Colleen M. Craig

Adipose Cell Size and Regional Fat Deposition as Predictors of Metabolic Response to Overfeeding in Insulin-Resistant and Insulin-Sensitive Humans

Obesity is associated with insulin resistance, but significant variability exists between similarly obese individuals, pointing to qualitative characteristics of body fat as potential mediators. To test the hypothesis that obese, insulin-sensitive (IS) individuals possess adaptive adipose cell/tissue responses, we measured subcutaneous adipose cell size, insulin suppression of lipolysis, and regional fat responses to short-term overfeeding in BMI-matched overweight/obese individuals classified as IS or insulin resistant (IR). At baseline, IR subjects exhibited significantly greater visceral adipose tissue (VAT), intrahepatic lipid (IHL), plasma free fatty acids, adipose cell diameter, and percentage of small adipose cells. With weight gain (3.1 ± 1.4 kg), IR subjects demonstrated no significant change in adipose cell size, VAT, or insulin suppression of lipolysis and only 8% worsening of insulin-mediated glucose uptake (IMGU). Alternatively, IS subjects demonstrated significant adipose cell enlargement; decrease in the percentage of small adipose cells; increase in VAT, IHL, and lipolysis; 45% worsening of IMGU; and decreased expression of lipid metabolism genes. Smaller baseline adipose cell size and greater enlargement with weight gain predicted decline in IMGU, as did increase in IHL and VAT and decrease in insulin suppression of lipolysis. Weight gain in IS humans causes maladaptive changes in adipose cells, regional fat distribution, and insulin resistance. The correlation between development of insulin resistance and changes in adipose cell size, VAT, IHL, and insulin suppression of lipolysis highlight these factors as potential mediators between obesity and insulin resistance.

Critical role for GLP-1 in symptomatic post-bariatric hypoglycaemia

Aims/hypothesisPost-bariatric hypoglycaemia (PBH) is a rare, but severe, metabolic disorder arising months to years after bariatric surgery. It is characterised by symptomatic postprandial hypoglycaemia, with inappropriately elevated insulin concentrations. The relative contribution of exaggerated incretin hormone signalling to dysregulated insulin secretion and symptomatic hypoglycaemia is a subject of ongoing inquiry. This study was designed to test the hypothesis that PBH and associated symptoms are primarily mediated by glucagon-like peptide-1 (GLP-1).MethodsWe conducted a double-blinded crossover study wherein eight participants with confirmed PBH were assigned in random order to intravenous infusion of the GLP-1 receptor (GLP-1r) antagonist. Exendin (9-39) (Ex-9), or placebo during an OGTT on two separate days at the Stanford University Clinical and Translational Research Unit. Metabolic, symptomatic and pharmacokinetic variables were evaluated. Results were compared with a cohort of BMI- and glucose-matched non-surgical controls (NSCs).ResultsInfusion of Ex-9 decreased the time to peak glucose and rate of glucose decline during OGTT, and raised the postprandial nadir by over 70%, normalising it relative to NSCs and preventing hypoglycaemia in all PBH participants. Insulin AUC and secretion rate decreased by 57% and 71% respectively, and peak postprandial insulin was normalised relative to NSCs. Autonomic and neuroglycopenic symptoms were significantly reduced during Ex-9 infusion.Conclusions/interpretationGLP-1r blockade prevented hypoglycaemia in 100% of individuals, normalised beta cell function and reversed neuroglycopenic symptoms, supporting the conclusion that GLP-1 plays a primary role in mediating hyperinsulinaemic hypoglycaemia in PBH. Competitive antagonism at the GLP-1r merits consideration as a therapeutic strategy.Trial registration :ClinicalTrials.gov NCT02550145

In vivo 2H2O administration reveals impaired triglyceride storage in adipose tissue of insulin-resistant humans

Indirect evidence suggests that impaired triglyceride storage in the subcutaneous fat depot contributes to the development of insulin resistance via lipotoxicity. We directly tested this hypothesis by measuring, in vivo, TG synthesis, de novo lipogenesis (DNL), adipocyte proliferation, and insulin suppression of lipolysis in subcutaneous adipose tissue of BMI-matched individuals classified as insulin resistant (IR) or insulin sensitive (IS). Nondiabetic, moderately obese subjects with BMI 25–35 kg/m2, classified as IR or IS by the modified insulin suppression test, consumed deuterated water (2H2O) for 4 weeks. Deuterium incorporation into glycerol, palmitate, and DNA indicated TG synthesis, DNL, and adipocyte proliferation, respectively. Net TG synthesis and DNL in adipose cells were significantly lower in IR as compared with IS subjects, whereas adipocyte proliferation did not differ significantly. Plasma FFAs measured during an insulin suppression test were 2.5-fold higher in IR subjects, indicating resistance to insulin suppression of lipolysis. Adipose TG synthesis correlated directly with DNL but not with proliferation. These results provide direct in vivo evidence for impaired TG storage in subcutaneous adipose tissue of IR as compared with IS. Relative inability to store TG in the subcutaneous depot may represent a mechanism contributing to the development of insulin resistance in the setting of obesity.

Integrative Personal Omics Profiles during Periods of Weight Gain and Loss

Advances in omics technologies now allow an unprecedented level of phenotyping for human diseases, including obesity, in which individual responses to excess weight are heterogeneous and unpredictable. To aid the development of better understanding of these phenotypes, we performed a controlled longitudinal weight perturbation study combining multiple omics strategies (genomics, transcriptomics, multiple proteomics assays, metabolomics, and microbiomics) during periods of weight gain and loss in humans. Results demonstrated that: (1) weight gain is associated with the activation of strong inflammatory and hypertrophic cardiomyopathy signatures in blood; (2) although weight loss reverses some changes, a number of signatures persist, indicative of long-term physiologic changes; (3) we observed omics signatures associated with insulin resistance that may serve as novel diagnostics; (4) specific biomolecules were highly individualized and stable in response to perturbations, potentially representing stable personalized markers. Most data are available open access and serve as a valuable resource for the community.

THE USE OF GASTROSTOMY TUBE FOR THE LONG-TERM REMISSION OF HYPERINSULINEMIC HYPOGLYCEMIA AFTER ROUX-EN-Y GASTRIC BYPASS: A CASE REPORT

ABSTRACT Objective: Hyperinsulinemic hypoglycemia is an increasingly reported complication of Roux-en-Y gastric bypass surgery (RYGB), for which there is currently no acceptable treatment. We present a case of the reversal of severe hyperinsulinemic hypoglycemia through gastrostomy tube (GT) feeding to the remnant stomach and uniquely report the durable resolution of neuroglycopenic symptoms 3 years after GT placement. Methods: The case subject underwent standardized postprandial measurement of plasma glucose, insulin, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon concentrations after oral or GT administration of a standardized liquid meal. Results: Hypersecretion of insulin, GLP-1, and glucagon elicited by oral administration of the liquid meal were reversed with GT feeding. GIP was not secreted in excess of normal after the oral meal. Conclusion: This case of reversible hyperinsulinemic hypoglycemia through GT feeding illustrates the physiology of this dis...

High Frequency Actionable Pathogenic Exome Mutations in an Average-Risk Cohort

Whole exome sequencing (WES) is increasingly utilized in both clinical and non-clinical settings, but little is known about the utility of WES in healthy individuals. In order to determine the frequency of both medically actionable and non-actionable but medically relevant exome findings in the general population we assessed the exomes of 70 participants who have been extensively characterized over the past several years as part of a longitudinal integrated multi-omics profiling study at Stanford University. We assessed exomes for rare likely pathogenic and pathogenic variants in genes associated with Mendelian disease in the Online Mendelian Inheritance in Man (OMIM) database. We used American College of Medical Genetics (ACMG) guidelines were used for the classification of rare sequence variants, and additionally we assessed pharmacogenetic variants. Twelve out of 70 (17%) participants had medically actionable findings in Mendelian disease genes, including 6 (9%) with mutations in genes not currently included in the ACMG’s list of 59 actionable genes. This number is higher than that reported in previous studies and suggests added benefit from utilizing expanded gene lists and manual curation to assess actionable findings. A total of 60 participants (89%) had non-actionable findings identified including 57 who were found to be mutation carriers for recessive diseases and 21 who have increased Alzheimer’s disease risk due to heterozyg ous or homozygous APOE e4 alleles (18 participants had both). These results suggest that exome sequencing may have considerably more utility for health management in the general population than previously thought.

Adipose tissue macrophages impair preadipocyte differentiation in humans

Aim The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation. Methods Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified. Results Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001). With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance. Conclusions The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

Efficacy and pharmacokinetics of subcutaneous exendin (9-39) in patients with post-bariatric hypoglycaemia

To evaluate the efficacy, pharmacokinetic (PK) profile and tolerability of subcutaneous (s.c.). exendin 9‐39 (Ex‐9) injection in patients with post‐bariatric hypoglycaemia (PBH).

Pasireotide Induced Adrenal Insufficiency

Dear Editor, We report the case of secondary adrenal insufficiency in a 56year-old woman with a history of post-Roux-en-Y gastric bypass hyperinsulinaemic hypoglycaemia, undergoing experimental treatment with pasireotide. Pasireotide (SOM230) is a somatostatin analogue with a broader somatostatin receptor binding profile than other somatostatin analogs, like octreotide. Pasireotide binds somatostatin receptor (SSTR) 1,2,3 and 5 with high affinity. Pasireotide was shown to have long-lasting inhibitory effects on growth hormone and IGF-I release, as well as ACTH (adrenocorticotropic hormone), insulin, and to a lesser degree, glucagon. Pasireotide binds SSTR5, the receptor expressed predominantly by corticotroph adenomas, with 40-times greater affinity than octreotide, which is consistent with the observation that pasireotide is more potent than octreotide in suppressing basal ACTH release by corticotroph adenomas. Administration of subcutaneous pasireotide at 600–900 mg twice daily to humans with Cushing’s disease decreases tumour size and cortisol levels. The drug Signafor is approved for treatment of Cushing’s disease in Europe and the United States and is undergoing trials for treatment of acromegaly. Pasireotide has a half-life of 12 h, and pasireotide LAR (long-acting release) has a half-life of 16 days. A side effect of pasireotide, observed in healthy, acromegalic and Cushing’s patients, is hyperglycaemia. Colao, et al. reported 73% of acromegalic patients receiving pasireotide experienced hyperglycaemia. Thus, the drug is undergoing study for the treatment of hypoglycaemia in patients with dumping syndrome. Octreotide has been used to treat dumping syndrome. Due to its affinity for SSTR2, the sole regulator of glucagon secretion, octreotide poses a risk for worsening hypoglycaemia by blocking glucagon, inhibiting one of the main counterregulatory responses preventing hypoglycaemia. Alternatively, pasireotide has a strong affinity for SSTR5, which inhibits insulin but not glucagon secretion, and is hypothetically more effective in raising blood glucose. The glucoregulatory effect of pasireotide was evaluated in rats, demonstrating that pasireotide had a weaker inhibitory effect on glucagon secretion and a greater glucoseraising effect than octreotide. A 56-year-old woman with a history of obesity without diabetes mellitus underwent Roux-en-Y gastric bypass in 2007 at age 49, with weight loss of 125 pounds. Approximately 5 years after surgery, she began experiencing episodes of severe symptomatic hyperinsulinaemic hypoglycaemia, with measured blood glucose of 2 1 mmol/l and insulin of 111 12 pmol/l after an oral glucose load. Medications included ferrous sulphate, zoledronic acid, amitriptyline and oxycodone–acetaminophen (stable dose for 1 year prior and through ensuing treatment). She had never previously been prescribed exogenous steroids or received steroid injections. Other causes of hypoglycaemia were eliminated with negative sulfonylurea screen, negative anti-insulin antibodies, and normal IGF-2, plasma ACTH (4 4 pmol/l), renal and liver function tests. The patient was diagnosed with postbariatric hyperinsulinaemic hypoglycaemia. She was enrolled in a clinical trial (NCT01637272) with pasireotide 50mcg subcutaneous injection three times daily beginning July 2014 (Fig. 1). At nine weeks after first study drug injection, her dose was escalated to 100mcg three times daily. She had resolution of her hypoglycaemia. At thirteen weeks, she was transitioned to pasireotide LAR at a dose of 10 mg intramuscular injection monthly. She received her third dose of LAR injection at week 22 and reported feeling well. At week 24, she was seen in endocrinology clinic (Fig. 1, endocrine visit #1) with complaints of severe fatigue, exercise intolerance, and recurrent episodes of hypoglycaemia. Blood tests revealed normal electrolytes, haematocrit of 32% (unchanged from blood work prior to study enrolment), and normal thyroid function tests. An echocardiogram and chest X-ray were normal. She had a cosyntropin stimulation test (250 lg cosyntropin) at 9:00am. Serum cortisol values prior to and 1 h after stimulation were <27 nmol/l (110-662 nmol/l) and 157 nmol/l, respectively. Fasting ACTH concentration was <1 1 pmol/l (2 2-13 2 pmol/l). The patient was diagnosed with adrenal insufficiency, removed from the trial (events reported per study protocol), and started on hydrocortisone 10 mg in the morning and 5 mg in the evening. At 26 weeks, she was seen in endocrine clinic (Fig. 1, endocrine clinic visit #2), where a random serum cortisol (at 11:00am, after cessation of hydrocortisone for 24 h) was <27 6 nmol/l and ACTH was <1 1 pmol/l. Her dose of hydrocortisone was increased to 20 mg in the morning and 10 mg in the evening. At 33 weeks (11 weeks after her last injection of pasireotide LAR, Fig. 1, endocrine visit #3), after stopping her hydrocortisone dose for 24 h, she had a cosyntropin stimulation test (250 lg cosyntropin) at 9:00am. Serum cortisol prior to and 1 h after stimulation were 386 and 469 nmol/l, respectively. She reported feeling at baseline energy level, exercise tolerance, and pattern of hypoglycaemia. To confirm resolution of her secondary adrenal insufficiency, cosyntropin stimulation and plasma ACTH were performed at 69 weeks. Serum cortisol prior to and 1 h after stimulation were 497 and 745 nmol/l, respectively. ACTH had corrected to 5 06 pmol/l. We report herein the development of adrenal insufficiency in association with LAR pasireotide administration to a non-Cushing’s human subject. Colao, et al. reported hypocortisolismrelated adverse events in 8% of patients when evaluating pasireotide for the treatment of Cushing’s disease. Outside of that report, no further cases have been described. Our patient developed symptoms of hypocortisolism 24 weeks after initiating

Metabolic markers, regional adiposity, and adipose cell size: relationship to insulin resistance in African-American as compared with Caucasian women

Background / objectivesAfrican-American women have the greatest prevalence of obesity in the United States, and higher rates of type 2 diabetes than Caucasian women, yet paradoxically lower plasma triglycerides (TG), visceral fat and intrahepatic fat, and higher high-density lipoprotein (HDL)-cholesterol. Visceral fat has not been evaluated against insulin resistance in African-American women, and TG/HDL-cholesterol has been criticized as a poor biomarker for insulin resistance in mixed-sex African-American populations. Adipocyte hypertrophy, reflecting adipocyte dysfunction, predicts insulin resistance in Caucasians, but has not been studied in African-Americans. Our goal was to assess whether traditional correlates of insulin resistance, measures of adiposity and adipocyte characteristics similarly predict peripheral insulin resistance in African-American and Caucasian women.Subjects / methodsThirty-four healthy African-American (n = 17) and Caucasian (n = 17) women, matched for age (mean = 53.0 yrs) and body mass index (BMI) (mean = 30 kg/m2), underwent a steady-state plasma glucose test to measure insulin sensitivity; computed tomography (fat distribution); and a periumbilical scalpel biopsy (adipocyte characterization). By-race analyzes utilized analysis of covariance; linear regressions evaluated relationships between metabolic/adipose variables. All analyses adjusted for BMI and menopausal status.ResultsInsulin sensitivity did not differ between groups (p = 0.65). Neither BMI, nor %body fat or thigh fat predicted insulin resistance in African-American women. Fasting TG (p = 0.046), HDL-cholesterol (p = 0.0006) and TG/HDL-cholesterol ratio (p = 0.009) strongly predicted insulin resistance in African-American women. Despite being lower in African-American women, hepatic fat and visceral adipose tissue (VAT) correlated with insulin resistance in both groups, as did fasting glucose, VAT/SAT (subcutaneous adipose tissue) ratio, and %SAT (inverse).ConclusionsTotal adiposity measures and adipocyte hypertrophy did not predict insulin resistance in African-American women, but did in Caucasian women. Plasma TG and HDL-cholesterol were significant predictors of insulin resistance in African-American women. Our findings demonstrate the need to identify race and sex-specific biomarkers for metabolic risk profiling.