The development of the immunoblot to detect and characterize a protein with an antisera, even in a crude mixture, was a breakthrough with wide-ranging and unpredictable applications across physiology and medicine. Initially, this technique was viewed as a tool for qualitative, not quantitative, analyses of proteins because of the high number of variables between sample preparation and detection with antibodies. Nonetheless, as the immunoblot method was streamlined and improved, investigators pushed it to quantitate protein abundance in unpurified samples as a function of treatment, genotype, or pathology. This short review, geared at investigators, reviewers, and critical readers, presents a set of issues that are of critical importance for quantitative analysis of protein abundance: 1) Consider whether tissue samples are of equivalent integrity and assess how handling between collection and assay influences the apparent relative abundance. 2) Establish the specificity of the antiserum for the protein of interest by providing clear images, molecular weight markers, positive and negative controls, and vendor details. 3) Provide convincing evidence for linearity of the detection system by assessing signal density as a function of sample loaded. 4) Recognize that loading control proteins are rarely in the same linear range of detection as the protein of interest; consider protein staining of the gel or blot. In summary, with careful attention to sample integrity, antibody specificity, linearity of the detection system, and acceptable loading controls, investigators can implement quantitative immunoblots to convincingly assess protein abundance in their samples.

Renin-angiotensin system blockade improves glucose intolerance and insulin resistance, which contribute to the development of metabolic syndrome. However, the contribution of impaired insulin secretion to the pathogenesis of metabolic syndrome is not well defined. To assess the contributions of angiotensin receptor type 1 (AT₁) activation and high glucose intake on pancreatic function and their effects on insulin signaling in skeletal muscle and adipose tissue, an oral glucose tolerance test (oGTT) was performed in five groups (n = 10/group) of rats: 1) lean strain-control 2) obese Otsuka Long-Evans Tokushima Fatty (OLETF), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg/kg · d olmesartan for 6 wk; OLETF ARB), 4) OLETF + 5% glucose water (HG) for 6 wk (OLETF HG), and 5) OLETF + HG + ARB (OLETF HG/ARB). The glucose response to the oGTT increased 58% in OLETF compared with lean-strain control, whereas glucose supplementation increased it an additional 26%. Blockade of angiotensin receptor reduced the oGTT response 19% in the ARB-treated groups and increased pancreatic insulin secretion 64 and 113% in OLETF ARB and OLETF HG/ARB, respectively. ARB treatment in OLETF ARB and OLETF HG/ARB did not have an effect on insulin signaling proteins in skeletal muscle; however, it reduced pancreatic AT₁ protein expression 20 and 27%, increased pancreatic glucagon-like peptide-1 (GLP-1) receptor protein expression 41 and 88%, respectively, and increased fasting plasma GLP-1 approximately 2.5-fold in OLETF ARB. The results suggest that improvement of glucose intolerance is independent of an improvement in muscle insulin signaling, but rather by improved glucose-stimulated insulin secretion associated with decreased pancreatic AT₁ activation and increased GLP-1 signaling.

Obesity is associated with the inappropriate activation of the renin-angiotensin system (RAS), which increases arterial pressure, impairs insulin secretion and decreases peripheral tissue insulin sensitivity. RAS blockade reverses these detriments; however, it is not clear whether the disease state of the organism and treatment duration determine the beneficial effects of RAS inhibition on insulin secretion and insulin sensitivity. Therefore, the objective of this study was to compare the benefits of acute vs chronic angiotensin receptor type 1 (AT) blockade started after the onset of obesity, hyperglycemia and hypertension on pancreatic function and peripheral insulin resistance. We assessed adipocyte morphology, glucose intolerance, pancreatic redox balance and insulin secretion after 2 and 11 weeks of AT blockade in the following groups of rats: (1) untreated Long-Evans Tokushima Otsuka (lean control; = 10), (2) untreated Otsuka Long-Evans Tokushima Fatty (OLETF; = 12) and (3) OLETF + ARB (ARB; 10 mg olmesartan/kg/day by oral gavage; = 12). Regardless of treatment duration, AT blockade decreased systolic blood pressure and fasting plasma triglycerides, whereas chronic AT blockade decreased fasting plasma glucose, glucose intolerance and the relative abundance of large adipocytes by 22, 36 and 70%, respectively. AT blockade, however, did not improve pancreatic oxidative stress or reverse impaired insulin secretion. Collectively, these data show that AT blockade after the onset of obesity, hyperglycemia and hypertension improves peripheral tissue insulin sensitivity, but cannot completely reverse the metabolic derangement characterized by impaired insulin secretion once it has been compromised.

Angiotensin II (Ang II) and aldosterone contribute to hypertension, oxidative stress and cardiovascular damage, but the contributions of aldosterone during Ang II-dependent hypertension are not well defined because of the difficulty to assess each independently. To test the hypothesis that during Ang II infusion, oxidative and nitrosative damage is mediated through both the mineralocorticoid receptor (MR) and angiotensin type 1 receptor (AT1), five groups of Sprague-Dawley rats were studied: 1) control, 2) Ang II infused (80 ng/min × 28d), 3) Ang II + AT1 receptor blocker (ARB; 10 mg losartan/kg/d × 21d), 4) Ang II + mineralocorticoid receptor (MR) antagonist (Epl; 100 mg eplerenone/d × 21d) and 5) Ang II + ARB + Epl (Combo; × 21d). Both ARB and combination treatments completely alleviated the Ang II-induced hypertension, whereas eplerenone treatment only prolonged the onset of the hypertension. Eplerenone treatment exacerbated the Ang II-mediated increase in plasma and heart aldosterone 2.3- and 1.8-fold, respectively, while ARB treatment reduced both. Chronic MR blockade was sufficient to ameliorate the AT1-mediated increase in oxidative damage. All treatments normalized protein oxidation (nitrotyrosine) levels; however, only ARB and Combo treatments completely reduced lipid peroxidation (4-hydroxynonenal) to control levels. Collectively, these data suggest that receptor signaling, and not the elevated arterial blood pressure, is the principal culprit in the oxidative stress-associated cardiovascular damage in Ang II-dependent hypertension.

Angiotensin II (Ang II) and aldosterone (aldo) contribute to cardiovascular damage via oxidative stress, but their respective contributions remain elusive. To independently assess the contributions of Ang II and aldo during Ang II‐dependent hypertension, five groups of Sprague Dawley rats (n =11‐14/group) were studied: 1) Control, 2) Ang II infused (80 ng/min x 28d), 3) Ang II + angiotensin receptor blocker (ARB; 10 mg losartan/kg/d x 21d), 4) Ang II + mineralocorticoid receptor antagonist (Epl; 100 mg eplerenone/d x 21d), and 5) Ang II + ARB + Epl (Combo). ARB and Combo reduced the 65% Ang II‐induced increase in systolic blood pressure (SBP), but Epl had no effect. Epl exacerbated the increase in plasma and heart aldo while ARB reduced both. Ang II increased relative heart mass 19% and heart cardiotrophin‐1 content 57%. All treatments prevented these increases. Ang II reduced mean glutathione peroxidase activity 29%, while ARB and Combo recovered levels beyond Control by 25 and 28%, respectively. Ang II also increased superoxide dismutase (SOD) activity 20% whereas all treatments reduced SOD. Ang II increased mean heart nitrotyrosine (NT) content 3‐fold and mean 4‐hydroxynonenal (HNE) content 26%. All treatments improved NT, but Epl had no effect on HNE levels. Despite the inability to reduce SBP, chronic MR blockade prevented oxidative stress suggesting aldo as a therapeutic target for AT1‐mediated oxidative damage. Grant Funding Source: Supported in parts by: NHLBI K02HL103787/R01HL091767, NIH NIMHD T37MD001480, NIH

Activation of the renin‐angiotensin system (RAS) has been shown to contribute to metabolic syndrome. Hyptertension is a key component of metabolic syndrome and is regulated by RAS. The Otsuka Long‐Evans Tokushima Fatty (OLETF) rat is a relatively new and highly relevant model because its pathogenesis of insulin resistance, metabolic syndrome, and type 2 diabetes closely resembles that of the progression of the human condition. However, the contribution of adipose RAS to metabolic syndrome is not well defined. To address the hypothesis that adipose RAS activation is increased with insulin resistance, rats were divided into two groups (n=10–12/group): 1) lean strain‐control LETO and 2) obese insulin resistant OLETF. Systolic blood pressure (SBP) was measured weekly for 24 weeks and insulin resistance index (IRI) was determined at 9 and 24 weeks. Tissue samples were collected from each group (n=5–6/group) at 15 and 24 weeks. Mean SBP from OLETF was 28% and 27% greater compared to that of the LETO after 15 and 24 wk respectively. Mean IRI was greater at both time periods and was exacerbated in 24 wk OLETF compared to 15 wk. By 24 wk, adipose angiotensinogen (angiotensin precursor) expression had increased in 24 wk OLETF suggesting that activation of adipose RAS was increased. The data suggest that increased activation of adipose RAS may contribute to the increased metabolic disorders (SBP and IRI) commonly associated with metabolic syndrome.

High fat diet (HFD) accelerates diabetic nephropathy via impaired sodium (Na+) regulation and improper insulin signaling. Angiotensin II (AngII) receptor (AT1) blockers (ARBs) ameliorate insulin resistance, antinatriuresis, and elevated systolic blood pressure (SBP). However, the renoprotective effects of ARB during HFD remain elusive. To assess the role of AT1 activation to improper Na+ handling in a model of metabolic syndrome (OLETF) supplemented a HFD, seven groups were studied: 1) untreated, lean LETO, 2) LETO HFD (62% fat), 3) LETO HFD + ARB (10 mg olmesartan/kg/d), 4) untreated, obese OLETF, 5) OLETF HFD, 6) OLETF ARB, and 7) OLETF HFD + ARB. ARB reduced SBP in both OLETF (151 ± 3 mmHg vs 114 ± 3) and OLETF HFD (151 ± 2 vs 101 ± 6). Creatinine clearance decreased 35% in OLETF compared to LETO and increased 2‐fold in OLETF HFD compared to OLETF. Fractional excretion of Na+ (FENa) decreased 40% in OLETF compared to LETO. HFD decreased FENa 50‐75%, whereas ARB increased FENa in all cases. HFD increased urine aldosterone excretion 35% in both LETO and OLETF. Also, HFD increased potassium FE (FEK) 2 and 4‐fold in OLETF and LETO, respectively. ARB reduced FEK levels in all groups. Also, HFD increase levels of cleaved γ‐Epithelial Na+ Channel (ENaC) content 31% in OLETFs. Collectively, these data indicate that HFD exacerbates impaired renal function by increased ENaC abundance during Ang II‐mediated hypertension. Grant Funding Source: Supported by: NIH MHIRT and USDA Minority Research Training Fellowship

High fat diet (HFD) is known to accelerate diabetic nephropathy (DN) while blockade of the renin angiotensin system (RAS) improves the proteinuria and antinatriuresis associated with DN. However, whether RAS blockade can improve defects in sodium (Na+) handling resulting from HFD induced insulin resistance remains unknown. To assess the effect of HFD on the epithelial Na+ channel (ENaC), which regulates Na+ transport and balance and is regulated by the insulin‐signaling pathway, we performed Western blot and urinary analyses on the following groups (6 weeks of treatment): 1) untreated, lean LETO, 2) LETO + HFD, 3) LETO HFD + ARB, 4) untreated OLETF, 5) OLETF + HFD, 6) OLETF + ARB, and 7) OLETF + HFD + ARB. ARB reduced SBP in both OLETF (114 ± 3 vs 151 ± 3 mmHg) and OLETF HFD (101 ± 6 vs 151 ± 2). Urine Na+ excretion (UNaV) decreased 50% in OLETFs compared to LETOs. These data suggest that impaired UNaV and overactivation of AT1 may contribute to the manifestation of hypertension in insulin resistant rats. Treatment with ARB increased UNaV in all treatment groups by 3 to 4 fold and HFD decreased UNaV by 70 and 30% in LETOs and OLETFs, suggesting that a HFD may contribute to activation of AT1 in the kidney, resulting in impaired Na+ regulation. Levels of cleaved γ‐ENaC (60 kDa) negatively correlated with UNaV indicating that cleavage in vivo is likely a key component in the regulation of UNaV and blood pressure in insulin resistant conditions.