chen, Aaron M. Cypess
BACKGROUND. Residual C-peptide is detected in many people for years following the diagnosis of type 1 diabetes; however, the physiologic significance of low levels of detectable C-peptide is not known. METHODS. We studied sixty-three adults with type 1 diabetes classified by peak mixed-meal tolerance test (MMTT) C-peptide as negative (<0.007; n =15), low (0.017–0.200; n =16), intermediate (>0.200–0.400; n =15), or high (>0.400 pmol/mL; n =17). We compared the groups’ glycemia from continuous glucose monitoring (CGM), β-cell secretory responses from a glucose-potentiated arginine (GPA) test, insulin sensitivity from a hyperinsulinemia euglycemic (EU) clamp, and glucose counterregulatory responses from a subsequent hypoglycemic (HYPO) clamp. RESULTS. Low and intermediate MMTT C-peptide groups did not exhibit β-cell secretory responses to hyperglycemia, whereas the high C-peptide group showed increases in both C-peptide and proinsulin (P ≤0.01). All groups with detectable MMTT C-peptide demonstrated acute C-peptide and proinsulin responses to arginine that were positively correlated with peak MMTT C-peptide (P <0.0001 for both analytes). During the EU-HYPO clamp, C-peptide levels were proportionately suppressed in the low, intermediate, and high C-peptide compared to the negative group (P ≤0.0001), whereas glucagon increased from EU to HYPO only in the high C-peptide group compared to negative (P =0.01). CGM demonstrated lower mean glucose and more time-in-range for the high C-peptide group. CONCLUSION. These results indicate that in adults with type 1 diabetes, β-cell responsiveness to hyperglycemia and α-cell responsiveness to hypoglycemia are only observed at high levels of residual C-peptide that likely contribute to glycemic control.
Michael R. Rickels, Carmella Evans-Molina, Henry T. Bahnson, Alyssa Ylescupidez, Kristen J. Nadeau, Wei Hao, Mark A. Clements, Jennifer L. Sherr, Richard E. Pratley, Tamara S. Hannon, Viral N. Shah, Kellee M. Miller, Carla J. Greenbaum
Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone–producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.
Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi
The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin–angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.
Matthew J. Taylor, Matthew R. Ullenbruch, Emily C. Frucci, Juilee Rege, Mark S. Ansorge, Celso E. Gomez-Sanchez, Salma Begum, Edward Laufer, David T. Breault, William E. Rainey
Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40% to 50% of adults with CF. The age-at-onset of CF-related diabetes (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age-at-onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that two common DNA haplotypes formed by the risk variants account for the association with diabetes (high risk, P-value: 4.34E-3; low risk, P-value: 1.14E-3). Single-cell RNA (scRNA) sequencing indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells, and frequently co-expressed with CFTR along with transcription factors that have binding sites 5′ of SLC26A9. These findings replicated upon re-analysis of scRNA data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9 bearing variants from the low risk haplotype generated 12% to 20% higher levels of expression in PANC-1 and CFPAC-1 cells compared to the high risk haplotype (P-values: 2.00E-3 to 5.15E-9). Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age-at-onset of diabetes, thereby suggesting a CFTR-agnostic treatment for a major complication of CF.
Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting
In order to determine whether the glucose-alanine cycle regulates rates of hepatic mitochondrial oxidation in humans, we applied positional isotopomer NMR tracer analysis (PINTA) to assess rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in healthy volunteers following both an overnight (12 hours) and a 60-hour fast. Following the 60-hour fast, rates of endogenous glucose production and mitochondrial oxidation decreased, whereas rates of hepatic pyruvate carboxylase flux remained unchanged. These reductions were associated with reduced rates of alanine turnover, assessed by [3-13C]alanine, in a subgroup of participants under similar fasting conditions. In order to determine whether this reduction in alanine turnover was responsible for the reduced rates of hepatic mitochondrial oxidation, we infused unlabeled alanine into another subgroup of 60-hour fasted subjects to increase rates of alanine turnover, similar to what was measured after a 12-hour fast, and found that this perturbation increased rates of hepatic mitochondrial oxidation. Taken together, these studies demonstrate that 60 hours of starvation induce marked reductions in rates of hepatic mitochondrial oxidation, which in turn can be attributed to reduced rates of glucose-alanine cycling, and reveal a heretofore undescribed role for glucose-alanine in the regulation of hepatic mitochondrial oxidation in humans.
Kitt Falk Petersen, Sylvie Dufour, Gary W. Cline, Gerald I. Shulman
Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with antiinflammatory and antidiabetic effects. PAHSAs reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin-resistant aged chow- and high-fat diet–fed (HFD-fed) mice. Here, we aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. Both acute and chronic PAHSA treatment enhanced the action of insulin to suppress endogenous glucose production (EGP) in chow- and HFD-fed mice. Moreover, chronic PAHSA treatment augmented insulin-stimulated glucose uptake in glycolytic muscle and heart in HFD-fed mice. The mechanisms by which PAHSAs enhanced hepatic insulin sensitivity included direct and indirect actions involving intertissue communication between adipose tissue and liver. PAHSAs inhibited lipolysis directly in WAT explants and enhanced the action of insulin to suppress lipolysis during the clamp in vivo. Preventing the reduction of free fatty acids during the clamp with Intralipid infusion reduced PAHSAs’ effects on EGP in HFD-fed mice but not in chow-fed mice. Direct hepatic actions of PAHSAs may also be important, as PAHSAs inhibited basal and glucagon-stimulated EGP directly in isolated hepatocytes through a cAMP-dependent pathway involving Gαi protein–coupled receptors. Thus, this study advances our understanding of PAHSA biology and the physiologic mechanisms by which PAHSAs exert beneficial metabolic effects.
Peng Zhou, Anna Santoro, Odile D. Peroni, Andrew T. Nelson, Alan Saghatelian, Dionicio Siegel, Barbara B. Kahn