Symptoms and Dietary Impact in Hypertriglyceridemia-Associated Pancreatitis: Development and Content Validity of Two New Measures.

BACKGROUND: Severe hypertriglyceridemia (sHTG) is a rare condition, complicated by episodes of acute pancreatitis (AP), which can cause pain and/or life-threatening multi-organ dysfunction. Currently, there are no disease-specific patient-reported outcome (PRO) measures evaluating symptoms or dietary impact for this condition. OBJECTIVE: The objective of this study was to explore patient-reported symptoms and impacts of sHTG and AP and develop new measures to capture the symptoms and dietary impacts of this condition using patient language. METHODS: In-depth, semi-structured concept elicitation interviews were conducted with 12 US-based participants to explore their experience and identify key symptoms and impact on dietary behavior, both during and between episodes of AP. Participants had a range of AP severity with a previous triglyceride reading > 1000 mg/dL, and at least one attack of AP within the last 12 months. Transcripts were coded using thematic analysis. RESULTS: Qualitative data analysis revealed the substantial burden of AP associated with sHTG. Participants reported experiencing symptoms, especially abdominal pain, both during and between attacks of AP, and discussed considerable diet changes to prevent or minimize future attacks. A conceptual model was refined, based on patient input, and reviewed by clinical experts to determine key concepts for inclusion within two PRO measures, one evaluating symptoms and another evaluating impact on dietary behavior. Items were drafted using patient-derived language. A 19-item symptoms measure [Hypertriglyceridemia and Acute Pancreatitis Symptom Scale (HAP-SS)] and a 6-item dietary impact measure (Hypertriglyceridemia and Acute Pancreatitis Dietary Behavior (HAP-DB) measure) were developed, both with a 24-h recall period. CONCLUSIONS: The qualitative analysis confirmed the substantial burden of AP associated with sHTG. This research resulted in development of two disease-specific PRO measures for use during and between attacks of AP. These measures are being utilized in a clinical trial, which will confirm content, structure, and psychometric properties.

Induction of human beta-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6.

OBJECTIVE: Most knowledge on human beta-cell cycle control derives from immunoblots of whole human islets, mixtures of beta-cells and non-beta-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules-cyclins D1-3 and cdk 4 and 6-in the adult human beta-cell. RESEARCH DESIGN AND METHODS: Immunocytochemistry for the five molecules and their relative abilities to drive human beta-cell replication were examined. Human beta-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse. RESULTS: Human beta-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human beta-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control beta-cells). A single molecule, cdk6, proved to be capable of driving human beta-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin. CONCLUSIONS: Human beta-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent beta-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal beta-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human beta-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human beta-cell proliferation and function in vivo.

Survey of the human pancreatic beta-cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta-cell replication and function in vivo.

OBJECTIVES: To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human beta-cell replication, to determine whether human beta-cell replication can be demonstrated in an in vivo model, and to enhance human beta-cell function in vivo. RESEARCH DESIGN AND METHODS: Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human beta-cells were studied in both in vitro and in vivo models. RESULTS: Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human beta-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study. CONCLUSIONS: The human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human beta-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human beta-cell replication can be induced and studied in vivo.

Lessons from the first comprehensive molecular characterization of cell cycle control in rodent insulinoma cell lines.

OBJECTIVE: Rodent insulinoma cell lines may serve as a model for designing continuously replicating human beta-cell lines and provide clues as to the central cell cycle regulatory molecules in the beta-cell. RESEARCH DESIGN AND METHODS: We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics. RESULTS: 1) Despite their common T-antigen-derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat beta-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate beta-cell differentiation and function. CONCLUSIONS: These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human beta-cells.